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Hydrogen sulfide protects against endoplasmic reticulum stress and mitochondrial injury in nucleus pulposus cells and ameliorates intervertebral disc degeneration
Accepted Manuscript Title: Hydrogen sulfide protects against endoplasmic reticulum stress and mitochondrial injury in nucleus pulposus cells and ameliorates intervertebral disc degeneration Author: Daoliang Xu Haiming Jin Jianxia Wen Jiaoxiang Chen Deheng Chen Ningyu Cai Yongli Wang Jianle Wang Yu Chen Xiaolei Zhang Xiangyang Wang PII: DOI:
S1043-6618(16)31163-X http://dx.doi.org/doi:10.1016/j.phrs.2017.01.005
Reference:
YPHRS 3466
To appear in:
Pharmacological Research
Received date: Revised date: Accepted date:
2-11-2016 17-12-2016 5-1-2017
Please cite this article as: Xu Daoliang, Jin Haiming, Wen Jianxia, Chen Jiaoxiang, Chen Deheng, Cai Ningyu, Wang Yongli, Wang Jianle, Chen Yu, Zhang Xiaolei, Wang Xiangyang.Hydrogen sulfide protects against endoplasmic reticulum stress and mitochondrial injury in nucleus pulposus cells and ameliorates intervertebral disc degeneration.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2017.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
H2S inhibits apoptosis of NP cells
1
Graphical abstract
2 3
1 / 42
H2S inhibits apoptosis of NP cells
4
Hydrogen sulfide protects against endoplasmic reticulum stress and mitochondrial
5
injury in nucleus pulposus cells and ameliorates intervertebral disc degeneration
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Ttile Page
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Daoliang Xu1, Haiming Jin1, Jianxia Wen2, Jiaoxiang Chen1, Deheng Chen1, Ningyu Cai1,
8
Yongli Wang4, Jianle Wang1, Yu Chen1, Xiaolei Zhang*1,3, Xiangyang Wang*1
9
1
Department of Orthopaedic Surgery, the Second Affiliated Hospital of Wenzhou Medical
10
University, Wenzhou, Zhejiang, China
11
2
12
University, Wenzhou, Zhejiang, China
13
3
Zhejiang Provincial Key Laboratory of Orthopaedics, Wenzhou, Zhejiang Province, China.
14
4
Department of Orthopaedic Surgery, Huzhou Central Hospital, Huzhou, China
15
*Correspondence to: Xiangyang Wang, 1Department of Orthopaedic Surgery, The Second
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Affiliated Hospital of Wenzhou Medical University, 109 Xueyuan Xi Road, Wenzhou,
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Zhejiang, 325027, China
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Phone: 86-577-88829799
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Fax: 86-577-88879123
20
Email: [email protected]
21
*Correspondence to: Xiaolei Zhang, 1Department of Orthopaedic Surgery, The Second
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Affiliated Hospital of Wenzhou Medical University, 109 Xueyuan Xi Road, Wenzhou,
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Zhejiang, 325027, China
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Phone: 86-577-88002823
Department of Gastrointestinal Surgery, the First Affiliated Hospital of Wenzhou Medical
2 / 42
H2S inhibits apoptosis of NP cells
25
Fax: 86-577-88002823
26
Email: [email protected]
27
Abstract
28
It has been suggested that excessive apoptosis in intervertebral disc cells induced by
29
inflammatory cytokines, such as interleukin (IL)-1β, is related to the process of intervertebral
30
disc degeneration (IVDD). Hydrogen sulfide (H2S), a gaseous signaling molecule, has drawn
31
attention for its anti-apoptosis role in various pathophysiological processes in degenerative
32
diseases. To date, there has been no investigation of the correlation of H2S production and
33
IVDD or of the effects of H2S on IL-1β-induced apoptosis in nucleus pulposus (NP) cells.
34
Here, we found that the expression levels of cystathionine β-synthase (CBS) and
35
cystathionine γ-lyase (CSE), two key enzymes in the generation of H2S, were significantly
36
decreased in human degenerate NP tissues as well as in IL-1β-treated NP cells. NaHS (H2S
37
donor) administration showed a protective effect by inhibiting the endoplasmic reticulum (ER)
38
stress response and mitochondrial dysfunction induced by IL-1β stimulation in vitro, the
39
effect was related to activation of the PI3K/Akt and ERK1/2 signaling pathways. Suppression
40
of these pathways by specific inhibitors, LY294002 and PD98059, partially reduced the
41
protective effect of NaHS. Moreover, in the percutaneous needle puncture disc degeneration
42
rat tail model, disc degeneration was partially reversed by NaHS administration. Taken
43
together, our results suggest that H2S plays a protective role in IVDD and the underlying
44
mechanism involves PI3K/Akt and ERK1/2 signaling pathways-mediated suppression of ER
45
stress and mitochondrial dysfunction in IL-1β-induced NP cells. 3 / 42
H2S inhibits apoptosis of NP cells
46
Key words: Hydrogen sulfide, intervertebral disc degeneration, endoplasmic reticulum stress,
47
mitochondrial injury, apoptosis.
48 49
1. Introduction
50
Intervertebral disc degeneration (IVDD) is a spinal disorder whose pathogenesis has not
51
been fully elucidated. It has been suggested that nucleus pulposus (NP) cells which produce
52
cartilage-specific extracellular matrix components play an important role in IVDD.[1]
53
Aberrant apoptosis [2-4] of NP cells has been considered to be the major cellular process
54
associated with IVDD. Studies have demonstrated that several pathological conditions may
55
lead to apoptosis of NP cells, among which abnormally increased inflammatory cytokines,
56
such as IL-1β and tumor necrosis factor-alpha, were considered to play a crucial role in the
57
process of IVDD. Through a series of signaling networks, inflammatory cytokines may
58
induce NP cell apoptosis, which further result in progressive IVDD.[5]
59
The endoplasmic reticulum (ER) is an important organelle responsible for cellular
60
homeostasis. When exposed to prolonged or strong intracellular and extracellular stimuli, the
61
ER stress-related proteins such as caspase 12 will be activated, which may ultimately lead to
62
apoptotic cell death, therefore ER stress is closely related to apoptosis. Another major event in
63
apoptosis is mitochondrial process, and the Bcl-2 protein family is involved not only in the
64
alteration of mitochondrial membrane potential but also in the release of mitochondrial
65
apoptotic factors.[6] Recently, intervertebral disc cell apoptosis was shown to be mediated by
66
both the endoplasmic reticulum (ER) and the mitochondrial pathways in rats.[7] Zhang et al. 4 / 42
H2S inhibits apoptosis of NP cells
67
reported that annular cell apoptosis induced by cyclic stretch was partially reversed by the
68
inhibition of ER stress in rats.[8] And, there is evidence showing that mitochondrial pathway
69
contribute to fas-mediated apoptosis of human lumbar disc cells.[9] These studies suggest an
70
emerging picture of the importance of the mitochondria and ER in IVDD.
71
Hydrogen sulfide (H2S) is synthesized from L-cysteine primarily by two key enzymes:
72
cystathionine-c-lyase (CSE) in the peripheral tissues and cystathionine-b-synthetase (CBS) in
73
the central nervous system. Recognized as the third endogenously produced gaseous
74
messenger, along with nitric oxide and carbon monoxide, H2S regulates a variety of biological
75
functions including anti-inflammatory[10] and anti-apoptotic[11] effects, affecting several key
76
mechanisms and pathways both in vitro and in vivo. Recent studies have suggested possible
77
roles for H2S in modulating ER stress-induced cardiovascular disease. In a rat model of
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hyperhomocysteinemia-induced cardiomyocytic ER stress injury and in doxorubicin-induced
79
H9c2 cells, H2S supplementation was shown to antagonize cytotoxicity and apoptosis through
80
inhibiting ER stress-associated proteins.[12, 13] Similarly, a study on formaldehyde-induced
81
neurotoxicity in PC12 cells showed that H2S pre-treatment significantly attenuated the
82
increases in the expression levels of ER stress markers.[14] Additionally, it was recently
83
demonstrated that H2S may reconcile Akt2 knockout-induced myocardial contractile defects
84
via attenuation of mitochondrial injury and apoptosis.[15] However, to our knowledge,
85
whether the production of H2S is correlated with IVDD and whether H2S can ameliorate ER
86
stress and mitochondrial dysfunction-induced apoptosis in human NP cells remain
87
unanswered. 5 / 42
H2S inhibits apoptosis of NP cells
88
This study aimed to illustrate the role of H2S in IVDD as well as its working mechanism.
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Our results suggest that H2S protects against apoptosis in NP cells in vitro and ameliorates
90
disc degeneration in rats in vivo. We believe that this study may help us to better understand
91
the role of H2S in pathogenesis of IVDD.
92 93
2. Materials and Methods
94
2.1 Ethics statement
95
All surgical interventions, treatments and postoperative animal care procedures were
96
performed in strict accordance with the Animal Care and Use Committee of Wenzhou
97
Medical University (wydw2014-0129).
98 99
2.2 Nucleus Pulposus Cell Isolation and Culture
100
The study was approved by the Second Affiliated Hospital of Wenzhou Medical University
101
Ethics Committee. Patients, between 11 years and 76 years old (A total of 43 disc samples
102
were collected from 38 patients, in total of 17 female and 21 male patients with scoliosis or
103
disc herniation or vertebral fracture), signed an informed consent form allowing the
104
researchers to use NP tissues obtained during spinal surgery. 30 disc samples (Pfirrmann <
105
grade III, and 4 were defined as grade I) were classified as non-degenerative IVD tissue
106
samples according to the Pfirrmann grading scale[16] determined by magnetic resonance
107
imaging (MRI), total 17 disc samples were used in Pfirrmann grading scale（2 male and 2
108
female, age from 19-45, average 34.3± 10.9 in grade Ⅰ; 3 male and 3 female, age from 25-46, 6 / 42
H2S inhibits apoptosis of NP cells
109
average 35.8± 7.2 in grade Ⅲ; 4 male and 3 female, age from 38-47, average 42.2± 4.8 in
110
grade Ⅴ）, which were only used for gene analysis and western blot. And there was no
111
significant difference among the three Pfirrman grade groups in age or sex. In this study, no
112
other complications related to IVDD, such as systemic disease (Diabetes, etc.) were found in
113
patients that consented to donate their NP tissues. The gel-like NP tissues were digested in
114
0.25% trypsin and 0.2% type II collagenase (Gibco) for approximately 3 hrs at 37°C. Then,
115
the digested tissues were transferred as explants to Dulbeccomodified Eagle medium (DMEM;
116
Gibco, Invitrogen, Grand Island, NY) with 10% fetal bovine serum (FBS; Hyclone, Thermo
117
Scientific, Logan, UT) and antibiotics (1% penicillin/ streptomycin) in a 5% CO2 incubator.
118
When confluent, the cells were passaged by using 0.25% Trypsin-EDTA (Gibco, Invitrogen),
119
and subcultured in 60-mm culture dish at the appropriate density. During passaging, no
120
significant changes in morphology of cells between primary cells (passage 0) and later
121
passage cells (passage 2) were noticed.
122 123
2.3 Cell Culture Treatment Protocols
124
To determine whether degeneration induced changes in H2S-generating and -metabolizing
125
enzymes, NP cells were treated with IL-1β (10 ng/ml) (PeproTech, Rocky Hill, USA) for 24 or
126
48hrs. In order to evaluate the effect of NaHS（Sigma-Aldrich, St. Louis, MO) on the viability
127
of NP cells, cells were incubated for 24 hrs with increasing concentrations (0.06–1 mM) of
128
NaHS. To establish the apoptosis model of NP cells, different concentrations of IL-1β (10 -
129
100 ng/ml) were added into the culture medium for 24 hrs. Cells were pre-treated with 7 / 42
H2S inhibits apoptosis of NP cells
130
different concentrations of NaHS for 1 hr before the addition of IL-1β (75 ng/ml) to
131
investigate its effect on cell apoptosis. To study the role of PI3K/Akt and ERK1/2 signal
132
pathways in NaHS-induced cell protection, NP cells were pre-treated with 1mM NaHS and 50
133
μM LY294002 (PI3K/Akt inhibitor; Cell Signaling Technology, MA, USA) or PD98059
134
(ERK1/2 inhibitor; Beyotime, Shanghai, China) for 1hr before they received IL-1β
135
administration.
136 137
2.4 Cell Viability Assay
138
Cell viability was assayed with the cell counting kit-8 (CCK-8; Dojindo Co, Kumamoto,
139
Japan) according to the manufacture’s protocol. NP cells were treated with NaHS, IL-1β,
140
LY294002 and PD98059 as described above. After treatment the cells were washed with PBS,
141
then 100 μl of DMEM containing 10 μl of CCK-8 solution was added to each well, and the
142
plate was incubated for an additional 1 hr. The absorbance of wells was then measured at 450
143
nm by a micro-plate reader.
144 145
2.5 Western Blot Assay
146
The human NP tissues and cells were lysed in ice-cold radio-immuneprecipitation assay
147
(RIPA) buffer, supplemented with phosphatase inhibitors and protease inhibitor cocktail
148
(Biotech Well, Shanghai, China). Protein concentration was measured by the BCA protein
149
assay kit (Beyotime, Shanghai, China), and equivalent amounts of protein were separated by
150
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 8 / 42
H2S inhibits apoptosis of NP cells
151
polyvinylidenedifluoride (PVDF) membrane (BIO-RAD, USA). Then the protein was probed
152
with antibody specific to CBS, CSE, CHOP, cytochrome c (Santa Cruz Biotechnology, CA,
153
USA), ERK, p-ERK, Akt, p-Akt, cleaved-caspase3, Bax, Bcl-2 (Cell Signaling Technology,
154
Beverly, MA, USA), ATF-6 (Bioworld, MN, USA), GAPDH (Arigo, Taiwan, ROC), GRP78
155
and Caspase12 ( Abcam, Cambridge, UK) followed by ECL signal detection (Invitrogen).
