mitophagy and attenuates oxidative damage in intestinal epithelial cells

Journal Pre-proof SIRT1/PGC-1 pathway activation triggers autophagy/mitophagy and attenuates oxidative damage in intestinal epithelial cells Danyang Liang, Yisha Zhuo, Zeheng Guo, Lihua He, Xueyi Wang, Yulong He, Lexing Li, Hanchuan Dai PII:

S0300-9084(19)30359-1

DOI:

https://doi.org/10.1016/j.biochi.2019.12.001

Reference:

BIOCHI 5802

To appear in:

Biochimie

Received Date: 22 May 2019 Accepted Date: 6 December 2019

Please cite this article as: D. Liang, Y. Zhuo, Z. Guo, L. He, X. Wang, Y. He, L. Li, H. Dai, SIRT1/PGC-1 pathway activation triggers autophagy/mitophagy and attenuates oxidative damage in intestinal epithelial cells, Biochimie, https://doi.org/10.1016/j.biochi.2019.12.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

ABSTRACT Oxidative stress leads to intestinal epithelial cells damage, which induces tight junction injury and systemic endogenous stress syndrome. The evidence suggests that SIRT1/PGC-1α pathway is closely associated with oxidative damage. However, the mechanism in protecting intestinal epithelial cells against oxidative stress dependant on autopahgy/mitophagy remains to be elucidated. In the current study, we investigated the functional role of SIRT1/PGC-1α pathway on regulation of autopahgy/mitophagy and tight junction expression underlying the oxidative dysfunction in porcine intestinal epithelial cells (IPEC-1). Results demonstrated that H2O2 exposure caused high accumulation of ROS, with a decrease of mitochondrial membrane potential and an inhibition of the tight junction molecules in IPEC-1 cells. Also, COX IV mRNA expression and SIRT1/PGC-1α pathway were suppressed. Autophagy and PINK1/Parkin dependant-mitophagy were activated following H2O2 treatment. Further research indicated that activation of SIRT1/PGC-1α pathway caused by specific activator SRT 1720 resulted in elevating autophagy/mitophagy related markers and SIRT1 inhibitor EX 527 reversed these effects. Additionally, SIRT1 activation significantly suppressed the ROS generation, leading to increase mitochondrial membrane potential and COX IV expression. Most importantly, the expression of tight junction molecules contributing

to

maintain

intestinal

barrier

integrity

was

significantly

up-regulated. Collectively, these findings indicated that autophagy/mitophagy

elevation caused by SIRT1/PGC-1α pathway activation might be a protective mechanism

to

increase

tight

junction

integrity

stress-mediated ROS production in IPEC-1 cells.

against

oxidative

1

SIRT1/PGC-1

pathway

activation

triggers

autophagy/mitophagy

2

attenuates oxidative damage in intestinal epithelial cells

and

3 4

Danyang Liang, Yisha Zhuo, Zeheng Guo, Lihua He, Xueyi Wang, Yulong He,

5

Lexing Li, Hanchuan Dai

6

Danyang Liang: ORCID, 0000-0003-1649-3666

7

Yisha Zhuo: ORCID, 0000-0001-6610-5727

8

Zeheng Guo: ORCID, 0000-0002-8233-5714

9

Lihua He: ORCID, Email, 0000-0001-6629-2075

10

Xueyi Wang: ORCID, 0000-0001-7273-5792

11

Yulong He: ORCID, 0000-0002-2043-3896

12

Lexing Li: ORCID, 0000-0001-7939-7407 Hanchuan Dai: ORCID, 0000-0001-6753-9703

13 14

College of Veterinary Medicine, Huazhong Agricultural University, Wuhan,

15

Hubei 430070, China

16 17

Corresponding Author:

18

Hanchuan Dai

19

College of Veterinary Medicine, Huazhong Agricultural University,

20

No.1 Shizishan Street, Wuhan 430070, Hubei, China

21

Tel. +8602787280408, E-mail. [email protected] 1

22

ABSTRACT

23

Oxidative stress leads to intestinal epithelial cells damage, which induces tight

24

junction injury and systemic endogenous stress syndrome. The evidence

25

suggests that SIRT1/PGC-1α pathway is closely associated with oxidative

26

damage. However, the mechanism in protecting intestinal epithelial cells

27

against oxidative stress dependant on autopahgy/mitophagy remains to be

28

elucidated. In the current study, we investigated the functional role of

29

SIRT1/PGC-1α pathway on regulation of autopahgy/mitophagy and tight

30

junction expression underlying the oxidative dysfunction in porcine intestinal

31

epithelial cells (IPEC-1). Results demonstrated that H2O2 exposure caused

32

high accumulation of ROS, with a decrease of mitochondrial membrane

33

potential and an inhibition of the tight junction molecules in IPEC-1 cells. Also,

34

COX IV mRNA expression and SIRT1/PGC-1α pathway were suppressed.

