Rutin attenuates neuroinflammation in spinal cord injury rats

Accepted Manuscript Rutin Attenuates Neuroinflammation in Spinal Cord Injury Rats Jiang Wu, MD, Li Maoqiang, MD, He Fan, MD, Bian Zhenyu, MD, He Qifang, MD, Wang Xuepeng, MD, Zhu Liulong, MD PII:

S0022-4804(16)00096-2

DOI:

10.1016/j.jss.2016.02.041

Reference:

YJSRE 13679

To appear in:

Journal of Surgical Research

Received Date: 22 November 2015 Revised Date:

22 January 2016

Accepted Date: 26 February 2016

Please cite this article as: Wu J, Maoqiang L, Fan H, Zhenyu B, Qifang H, Xuepeng W, Liulong Z, Rutin Attenuates Neuroinflammation in Spinal Cord Injury Rats, Journal of Surgical Research (2016), doi: 10.1016/j.jss.2016.02.041. 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.

ACCEPTED MANUSCRIPT Revised

2016.01.22

Title: Rutin Attenuates Neuroinflammation in Spinal Cord Injury Rats

Wu Jiang,MD, a

a

Fan He,MD,a

Zhenyu Bian,MD,a

Qifang

Xuepeng Wang,MD,a Liulong Zhu,MD,a,*

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He,MD,b

Maoqiang Li, MD,a

Department of Orthopedics, Nanjing Medical University, Affiliated Hangzhou

Hospital (Hangzhou First People's Hospital), No.261 Huansha Road, Shangcheng

b

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District, Hangzhou 310006,China

Department of Orthopedics, Shanghai Jiao Tong University affiliated Sixth

*

Corresponding author

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People's Hospital, No. 600 Yi Shan Road, Shanghai 200233, China. Liulong Zhu, Department of Orthopedics, Nanjing

Medical University, Affiliated Hangzhou Hospital (Hangzhou First People's Hospital), No.261 Huansha Road, Shangcheng District, Hangzhou, China, Fax number : +86057188146489, Telephone number: +8618758073880, E-mail

Acknowledgment

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address:[email protected]

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This study was supported by the Traditional Chinese Medicine Science and Technology Project of Zhejiang (grant no.2011ZA078) and National Nature Science Foundation of China (grant no. 81472511).

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W. J. and L.Z. conceived the study; W. J. participated in the design and drafted

the manuscript; L.Z. and M.L. participated in the design; Z.B.participated in established animal models, performed the statistical analysis; Q.H.performed the statistical analysis; F.H. performed H&E staining and microscopic examination; X. W. performed the Western blotting and ELISA assays; All authors read and approved the final manuscript.

Disclosure The authors declare that they have no conflict of interest.

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Abstract Background: Neuroinflammatory responses involve the activation of the interleukin (IL) -1β and IL-18. Processing and activation of the pro-inflammatory IL require

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NLRP3 inflammasome activation. Rutin can protect spinal cord against damange, but the potential mechanisms underlying remains unknown. Here, we investigated the molecular mechanisms of rutin-mediated neuroprotection in a rat model of spinal cord injury(SCI).

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Materials and methods: One hundred and twenty female Sprague–Dawley rats were randomly assigned to four groups: sham group, SCI group, SCI + Rutin50 group, and

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the SCI + Rutin100 group. The influences of rutin on inflammatory marker levels, histological alterations, and locomotion scale were analysed.

Results: SCI significantly increased the expression of the NLRP3, ASC, IL-1β, IL-18 and tumor necrosis factor-a (TNF-a). Rutin significantly reduced the levels of reactive oxygen species (ROS) ,malondialdehyde (MDA), NLRP3,ASC,caspase-1,

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IL-1β, IL-18, and TNF-a. Furthermore, rutin administration significantly attenuated histological alteration and improved locomotion recovery. Conclusions: Our data provide clear evidence that rutin attenuates tissue damage and

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improves locomotion recovery, and the mechanism may be related to the alleviation of inflammation and oxidative stress.