156 157
2.6 Real-time PCR
158
Total RNA was extracted from NP cells by Trizol reagent (Invitrogen, USA). One
159
microgram of total RNA was used to synthesize cDNA (MBI Fermantas, Germany).
160
Real-time polymerase chain reaction reactions were done in triplicate in 96-well plates using a
161
SYBR Premix Ex Taq Kit (Takara, Japan) in a final volume of 20 μl. The reaction and
162
detection were conducted in a light-cycle (Roche, Mannheim, Germany). The cycle threshold
163
(Ct) values were collected and normalized to the level of the housekeeping gene GAPDH. The
164
△△Ct method was used to calculate the relative mRNA levels of each target gene. The
165
primers
166
CBS(F)5’-GACCAAGTTCCTGAGCGACA-3’,(R)5’-CGGAGGATCTCGATGGTGTG-3’;C
167
SE(F)5’-GGCTCTACCTGCGTGCTTTA-3’,(R)5’-GCGAAATGTTGGAAGTGTGG-3’.
of
CBS
and
CSE
were
listed
as
follows:
168 169
2.7 siRNA Transfection
170
According to the manufacturer’s instructions, human nucleus pulposus cells were
171
transfected with CBS or CSE siRNA (RiboBio, Guangzhou, China) using Lipofectamine 9 / 42
H2S inhibits apoptosis of NP cells
172
RNAiMAX reagent (Invitrogen). Following further treatments, the transfected cells for
173
subsequent analysis.
174 175
2.8 mPTP Opening
176
To ascertain the role of NaHS in IL-1β-induced mitochondrial dysfunction, mitochondrial
177
permeability transition pore (mPTP) was determined by using the calcein AM/CoCl2
178
fluorescence-quenching assay according to the manufacturer’s protocol. Initially, after the
179
treatment on NP cells as described above, cells were treatment with calcein AM in the
180
absence of cobalt, resulting in the calcein fluorescence diffuse throughout the whole cell.
181
IL-1βstimuli can lead to translocation of Ca2+ from cytoplasm to mitochondria, resulting in
182
mPTP opening. Thus, only the calcein fluorescence outside of mitochondria should be
183
quenched with the addition of cobat in cells with mitochondrial dysfunction.
184 185
2.9 MMP Assay
186
The mitochondrial transmembrane potential (MMP) was assessed by using MitoTracker
187
Red CMXRos (Molecular Probes™, Thermo Fisher Scientific Inc.), a red-fluorescent dye,
188
which accumulation is dependent upon membrane potential, stains mitochondria in live cells.
189
NP cells after 24hrs treatment were stained at a final concentration of of 50 nM for 30 min at
190
37 °C, and nuclei were stained with Hoechst 33258 dye. Red fluorescence images of at least
191
three random microscopic fields were acquired per slide for microscopic observation with a
192
fluorescence microscope (Olympus Inc., Tokyo, Japan), and fluorescence intensity was 10 / 42
H2S inhibits apoptosis of NP cells
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measured using Image J software 2.1 (Bethesda, MD, USA) by observers who were blinded
194
to the experimental groups
195 196
2.10 ATP Assay
197
The ATP-Glo™ Bioluminometric Cell Viability Assay (Biotium, Hayward, CA, USA), was
198
used to assessed the cell ATP levels according to the manufacturer’s protocol. Data were
199
collected from multiple replicate wells for each experiment.
200 201
2.11 Transmission Electron Microscopy (TEM)
202
NP cells were fixed in 2.5% glutaraldehyde at 4r overnight, then postfixed in 2% osmium
203
tetroxide for 1 hr and stained with 2% uranyl acetate for 1 hr. After dehydration in an
204
ascending series of acetone, samples were embedded into araldite to cut into semi-thin
205
sections, then, stained with toluidine blue to locate cells. Sections were examined with a
206
transmission electron microscope (Hitachi, Tokyo, Japan).
207 208
2.12 Immunofluorescence
209
For Drp 1 (Abcam, Cambridge, UK) and Tom20 (Abcam, Cambridge, UK) staining, cells
210
were fixed with the 4% paraformaldehyde and blocked in PBS containing Triton X-100 for 10
211
mins, and then pretreated with blocking solution (5% bovine serum albumin) for 30 mins,
212
cells were then incubated with a cocktai of anti-Drp 1 (1:250) and anti-Tom20 (1:200)
213
overnight at 4 °C in a humidified chamber. On the next day, cells were washed and incubated 11 / 42
H2S inhibits apoptosis of NP cells
214
with fluorescein isothiocyanate- or tetramethyl rhodamine isothiocyanate-conjugated second
215
antibodies for 1 hr and labeled with DAPI for 5 mins. Finally, three fields of each slide were
216
chosen randomly for microscopic observation with a fluorescence microscope (Olympus Inc.,
217
Tokyo, Japan).
218 219
2.13 JC-1 Assay
220
Following the above treatment, NP cells were stained with 10 µM of JC-1 according to the
221
manufacturer instructions (Beyotime, Shanghai, China). Then, cells were analyzed using a
222
flow cytometer. Results were expressed as the percentage of cells that were double positive.
223 224
2.14 TUNEL Method
225
The terminal deoxynucleotidyltransferase (TdT) dUTP nick end labeling (TUNEL) method
226
is a technique for measuring apoptotic DNA fragmentations. After fixed with a freshly
227
prepared 4% paraformaldehyde for 1 hr, NP cells were incubated with 3% H2O2 and 0.1%
228
Triton X-100 for 10mins and washed with PBS for three times in every step. According to
229
manufacturer’s instructions, cells were stained with in situ cell death detection kit (F.
230
Hoffmann-La Roche Ltd., Basel, Switzerland) and 40, 6-diamidino-2-phenylindole (DAPI).
231
Finally，three random microscopic fields were acquired per slide for microscopic observation
232
with a fluorescence microscope (Olympus Inc., Tokyo, Japan).
233 234
2.15 Surgical Procedure 12 / 42
H2S inhibits apoptosis of NP cells
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A total of 48, 3-month-old male Sprague Dawley rats were randomly divided into three
236
groups (control group, saline group and NaHS group), then weighed and injected
237
intraperitoneally with 2% (w/v) pentobarbital (40 mg/kg). The saline and NaHS group went
238
through the model operation. As described in the previous study[17], the experimental level
239
rat tail disc (Co7/8) was located by digital palpation on the coccygeal vertebrae and confirmed
240
by counting the vertebrae from the sacral region in a trial radiograph. Needles (20G, about
241
4mm in length) were used to puncture the whole layer of annulus fibrosus (AF) though the tail
242
skin perpendicularly. All the needles were rotated 360° and held in position for 30 seconds
243
before extraction. After surgery, in the NaHS group, the NaHS solution was immediately
244
injected intraperitoneally to deliver a dose of 100 μmol/kg/day until the rats were sacrificed.
245
Daily monitoring of the rats was carried out to ensure their well-being and all animals were
246
allowed free unrestricted weight bearing and activity.
247 248 249 250
2.16 Magnetic Resonance Imaging Method. Before modeling and after 4 or 8 weeks of puncture, the animals were given the MRI examination.
251
Magnetic resonance imaging was performed on all rats to evaluate the signal and structural
252
changes in sagittal T2-weighted images using a 3.0 T clinical magnet (Philips Intera Achieva
253
3.0MR). T2-weighted sections in the sagittal plane were obtained in the following settings:
254
fast-spin echo sequence with time to repetition (TR) of 5400 ms and time to echo (TE) of 920
255
ms; 320 (h) 9 256 (v) matrix; field of view of 260; and 4 excitations. The section thickness 13 / 42
H2S inhibits apoptosis of NP cells
256
was 2 mm with a 0-mm gap. The MRIs were evaluated by another blinded orthopedic
257
researcher using the classification of intervertebral disk degeneration as reported by Pfirrmann
258
et al.[16] (1 point = Grade I, 2 points = Grade II, 3 points = Grade III, 4 points = Grade IV).
259 260
2.17 Histopathologic Analysis
261
The rats were sacrificed by an intraperitoneal overdosage injection of 10% chloral hydrate
262
and the tails were harvested on week 4 and 8 after surgery. The specimens were fixed in
263
formaldehyde and decalcified, then dehydrated and embedded in paraffin. The tissues were
264
cut into 5 μm sections. Two midsagittal sections of each disc were stained with safranin o-fast
265
green and hematoxylin, and hematoxylin and eosin respectively. The cellularity and
266
morphology of NP were examined by another experienced histology researchers in a blinded
267
fashion using a microscope, and evaluated by using a grading scale, as described
268
previously[17]. The histologic score was 5 for normal disc, 6–11 for moderately degenerated
269
disc, and 12–14 for severely degenerated disc.
270 271
2.18 Statistical Analysis
272
The experiments were performed at least three times. The results were presented as mean ±
273
SD. Statistical analyses were performed using SPSS statistical software program 20.0. Data
274
were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s test for
275
comparison between control and treatment groups. Nonparametric data (Pfirrmann grading)
276
were analyzed by the Kruskal–Wallis H test. Statistical significance was set at p < 0.05. 14 / 42
H2S inhibits apoptosis of NP cells
277
Statistical significance was set at p < 0.05.
278 279
3. Results
280
3.1 H2S generation is decreased in degenerate disc tissues and in IL-1β-treated NP cells
281
CBS and CSE are two major enzymes involved in the endogenous generation of
282
H2S.[18-20] Sulfide:quinone oxidoreductase (SQOR) is a membrane-bound enzyme that
283
catalyzes the first step in the mitochondrial metabolism of H2S.[21] To determine whether
284
degeneration induced changes in H2S-generating and -metabolizing enzymes in NP cells,
285
human NP tissues (Fig. 1A, Pfirrmann grade I to gradeⅤ) and cells were harvested for the
286
measurement of three enzymes by Western blotting and real-time PCR. We found that CBS
287
and CSE protein levels in intervertebral disc tissues decreased significantly with the degree of
288
degradation in vivo (Fig. 1B), and the results from IL-1β-induced NP cells degeneration in
289
vitro corroborated previous findings in degenerated human disc in a time-dependent manner
290
(Fig. 1C). Similarly, IL-1β exposure led to decreased gene expression (Fig. 1D, data not
291
shown for NP tissues), whereas there was no significant change in the SQOR protein or
292
mRNA level (data not shown). These results suggest that endogenous H2S may play an
293
important role in maintaining the normal function of NP cells under physiological conditions.
294
Fig.1
15 / 42
H2S inhibits apoptosis of NP cells
295 16 / 42
H2S inhibits apoptosis of NP cells
296
3.2 Inhibition of CBS or CSE by siRNA significantly increased IL-1β-induced apoptosis
297
in NP cells.
298
Since the level of the gasotransmitter hydrogen sulfide involved in physiological and
299
pathological responses is micro, and the dose of exogenous hydrogen sulfide used in vivo or
300
vitro seems to be too high. To further investigate the role of endogenous hydrogen sulfide in
301
IL-1β-induced apoptosis in NP cells, cells were transfected with CBS or CSE-siRNA before
302
stimuli. CBS or CSE-siRNA transfection into human NP cells led to the expression of CBS
303
and CSE significantly decreased in gene level (Fig.2A), and the result was further confirmed
304
by western blotting (Fig.2B,C). Compared with control siRNA group, the inhibitory of the
305
synthetase further exacerbated IL-1β-induced apoptosis in siRNA group (Fig2.D-F). These
306
results suggested that H2S is involved in the pathological process of IVDD.
307
Fig.2
308 17 / 42
H2S inhibits apoptosis of NP cells
309
3.3 NaHS treatment inhibits apoptosis in NP cells
310
Because H2S is toxic at high concentrations, a dose–response study with varying doses (0 –
311
4 mmol/L) of NaHS was performed to investigate the susceptibility of NP cells to exogenous
312
H2S. As shown in Figure 3A, low concentrations of NaHS did not alter cell viability, while
313
cellular viability decreased significantly at concentrations of 2 mmol/l and higher. Indeed,
314
doses of NaHS from 0.5 to 1 mmol/L slightly promoted cell proliferation, leading to an
315
increase in cell viability, peaking at 1 mmol/L. IL-1β (75 ng/mL) resulted in a marked
316
decrease in cell viability (Fig. 3B). This reduction in cell viability could be effectively
317
increased by pre-treatment with 1 mmol/L NaHS (Fig. 3C). To further assess whether NaHS
318
modulated cellular apoptosis, levels of cleaved caspase3, Bax, and Bcl-2 were analyzed by
319
western blot. The results showed that NaHS significantly reversed the increasing level of
320
cytochrome c and cleaved caspase 3 due to IL-1β (Fig. 3D, E). Similarly, levels of the
321
pro-apoptotic protein Bax were down-regulated, whereas the anti-apoptotic protein Bcl-2 was
322
up-regulated by pre-treatment with NaHS.
323
Fig.3
18 / 42
H2S inhibits apoptosis of NP cells
324 325
3.4 NaHS prevents IL-1β-induced mitochondrial dysfunction
326
IL-1β can trigger sustained mitochondrial permeability transition pore opening, alter
327
mitochondrial membrane potential and ATP content via mitochondrial Ca2+ uptake[22].To
328
examine the protective effect of NaHS on IL-1β-induced mitochondria damage, mPTP
329
opening, ATP and mitochondrial membrane potential were determined after IL-1β
330
administration. As shown in Fig.4A, calcein fluorescence was found to diffuse throughout the
331
entire NP cells without cobalt in control group, while calcein fluorescence outside of
332
mitochondria was quenched in control cells for the addition of cobalt. IL-1β treatment
333
significantly increased the fluorescence within mitochondria, suggesting the activation of
334
mitochondrial Ca2+ uptake and mPTP opening which should take the blame for mitochondrial
335
dysfunction. However, NaHS pretreatment led to a significant decrease in mitochondrial
336
fluorescence, compare to IL-1β group.