35

Autophagy and PINK1/Parkin dependant-mitophagy were activated following

36

H2O2 treatment. Further research indicated that activation of SIRT1/PGC-1α

37

pathway caused by specific activator SRT 1720 resulted in elevating

38

autophagy/mitophagy related markers and SIRT1 inhibitor EX 527 reversed

39

these effects. Additionally, SIRT1 activation significantly suppressed the ROS

40

generation, leading to increase mitochondrial membrane potential and COX

41

IV expression. Most importantly, the expression of tight junction molecules

42

contributing to maintain intestinal barrier integrity was significantly up-

2

43

regulated. Collectively, these findings indicated that autophagy/mitophagy

44

elevation caused by SIRT1/PGC-1α pathway activation might be a protective

45

mechanism to increase tight junction integrity against oxidative stress-

46

mediated ROS production in IPEC-1 cells.

47 48

KEYWORDS: SIRT1/PGC-1α; Oxidative damage; Intestinal epithelial cells;

49

Autophagy; Mitophagy

50 51

Abbreviations: IPEC, Porcine intestinal epithelial cells; ROS, Reactive

52

oxygen species; SIRT1, Silent information regulator 1; PGC-1α, Proliferator-

53

activated receptor γ coactivator 1α; LC3, Microtubule associated protein 1

54

light chain 3; ATG5, Autophagy-related proteins 5; qPCR, Quantity

55

Polymerase Chain Reactiton; DCFH-DA, 2',7'- Dichlorodi hydro fluorescein

56

diacetate; NAD+, Nicotinamide adenine dinucleotide; ZO-1, Zonula occludens-

57

1; FBS, Fetal bovine serum; HRP, Horseradish Peroxidase; DEPC, Diethyl

58

Pyrocarbonate; PVDF, Polyvinylidene fluoride.

59 60 61 62 63

3

64

1. Introduction

65

Intestinal epithelial cells are essential to the maintenance of the symbiotic

66

relationship between gut microbiota and the host by constructing mucosal

67

barriers, secreting various immunological mediators and delivering bacterial

68

antigens [1, 2]. The intestinal mucosal epithelial barrier is the histological

69

basis for selective permeability of the intestinal mucosa, which prevents

70

conflict between gut microbiota and host immune cells that would result in

71

intestinal inflammation and oxidative stress [3]. The intestine has been

72

characterized as the motor of multiple organ dysfunction syndrome (MODS) [4]

73

and is a key source of ROS. Various intestinal diseases and dysfunction have

74

been attributed to the excess production of ROS [5]. Oxidative stress caused

75

by ROS contributes to impairment of intestinal barrier integrity, eventually

76

leading to the pathogenesis of systemic bowel-origin stress syndrome [6, 7].

77

Recently, targeting cellular stress signaling and ROS have been proposed as

78

new therapies for intestinal diseases [7].

79

Oxidative stress is regarded as a defense mechanism and strongly related

80

with autophagy [8, 9]. As an evolutionarily material degradation and turnover

81

process, autophagy may decrease cellular oxidative stress by clearance of

82

reactive species generating organelles, damaged proteins, or alternatively

83

decrease specific antioxidants to maintain cellular homeostasis in response

84

to different stresses [10, 11]. Cellular oxidative and accumulation of ROS are

4

85

mediators of mitochondrial damage. Mitochondrial dysfunction and oxidative

86

damage may contribute to pathogenesis of a variety of diseases [12].

87

Evidences suggested that PINK1/Parkin-mediated mitophagy is one of the

88

main pathways to eliminate the excessive mitochondria or damaged

89

mitochondria, which leads to mitochondrial quality control and may be

90

detrimental to cell survival [13]. PINK1 can recruit Parkin to the impaired

91

mitochondria as well as resulting in the recruitment of p62/SQSTM1 and

92

ubiquitinated mitochondria or other autophagy related proteins and induce

93

mitophagy.

94

autophagosomes and then degraded by autolysosomes to maintain the

95

homeostasis of mitochondria [14, 15].

Subsequently,

mito-aggresomes

are

phagocytosed

by

96

SIRT1 (silent information regulator of transcription 1) is a highly conserved

97

member of the family of NAD+-dependent Sir2 histone deacetylases, which

98

deacetylates downstream PGC-1α and consequently increases its activity [16].

99

SIRT1/PGC-1α has been reported to involve in the regulation of various

100

pathological processes related to oxidative stress, anti-aging, cell survival,

101

intestinal homeostasis [17-19]. SIRT1 can be activated by SRT 1720 and

102

resveratrol. The half-life of SRT 1720 is longer than that of resveratrol and its

103

affinity with SIRT1 is approximately 1000 times as strong as that with

104

resveratrol [20]. SIRT1 also has been demonstrated to improve mitochondrial

105

oxidative metabolism [21] and positively regulate autophagy and mitochondria

5

106

function under oxidative stress [22, 23]. Overexpression of SIRT1 stimulates

107

the formation of autophagosomes and elevates the basal levels of autophagy,

108

while SIRT1 deficiency arrests autophagy in response to nutrient deprivation

109

[13, 22, 24].