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Keywords: rutin; spinal cord injury; reactive oxygen species; NLRP3 inflammasome; inflammation

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1.Introduction Traumatic spinal cord injury (SCI) is a devastating condition which leads to a

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progressive state of nerve degeneration with accompanying physiologic, biochemical, and structural changes [1-3]. The pathophysiology of SCI consists of two major mechanisms:

primary

injury

and

secondary

injury,

induced

by

diverse

pathophysiologic mechanisms including inflammation and apoptosis [4-6]. Previous

nerve functional recovery in rat models of SCI [7]. responses

include

maturation

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Neuroinflammatory

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research has demonstrated that targeting the inflammatory response can improve

and

secretion

of

pro-inflammatory cytokines interleukin(IL)-1β and IL-18, which induce cell death[8]. The maturation and secretion of pro- IL-1β and pro-IL-18 requires the activation of proteolytic

enzyme

caspase-1,which

is

mediated

by

the

activation

of

nucleotide-binding domain -like receptor protein 3 (NLRP3) and subsequently the

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recruitment of apoptosisassociated speck-like protein(ASC)[9,10].The NLRP3 inflammasome ,a kind of cytosolic protein signaling complex, consists of NLRP3, ASC, and caspase-1, and is assembled after endogenous “danger”[11,12]. The NLRP3

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inflammasome can be activated by a variety of stimulating factors including reactive oxygen species (ROS)[13].The NLRP3 inflammasome regulates the maturation and release of IL-1β and IL-18, and targeting of the NLRP3 inflammasome can exert

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neuroprotection in SCI rats[8]. Rutin, a flavonoid obtained from foods and plants, is one of important

compounds and has been shown to be effective in some other disease conditions involving inflammation[14] and oxidative stress[15]. For example, in rats, rutin has been shown to reduce neural damage after intracerebral hemorrhage in rats [16].Previous study have shown that rutin can exert neuroprotective effects through enhancing the neurotrophy in SCI rats[17]. There are other studies that show the rutin-mediated effect, which further inhibits NLRP3 inflammasome activation[17,18]. However, this mechanism of inhibition of NLRP3 inflammasome remains unknown in

ACCEPTED MANUSCRIPT the case of SCI.

Based on above considerations, we investigated whether rutin could inhibit

2. Materials and Methods 2.1. Animal Preparation and Grouping

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NLRP3 inflammasome activation with neuroprotection in a rat model of SCI.

Adult female Sprague-Dawley rats weighing 250–300 g were purchased from

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Beijing Haidian Thriving Experimental Animal Centre (Beijing, China). All procedures for these experiments complied with the guidelines of the Animal Ethics

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Committee of Hangzhou First People's Hospital (Hangzhou, China). The rats were housed in a standard animal room with a 12 h light/dark cycle. One hundred and twenty rats were randomly assigned into four equal groups via a random number table: (1) sham group, where the rats only underwent laminectomy and intraperitoneal injection with 1 ml dimethyl sulfoxide(DMSO)

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immediately after SCI; (2) SCI group, where rats underwent SCI and intraperitoneal injection with 1 ml DMSO immediately after SCI; (3) SCI + Rutin50 group: where 50 mg/kg rutin (Sigma-Aldrich, St.Louis, MO, USA) in 1 ml DMSO was

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intraperitoneally injected immediately after SCI;(4) SCI + Rutin100 group: where 100 mg/kg rutin in 1 ml DMSO was intraperitoneally injected immediately after SCI. All animals in the four groups underwent an intraperitoneal injection daily for 3 days (3

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times totally). The dose and timing of rutin were based on previous studies[18].

2.2. Establishment of the SCI Model SCI was induced by a model described by Farsi[20]. Rats were anesthetized

with an intraperitoneal injection of 3.0 mL/kg 10% chloral hydrate. Laminectomy was performed to expose the spinal cord at the vertebral T9–T11 segment without damage to the dura. The spinal cord at the vertebral T10 segment (spinal T9) underwent a 1 min compression with an aneurysm clip, horizontally. Rats were administered an intramuscular injections of penicillin (400,000 unit/animal/d) and buprenorphine to

ACCEPTED MANUSCRIPT prevent infection and relieve pain postoperatively. In addition, rats underwent manual bladder emptying twice a day.

2.3. Evaluation of Locomotor Deficit

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Locomotion deficit was evaluated using the Basso-Beattie-Bresnahan (BBB) locomotion rating scale, as previously described [8]. The rating scale was from 0 to 21 (0=complete paralysis, 21=normal). The rats (n=5 for each group) were assessed with this scale at days 1, 3, 7, and 14 after SCI by two independent investigators who were

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blinded to group assignment.