337
To detect whether the sustained mPTP opening can dissipate MMP in NP cells, the 19 / 42
H2S inhibits apoptosis of NP cells
338
membrane potential-dependent MitoTracker was used to assess the MMP. Our results showed
339
that the red fluorescence significantly reduced after a 24hrs exposure to IL-1β, while NaHS
340
markedly abrogated the decrease of fluorescence, compare with IL-1β-treated cells (Fig.4B,C).
341
As expected, similar beneficial effect was also observed in ATP levels, we found that IL-1β
342
treatment prominently increased cellular ATP depletion, which was reverted by NaHS
343
pretreatment. (Fig.4D)
344
Transmission electron microscopy (TEM) was used to examine mitochondrial ultrastructure.
345
In our experiment, we observed aberrant mitochondrial fragmentation and swollen
346
mitochondria in IL-1β treated NP cells, indicating the mitochondrial morphological
347
deterioration in IL-1β treated NP cells, while NaHS pretreatment partly reversed the
348
mitochondrial morphological alterations. (Fig.4E) Taking together, these data demonstrate
349
that NaHS prevents mitochondrial dysfunction induced by IL-1β in NP cells.
350
Fig.4
20 / 42
H2S inhibits apoptosis of NP cells
351 352
3.5 IL-1β-induced ER stress is decreased in NaHS-treated NP cells
353
To determine whether ER stress was involved in the anti-apoptotic effect of NaHS in NP
354
cells, the ER stress-related proteins were assessed by western blot. As shown in Figure 5, we
355
found significant increases in the levels of CHOP, cleaved caspase 12, GRP-78, and ATF-6 in 21 / 42
H2S inhibits apoptosis of NP cells
356
the IL-1β-treated group versus the control group, whereas these increases were attenuated
357
significantly in the cells with NaHS pre-treatment. These findings provide evidence that the
358
protective role of NaHS may involve the inhibition of ER stress-related proteins.
359
Fig.5
360 361
3.6 NaHS activates PI3K/Akt and ERK1/2 pathways
362
Based on previous studies showing that PI3K/Akt and ERK1/2 triggered and mediated the
363
cardioprotective effects of H2S preconditioning,[23] we further explored whether the
364
PI3K/Akt and ERK1/2 pathways were involved in the NaHS-mediated inhibition of ER stress. 22 / 42
H2S inhibits apoptosis of NP cells
365
Western blot was used to detect proteins levels of p-Akt, Akt, p-ERK1/2, and ERK1/2
366
(Fig.6A). Compared with the control group, significantly higher levels of expression of
367
p-ERK1/2/ERK1/2 and p-Akt/Akt (Fig. 6B, C) were observed in cells exposed to IL-1β,
368
indicating activation of the PI3K/Akt and ERK1/2 pathways. The activation of the PI3K/Akt
369
and ERK1/2 pathways in NP cells was further increased by the pretreatment with NaHS.
370
These data suggest that the protective effect of NaHS is involved in the PI3K/Akt as well as
371
the ERK1/2 pathways.
372
Fig.6
373 23 / 42
H2S inhibits apoptosis of NP cells
374
3.7 Inhibition of the PI3K/Akt and ERK1/2 pathways partially reverses the protective
375
effect of NaHS
376
To evaluate the roles of PI3K/Akt and ERK1/2 activation by NaHS, two specific signal
377
pathway inhibitors, LY294002 for PI3K/Akt and PD98059 for ERK1/2, were added to the
378
medium. No effect of these inhibitors on cell death was observed (data not shown). LY294002
379
and PD98059 inhibited the activation of Akt and ERK1/2, respectively, compared with the
380
NaHS group (Fig. 7A, B). However, the inhibitory effect of NaHS on the ER stress-related
381
protein levels of CHOP, GPR78, and cleaved caspase 12 was reversed by the addition of
382
LY294002 and PD98059 (Fig. 7A, C). To further confirm the molecular mechanism of NaHS
383
in mitochondrial injury-induced apoptosis in vitro, we carried out immunocytochemical
384
studies on Drp 1 translocation that participates in mitochondrial fission, and mitochondrial
385
probe dye JC-1, was used to measure the descent in mitochondrial transmembrane potential
386
that occurs early in apoptosis. Compared with IL-1β treatment cells, NaHS pretreatment
387
significantly decreased the mitochondrial membrane location of Drp1 in NP cells. However,
388
when ERK1/2 and Akt pathway were inhibited by LY294002 and PD98059, the protective
389
effect of NaHS on mitochondrial function was partially abolished, suggesting that NaHS
390
protected mitochondrial dysfunction through ERK1/2 and Akt pathway. (Fig.8 A) In line with
391
this result, exposure to LY294002 and PD98059 aggravated the reduction of mitochondrial
392
membrane potential by flow cytometry analysis, compare with the NaHS pretreatment cells.
393
(Fig.8 B) As shown in Fig.8E-G, LY294002 and PD98059 abolished the inhibitory effect of
394
NaHS on the expression of apoptotic proteins cleaved caspase 3, Bax, and Bcl-2. Based on an 24 / 42
H2S inhibits apoptosis of NP cells
395
analysis of band density, we found that, compared with NaHS treatment, the levels of cleaved
396
caspase 3 and Bax increased with exposure to LY294002 and PD98059, whereas that of the
397
anti-apoptotic protein Bcl-2 decreased. These results were further confirmed in TUNEL test,
398
the addition of LY294002 or PD98059 significantly increased cell apoptosis in comparison
399
with the NaHS group (Fig. 8C, D). Taken together, these results provide evidence that ER
400
stress and mitochondrial injury-induced apoptosis was aggravated by the addition of Akt or
401
ERK inhibitors and that the protective effect of NaHS was mediated by both the PI3K/Akt
402
and the ERK1/2 signaling pathways.
403
Fig.7
404 405
Fig.8
25 / 42
H2S inhibits apoptosis of NP cells
406 407
3.8 NaHS ameliorates rat intervertebral disc degeneration in vivo 26 / 42
H2S inhibits apoptosis of NP cells
408
To determine the therapeutic effect of NaHS on degenerative rat discs, the magnetic
409
resonance imaging (MRI) and the Pfirrmann MRI grade scores MR images were obtained at 4
410
and 8 weeks after puncture, which showed stronger T2-weighted signal intensities in the
411
NaHS group than in the saline group. Similar results were also observed at 8 weeks (Fig. 9A).
412
Moreover, the Pfirrmann MRI grade scores, which indicate the degree of disc degeneration,
413
were significantly lower in the NaHS treated rat than in the saline group at 4 weeks and 8
414
weeks (Fig. 9C). The beneficial effects were confirmed by Safranin O-Fast Green and
415
hematoxylin and eosin staining at 4 and 8 weeks (data not shown) in the degenerative model.
416
(Fig. 9B) The nuclear cells in control discs were mostly stellar-shaped and distributed evenly
417
in the NP, with the proteoglycan matrix organized in slender streaks among them (Fig. 9B
418
a–d). Compared with the control group, the size of the NP in the saline group decreased
419
progressively and the nuclear cells became larger, rounded, grouped into clusters, and
420
separated by dense areas of proteoglycan matrix, indicating severe degeneration of the NP
421
cells (Fig. 9E–H). NaHS treatment significantly alleviated the decrease of nucleus pulposus
422
tissue and and reversed morphological changes compared with the saline group (Fig. 9B i–l).
423
And, the beneficial effect of NaHS treatment was more significant at 4 weeks post operation
424
than 8 weeks. (Fig. 9D)
425
Fig.9
27 / 42
H2S inhibits apoptosis of NP cells
426 427
4. Discussion
428
IVDD is the leading cause of the most common disorders, however there is no effective
429
therapies for IVDD because the pathogenesis of IVDD is still unclear. Our study
430
demonstrated that the expression levels of CBS and CSE was decreased in both degenerative
431
intervertebral disc tissues and pro-inflammatory cytokine IL-1β-induced NP cells, indicating a
432
reduced ability to generate cellular H2S, which suggested a essential role of endogenous H2S 28 / 42
H2S inhibits apoptosis of NP cells
433
in the process of IVDD (Fig. 1). These finding were further confirmed in endogenous H2S
434
system by the CBS or CSE-siRNA transfection into NP cells. The impaired endogenous H2S
435
generation system made NP cells more vulnerable to inflammatory stimuli.(Fig.2)
436
There is accumulating evidence that, alongside nitric oxide (NO) and carbon oxide (CO),
437
H2S is an important endogenous gasotransmitter signaling molecule. The role of H2S in cell
438
viability is complex, with both pro- and anti-apoptotic effects. The data presented here
439
demonstrated that short-term exposure to 1 mM NaHS markedly ameliorated the decreased
440
viability of NP cells and increased expression of apoptosis-related proteins (Fig. 3). Similarly,
441
a recent study reported that NaHS treatment may increase cell viability and decrease apoptosis
442
in recovery of cardioprotection from ischemic.[24] However, studies have showed the
443
opposite effect of H2S in regulation of apoptosis. H2S exacerbated cisplatin-induced
444
nephrotoxicity in mice, possibly through promoting an apoptotic response.[25] A similar
445
effect of H2S was reported in INS-1E cells, where the increased release of endogenous H2S
446
not only aggravated cellular stress but also stimulated apoptosis.[26] These contrasting results
447
may be attribute to different durations of H2S treatment, such that exposure to an appropriate
448
concentration of H2S attenuates apoptotic responses, whereas higher concentration of H2S can
449
result in increased apoptosis, even necrosis.
450
Due to mitochondria are very sensitive to the alterations of intracellular environment, the
451
role of mitochondria can switch from being supporters of life to facilitator of cell death
452
quickly. Thus, mitochondrial dysfunction is considered to be associated with the loss of NP
453
cells and the subsequent development of IVDD. Previous studies have shown that 29 / 42
H2S inhibits apoptosis of NP cells
454
mitochondrial pathways[27] were involved in IVD cell apoptosis other than death receptor[28,
455
29]Mitochondrial mediated apoptosis is induced by permeabilization of the outer
456
mitochondrial membrane, and pro-apoptotic Bcl-2 family members such as Bax and Bak[6]
457
are activated which lead to the release of cytochrome c[30]. In addition to Bcl-2 protein
458
family, cleaved caspase 3 is another important protein involved in apoptosis, and activation of
459
cleaved caspase 3 has been identified as the final executor of apoptosis.[31] In the present
460
study, we assessed the levels of these mitochondria apoptosis related proteins, mitochondrial
461
permeability transition pore (mPTP) opening, and mitochondrial membrane potential and ATP
462
levels after IL-1β stimulation to investigate the molecular mechanism of H2S in mitochondrial
463
dysfunction induced NP cells apoptosis. Our results showed that the expression of
464
cytochrome c, cleaved caspase 3, and Bax were upregulated by the administration of IL-1β,
465
while the level of Bcl2 was downregulated, NaHS pretreatment attenuated cytochrome c,
466
cleaved caspase 3 and Bax activation as well as Bcl2 inhibition (Fig.3 D, E). IL-1β treatment
467
induced mitochondrial dysfunction through enhanced mPTP opening, mitochondrial
468
membrane potential collapse, and decreased ATP levels, eventually leading to related proteins
469
release and apoptosis, which were significantly reversed by NaHS treatment.(Fig.4) These
470
findings indicate that the protective role of H2S against NP cells apoptosis was related to the
471
inhibition of mitochondrial dysfunction, were consistent with previous findings in the
472
cardiovascular system.[15, 32]
473
Inhibitors of the mitochondrial apoptotic pathway failed to completely suppress apoptosis
474
in cultured disc cells,[9, 27] indicating that disc cell apoptosis may be also mediated by other 30 / 42
H2S inhibits apoptosis of NP cells
475
signal transduction pathways, such as the ER pathway. In addition to the function of synthesis
476
and formation of tertiary structure of proteins, ER also acts as a dynamic Ca2+ reservoir and
477
responsible for rapid physiological signal transduction. The ER stress is activated by various
478
cellular stresses that may induce apoptosis.[33] GRP78, ATF6, CHOP and caspase 12 are
479
proteins that up-regulate in response to ER stress and are considered to be mediators of the
480
apoptosis pathways induced by ER stress.[34] Increasing evidence indicates that activation of
481
the ER stress-induced apoptosis pathway contributes to various degenerative diseases,
482
including Alzheimer’s disease,[35] Parkinson’s disease[36] and osteoarthritis.[37] A recent
483
study demonstrated that ER stress was also related to the process of IVDD. In a disc cell
484
apoptosis model induced by cyclic stretch, the suppression of CHOP significantly inhibited
485
excessive apoptosis and attenuated disc degeneration.[8] Similarly, Zhao et al. found that the
486
ER and mitochondria were both involved in disc cell apoptosis and IVDD in rats.[7]
487
Consistent with previous studies, our results showed that levels of GRP78, ATF-6, CHOP and
488
caspase 12 were increased after IL-1β administration. NaHS pre-treatment reduced the
489
accumulation of these ER stress-related proteins so as to inhibit apoptosis (Fig. 3). These
490
results indicated that the protective role of H2S in NP cells may attribute to the inhibition of
491
ER stress.
492
We further explored the underlying mechanism of the protective effect of H2S against
493
mitochondrial dysfunction and ER stress. It has been reported that H2S could induce
494
activation of Akt and ERK1/2 pathways in various cells.[23, 38] While Akt was reported to
495
inhibit the formation of the pro-apoptotic proteins such as Bad, Bax and caspase.[39] Thus, 31 / 42
H2S inhibits apoptosis of NP cells
496
the PI3K signaling cascade was supposed to contribute to the recruitment of endogenous
497
cytoprotective pathways to reduce NP cells apoptosis after stress stimuli. Evidence also
498
showed that both the PI3K/Akt and ERK1/2 pathways are involved in leptin-induced human
499
NP cells cyclin D1 expression and proliferation.[40] In the present study, we found that the
500
protective effect of NaHS preconditioning in NP cells was also related to the activation of
501
both the PI3K/Akt and ERK1/2 pathways (Fig. 6), which was consistent with previous
502
findings. To further confirm that these two pathways are essential for the protective effects of
503
NaHS, the PI3K/Akt inhibitor LY294002 and the ERK1/2 inhibitor PD98059 were used to
504
treat NP cells. The results showed that apoptosis induced by IL-1β was inhibited by NaHS
505
pretreatment, while the combination of inhibitors abolished the protective effect and
506
aggravated mitochondrial damage, further suggesting that H2S protects against endoplasmic
507
reticulum stress and mitochondrial injury via PI3K/Akt and ERK1/2 pathways in NP cells .