110

To explore the mechanism of SIRT1/PGC-1α pathway activation against

111

cell damage, we performed a series of biochemical assays in IPEC-1 cells.

112

Our results elucidated a pathway whereby SIRT1 might function to decrease

113

oxidative stress injury and maintain intestinal epithelium integrity via

114

autophagy/mitophagy activation.

115 116

2 Materials and methods

117

2.1. Cell culture and treantment

118

IPEC-1 cells were cultured in DMEM/F12 (1:1) (Cat. no: SH30023.01B,

119

Hyclone, USA) medium supplemented with 5% fetal bovine serum (1027-106,

120

Gibco, USA), 1% Penicillin-Streptomycin (15140122, Gibco, USA), 1‰ Insulin,

121

Transferrin, Selenium, Ethanolamine Solution (ITS-X) (51500056, Gibco,

122

USA), 5 µg/L EGF recombinant human protein (PHG0313, Gibco, USA), and

123

maintained in an atmosphere of 5% CO2 at 37 °C (Thermo Fisher Scientific,

124

Loughborough, UK). Cells were seeded in 6- or 96-well plates (Corning, USA)

125

at 1 × 104 or 5×105 cells/well, respectively, and then stimulated with 300 µM

126

H2O2 [25] at a confluence of 70-80%.

6

127

To evaluate the functional role of SIRT1 on oxidative, autophagy and

128

mitophagy, the optimal working concentrations of specific activator SRT 1720

129

(925434-55-5, MCE, USA) and inhibitor EX 527 (49843-98-3, MCE, USA)

130

were screened. Cells were first treated with activator and inhibitor for 12 h,

131

and co-incubated with H2O2 for 12 h under the same conditions cited above.

132 133

2.2. ROS generation measurement

134

ROS concentration in IPEC-1 cells was measured by loading with the

135

fluorescent probe 2ʹ, 7ʹ-dichlorofluorescindiacetate (DCFH-DA) (Jiancheng,

136

Nanjing, China). Briefly, IPEC-1 cells were cultured in 96 well plate at a

137

concentration of 5 × 105 cells/well for 18 h. Supernatants were removed and

138

incubated with HBSS containing 10 µmol/L of the DCFH-DA and incubated at

139

37 °C for 30 min in dark. The cells were treated with 300 µmol/L H2O2 for 1 h.

140

ROS generation was detected by measuring fluorescence at 500/525 (Ex/Em)

141

wave lengths using fluorescence microplate (PE, USA).

142 143 144

2.3. Mitochondrial membrane potential detection 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidazolylcarbocyanine iodide (JC-1)

145

probe was employed to measure mitochondrial depolarization in IPEC-1 cells

146

with mitochondrial membrane potential assay kit (C2006, Beyotime, China).

147

IPEC-1 cells were stained with JC-1 for 15 min at 37 °C according to the

7

148

instruction of manufacturer. The JC-1 monomer and JC-1 polymer in the

149

mitochondria were detected by fluorescence microplate (PE, USA). The green

150

and red fluorescence values were collected after treating with JC-1 staining

151

working solution, and the change of mitochondrial membrane potential was

152

detected by the conversion ratio of fluorescence color.

153 154

2.4. RNA extraction and Real-time quantitative PCR (qPCR) analysis

155

Total RNAs from cells were extracted with Trizol reagent (Invitrogen,

156

Carlsbad, CA, USA) and the reverse transcription reaction was carried out

157

using first strand cDNA synthesis kit (FSK-101, Toyobo, Japan). qPCR was

158

performed using a standard protocol from the SYBR Green Realtime PCR

159

Master kit (QPK-201, Toyobo, Japan) on the LightCycler® 96 detection

160

system (Roche, Switzerland). Comparative quantification was calculated

161

utilizing the 2–∆∆Ct (cycle threshold) method and normalized to GAPDH levels.

162

All samples were performed in triplicate. The primers (Qingke, China) used

163

are listed in the Table 1.