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2.4. Histologic study

72 h after injury, rats (n=5 in each group) were perfused with 0.9% saline and subsequently with 4% paraformaldehyde. For the histologic analyses, some paraffin spinal cord sections were stained with hematoxylin-eosin (HE) reagent. Histologic scoring was on the basis of (1) edema, (2) neutrophil infiltration, and (3) hemorrhage.

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The score representing severity of spinal cord injury was recorded as follows: 0, none or minor; 1, limited; 2, intermediate; 3, prominent; and 4, widespread [21].

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2.5. Biochemical Analysis

After the spinal cord samples at the damaged area (10 mm, n=5 for each group) were removed 72 h after injury, they were immediately homogenized in phosphate

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buffered saline(PBS) and centrifuged at 1000 rpm for 15 min at 4° C. IL-1β, IL-18, and tumor necrosis factor-alpha (TNF-a) concentrations in the collected supernatants were determined through enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA).

2.6. Protein Extraction and Western Blot Analysis The spinal cord samples at the damaged area (10 mm, n=5 for each group) were removed 24 h after injury and stored at -80° C until further use. Specimens were homogenized in Radio-Immunoprecipitation Assay(RIPA) buffer and then centrifuged

ACCEPTED MANUSCRIPT at 12,000 rpm for 30 min at 4° C. Protein concentration in the supernatant was quantified via the BCA method. Total protein (20 µg) was separated with 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Membranes were

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blocked with 5% skimmed milk, and subsequently incubated with specific primary antibodies overnight at 4° C. Primary antibodies contained anti-NLRP3, anti-ASC, anti-caspase-1, and anti- β-actin (all 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with PBST, the membranes were incubated with a

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horseradish peroxidase-coupled secondary antibody (1:1000; Millipore). Detection of proteins was performed using the enhanced chemiluminescence kit (Thermo

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Scientific, Rockford, IL, USA). Protein levels were analyzed via imaging software (Quantity One; Bio-Rad Co. Ltd., Hercules, CA).

2.7. Measurement of ROS and MDA (Malondialdehyde)Levels The measurement of ROS production in spinal cord sample (n=5 for each group)

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was performed through the oxidative fluorescent dye dihydroethidium(DHE) 72h after SCI. Some 10- µ m spinal cord cryosections were equilibrated with PBS for half an hour at 37 °C, and then incubated with DHE for half an hour at 37 °C . Oxidized

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DHE was determined by fluorescence microscope (TE2000, Nikon, Tokyo, Japan). 72 h after injury, the measurement of spinal cord sample (n=5 for each group) tissue MDA level was performed with a commercial kit (Jiancheng Co, Nanjing,

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China) according to the manufacturer’s instructions. The MDA level was expressed as nanomole / milligram protein.

2.8. Statistical Analysis Data in the study are expressed as the mean ± standard error of the mean (SEM) and were analyzed using SPSS software version 16.0 (SPSS Inc, Chicago, IL). Comparisons between different animal groups was performed by one-way analysis of variance and the Dunnett post hoc test. A p-value of less than 0.05 was considered to be statistically significant.

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3. Results

3.1. Rutin Administration Promotes Functional Recovery

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The BBB score was used to assess the influence of rutin on the progression of locomotion recovery after SCI in rats (Figure 1). We demonstrated that after SCI, locomotion of all rats exhibited partial recovery after days. In comparison with the SCI group, rats in the SCI + Rutin50 (Figure 1;p<0.01) and SCI+Rutin100 (Figure

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1;p<0.001) groups showed a significant improvement in functional recovery from day 3 onwards. However, there was no significant difference between the Rutin50 and

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Rutin100 groups with regards to locomotion recovery.

3.2. Rutin Administration Reduces Histopathologic Damage Histopathologic changes of the spinal cord were used to assay the protective effect of rutin on rats with SCI. Compared with no change in the sham group(Figure

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2A), congestion, edema, neutrophil infiltration and structural disruption were shown in the SCI group (Figure 2B;p<0.001). Nevertheless, the changes were inhibited significantly by rutin administration in the SCI + Rutin50 (Figure 2C;p<0.01) and

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SCI+Rutin100 (Figure 2D;p<0.001) groups, as compared with the SCI group. Histopathologic scores were calculated and are shown in Figure 2E. However, there was no significant difference between the Rutin50 and Rutin100 groups with regards

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to histopathologic changes.