508
(Figs. 7, 8) This is consistent with previous findings which revealed the protective effects of
509
H2S in cardiomyocytes[41]. However, recently Xie et al.[38] reported that H2S exerted its
510
protective effects by activating Akt but not ERK1/2 pathway in SH-SY5Y cells. These
511
inconsistent studies demonstrated that H2S may activate different signaling pathways in
512
various cells. To further assess the therapeutic effects of NaHS in vivo, needle
513
puncture-induced degenerative rat discs were evaluated after 4 and 8 weeks of successful
514
modeling. Our results showed the appearance of severely degenerated intervertebral discs in
515
the saline group and discs with less severe degeneration in the NaHS group, indicating that
516
NaHS could ameliorate disc degeneration in vivo (Fig. 9). Although the therapeutic effects of 32 / 42
H2S inhibits apoptosis of NP cells
517
H2S in ameliorating IVDD were also observed after 8 weeks, it was diminishing after 4 weeks,
518
which may be attributable to the loss of a large number of NP cells caused by the further
519
destruction of the intervertebral disc structure in the advanced stages of IVDD.
520
There are some limitations in our study. Firstly, although the differential expression of CBS
521
and CSE have been evaluated in our study, due to technical limitation of our laboratory, the
522
physiological and pathological concentration of H2S in vivo has not yet been detected, which
523
warrant further studies. Secondly, other factors such as oxidative and mechanical stress, are
524
relevant in vivo and may induce NP cells apoptosis haven’t yet been investigated in this study,
525
which should be considered in further studies on the promotion of NP cells apoptosis. Finally,
526
PI3K/Akt and ERK1/2 signaling pathways may mediate multiple complex functions in cells,
527
potentially affect cellular proliferation, differentiation, migration, and apoptosis. Thus,
528
activation of these signaling pathways may affect other physiological functions within the cell
529
that are unrelated to the focus of our study, this should be considered in interpreting our
530
results.
531
As shown to exert protective effect for IVDD in our study, H2S may be a potential medicine
532
for IVDD therapy. However, the half-life of NaHS in aqueous solution is very short, which
533
may result in the loss of its biological activity. Thus, a delivery system to increase the stability
534
of NaHS may contribute to the successful application of H2S.
535
In conclusion, this study provides novel evidence that H2S may play a role in the
536
pathogenesis of IVDD by affecting the apoptosis of NP cells. We found that the protective
537
role of H2S involves the suppression of ER stress and mitochondrial dysfunction via 33 / 42
H2S inhibits apoptosis of NP cells
538
activation of the PI3K/Akt and ERK1/2 signaling pathways. Our study may help to better
539
understand the role of H2S in pathogenesis of IVDD.
540 541
Abbreviations: IL-1β, interleukin (IL)-1β; IVDD, Intervertebral disc degeneration; IVD,
542
Intervertebral disc; H2S, Hydrogen sulfide; NP, nucleus pulposus; CBS, cystathionine
543
β-synthase; CSE, cystathionine γ-lyase; ER, endoplasmic reticulum; siRNA, small interfering
544
RNA; MRI, Magnetic resonance imagings; mPTP, mitochondrial permeability transition pore;
545
MMP, mitochondrial transmembrane potential.
546 547
Acknowledgments
548
This work is supported by grant from National Nature Foundation of China (Grant no.
549
81371988, 81401871 and 81401162), Natural Science Foundation of Zhejiang Province for
550
Distinguished Young Scholars (LR12H06001); Major scientific and technological project of
551
medical and health in Zhejiang Province (WKJ-ZJ-1527); Zhejiang Undergraduate Talent
552
Project (2016R413072).
553 554
Author Contributions
555
D.X., X.Z. and X.W. contributed to the design of the study. H.J., J.C., X.C., J.W. and J.C.
556
contributed to the collection and assembly of data. D.C., N.C., J.W., Yu C., N.T. and Z.Z.
557
contributed reagents/materials/analysis tools. D.X. and X.W. wrote the main manuscript text.
558
All authors contributed in revising the manuscript and approved the final version to be 34 / 42
H2S inhibits apoptosis of NP cells
559
submitted.
560 561
Conflict of Interest
562
The authors declare no conflict of interest.
563 564
References
565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593
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(2008).
5. Figure Legends 37 / 42
H2S inhibits apoptosis of NP cells
671
5.1 Fig. 1 H2S generation is decreased in degenerate disc tissues and in IL-1β-treated NP
672
cells
673
(A) a: Pfirrmann grade I patient, male, 22 years old, Lumbar burst fracture caused by accident;
674
b: Pfirrmann grade Ⅲ patient, 18 years old, lumbar disc herniation; c: Pfirrmann
675
gradePfirrmann 76 years old, lumbar disc herniation and spinal stenosis. (B)Representative
676
western blots and bar diagram of CBS and CSE in each human NP tissues group (Pfirrmann
677
grade I to grade Ⅴto (C) NP cells were incubated with 10ng/ml IL-1β for 24hrs and 48 hrs,
678
then, harvested for the measurement of CBS, CSE, and SQOR by western blot. Bar diagram
679
of CBS and CSE expression from three Western blot analyses. (D) CBS, CSE, and SQOR
680
cellular levels were also measured by real-time PCR (RT-PCR). Bar diagram of CBS and CSE
681
expression from three RT-PCR analyses. NP tissues from nine different patients (three in each
682
grade) were used for tissuse gene analysis and western blotting. NP cells from four different
683
non-degenerative IVD tissues were used in cellar experiments. All experiments were
684
performed three times in duplicates. *P < 0.05, **P < 0.01 compared with the control group.
685 686
5.2 Fig.2 Inhibition of CBS or CSE by siRNA significantly increased IL-1β-induced
687
apoptosis in NP cells.
688
(A-B) After CBS or CSE-siRNA transfection, NP cells were harvested to analyse the
689
expression of CBS and CSE in both gene and protein levels. (C) Bar diagram of CBS and
690
CSE levels. (D) The NP cells were incubated 75ng/ml IL-1β for 24hrs. The cell lysates were
691
analysed by western blotting to detect the expression of Cyt c, cleaved caspase3, Bax and 38 / 42
H2S inhibits apoptosis of NP cells
692
Bcl-2. (E-F) Bar diagram of the Cyt c, cleaved caspase3, Bax and Bcl-2 expression from three
693
Western blot analyses. NP cells from four different non-degenerative IVD tissues were used
694
for evaluation three times in duplicates. *P < 0.05, **P < 0.01 compared with the control
695
group. #P < 0.05, ##P < 0.01 versus the IL-1β+Con-siRNA group.
696 697
5.3 Fig. 3 NaHS treatment inhibits apoptosis in NP cells
698
(A and B) NP cells were treated with different doses of NaHS or IL-1β for 24 h. (C) NP cells
699
were pre-treated with 1 mM NaHS for 1 hr, then, 75 ng/ml IL-1β was added for an additional
700
24hrs. (D)The cell lysates were analysed by western blotting to detect the expression of Cyt c,
701
cleaved caspase3, Bax and Bcl-2. (E) Bar diagram of the Cyt c, cleaved caspase3, Bax and
702
Bcl-2 expression from three Western blot analyses. GAPDH was used as a protein loading
703
control and for band density normalization. Quantitative evaluation of three independent
704
experiments employing NP cells from three different non-degenerative IVD tissues tested in
705
duplicates. *P < 0.05, **P < 0.01 compared with the control group, #P < 0.05, ##P < 0.01
706
versus the IL-1β group.
707 708
5.4 Fig. 4 NaHS prevents IL-1β-induced mitochondrial dysfunction
709
NP cells were pre-treated with 1 mM NaHS for 1 hrs, and then 75 ng/ml IL-1β was added for
710
an additional 24 hrs. (A) The mPTP opening was detected by co-loading with calcein-AM and
711
CoCl2. Scale bar, 20 μm. (B) Mitochondrial membrane potential was detected using
712
Mitotracker Red CMXRos, and nuclei were stained with Hoechst. Scale bar, 10 μm. (C) 39 / 42
H2S inhibits apoptosis of NP cells
713
ImageJ software was used to measure relative red fluorescence per cell. (D) ATP content was
714
assessed by ATP-Glo Bioluminometric Cell Viability Assay. (E) Mitochondrial morphology
715
was detected by transmission electron microscopy (Viability NP cells. Arrow: swollen
716
mitochondria; triangle: mitochondrial fragmentation; star: fragmented mitochondria in
717
lysosome). In all experiments NP cells from two different non-degenerative IVD tissues were
718
tested three times in duplicates. *P < 0.05, **P < 0.01, versus the control group, #P < 0.05,
719
##P < 0.01 compared with the IL-1β group.
720 721
5.5 Fig. 5 IL-1β-induced ER stress is decreased in NaHS-treated NP cells
722
(A) NP cells were treated with either no addition, 1 mM NaHS and/or 75 ng/ml IL-1β,
723
asdescribed in the “Materials and methods” section. The cell lysates were analysed for the
724
expression of CHOP, GRP-78, ATF-6 and caspase-12 by western blotting. Bar diagram of (B)
725
CHOP, ATF-6, (C) GRP-78 and (D) caspase-12 expression from three Western blot analyses.
726
NP cells from three different non-degenerative IVD tissues were used in these experiments.
727
All results were repeated three times in duplicates. *P < 0.05, **P < 0.01, versus the control
728
group, #P < 0.05, ##P < 0.01 compared with the IL-1β group.
729 730
5.6 Fig. 6 NaHS activates PI3K/Akt and ERK1/2 in NP cells
731
(A) NP cells were pre-treated with 1 mM NaHS for 1 hrs, and then 75 ng/ml IL-1β was added
732
for an additional 24 hrs. The cell lysates were analysed for the expression of phospho-Akt,
733
Akt, phospho-ERK and ERK by western blotting. (B) Bar diagram of phospho-Akt/Akt and 40 / 42
H2S inhibits apoptosis of NP cells
734
phospho-ERK/ERK levels from three Western blot analyses. GAPDH was used as a protein
735
loading control and for band density normalization. NP cells from twelve different
736
non-degenerative IVD tissues were used in these experiments. All results were repeated three
737
times in duplicates. *P < 0.05, **P < 0.01, compared with the control group, #P < 0.05, ##P <
738
0.01 versus the IL-1β group.
739 740
5.7 Fig. 7 Inhibition of the PI3K/Akt and ERK1/2 pathways partially attenuates the
741
NaHS-mediated reduction in the endoplasmic reticulum (ER) stress effects in NP cells
742
NP cells were pre-treated with 1 mM NaHS with or without the specific inhibitors LY294002
743
(50 μM) and PD98059 (50 μM) for 1 hr, and then 75 ng/ml IL-1β was added for an additional
744
24 hrs. The cell lysates were analysed by western blotting to detect the expression of
745
phospho-Akt, phospho-ERK and ERK, CHOP, GRP-78, ATF-6 and caspase-12. Bar diagram
746
of the (B) phospho-Akt/Akt ratio, phospho-ERK/ERK ratio, (C) CHOP, GRP-78, ATF-6 and
747
caspase-12 expression from three Western blot analyses. GAPDH was used as a protein
748
loading control and for band density normalization. NP cells from twelve different
749
non-degenerative IVD tissues were used in these experiments. All results were repeated three
750
times in duplicates. *P < 0.05, **P < 0.01 versus the control group, #P < 0.05, ##P < 0.01
751
versus the IL-1βgroup.
752 753
5.8 Fig. 8 Inhibition of the PI3K/Akt and ERK1/2 pathways partially reverses the
754
protective effect of NaHS and aggravates mitochondrial dysfunction 41 / 42
H2S inhibits apoptosis of NP cells
755
NP cells were pre-treated with 1 mM NaHS with or without the specific inhibitors LY294002
756
(50 μM) and PD98059 (50 μM) for 1 hr, and then 75 ng/ml IL-1β was added for an additional
757
24hrs. (A) Immunofluorescence double-labeled staining for co-localization of Drp1 with
758
Tom20 in NP cells, scale bar, 10μm. (B) JC-1 mitochondrial membrane potential assay was
759
analyzed by flow cytmetry. (C) Detection of apoptotic cells by TUNEL (green) and DAPI
760
(blue) staining assay. (D) Bar diagram of apoptotic cell rates from three separate experiments.