164 165 166 167 168

8

Table1 Primers sequences of qPCR

169 Gene name

Forward primer

Reverse primer

GAPDH

GGTGAAGGTCGGAGTGAA

COX IV

TGG GCAGCAGTGGCAGAATGT

CCCGAAGGCACACCGAAGTAGA

SIRT1

ATTCTTGTGAAAGTGATGAGGATG

ATTGTTCGAGGATCTGTGCC

PGC-1α

GTGTCGCCTTCTTGTTCTTCTTTT

CGCATCCTTTGGGGTCTTT

PINK1

CTCTGGTCGACTACCCCGAT

ATGACGAGGAAGAGTGTCCG

Parkin

CCAAACCGGATGAGTGGTGA

CTTGTCAGAGGTCGGGTGTG

LC3 B

CCGAACCTTCGAACAGAGAG

AGGCTTGGTTAGCATTGAGC

Beclin1

AGGAGCTGCCGTTGTACTGT

CACTGCCTCCTGTGTCTTCA

p62

AGTGTCCGTGTTTCACCTTCC

TGCCCAGACTACGACTTGTG

ATG5

CCCTCTTGGGGTACATGTCT

CGTCCAAACCACACATCTCG

ZO-1

CGGCGAAGGTAATTCAGTGT

TCTTCTCGGTTTGGTGGTCT

Claudin-1

AGATTTACTCCTACGCTGGTGAC

GCAAAGTGGTGTTCAGATTCAG

Occludin

ATGCTTTCTCAGCCAGCGTA

AAGGTTCCATAGCCTCGGTC

CAGAAGGGGCAGAGATGA

170 171

2.5. Protein extraction and western blotting

172

The cells were lysed in RIPA buffer (cat. no. PP1202, Aidelai, China) for 30

173

min on ice. Protein concentration was determined by the bicinchoninic acid

174

method (cat. no. PP0101, Aidelai, China). The proteins were separated by 8%

175

or 12% polyacrylamide gel electrophoresis containing 0.1% SDS and

176

transferred to PVDF membranes. The membranes were incubated for 2 h at

177

room temperature in blocking buffer (20 mM Tris-HCl, 137 mM NaCl, pH 8.0,

178

containing 0.1% Tween and 5% non-fat dry milk) and probed with antibodies 9

179

against LC3 (cat. no. 12741; 1:1000 dilution; CST, USA), Beclin1 (cat. no.

180

3495; 1:1000 dilution; CST, USA), p62 (cat. no. 5114S; 1:1000 dilution; CST,

181

USA), ATG5（cat. no. 12994S; 1:1000 dilution; CST, USA）, PINK1 (cat. no.

182

6946S; 1:1000 dilution; CST, USA), Parkin (cat. no. 14060-1-AP; 1:1000

183

dilution; Proteintech, USA), SIRT1 (cat. no. 9475S; 1:1000 dilution; CST,

184

USA), PGC-1α (cat. no. ab106814; 1:1000 dilution; Abcam, USA), Claudin-1

185

(cat. no. D3H7C; 1:1000 dilution; CST, USA), GAPDH (cat. no. GB13002;

186

1:3000 dilution; Servicebio, China) at 4 °C overnight. After being washed 3

187

times with TBST, the membranes were incubated with HRP-conjugated anti-

188

IgG (cat. no. GB23303; 1:3000 dilution; Servicebio, China) at room

189

temperature for 2 h. GAPDH was used as an internal control. The reacted

190

proteins were visualized using an electrochemiluminescence (ECL) system

191

(Biotanon, China) and protein ratios were calculated following Image J

192

densitometric analysis.

193 194

2.6. Statistical analysis

195

Data were expressed as the mean ± SEM of at least three independent

196

experiments for each cellular experimental group. We evaluated the data by

197

two-tailed student's t-test with Graphpad Prism version 5.0 (Graphpad

198

software, USA). P value of less than 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001)

199

was considered as statistically significant difference.

10

200

3. Results

201

3.1. H2O2 exposure induces oxidative damage in IPEC-1 cells

202

In order to explore the effect of oxidative stress in IPEC-1 cell, cells were

203

treated with 300 µM H2O2 for 12 h. Firstly, ROS levels, mitochondrial

204

membrane potential and COX IV mRNA expression were investigated by

205

using DCFH-DA fluorescent probe, JC-1 fluorescent probe and qPCR. Results

206

showed that ROS levels in IPEC-1 cells were increased after stimulation with

207

H2O2 (Fig. 1A). Meanwhile, the mitochondrial membrane potential (Fig. 1B)

208

and COX IV mRNA expression (Fig. 1C) were significantly suppressed

209

following treatment with H2O2,, which indicated that the oxidative stress might

210

contribute to mitochondrial dysfunction in IPEC-1 cells. Additionally, tight

211

junction molecules including Occludin, ZO-1, Claudin-1 mRNA expression and

212

Claudin-1 protein levels were exerted. The data showed that the mRNA

213

expression levels of Occludin (Fig. 1D), ZO-1 (Fig. 1E) and Claudin-1 (Fig. 1F)

214

were significantly down-regulated. Also, Claudin-1 Protein expression was

215

inhibited (Fig. 1G-H). These findings suggested that oxidative stress resulted

216

in intestinal epithelial cells oxidative damage and triggered intestinal mucosal

217

barrier dysfunction.

11

218 219 12

220

Fig. 1. H2O2 exposure induced oxidative damage in IPEC-1 cells. QPCR and

221

western blotting were performed for detecting COX IV and tight junction

222

molecules including Occludin, ZO-1, Claudin-1 mRNA expression and

223

Claudin-1 protein. A. ROS levels were detected by DCFH-DA fluorescent

224

probe; B. The membrane potential was investigated by JC-1 fluorescent probe;

225

C. COX IV mRNA expression; D. Occludin mRNA expression; E. ZO-1 mRNA

226

expression; F. Claudin-1 mRNA expression; G and H. Claudin-1 protein

227

expression. Data were shown as mean ± SEM of three independent

228

experiments. Significant difference from control (**P < 0.01, ***P < 0.001).

229 230

3.2. Oxidative stress triggers autophagy/mitophagy in IPEC-1 cells

231

Autophagy/mitophagy promotes cell survival under various stressors. To

232

further confirm the oxidative stress-induced autophagy/mitophagy in IPEC-1

233

cells, we measured the expression of autophagy/mitophagy associated

234

markers. The results demonstrated that the mRNA and protein expression of

235

LC3 and Beclin1 were elevated following H2O2 treatment (Fig. 2A-C).

236

The p62 protein, also called sequestosome 1 (SQSTM1), is one of the

237

selective substrates for autophagy, which can bind directly to LC3 and

238

GABARAP family proteins via a specific sequence motif. The results

239

demonstrated that p62/SQSTM1 is negatively correlated with autophagy

240

activity (Fig. 2A-C). It was suggested that PINK1/Parkin mediated mitophagy

13

241

appears to be the best understanding pathway in regulating mitochondria

242

homeostasis in oxidative stress. Thus, we tried to explore the expression of

243

PINK1 and Parkin. The data showed that the mRNA and protein expressions

244

of PINK1 and Parkin were significantly increased (Fig. 2D-F). These findings

245

indicated that oxidative stress might induce IPEC-1 cells autophagy and lead

246

to activate mitophagy process through PINk1/Parkin pathway.

247 248

Fig. 2. Oxidative stress activated autophagy/mitophagy program in IPEC-1

249

cells.

QPCR

and

western

blotting

14

were

performed

for

detecting

250

autophagy/mitophagy related markers including LC3, Beclin1, p62, PINK1 and

251

Parkin. A. LC3, Beclin1 and p62 mRNA expression; B and C. LC3, Beclin1

252

and p62 protein expression; D. PINK1 and Parkin mRNA expression; E and F.

253

PINK1 and Parkin protein expression. Data were shown as mean ± SEM of

254

three independent experiments. Significant difference from control (*P < 0.05,

255

**P < 0.01, ***P < 0.001).

256 257

3.3. SIRT1/PGC-1α pathway is activated by SIRT1 activator SRT 1720

258

SIRT1, known as NAD+-dependent deacetylase sirtuin 1, deacetylates

259

proteins which contribute to cellular regulation of stressors and regulates the

260

activity of the PGC-1α. We investigated the expression of SIRT1/ PGC-

261

1αunder oxidative stress condition in IPEC-1 cells. The results showed that

262

the mRNA (Fig. 3A) and protein (Fig. 3C-D) expression of SIRT1 were down-

263

regulated in IPEC-1 cells after H2O2 treatment. Meanwhile, the levels of PGC-

264

1αmRNA (Fig. 3B) and protein (Fig. 3C-D) were also significantly decreased.

265

These data indicated that oxidative stress could inhibit SIRT1/PGC-1α

266

pathway in IPEC-1 cells. To further confirm the specific role of SIRT1 on

267

autophagy/mitophagy in IPEC-1 cell, cells were treated with SIRT1 activator

268

SRT 1720 (0 µM、1.25 µM、2.5 µM and 5 µM) and inhibitor EX 527 (0 µM、1

269

µM、5 µM、10 µM and 20 µM) to screen the optimal working concentration

270

on the expression of SIRT1 and PGC-1α. QPCR results preliminary illustrated

15

271

that the mRNA expression of SIRT1 (Fig. 4A-B) and PGC-1α (Fig. 4C-D) was

272

significantly increased or inhibited respectively along with the different

273

concentration of the activator and inhibitor. At a concentration of 1.25 µM SRT

274

1720 and 1 µM EX 527, the activation and inhibition were the most

275

pronounced. Next, western blotting was carried out to further verify the effects.

276

We found that 1.25 µM SRT 1720 has significant activation on the SIRT1 and

277

PGC-1α protein expression, and 1 µM EX 527 showed the strong inhibition

278

ability under oxidative stress conditions (Fig. 4E-H). These results indicated

279

that inhibition of SIRT1/PGC-1α pathway caused by oxidative stress could be

280

elevated by SRT 1720 and reversed by EX 527.