761
(E) The cell lysates were analysed for the expression of Cyt c, C-caspase3, Bax and Bcl-2 by
762
western blotting. Bar diagram of (F) Cyt c and C-caspase3, (G) Bax and (F) Bcl-2 expression
763
from three Western blot analyses. NP cells from four different non-degenerative IVD tissues
764
were used in these experiments. All results were repeated three times in duplicates. *P < 0.05,
765
**P < 0.01, versus the control group, #P < 0.05, ##P < 0.01 versus the IL-1β group.
766 767
5.9 Fig. 9 NaHS ameliorates rat intervertebral disc degeneration in vivo
768
(A) T2-weighted MRI of a rat tail with a needle-punctured disc at 4 and 8 weeks post surgery
769
(white arrows). (C) The Pfirrmann MRI grade scores in three groups at week 4 and week 8. (B)
770
Representative safranin o-fast green and hematoxylin, and hematoxylin and eosin staining of
771
of disc samples from different experimental groups at 4 and 8 weeks post surgery. Scale bars
772
are 200 μm (40×) and 50 μm (200×), respectively. (D) The histological grades evaluated at
773
week 4 and week 8 in three groups. Samples from 48 rats (sixteen in each group ) were used for
774
imageological and histopathologic analysis.*P < 0.05, **P < 0.01, versus the control group,
775
#P < 0.05, ##P < 0.01 versus the saline group. 42 / 42
S1043-6618(16)31163-X http://dx.doi.org/doi:10.1016/j.phrs.2017.01.005
Reference:
YPHRS 3466
To appear in:
Pharmacological Research
Received date: Revised date: Accepted date:
2-11-2016 17-12-2016 5-1-2017
Please cite this article as: Xu Daoliang, Jin Haiming, Wen Jianxia, Chen Jiaoxiang, Chen Deheng, Cai Ningyu, Wang Yongli, Wang Jianle, Chen Yu, Zhang Xiaolei, Wang Xiangyang.Hydrogen sulfide protects against endoplasmic reticulum stress and mitochondrial injury in nucleus pulposus cells and ameliorates intervertebral disc degeneration.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2017.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
H2S inhibits apoptosis of NP cells
1
Graphical abstract
2 3
1 / 42
H2S inhibits apoptosis of NP cells
4
Hydrogen sulfide protects against endoplasmic reticulum stress and mitochondrial
5
injury in nucleus pulposus cells and ameliorates intervertebral disc degeneration
6
Ttile Page
7
Daoliang Xu1, Haiming Jin1, Jianxia Wen2, Jiaoxiang Chen1, Deheng Chen1, Ningyu Cai1,
8
Yongli Wang4, Jianle Wang1, Yu Chen1, Xiaolei Zhang*1,3, Xiangyang Wang*1
9
1
Department of Orthopaedic Surgery, the Second Affiliated Hospital of Wenzhou Medical
10
University, Wenzhou, Zhejiang, China
11
2
12
University, Wenzhou, Zhejiang, China
13
3
Zhejiang Provincial Key Laboratory of Orthopaedics, Wenzhou, Zhejiang Province, China.
14
4
Department of Orthopaedic Surgery, Huzhou Central Hospital, Huzhou, China
15
*Correspondence to: Xiangyang Wang, 1Department of Orthopaedic Surgery, The Second
16
Affiliated Hospital of Wenzhou Medical University, 109 Xueyuan Xi Road, Wenzhou,
17
Zhejiang, 325027, China
18
Phone: 86-577-88829799
19
Fax: 86-577-88879123
20
Email: [email protected]
21
*Correspondence to: Xiaolei Zhang, 1Department of Orthopaedic Surgery, The Second
22
Affiliated Hospital of Wenzhou Medical University, 109 Xueyuan Xi Road, Wenzhou,
23
Zhejiang, 325027, China
24
Phone: 86-577-88002823
Department of Gastrointestinal Surgery, the First Affiliated Hospital of Wenzhou Medical
2 / 42
H2S inhibits apoptosis of NP cells
25
Fax: 86-577-88002823
26
Email: [email protected]
27
Abstract
28
It has been suggested that excessive apoptosis in intervertebral disc cells induced by
29
inflammatory cytokines, such as interleukin (IL)-1β, is related to the process of intervertebral
30
disc degeneration (IVDD). Hydrogen sulfide (H2S), a gaseous signaling molecule, has drawn
31
attention for its anti-apoptosis role in various pathophysiological processes in degenerative
32
diseases. To date, there has been no investigation of the correlation of H2S production and
33
IVDD or of the effects of H2S on IL-1β-induced apoptosis in nucleus pulposus (NP) cells.
34
Here, we found that the expression levels of cystathionine β-synthase (CBS) and
35
cystathionine γ-lyase (CSE), two key enzymes in the generation of H2S, were significantly
36
decreased in human degenerate NP tissues as well as in IL-1β-treated NP cells. NaHS (H2S
37
donor) administration showed a protective effect by inhibiting the endoplasmic reticulum (ER)
38
stress response and mitochondrial dysfunction induced by IL-1β stimulation in vitro, the
39
effect was related to activation of the PI3K/Akt and ERK1/2 signaling pathways. Suppression
40
of these pathways by specific inhibitors, LY294002 and PD98059, partially reduced the
41
protective effect of NaHS. Moreover, in the percutaneous needle puncture disc degeneration
42
rat tail model, disc degeneration was partially reversed by NaHS administration. Taken
43
together, our results suggest that H2S plays a protective role in IVDD and the underlying
44
mechanism involves PI3K/Akt and ERK1/2 signaling pathways-mediated suppression of ER
45
stress and mitochondrial dysfunction in IL-1β-induced NP cells. 3 / 42
H2S inhibits apoptosis of NP cells
46
Key words: Hydrogen sulfide, intervertebral disc degeneration, endoplasmic reticulum stress,
47
mitochondrial injury, apoptosis.
48 49
1. Introduction
50
Intervertebral disc degeneration (IVDD) is a spinal disorder whose pathogenesis has not
51
been fully elucidated. It has been suggested that nucleus pulposus (NP) cells which produce
52
cartilage-specific extracellular matrix components play an important role in IVDD.[1]
53
Aberrant apoptosis [2-4] of NP cells has been considered to be the major cellular process
54
associated with IVDD. Studies have demonstrated that several pathological conditions may
55
lead to apoptosis of NP cells, among which abnormally increased inflammatory cytokines,
56
such as IL-1β and tumor necrosis factor-alpha, were considered to play a crucial role in the
57
process of IVDD. Through a series of signaling networks, inflammatory cytokines may
58
induce NP cell apoptosis, which further result in progressive IVDD.[5]
59
The endoplasmic reticulum (ER) is an important organelle responsible for cellular
60
homeostasis. When exposed to prolonged or strong intracellular and extracellular stimuli, the
61
ER stress-related proteins such as caspase 12 will be activated, which may ultimately lead to
62
apoptotic cell death, therefore ER stress is closely related to apoptosis. Another major event in
63
apoptosis is mitochondrial process, and the Bcl-2 protein family is involved not only in the
64
alteration of mitochondrial membrane potential but also in the release of mitochondrial
65
apoptotic factors.[6] Recently, intervertebral disc cell apoptosis was shown to be mediated by
66
both the endoplasmic reticulum (ER) and the mitochondrial pathways in rats.[7] Zhang et al. 4 / 42
H2S inhibits apoptosis of NP cells
67
reported that annular cell apoptosis induced by cyclic stretch was partially reversed by the
68
inhibition of ER stress in rats.[8] And, there is evidence showing that mitochondrial pathway
69
contribute to fas-mediated apoptosis of human lumbar disc cells.[9] These studies suggest an
70
emerging picture of the importance of the mitochondria and ER in IVDD.
71
Hydrogen sulfide (H2S) is synthesized from L-cysteine primarily by two key enzymes:
72
cystathionine-c-lyase (CSE) in the peripheral tissues and cystathionine-b-synthetase (CBS) in
73
the central nervous system. Recognized as the third endogenously produced gaseous
74
messenger, along with nitric oxide and carbon monoxide, H2S regulates a variety of biological
75
functions including anti-inflammatory[10] and anti-apoptotic[11] effects, affecting several key
76
mechanisms and pathways both in vitro and in vivo. Recent studies have suggested possible
77
roles for H2S in modulating ER stress-induced cardiovascular disease. In a rat model of
78
hyperhomocysteinemia-induced cardiomyocytic ER stress injury and in doxorubicin-induced
79
H9c2 cells, H2S supplementation was shown to antagonize cytotoxicity and apoptosis through
80
inhibiting ER stress-associated proteins.[12, 13] Similarly, a study on formaldehyde-induced
81
neurotoxicity in PC12 cells showed that H2S pre-treatment significantly attenuated the
82
increases in the expression levels of ER stress markers.[14] Additionally, it was recently
83
demonstrated that H2S may reconcile Akt2 knockout-induced myocardial contractile defects
84
via attenuation of mitochondrial injury and apoptosis.[15] However, to our knowledge,
85
whether the production of H2S is correlated with IVDD and whether H2S can ameliorate ER
86
stress and mitochondrial dysfunction-induced apoptosis in human NP cells remain
87
unanswered. 5 / 42
H2S inhibits apoptosis of NP cells
88
This study aimed to illustrate the role of H2S in IVDD as well as its working mechanism.
89
Our results suggest that H2S protects against apoptosis in NP cells in vitro and ameliorates
90
disc degeneration in rats in vivo. We believe that this study may help us to better understand
91
the role of H2S in pathogenesis of IVDD.
92 93
2. Materials and Methods
94
2.1 Ethics statement
95
All surgical interventions, treatments and postoperative animal care procedures were
96
performed in strict accordance with the Animal Care and Use Committee of Wenzhou
97
Medical University (wydw2014-0129).
98 99
2.2 Nucleus Pulposus Cell Isolation and Culture
100
The study was approved by the Second Affiliated Hospital of Wenzhou Medical University
101
Ethics Committee. Patients, between 11 years and 76 years old (A total of 43 disc samples
102
were collected from 38 patients, in total of 17 female and 21 male patients with scoliosis or
103
disc herniation or vertebral fracture), signed an informed consent form allowing the
104
researchers to use NP tissues obtained during spinal surgery. 30 disc samples (Pfirrmann <
105
grade III, and 4 were defined as grade I) were classified as non-degenerative IVD tissue
106
samples according to the Pfirrmann grading scale[16] determined by magnetic resonance
107
imaging (MRI), total 17 disc samples were used in Pfirrmann grading scale（2 male and 2
108
female, age from 19-45, average 34.3± 10.9 in grade Ⅰ; 3 male and 3 female, age from 25-46, 6 / 42
H2S inhibits apoptosis of NP cells
109
average 35.8± 7.2 in grade Ⅲ; 4 male and 3 female, age from 38-47, average 42.2± 4.8 in
110
grade Ⅴ）, which were only used for gene analysis and western blot. And there was no
111
significant difference among the three Pfirrman grade groups in age or sex. In this study, no
112
other complications related to IVDD, such as systemic disease (Diabetes, etc.) were found in
113
patients that consented to donate their NP tissues. The gel-like NP tissues were digested in
114
0.25% trypsin and 0.2% type II collagenase (Gibco) for approximately 3 hrs at 37°C. Then,
115
the digested tissues were transferred as explants to Dulbeccomodified Eagle medium (DMEM;
116
Gibco, Invitrogen, Grand Island, NY) with 10% fetal bovine serum (FBS; Hyclone, Thermo
117
Scientific, Logan, UT) and antibiotics (1% penicillin/ streptomycin) in a 5% CO2 incubator.
118
When confluent, the cells were passaged by using 0.25% Trypsin-EDTA (Gibco, Invitrogen),
119
and subcultured in 60-mm culture dish at the appropriate density. During passaging, no
120
significant changes in morphology of cells between primary cells (passage 0) and later
121
passage cells (passage 2) were noticed.
122 123
2.3 Cell Culture Treatment Protocols
124
To determine whether degeneration induced changes in H2S-generating and -metabolizing
125
enzymes, NP cells were treated with IL-1β (10 ng/ml) (PeproTech, Rocky Hill, USA) for 24 or
126
48hrs. In order to evaluate the effect of NaHS（Sigma-Aldrich, St. Louis, MO) on the viability
127
of NP cells, cells were incubated for 24 hrs with increasing concentrations (0.06–1 mM) of
128
NaHS. To establish the apoptosis model of NP cells, different concentrations of IL-1β (10 -
129
100 ng/ml) were added into the culture medium for 24 hrs. Cells were pre-treated with 7 / 42
H2S inhibits apoptosis of NP cells
130
different concentrations of NaHS for 1 hr before the addition of IL-1β (75 ng/ml) to
131
investigate its effect on cell apoptosis. To study the role of PI3K/Akt and ERK1/2 signal
132
pathways in NaHS-induced cell protection, NP cells were pre-treated with 1mM NaHS and 50
133
μM LY294002 (PI3K/Akt inhibitor; Cell Signaling Technology, MA, USA) or PD98059
134
(ERK1/2 inhibitor; Beyotime, Shanghai, China) for 1hr before they received IL-1β
135
administration.
136 137
2.4 Cell Viability Assay
138
Cell viability was assayed with the cell counting kit-8 (CCK-8; Dojindo Co, Kumamoto,
139
Japan) according to the manufacture’s protocol. NP cells were treated with NaHS, IL-1β,
140
LY294002 and PD98059 as described above. After treatment the cells were washed with PBS,
141
then 100 μl of DMEM containing 10 μl of CCK-8 solution was added to each well, and the
142
plate was incubated for an additional 1 hr. The absorbance of wells was then measured at 450
143
nm by a micro-plate reader.
144 145
2.5 Western Blot Assay
146
The human NP tissues and cells were lysed in ice-cold radio-immuneprecipitation assay
147
(RIPA) buffer, supplemented with phosphatase inhibitors and protease inhibitor cocktail
148
(Biotech Well, Shanghai, China). Protein concentration was measured by the BCA protein
149
assay kit (Beyotime, Shanghai, China), and equivalent amounts of protein were separated by
150
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 8 / 42
H2S inhibits apoptosis of NP cells
151
polyvinylidenedifluoride (PVDF) membrane (BIO-RAD, USA). Then the protein was probed
152
with antibody specific to CBS, CSE, CHOP, cytochrome c (Santa Cruz Biotechnology, CA,
153
USA), ERK, p-ERK, Akt, p-Akt, cleaved-caspase3, Bax, Bcl-2 (Cell Signaling Technology,
154
Beverly, MA, USA), ATF-6 (Bioworld, MN, USA), GAPDH (Arigo, Taiwan, ROC), GRP78
155
and Caspase12 ( Abcam, Cambridge, UK) followed by ECL signal detection (Invitrogen).
156 157
2.6 Real-time PCR
158
Total RNA was extracted from NP cells by Trizol reagent (Invitrogen, USA). One
159
microgram of total RNA was used to synthesize cDNA (MBI Fermantas, Germany).