281 282

Fig. 3. Oxidative stress inhibited SIRT1/PGC-1α pathway pathway in IPEC-1

16

283

cells. QPCR and Western blotting were performed for detecting the

284

expression of SIRT1 and its downstream PGC-1α. A. SIRT1 mRNA

285

expression; B. PGC-1α mRNA expression; C and D. SIRT1 and PGC-1α

286

protein expression. Data were shown as mean ± SEM of three independent

287

experiments. Significant difference from control (**P < 0.01, ***P < 0.001).

17

288 289

Fig. 4. SIRT1/PGC-1α expression in IPEC-1 cells treated with activator SRT

290

1720 and inhibitor EX 527. QPCR and Western blotting were performed for 18

291

detecting the expression of SIRT1/PGC-1α mRNA and protein. A and B.

292

SIRT1 mRNA expression; C and D. PGC-1α mRNA expression; E and F.

293

SIRT1 protein expression treated with 1.25 µM activator SRT 1720 and 1 µM

294

EX 527; G and H. PGC-1α protein expression treated with 1.25 µM activator

295

SRT 1720 and 1 µM EX 527. Data were shown as mean ± SEM of three

296

independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). NS: no

297

significant difference was observed.

298 299

3.4. Activation of SIRT1 enhances autophagy/mitophagy

300

To explore the role of SIRT1 on the regulation of autophagy/mitophagy in

301

IPEC-1 cells, we treated cells with the 1.25 µM activator SRT 1720 and 1 µM

302

inhibitor EX 527 respectively for 12 h and then co-incubated with H2O2 for

303

another 12 h. The mRNA and protein levels of LC3 and ATG5 were detected

304

by qPCR and Western Blotting. The data demonstrated that activation of

305

SIRT1 could significantly up-regulate mRNA and protein expression levels of

306

autophagy related markers LC3, but the activation was abolished by inhibitor

307

EX 527 (Fig. 5A-C). Notably, as a key protein involved in the extension of the

308

phagophoric membrane in autophagic vesicles, ATG5 contributed to gut

309

microenvironment [26]. The expression was also increased (Fig. 5D-F).

310

Afterwards, the effect of SIRT1 on the PINK1/Parkin dependant-mitophagy

311

was checked. Activation of SIRT1 also promoted PINK1 and Parkin mRNA

19

312

and protein expression (Fig. 6). It was concluded that activation of SIRT1

313

might enhance oxidative stress-induced autophagy and result in elevation of

314

PINK1/Parkin dependant-mitophagy.

315 316

Fig. 5. Evaluation of autophagy related gene after treatment with 1.25 µM

317

SRT 1720 and 1 µM EX 527. QPCR and western blotting were performed for

20

318

detecting autophagy related markers including LC3 and ATG5. A. LC3 mRNA

319

expression; B and C. LC3 protein expression; D. ATG5 mRNA expression; E

320

and F. ATG5 protein expression. Data were shown as mean ± SEM. of three

321

independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). NS: no

322

significant difference was observed.

323

324 21

325

Fig. 6. Evaluation of PINK1/Parkin dependant-mitophagy after treatment with

326

1.25 µM SRT 1720 and 1 µM EX 527. QPCR and western blotting were

327

performed for detecting mitophagy related markers including PINK1 and

328

Parkin. A. PINK1 mRNA expression; B and C. PINK1 protein expression; D.

329

Parkin mRNA expression; E and F. Parkin protein expression. Data were

330

shown as mean ± SEM of three independent experiments (*P < 0.05, **P <

331

0.01, ***P < 0.001). NS: no significant difference was observed.

332 333 334

3.5. Activation of SIRT1 ameliorates oxidative damage in IPEC-1 cells The previous results showed that SIRT1 is abnormally expressed in

335

oxidative

damage

IPEC-1

cells.

Activation

336

autophagy/mitophagy. To investigate whether SIRT1 could ameliorate

337

oxidative damage, IPEC-1 cells were treated with activator SRT 1720 and

338

inhibitor EX 527 for 12 h and coincubated with H2O2 for another 12 h. ROS,

339

mitochondrial membrane potential and COX IV mRNA expression were

340

detected with DCFH-DA fluorescence probe, JC-1 fluorescence probe and

341

qPCR. Results showed that, compared with the H2O2 group, the ROS level

342

was significantly decreased (Fig. 7A). The membrane potential level (Fig. 7B)

343

and COX IV mRNA expression were increased (Fig. 7C) following H2O2

344

treatment, while SIRT1 inhibitor treatment showed the opposite effect. Also,

345

intestinal mucosal tight junction molecule Occludin, ZO-1, Claudin-1 mRNA

22

of

SIRT1

can

enhance

346

and Claudin-1 protein were checked by qPCR or Western blotting. The results

347

indicated that ZO-1 (Fig. 7D), Occludin (Fig. 7E), and Claudin-1 (Fig. 7F)

348

mRNA levels were increased following SRT 1720 treatment on the basis of

349

H2O2 stimulation. Further research indicated that Claudin-1 protein expression

350

was increased, and the inhibitor blocked the effect. These data suggested that

351

activation of SIRT1 could repair the damage of the intestinal barrier integrity,

352

and contribute to a significant antioxidant effect in IPEC-1 cells.

23

353 354

Fig. 7. Activation of SIRT1 could ameliorate oxidative damage in IPEC-1 cells. 24

355

QPCR and western blotting were performed for detecting COX IV and tight

356

junction molecules including Occludin, ZO-1, Claudin-1 mRNA expression and

357

Claudin-1 protein; A. ROS level was investigated by DCFH-DA fluorescent

358

probe; B. The membrane potential was detected by JC-1 fluorescent probe; C.

359

COX IV mRNA expression; D. ZO-1 mRNA; E. Occludin mRNA expression; F.

360

Claudin-1 mRNA expression; G and H. Claudin-1 protein expression. Data

361

were shown as mean ± SEM of three independent experiments (*P < 0.05, **

362

P < 0.01, ***P < 0.001). NS: no significant difference was observed.

363 364

4. Discussion

365

Intestinal epithelial cells play a critical role in the maintenance of gut

366

homeostasis, and involve in multiple biological processes, including digestion

367

and absorption, secretion, immunity, signal recognition [2]. Effective

368

management of intestinal remains a significant challenge to health system.

369

Oxidative stress has been considered as a central mediator and one of the

370

main pathogenic mechanisms of intestinal epithelial cells damage, which is

371

known to be characterized by excessive ROS production and results in cell

372

damage and subsequent complications [27, 28]. These responses are

373

associated with many pathogenic diseases such as microbial and viral

374

infections, exposure to allergens, radiation and toxic chemicals, autoimmune

375

and chronic diseases, damaging many components including proteins,

25

376

DNA/RNA and lipids [29, 30]. Our results indicated oxidative stress caused by

377

H2O2 leaded to ROS accumulation and inhibition of mitochondrial membrane

378

potential. Also, COX IV and tight junction molecules mRNA or protein

379

expression were suppressed. These findings demonstrated that oxidative

380

stress resulted in intestinal epithelial cells oxidative damage and triggered

381

intestinal mucosal barrier dysfunction, which suggested that maintaining the

382

balance between oxidants and antioxidants was important to avoid oxidative

383

stress for alteration intestinal epithelial cells integrity.

384

SIRT1, the best-studied Sirtuin protein family member, is a nuclear and

385

cytoplasmic sirtuin involved in the control of histones a transcription factors

386

function [31]. SIRT1 may contribute to cellular function regulation by

387

deacetylating PGC-1α and involves in energy management, mitochondrial

388

biogenesis and various physiological processes including aging and stress

389

response [20, 32]. SIRT1 and PGC-1α are transcriptional coactivator of many

390

genes and play an important protective role against oxidative stress-related

391

diseases [32, 33]. As an NAD+-dependent deacetylase, SIRT1 can be

392

activated by a small molecule activator SRT 1270 and exerts multiple

393

pharmacological activities with beneficial health effects. It can repair DNA

394

damage, maintain the stability of the genome, regulate mitosis and control

395

oxidative stress [34]. In the present study, a performance of SIRT1/PGC-1α

396

pathway was noted following H2O2 treatment in IPEC-1 cells. The data

26

397

showed that the expression of SIRT1 and PGC-1α protein was decreased

398

under oxidative stress. SIRT1/PGC-1α pathway activation could be caused by

399

agonists SRT 1720, leading to a significant decrease of ROS and an elevation

400

of mitochondrial membrane potential and COX IV expression. However, this

401

activation could be reversed by SIRT1 antagonist, EX 527. These findings

402

suggested oxidative stress resulted in SIRT1/PGC-1α pathway malfunction,

403

and SIRT1/PGC-1α activity elevation caused by SRT 1720 might contribute to

404

antioxidant capacity and oxidative stress inhibition.

405

Accumulating evidences demonstrated that SIRT1 involved in oxidative

406

damage and might promote autophagosome formation through deacetylation

407

of key autophagy related molecules in the form of NAD+-dependence [35, 36].