160
Real-time polymerase chain reaction reactions were done in triplicate in 96-well plates using a
161
SYBR Premix Ex Taq Kit (Takara, Japan) in a final volume of 20 μl. The reaction and
162
detection were conducted in a light-cycle (Roche, Mannheim, Germany). The cycle threshold
163
(Ct) values were collected and normalized to the level of the housekeeping gene GAPDH. The
164
△△Ct method was used to calculate the relative mRNA levels of each target gene. The
165
primers
166
CBS(F)5’-GACCAAGTTCCTGAGCGACA-3’,(R)5’-CGGAGGATCTCGATGGTGTG-3’;C
167
SE(F)5’-GGCTCTACCTGCGTGCTTTA-3’,(R)5’-GCGAAATGTTGGAAGTGTGG-3’.
of
CBS
and
CSE
were
listed
as
follows:
168 169
2.7 siRNA Transfection
170
According to the manufacturer’s instructions, human nucleus pulposus cells were
171
transfected with CBS or CSE siRNA (RiboBio, Guangzhou, China) using Lipofectamine 9 / 42
H2S inhibits apoptosis of NP cells
172
RNAiMAX reagent (Invitrogen). Following further treatments, the transfected cells for
173
subsequent analysis.
174 175
2.8 mPTP Opening
176
To ascertain the role of NaHS in IL-1β-induced mitochondrial dysfunction, mitochondrial
177
permeability transition pore (mPTP) was determined by using the calcein AM/CoCl2
178
fluorescence-quenching assay according to the manufacturer’s protocol. Initially, after the
179
treatment on NP cells as described above, cells were treatment with calcein AM in the
180
absence of cobalt, resulting in the calcein fluorescence diffuse throughout the whole cell.
181
IL-1βstimuli can lead to translocation of Ca2+ from cytoplasm to mitochondria, resulting in
182
mPTP opening. Thus, only the calcein fluorescence outside of mitochondria should be
183
quenched with the addition of cobat in cells with mitochondrial dysfunction.
184 185
2.9 MMP Assay
186
The mitochondrial transmembrane potential (MMP) was assessed by using MitoTracker
187
Red CMXRos (Molecular Probes™, Thermo Fisher Scientific Inc.), a red-fluorescent dye,
188
which accumulation is dependent upon membrane potential, stains mitochondria in live cells.
189
NP cells after 24hrs treatment were stained at a final concentration of of 50 nM for 30 min at
190
37 °C, and nuclei were stained with Hoechst 33258 dye. Red fluorescence images of at least
191
three random microscopic fields were acquired per slide for microscopic observation with a
192
fluorescence microscope (Olympus Inc., Tokyo, Japan), and fluorescence intensity was 10 / 42
H2S inhibits apoptosis of NP cells
193
measured using Image J software 2.1 (Bethesda, MD, USA) by observers who were blinded
194
to the experimental groups
195 196
2.10 ATP Assay
197
The ATP-Glo™ Bioluminometric Cell Viability Assay (Biotium, Hayward, CA, USA), was
198
used to assessed the cell ATP levels according to the manufacturer’s protocol. Data were
199
collected from multiple replicate wells for each experiment.
200 201
2.11 Transmission Electron Microscopy (TEM)
202
NP cells were fixed in 2.5% glutaraldehyde at 4r overnight, then postfixed in 2% osmium
203
tetroxide for 1 hr and stained with 2% uranyl acetate for 1 hr. After dehydration in an
204
ascending series of acetone, samples were embedded into araldite to cut into semi-thin
205
sections, then, stained with toluidine blue to locate cells. Sections were examined with a
206
transmission electron microscope (Hitachi, Tokyo, Japan).
207 208
2.12 Immunofluorescence
209
For Drp 1 (Abcam, Cambridge, UK) and Tom20 (Abcam, Cambridge, UK) staining, cells
210
were fixed with the 4% paraformaldehyde and blocked in PBS containing Triton X-100 for 10
211
mins, and then pretreated with blocking solution (5% bovine serum albumin) for 30 mins,
212
cells were then incubated with a cocktai of anti-Drp 1 (1:250) and anti-Tom20 (1:200)
213
overnight at 4 °C in a humidified chamber. On the next day, cells were washed and incubated 11 / 42
H2S inhibits apoptosis of NP cells
214
with fluorescein isothiocyanate- or tetramethyl rhodamine isothiocyanate-conjugated second
215
antibodies for 1 hr and labeled with DAPI for 5 mins. Finally, three fields of each slide were
216
chosen randomly for microscopic observation with a fluorescence microscope (Olympus Inc.,
217
Tokyo, Japan).
218 219
2.13 JC-1 Assay
220
Following the above treatment, NP cells were stained with 10 µM of JC-1 according to the
221
manufacturer instructions (Beyotime, Shanghai, China). Then, cells were analyzed using a
222
flow cytometer. Results were expressed as the percentage of cells that were double positive.
223 224
2.14 TUNEL Method
225
The terminal deoxynucleotidyltransferase (TdT) dUTP nick end labeling (TUNEL) method
226
is a technique for measuring apoptotic DNA fragmentations. After fixed with a freshly
227
prepared 4% paraformaldehyde for 1 hr, NP cells were incubated with 3% H2O2 and 0.1%
228
Triton X-100 for 10mins and washed with PBS for three times in every step. According to
229
manufacturer’s instructions, cells were stained with in situ cell death detection kit (F.
230
Hoffmann-La Roche Ltd., Basel, Switzerland) and 40, 6-diamidino-2-phenylindole (DAPI).
231
Finally，three random microscopic fields were acquired per slide for microscopic observation
232
with a fluorescence microscope (Olympus Inc., Tokyo, Japan).
233 234
2.15 Surgical Procedure 12 / 42
H2S inhibits apoptosis of NP cells
235
A total of 48, 3-month-old male Sprague Dawley rats were randomly divided into three
236
groups (control group, saline group and NaHS group), then weighed and injected
237
intraperitoneally with 2% (w/v) pentobarbital (40 mg/kg). The saline and NaHS group went
238
through the model operation. As described in the previous study[17], the experimental level
239
rat tail disc (Co7/8) was located by digital palpation on the coccygeal vertebrae and confirmed
240
by counting the vertebrae from the sacral region in a trial radiograph. Needles (20G, about
241
4mm in length) were used to puncture the whole layer of annulus fibrosus (AF) though the tail
242
skin perpendicularly. All the needles were rotated 360° and held in position for 30 seconds
243
before extraction. After surgery, in the NaHS group, the NaHS solution was immediately
244
injected intraperitoneally to deliver a dose of 100 μmol/kg/day until the rats were sacrificed.
245
Daily monitoring of the rats was carried out to ensure their well-being and all animals were
246
allowed free unrestricted weight bearing and activity.
247 248 249 250
2.16 Magnetic Resonance Imaging Method. Before modeling and after 4 or 8 weeks of puncture, the animals were given the MRI examination.
251
Magnetic resonance imaging was performed on all rats to evaluate the signal and structural
252
changes in sagittal T2-weighted images using a 3.0 T clinical magnet (Philips Intera Achieva
253
3.0MR). T2-weighted sections in the sagittal plane were obtained in the following settings:
254
fast-spin echo sequence with time to repetition (TR) of 5400 ms and time to echo (TE) of 920
255
ms; 320 (h) 9 256 (v) matrix; field of view of 260; and 4 excitations. The section thickness 13 / 42
H2S inhibits apoptosis of NP cells
256
was 2 mm with a 0-mm gap. The MRIs were evaluated by another blinded orthopedic
257
researcher using the classification of intervertebral disk degeneration as reported by Pfirrmann
258
et al.[16] (1 point = Grade I, 2 points = Grade II, 3 points = Grade III, 4 points = Grade IV).
259 260
2.17 Histopathologic Analysis
261
The rats were sacrificed by an intraperitoneal overdosage injection of 10% chloral hydrate
262
and the tails were harvested on week 4 and 8 after surgery. The specimens were fixed in
263
formaldehyde and decalcified, then dehydrated and embedded in paraffin. The tissues were
264
cut into 5 μm sections. Two midsagittal sections of each disc were stained with safranin o-fast
265
green and hematoxylin, and hematoxylin and eosin respectively. The cellularity and
266
morphology of NP were examined by another experienced histology researchers in a blinded
267
fashion using a microscope, and evaluated by using a grading scale, as described
268
previously[17]. The histologic score was 5 for normal disc, 6–11 for moderately degenerated
269
disc, and 12–14 for severely degenerated disc.
270 271
2.18 Statistical Analysis
272
The experiments were performed at least three times. The results were presented as mean ±
273
SD. Statistical analyses were performed using SPSS statistical software program 20.0. Data
274
were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s test for
275
comparison between control and treatment groups. Nonparametric data (Pfirrmann grading)
276
were analyzed by the Kruskal–Wallis H test. Statistical significance was set at p < 0.05. 14 / 42
H2S inhibits apoptosis of NP cells
277
Statistical significance was set at p < 0.05.
278 279
3. Results
280
3.1 H2S generation is decreased in degenerate disc tissues and in IL-1β-treated NP cells
281
CBS and CSE are two major enzymes involved in the endogenous generation of
282
H2S.[18-20] Sulfide:quinone oxidoreductase (SQOR) is a membrane-bound enzyme that
283
catalyzes the first step in the mitochondrial metabolism of H2S.[21] To determine whether
284
degeneration induced changes in H2S-generating and -metabolizing enzymes in NP cells,
285
human NP tissues (Fig. 1A, Pfirrmann grade I to gradeⅤ) and cells were harvested for the
286
measurement of three enzymes by Western blotting and real-time PCR. We found that CBS
287
and CSE protein levels in intervertebral disc tissues decreased significantly with the degree of
288
degradation in vivo (Fig. 1B), and the results from IL-1β-induced NP cells degeneration in
289
vitro corroborated previous findings in degenerated human disc in a time-dependent manner
290
(Fig. 1C). Similarly, IL-1β exposure led to decreased gene expression (Fig. 1D, data not
291
shown for NP tissues), whereas there was no significant change in the SQOR protein or
292
mRNA level (data not shown). These results suggest that endogenous H2S may play an
293
important role in maintaining the normal function of NP cells under physiological conditions.
294
Fig.1
15 / 42
H2S inhibits apoptosis of NP cells
295 16 / 42
H2S inhibits apoptosis of NP cells
296
3.2 Inhibition of CBS or CSE by siRNA significantly increased IL-1β-induced apoptosis
297
in NP cells.
298
Since the level of the gasotransmitter hydrogen sulfide involved in physiological and
299
pathological responses is micro, and the dose of exogenous hydrogen sulfide used in vivo or
300
vitro seems to be too high. To further investigate the role of endogenous hydrogen sulfide in
301
IL-1β-induced apoptosis in NP cells, cells were transfected with CBS or CSE-siRNA before
302
stimuli. CBS or CSE-siRNA transfection into human NP cells led to the expression of CBS
303
and CSE significantly decreased in gene level (Fig.2A), and the result was further confirmed
304
by western blotting (Fig.2B,C). Compared with control siRNA group, the inhibitory of the
305
synthetase further exacerbated IL-1β-induced apoptosis in siRNA group (Fig2.D-F). These
306
results suggested that H2S is involved in the pathological process of IVDD.
307
Fig.2
308 17 / 42
H2S inhibits apoptosis of NP cells
309
3.3 NaHS treatment inhibits apoptosis in NP cells
310
Because H2S is toxic at high concentrations, a dose–response study with varying doses (0 –
311
4 mmol/L) of NaHS was performed to investigate the susceptibility of NP cells to exogenous
312
H2S. As shown in Figure 3A, low concentrations of NaHS did not alter cell viability, while
313
cellular viability decreased significantly at concentrations of 2 mmol/l and higher. Indeed,
314
doses of NaHS from 0.5 to 1 mmol/L slightly promoted cell proliferation, leading to an
315
increase in cell viability, peaking at 1 mmol/L. IL-1β (75 ng/mL) resulted in a marked
316
decrease in cell viability (Fig. 3B). This reduction in cell viability could be effectively
317
increased by pre-treatment with 1 mmol/L NaHS (Fig. 3C). To further assess whether NaHS
318
modulated cellular apoptosis, levels of cleaved caspase3, Bax, and Bcl-2 were analyzed by
319
western blot. The results showed that NaHS significantly reversed the increasing level of
320
cytochrome c and cleaved caspase 3 due to IL-1β (Fig. 3D, E). Similarly, levels of the
321
pro-apoptotic protein Bax were down-regulated, whereas the anti-apoptotic protein Bcl-2 was
322
up-regulated by pre-treatment with NaHS.
323
Fig.3
18 / 42
H2S inhibits apoptosis of NP cells
324 325
3.4 NaHS prevents IL-1β-induced mitochondrial dysfunction
326
IL-1β can trigger sustained mitochondrial permeability transition pore opening, alter
327
mitochondrial membrane potential and ATP content via mitochondrial Ca2+ uptake[22].To
328
examine the protective effect of NaHS on IL-1β-induced mitochondria damage, mPTP
329
opening, ATP and mitochondrial membrane potential were determined after IL-1β
330
administration. As shown in Fig.4A, calcein fluorescence was found to diffuse throughout the
331
entire NP cells without cobalt in control group, while calcein fluorescence outside of
332
mitochondria was quenched in control cells for the addition of cobalt. IL-1β treatment
333
significantly increased the fluorescence within mitochondria, suggesting the activation of
334
mitochondrial Ca2+ uptake and mPTP opening which should take the blame for mitochondrial
335
dysfunction. However, NaHS pretreatment led to a significant decrease in mitochondrial
336
fluorescence, compare to IL-1β group.
337
To detect whether the sustained mPTP opening can dissipate MMP in NP cells, the 19 / 42
H2S inhibits apoptosis of NP cells
338
membrane potential-dependent MitoTracker was used to assess the MMP. Our results showed
339
that the red fluorescence significantly reduced after a 24hrs exposure to IL-1β, while NaHS
340
markedly abrogated the decrease of fluorescence, compare with IL-1β-treated cells (Fig.4B,C).