408

Autophagy is usually considered as a protective process that prepares the cell

409

to survive under various stress conditions [37, 38]. Mitophagy dependant

410

PINK1/Parkin

411

eliminating damaged mitochondria, which is regarded as a defense

412

mechanism of mitochondrial function [39]. Autophagy/motophagy activation

413

contributes to remove the damaged or redundant organelles metabolites and

414

maintain the cell homeostasis [40]. In the present study, oxidative stress

415

significantly activated autophagy markers including LC3  and Beclin 1, and

416

p62 expression level was inhibited. Also, PINK1 and Parkin expression were

417

elevated after treatment with H2O2. The further results indicated autophagy

pathway is

thought

to

27

maintain

mitochondrial quality by

418

markers and PINK1/Parkin pathway were significantly increased compared

419

with H2O2 group. As suggested, LC3 and Beclin1 were autophagy markers

420

to monitor the autophagy flux, and p62 was an autophagic substrates to verify

421

the lysosome degradation [38]. PINK1/Parkin dependant-mitophagy is one of

422

the main pathways contributing to maintain the homeostasis and quality

423

control of mitochondria [41]. The data indicated that activation of SIRT1 could

424

further result in autophagy/motophagy activation based on the oxidative stress

425

in IPEC-1 cells.

426

Intestinal epithelial cells participate in digestion and absorption, secretion,

427

immunity, signal recognition and has been regarded as the center of stress

428

response. Dysfunctional Intestinal epithelial cells are often accompanied by

429

systemic inflammatory response syndrome, sepsis, and even organ

430

dysfunction [42]. Oxidative stress can easily induce intestinal mucosal

431

epithelial structure damage, intestinal flora disorder, increasing intestinal

432

permeability and cause the multiple diseases [43, 44]. Tight junctions

433

represented by Occludin, ZO-1 and Claudin-1 are considered as a

434

cornerstone of barrier integrity and barrier function [45]. We found that the

435

expression of Claudin-1, ZO-1, Occludin mRNA expression and the Claudin-1

436

protein level were suppressed following oxidative stress. SIRT1/PGC-1α

437

pathway activation caused by SRT 1720 resulted in expression elevation of

438

tight junction proteins. SIRT1 inhibitor EX 527 could reverse the effect. These

28

439

findings allowed us to infer that the imbalance of cellular redox system

440

promotes the production of reactive oxygen species (ROS) and contributes to

441

barrier dysfunction. SIRT1/PGC-1α pathway activation could effectively

442

protect the intestinal mucosal barrier from free oxygen radical damage.

443

In conclusion, H2O2 exposure induces IPEC-1 cells oxidative damage and

444

mitochondrial

dysfunction,

which

resulted

445

1αpathway activity and led to autophagy/motophagy activation. SIRT1/PGC-

446

1α pathway activation contributed to autophagy/mitophagy elevation and

447

ameliorated oxidative damage in IPEC-1 cells (Fig. 8). These results indicated

448

that autophagy/mitophagy elevation caused by SIRT1/PGC-1α pathway

449

activation would be a protective mechanism to increase tight junction integrity

450

against oxidative stress -mediated ROS production in IPEC-1 cells and

451

suggested that SIRT1/autophagy/mitophagy/oxidative stress might be an

452

effective potential target and research design idea for intestinal stress injury.

29

in

decreased

SIRT1/PGC-

453 454

Fig. 8 Schematic illustration of SIRT1/PGC-1α on oxidative atress, autophagy

455

and mitopahgy. Oxidative stress induces IPEC-1 cells integrity damage and

456

resulted

457

autophagy/motophagy elevation. SIRT1/PGC-1α pathway activation might

458

ameliorate oxidative damage in IPEC-1 cells through autophagy/mitophagy

459

process.

in

decreased

SIRT1/PGC-1α

pathway

460 461 462

Conflicts of interest The authors declare no competing financial interests.

463 464

Author contributions 30

activation,

and

465

Danyang Liang and Hanchuan Dai conceived and designed the study.

466

Danyang Liang, Yisha Zhuo, Zeheng Guo, Lexing Li performed the

467

experiments. Danyang Liang, Lihua He, Xueyi Wang, Yulong He and

468

Hanchuan Dai analyzed the experimental data. Danyang Liang and Hanchuan

469

Dai wrote the paper. All authors read and approved the final manuscript.

470 471

Acknowledgments

472

This work was sponsored by National key Research and Development

473

Program (Grant no. 2016YFD0501210), Natural Science Foundation of Hubei

474

(Grant no. 2018CFB444), the Fundamental Research Funds for Central

475

Universities of China (Grant no. 2011QC004).

476 477

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478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495

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34

Highlights

1.

H2O2 exposure

triggered

intestinal

mucosal

barrier

dysfunction,

autophagy/motophagy activation and suppressed SIRT1/PGC-1α pathway.

2. Activation of SIRT1/PGC-1α pathway enhanced autophagy and PINK1/Parkin -mediated mitophagy.

3. Activation of SIRT1/PGC-1α pathway contributed to ameliorate oxidative damage in intestinal epithelial cells.

Conflicts of interest The authors declare no competing financial interests.