341
As expected, similar beneficial effect was also observed in ATP levels, we found that IL-1β
342
treatment prominently increased cellular ATP depletion, which was reverted by NaHS
343
pretreatment. (Fig.4D)
344
Transmission electron microscopy (TEM) was used to examine mitochondrial ultrastructure.
345
In our experiment, we observed aberrant mitochondrial fragmentation and swollen
346
mitochondria in IL-1β treated NP cells, indicating the mitochondrial morphological
347
deterioration in IL-1β treated NP cells, while NaHS pretreatment partly reversed the
348
mitochondrial morphological alterations. (Fig.4E) Taking together, these data demonstrate
349
that NaHS prevents mitochondrial dysfunction induced by IL-1β in NP cells.
350
Fig.4
20 / 42
H2S inhibits apoptosis of NP cells
351 352
3.5 IL-1β-induced ER stress is decreased in NaHS-treated NP cells
353
To determine whether ER stress was involved in the anti-apoptotic effect of NaHS in NP
354
cells, the ER stress-related proteins were assessed by western blot. As shown in Figure 5, we
355
found significant increases in the levels of CHOP, cleaved caspase 12, GRP-78, and ATF-6 in 21 / 42
H2S inhibits apoptosis of NP cells
356
the IL-1β-treated group versus the control group, whereas these increases were attenuated
357
significantly in the cells with NaHS pre-treatment. These findings provide evidence that the
358
protective role of NaHS may involve the inhibition of ER stress-related proteins.
359
Fig.5
360 361
3.6 NaHS activates PI3K/Akt and ERK1/2 pathways
362
Based on previous studies showing that PI3K/Akt and ERK1/2 triggered and mediated the
363
cardioprotective effects of H2S preconditioning,[23] we further explored whether the
364
PI3K/Akt and ERK1/2 pathways were involved in the NaHS-mediated inhibition of ER stress. 22 / 42
H2S inhibits apoptosis of NP cells
365
Western blot was used to detect proteins levels of p-Akt, Akt, p-ERK1/2, and ERK1/2
366
(Fig.6A). Compared with the control group, significantly higher levels of expression of
367
p-ERK1/2/ERK1/2 and p-Akt/Akt (Fig. 6B, C) were observed in cells exposed to IL-1β,
368
indicating activation of the PI3K/Akt and ERK1/2 pathways. The activation of the PI3K/Akt
369
and ERK1/2 pathways in NP cells was further increased by the pretreatment with NaHS.
370
These data suggest that the protective effect of NaHS is involved in the PI3K/Akt as well as
371
the ERK1/2 pathways.
372
Fig.6
373 23 / 42
H2S inhibits apoptosis of NP cells
374
3.7 Inhibition of the PI3K/Akt and ERK1/2 pathways partially reverses the protective
375
effect of NaHS
376
To evaluate the roles of PI3K/Akt and ERK1/2 activation by NaHS, two specific signal
377
pathway inhibitors, LY294002 for PI3K/Akt and PD98059 for ERK1/2, were added to the
378
medium. No effect of these inhibitors on cell death was observed (data not shown). LY294002
379
and PD98059 inhibited the activation of Akt and ERK1/2, respectively, compared with the
380
NaHS group (Fig. 7A, B). However, the inhibitory effect of NaHS on the ER stress-related
381
protein levels of CHOP, GPR78, and cleaved caspase 12 was reversed by the addition of
382
LY294002 and PD98059 (Fig. 7A, C). To further confirm the molecular mechanism of NaHS
383
in mitochondrial injury-induced apoptosis in vitro, we carried out immunocytochemical
384
studies on Drp 1 translocation that participates in mitochondrial fission, and mitochondrial
385
probe dye JC-1, was used to measure the descent in mitochondrial transmembrane potential
386
that occurs early in apoptosis. Compared with IL-1β treatment cells, NaHS pretreatment
387
significantly decreased the mitochondrial membrane location of Drp1 in NP cells. However,
388
when ERK1/2 and Akt pathway were inhibited by LY294002 and PD98059, the protective
389
effect of NaHS on mitochondrial function was partially abolished, suggesting that NaHS
390
protected mitochondrial dysfunction through ERK1/2 and Akt pathway. (Fig.8 A) In line with
391
this result, exposure to LY294002 and PD98059 aggravated the reduction of mitochondrial
392
membrane potential by flow cytometry analysis, compare with the NaHS pretreatment cells.
393
(Fig.8 B) As shown in Fig.8E-G, LY294002 and PD98059 abolished the inhibitory effect of
394
NaHS on the expression of apoptotic proteins cleaved caspase 3, Bax, and Bcl-2. Based on an 24 / 42
H2S inhibits apoptosis of NP cells
395
analysis of band density, we found that, compared with NaHS treatment, the levels of cleaved
396
caspase 3 and Bax increased with exposure to LY294002 and PD98059, whereas that of the
397
anti-apoptotic protein Bcl-2 decreased. These results were further confirmed in TUNEL test,
398
the addition of LY294002 or PD98059 significantly increased cell apoptosis in comparison
399
with the NaHS group (Fig. 8C, D). Taken together, these results provide evidence that ER
400
stress and mitochondrial injury-induced apoptosis was aggravated by the addition of Akt or
401
ERK inhibitors and that the protective effect of NaHS was mediated by both the PI3K/Akt
402
and the ERK1/2 signaling pathways.
403
Fig.7
404 405
Fig.8
25 / 42
H2S inhibits apoptosis of NP cells
406 407
3.8 NaHS ameliorates rat intervertebral disc degeneration in vivo 26 / 42
H2S inhibits apoptosis of NP cells
408
To determine the therapeutic effect of NaHS on degenerative rat discs, the magnetic
409
resonance imaging (MRI) and the Pfirrmann MRI grade scores MR images were obtained at 4
410
and 8 weeks after puncture, which showed stronger T2-weighted signal intensities in the
411
NaHS group than in the saline group. Similar results were also observed at 8 weeks (Fig. 9A).
412
Moreover, the Pfirrmann MRI grade scores, which indicate the degree of disc degeneration,
413
were significantly lower in the NaHS treated rat than in the saline group at 4 weeks and 8
414
weeks (Fig. 9C). The beneficial effects were confirmed by Safranin O-Fast Green and
415
hematoxylin and eosin staining at 4 and 8 weeks (data not shown) in the degenerative model.
416
(Fig. 9B) The nuclear cells in control discs were mostly stellar-shaped and distributed evenly
417
in the NP, with the proteoglycan matrix organized in slender streaks among them (Fig. 9B
418
a–d). Compared with the control group, the size of the NP in the saline group decreased
419
progressively and the nuclear cells became larger, rounded, grouped into clusters, and
420
separated by dense areas of proteoglycan matrix, indicating severe degeneration of the NP
421
cells (Fig. 9E–H). NaHS treatment significantly alleviated the decrease of nucleus pulposus
422
tissue and and reversed morphological changes compared with the saline group (Fig. 9B i–l).
423
And, the beneficial effect of NaHS treatment was more significant at 4 weeks post operation
424
than 8 weeks. (Fig. 9D)
425
Fig.9
27 / 42
H2S inhibits apoptosis of NP cells
426 427
4. Discussion
428
IVDD is the leading cause of the most common disorders, however there is no effective
429
therapies for IVDD because the pathogenesis of IVDD is still unclear. Our study
430
demonstrated that the expression levels of CBS and CSE was decreased in both degenerative
431
intervertebral disc tissues and pro-inflammatory cytokine IL-1β-induced NP cells, indicating a
432
reduced ability to generate cellular H2S, which suggested a essential role of endogenous H2S 28 / 42
H2S inhibits apoptosis of NP cells
433
in the process of IVDD (Fig. 1). These finding were further confirmed in endogenous H2S
434
system by the CBS or CSE-siRNA transfection into NP cells. The impaired endogenous H2S
435
generation system made NP cells more vulnerable to inflammatory stimuli.(Fig.2)
436
There is accumulating evidence that, alongside nitric oxide (NO) and carbon oxide (CO),
437
H2S is an important endogenous gasotransmitter signaling molecule. The role of H2S in cell
438
viability is complex, with both pro- and anti-apoptotic effects. The data presented here
439
demonstrated that short-term exposure to 1 mM NaHS markedly ameliorated the decreased
440
viability of NP cells and increased expression of apoptosis-related proteins (Fig. 3). Similarly,
441
a recent study reported that NaHS treatment may increase cell viability and decrease apoptosis
442
in recovery of cardioprotection from ischemic.[24] However, studies have showed the
443
opposite effect of H2S in regulation of apoptosis. H2S exacerbated cisplatin-induced
444
nephrotoxicity in mice, possibly through promoting an apoptotic response.[25] A similar
445
effect of H2S was reported in INS-1E cells, where the increased release of endogenous H2S
446
not only aggravated cellular stress but also stimulated apoptosis.[26] These contrasting results
447
may be attribute to different durations of H2S treatment, such that exposure to an appropriate
448
concentration of H2S attenuates apoptotic responses, whereas higher concentration of H2S can
449
result in increased apoptosis, even necrosis.
450
Due to mitochondria are very sensitive to the alterations of intracellular environment, the
451
role of mitochondria can switch from being supporters of life to facilitator of cell death
452
quickly. Thus, mitochondrial dysfunction is considered to be associated with the loss of NP
453
cells and the subsequent development of IVDD. Previous studies have shown that 29 / 42
H2S inhibits apoptosis of NP cells
454
mitochondrial pathways[27] were involved in IVD cell apoptosis other than death receptor[28,
455
29]Mitochondrial mediated apoptosis is induced by permeabilization of the outer
456
mitochondrial membrane, and pro-apoptotic Bcl-2 family members such as Bax and Bak[6]
457
are activated which lead to the release of cytochrome c[30]. In addition to Bcl-2 protein
458
family, cleaved caspase 3 is another important protein involved in apoptosis, and activation of
459
cleaved caspase 3 has been identified as the final executor of apoptosis.[31] In the present
460
study, we assessed the levels of these mitochondria apoptosis related proteins, mitochondrial
461
permeability transition pore (mPTP) opening, and mitochondrial membrane potential and ATP
462
levels after IL-1β stimulation to investigate the molecular mechanism of H2S in mitochondrial
463
dysfunction induced NP cells apoptosis. Our results showed that the expression of
464
cytochrome c, cleaved caspase 3, and Bax were upregulated by the administration of IL-1β,
465
while the level of Bcl2 was downregulated, NaHS pretreatment attenuated cytochrome c,
466
cleaved caspase 3 and Bax activation as well as Bcl2 inhibition (Fig.3 D, E). IL-1β treatment
467
induced mitochondrial dysfunction through enhanced mPTP opening, mitochondrial
468
membrane potential collapse, and decreased ATP levels, eventually leading to related proteins
469
release and apoptosis, which were significantly reversed by NaHS treatment.(Fig.4) These
470
findings indicate that the protective role of H2S against NP cells apoptosis was related to the
471
inhibition of mitochondrial dysfunction, were consistent with previous findings in the
472
cardiovascular system.[15, 32]
473
Inhibitors of the mitochondrial apoptotic pathway failed to completely suppress apoptosis
474
in cultured disc cells,[9, 27] indicating that disc cell apoptosis may be also mediated by other 30 / 42
H2S inhibits apoptosis of NP cells
475
signal transduction pathways, such as the ER pathway. In addition to the function of synthesis
476
and formation of tertiary structure of proteins, ER also acts as a dynamic Ca2+ reservoir and
477
responsible for rapid physiological signal transduction. The ER stress is activated by various
478
cellular stresses that may induce apoptosis.[33] GRP78, ATF6, CHOP and caspase 12 are
479
proteins that up-regulate in response to ER stress and are considered to be mediators of the
480
apoptosis pathways induced by ER stress.[34] Increasing evidence indicates that activation of
481
the ER stress-induced apoptosis pathway contributes to various degenerative diseases,
482
including Alzheimer’s disease,[35] Parkinson’s disease[36] and osteoarthritis.[37] A recent
483
study demonstrated that ER stress was also related to the process of IVDD. In a disc cell
484
apoptosis model induced by cyclic stretch, the suppression of CHOP significantly inhibited
485
excessive apoptosis and attenuated disc degeneration.[8] Similarly, Zhao et al. found that the
486
ER and mitochondria were both involved in disc cell apoptosis and IVDD in rats.[7]
487
Consistent with previous studies, our results showed that levels of GRP78, ATF-6, CHOP and
488
caspase 12 were increased after IL-1β administration. NaHS pre-treatment reduced the
489
accumulation of these ER stress-related proteins so as to inhibit apoptosis (Fig. 3). These
490
results indicated that the protective role of H2S in NP cells may attribute to the inhibition of
491
ER stress.
492
We further explored the underlying mechanism of the protective effect of H2S against
493
mitochondrial dysfunction and ER stress. It has been reported that H2S could induce
494
activation of Akt and ERK1/2 pathways in various cells.[23, 38] While Akt was reported to
495
inhibit the formation of the pro-apoptotic proteins such as Bad, Bax and caspase.[39] Thus, 31 / 42
H2S inhibits apoptosis of NP cells
496
the PI3K signaling cascade was supposed to contribute to the recruitment of endogenous
497
cytoprotective pathways to reduce NP cells apoptosis after stress stimuli. Evidence also
498
showed that both the PI3K/Akt and ERK1/2 pathways are involved in leptin-induced human
499
NP cells cyclin D1 expression and proliferation.[40] In the present study, we found that the
500
protective effect of NaHS preconditioning in NP cells was also related to the activation of
501
both the PI3K/Akt and ERK1/2 pathways (Fig. 6), which was consistent with previous
502
findings. To further confirm that these two pathways are essential for the protective effects of
503
NaHS, the PI3K/Akt inhibitor LY294002 and the ERK1/2 inhibitor PD98059 were used to
504
treat NP cells. The results showed that apoptosis induced by IL-1β was inhibited by NaHS
505
pretreatment, while the combination of inhibitors abolished the protective effect and
506
aggravated mitochondrial damage, further suggesting that H2S protects against endoplasmic
507
reticulum stress and mitochondrial injury via PI3K/Akt and ERK1/2 pathways in NP cells .
508
(Figs. 7, 8) This is consistent with previous findings which revealed the protective effects of
509
H2S in cardiomyocytes[41]. However, recently Xie et al.[38] reported that H2S exerted its
510
protective effects by activating Akt but not ERK1/2 pathway in SH-SY5Y cells. These
511
inconsistent studies demonstrated that H2S may activate different signaling pathways in
512
various cells. To further assess the therapeutic effects of NaHS in vivo, needle
513
puncture-induced degenerative rat discs were evaluated after 4 and 8 weeks of successful
514
modeling. Our results showed the appearance of severely degenerated intervertebral discs in
515
the saline group and discs with less severe degeneration in the NaHS group, indicating that
516
NaHS could ameliorate disc degeneration in vivo (Fig. 9). Although the therapeutic effects of 32 / 42
H2S inhibits apoptosis of NP cells
517
H2S in ameliorating IVDD were also observed after 8 weeks, it was diminishing after 4 weeks,
518
which may be attributable to the loss of a large number of NP cells caused by the further
519
destruction of the intervertebral disc structure in the advanced stages of IVDD.
520
There are some limitations in our study. Firstly, although the differential expression of CBS
521
and CSE have been evaluated in our study, due to technical limitation of our laboratory, the
522
physiological and pathological concentration of H2S in vivo has not yet been detected, which
523
warrant further studies. Secondly, other factors such as oxidative and mechanical stress, are
524
relevant in vivo and may induce NP cells apoptosis haven’t yet been investigated in this study,
525
which should be considered in further studies on the promotion of NP cells apoptosis. Finally,
526
PI3K/Akt and ERK1/2 signaling pathways may mediate multiple complex functions in cells,
527
potentially affect cellular proliferation, differentiation, migration, and apoptosis. Thus,
528
activation of these signaling pathways may affect other physiological functions within the cell
529
that are unrelated to the focus of our study, this should be considered in interpreting our
530
results.
531
As shown to exert protective effect for IVDD in our study, H2S may be a potential medicine
532
for IVDD therapy. However, the half-life of NaHS in aqueous solution is very short, which
533
may result in the loss of its biological activity. Thus, a delivery system to increase the stability
534
of NaHS may contribute to the successful application of H2S.
535
In conclusion, this study provides novel evidence that H2S may play a role in the
536
pathogenesis of IVDD by affecting the apoptosis of NP cells. We found that the protective
537
role of H2S involves the suppression of ER stress and mitochondrial dysfunction via 33 / 42
H2S inhibits apoptosis of NP cells
538
activation of the PI3K/Akt and ERK1/2 signaling pathways. Our study may help to better
539
understand the role of H2S in pathogenesis of IVDD.
540 541
Abbreviations: IL-1β, interleukin (IL)-1β; IVDD, Intervertebral disc degeneration; IVD,
542
Intervertebral disc; H2S, Hydrogen sulfide; NP, nucleus pulposus; CBS, cystathionine
543
β-synthase; CSE, cystathionine γ-lyase; ER, endoplasmic reticulum; siRNA, small interfering
544
RNA; MRI, Magnetic resonance imagings; mPTP, mitochondrial permeability transition pore;
545
MMP, mitochondrial transmembrane potential.
546 547
Acknowledgments
548
This work is supported by grant from National Nature Foundation of China (Grant no.
549
81371988, 81401871 and 81401162), Natural Science Foundation of Zhejiang Province for
550
Distinguished Young Scholars (LR12H06001); Major scientific and technological project of
551
medical and health in Zhejiang Province (WKJ-ZJ-1527); Zhejiang Undergraduate Talent
552
Project (2016R413072).
553 554
Author Contributions
555
D.X., X.Z. and X.W. contributed to the design of the study. H.J., J.C., X.C., J.W. and J.C.
556
contributed to the collection and assembly of data. D.C., N.C., J.W., Yu C., N.T. and Z.Z.
557
contributed reagents/materials/analysis tools. D.X. and X.W. wrote the main manuscript text.
558
All authors contributed in revising the manuscript and approved the final version to be 34 / 42
H2S inhibits apoptosis of NP cells
559
submitted.
560 561
Conflict of Interest
562
The authors declare no conflict of interest.
563 564
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565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593
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(2008).
5. Figure Legends 37 / 42
H2S inhibits apoptosis of NP cells
671
5.1 Fig. 1 H2S generation is decreased in degenerate disc tissues and in IL-1β-treated NP
672
cells
673
(A) a: Pfirrmann grade I patient, male, 22 years old, Lumbar burst fracture caused by accident;
674
b: Pfirrmann grade Ⅲ patient, 18 years old, lumbar disc herniation; c: Pfirrmann
675
gradePfirrmann 76 years old, lumbar disc herniation and spinal stenosis. (B)Representative
676
western blots and bar diagram of CBS and CSE in each human NP tissues group (Pfirrmann
677
grade I to grade Ⅴto (C) NP cells were incubated with 10ng/ml IL-1β for 24hrs and 48 hrs,
678
then, harvested for the measurement of CBS, CSE, and SQOR by western blot. Bar diagram
679
of CBS and CSE expression from three Western blot analyses. (D) CBS, CSE, and SQOR
680
cellular levels were also measured by real-time PCR (RT-PCR). Bar diagram of CBS and CSE
681
expression from three RT-PCR analyses. NP tissues from nine different patients (three in each
682
grade) were used for tissuse gene analysis and western blotting. NP cells from four different
683
non-degenerative IVD tissues were used in cellar experiments. All experiments were
684
performed three times in duplicates. *P < 0.05, **P < 0.01 compared with the control group.
685 686
5.2 Fig.2 Inhibition of CBS or CSE by siRNA significantly increased IL-1β-induced
687
apoptosis in NP cells.
688
(A-B) After CBS or CSE-siRNA transfection, NP cells were harvested to analyse the
689
expression of CBS and CSE in both gene and protein levels. (C) Bar diagram of CBS and
690
CSE levels. (D) The NP cells were incubated 75ng/ml IL-1β for 24hrs. The cell lysates were
691
analysed by western blotting to detect the expression of Cyt c, cleaved caspase3, Bax and 38 / 42
H2S inhibits apoptosis of NP cells
692
Bcl-2. (E-F) Bar diagram of the Cyt c, cleaved caspase3, Bax and Bcl-2 expression from three
693
Western blot analyses. NP cells from four different non-degenerative IVD tissues were used
694
for evaluation three times in duplicates. *P < 0.05, **P < 0.01 compared with the control
695
group. #P < 0.05, ##P < 0.01 versus the IL-1β+Con-siRNA group.
696 697
5.3 Fig. 3 NaHS treatment inhibits apoptosis in NP cells
698
(A and B) NP cells were treated with different doses of NaHS or IL-1β for 24 h. (C) NP cells
699
were pre-treated with 1 mM NaHS for 1 hr, then, 75 ng/ml IL-1β was added for an additional
700
24hrs. (D)The cell lysates were analysed by western blotting to detect the expression of Cyt c,
701
cleaved caspase3, Bax and Bcl-2. (E) Bar diagram of the Cyt c, cleaved caspase3, Bax and
702
Bcl-2 expression from three Western blot analyses. GAPDH was used as a protein loading
703
control and for band density normalization. Quantitative evaluation of three independent
704
experiments employing NP cells from three different non-degenerative IVD tissues tested in
705
duplicates. *P < 0.05, **P < 0.01 compared with the control group, #P < 0.05, ##P < 0.01
706
versus the IL-1β group.
707 708
5.4 Fig. 4 NaHS prevents IL-1β-induced mitochondrial dysfunction
709
NP cells were pre-treated with 1 mM NaHS for 1 hrs, and then 75 ng/ml IL-1β was added for
710
an additional 24 hrs. (A) The mPTP opening was detected by co-loading with calcein-AM and
711
CoCl2. Scale bar, 20 μm. (B) Mitochondrial membrane potential was detected using
712
Mitotracker Red CMXRos, and nuclei were stained with Hoechst. Scale bar, 10 μm. (C) 39 / 42
H2S inhibits apoptosis of NP cells
713
ImageJ software was used to measure relative red fluorescence per cell. (D) ATP content was
714
assessed by ATP-Glo Bioluminometric Cell Viability Assay. (E) Mitochondrial morphology
715
was detected by transmission electron microscopy (Viability NP cells. Arrow: swollen
716
mitochondria; triangle: mitochondrial fragmentation; star: fragmented mitochondria in
717
lysosome). In all experiments NP cells from two different non-degenerative IVD tissues were
718
tested three times in duplicates. *P < 0.05, **P < 0.01, versus the control group, #P < 0.05,
719
##P < 0.01 compared with the IL-1β group.
720 721
5.5 Fig. 5 IL-1β-induced ER stress is decreased in NaHS-treated NP cells
722
(A) NP cells were treated with either no addition, 1 mM NaHS and/or 75 ng/ml IL-1β,
723
asdescribed in the “Materials and methods” section. The cell lysates were analysed for the
724
expression of CHOP, GRP-78, ATF-6 and caspase-12 by western blotting. Bar diagram of (B)
725
CHOP, ATF-6, (C) GRP-78 and (D) caspase-12 expression from three Western blot analyses.
726
NP cells from three different non-degenerative IVD tissues were used in these experiments.
727
All results were repeated three times in duplicates. *P < 0.05, **P < 0.01, versus the control
728
group, #P < 0.05, ##P < 0.01 compared with the IL-1β group.
729 730
5.6 Fig. 6 NaHS activates PI3K/Akt and ERK1/2 in NP cells
731
(A) NP cells were pre-treated with 1 mM NaHS for 1 hrs, and then 75 ng/ml IL-1β was added
732
for an additional 24 hrs. The cell lysates were analysed for the expression of phospho-Akt,
733
Akt, phospho-ERK and ERK by western blotting. (B) Bar diagram of phospho-Akt/Akt and 40 / 42
H2S inhibits apoptosis of NP cells
734
phospho-ERK/ERK levels from three Western blot analyses. GAPDH was used as a protein
735
loading control and for band density normalization. NP cells from twelve different
736
non-degenerative IVD tissues were used in these experiments. All results were repeated three
737
times in duplicates. *P < 0.05, **P < 0.01, compared with the control group, #P < 0.05, ##P <
738
0.01 versus the IL-1β group.
739 740
5.7 Fig. 7 Inhibition of the PI3K/Akt and ERK1/2 pathways partially attenuates the
741
NaHS-mediated reduction in the endoplasmic reticulum (ER) stress effects in NP cells
742
NP cells were pre-treated with 1 mM NaHS with or without the specific inhibitors LY294002
743
(50 μM) and PD98059 (50 μM) for 1 hr, and then 75 ng/ml IL-1β was added for an additional
744
24 hrs. The cell lysates were analysed by western blotting to detect the expression of
745
phospho-Akt, phospho-ERK and ERK, CHOP, GRP-78, ATF-6 and caspase-12. Bar diagram
746
of the (B) phospho-Akt/Akt ratio, phospho-ERK/ERK ratio, (C) CHOP, GRP-78, ATF-6 and
747
caspase-12 expression from three Western blot analyses. GAPDH was used as a protein
748
loading control and for band density normalization. NP cells from twelve different
749
non-degenerative IVD tissues were used in these experiments. All results were repeated three
750
times in duplicates. *P < 0.05, **P < 0.01 versus the control group, #P < 0.05, ##P < 0.01
751
versus the IL-1βgroup.
752 753
5.8 Fig. 8 Inhibition of the PI3K/Akt and ERK1/2 pathways partially reverses the
754
protective effect of NaHS and aggravates mitochondrial dysfunction 41 / 42
H2S inhibits apoptosis of NP cells
755
NP cells were pre-treated with 1 mM NaHS with or without the specific inhibitors LY294002
756
(50 μM) and PD98059 (50 μM) for 1 hr, and then 75 ng/ml IL-1β was added for an additional
757
24hrs. (A) Immunofluorescence double-labeled staining for co-localization of Drp1 with
758
Tom20 in NP cells, scale bar, 10μm. (B) JC-1 mitochondrial membrane potential assay was
759
analyzed by flow cytmetry. (C) Detection of apoptotic cells by TUNEL (green) and DAPI
760
(blue) staining assay. (D) Bar diagram of apoptotic cell rates from three separate experiments.
761
(E) The cell lysates were analysed for the expression of Cyt c, C-caspase3, Bax and Bcl-2 by
762
western blotting. Bar diagram of (F) Cyt c and C-caspase3, (G) Bax and (F) Bcl-2 expression
763
from three Western blot analyses. NP cells from four different non-degenerative IVD tissues
764
were used in these experiments. All results were repeated three times in duplicates. *P < 0.05,
765
**P < 0.01, versus the control group, #P < 0.05, ##P < 0.01 versus the IL-1β group.
766 767
5.9 Fig. 9 NaHS ameliorates rat intervertebral disc degeneration in vivo
768
(A) T2-weighted MRI of a rat tail with a needle-punctured disc at 4 and 8 weeks post surgery
769
(white arrows). (C) The Pfirrmann MRI grade scores in three groups at week 4 and week 8. (B)
770
Representative safranin o-fast green and hematoxylin, and hematoxylin and eosin staining of
771
of disc samples from different experimental groups at 4 and 8 weeks post surgery. Scale bars
772
are 200 μm (40×) and 50 μm (200×), respectively. (D) The histological grades evaluated at
773
week 4 and week 8 in three groups. Samples from 48 rats (sixteen in each group ) were used for
774
imageological and histopathologic analysis.*P < 0.05, **P < 0.01, versus the control group,
775
#P < 0.05, ##P < 0.01 versus the saline group. 42 / 42