3.3. Rutin Administration Reduces Proinflammatory Cytokine Levels SCI significantly increased proinflammatory cytokines IL-1β, IL-18, and TNF-α

production 72 h after injury in the SCI group (Figure 3A, Figure 3B and Figure 3C;IL-1β:p<0.001;IL-18:p<0.001; TNF-α:p<0.001), as compared with the sham group. However, rutin administration significantly reduced inflammatory cytokine levels in the SCI + Rutin50 (Figure 3A, Figure 3B and Figure 3C;IL-1β:p<0.01; IL-18:p<0.01; TNF-α: p<0.05) and SCI+Rutin100 (Figure 3A, Figure 3B and Figure

ACCEPTED MANUSCRIPT 3C;IL-1β:p<0.001; IL-18:p<0.001; TNF-α: p<0.01) groups as compared with the SCI group. Furthermore, there was no significant difference between the Rutin50 and Rutin100 groups, sham and Rutin100 groups with regards to proinflammatory cytokines level. However, there was significant difference between sham and Rutin50

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groups(IL-1β:p<0.05; IL-18:p<0.01; TNF-α: p<0.05).

3.4. Rutin Administration Inhibits NLRP3 Inflammasome Activation

SCI induced significant upregulated protein expression of NLRP3, ASC, and

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active-caspase-1 in the SCI group (Figure 4;NLRP3:p<0.01; ASC:p<0.001; active-caspase-1: p<0.001), as compared with the sham group. Nevertheless, rutin

Rutin50

(Figure

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administration reduced the NLRP3, ASC, and active-caspase-1 levels in the SCI + 4;NLRP3:p<0.01;ASC:p<0.01;active-caspase-1:p<0.001)

and

SCI+Rutin100 (Figure 4;NLRP3:p<0.01; ASC: p<0.001;active-caspase-1:p<0.001) groups as compared with the SCI group. However, there was no significant difference between the Rutin50 and Rutin100 groups with regards to protein expression of

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NLRP3, ASC, and active-caspase-1.

3.5. Rutin Administration Reduces ROS and MDA Levels

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ROS and MDA productions were significantly increased in the SCI group (Figure 5A and Figure 5B;ROS: p<0.001;MDA p<0.001) compared with the sham group.

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Furthermore, ROS and MDA productions were markedly decreased in the SCI + Rutin50 (Figure 5A and Figure 5B;ROS: p<0.05;MDA p<0.05) and SCI+Rutin100 (Figure 5A and Figure 5B;ROS: p<0.01;MDA p<0.01) as compared with SCI group. However, there was no significant difference between the Rutin50 and Rutin100 groups with regards to ROS and MDA productions.

4. Discussion The positive effects of Chinese herbal medicine in SCI are widely recognized

ACCEPTED MANUSCRIPT [21,22]. Rutin, a flavonoid, has been shown to improve neurologic recovery in some models owing to its multiple beneficial pharmacologic effects [23]. In the present study, we found that rutin administration could improve neurologic recovery, which is in agreement with previous studies [19]. Furthermore, we showed that rutin

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administration reduced ROS and MDA productions and suppressed NLRP3 inflammasome activation with decrease of IL-1β, IL-18, and TNF-α levels and attenuation of histopathology 72 h after SCI. However, there was no significant difference between the Rutin50 and Rutin100 groups with regards to these markers. exhibits

anti-inflammatory

neuroprotective

properties.

In

effects

vitro,

rutin

through

antioxidative

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Rutin

reduced

nitric

oxide

and and

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pro-inflammatory cytokines production in SH-SY5Y neuroblastoma cells[24]. Furthermore, rutin attenuated 6-OHDA-induced neurotoxicity via improving antioxidant enzyme levels and reducing lipid peroxidation in PC-12 cells[25]. Moreover, in vivo, rutin has been demonstrated to enhance the neurotrophic effect by reduction of MIP-2 and p-Akt expression, and MMP-9 activation in SCI rats[17]. Our

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data showed that rutin controlled oxidative stress and inflammation with inhibition of NLRP3 inflammasome activation in rat model of SCI. IL-1β plays a key role in spinal cord damage [26,27], which is involved in

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increasing other proinflammatory cytokines such as TNF-α,IL-6 [28,29], enhancing vascular permeability [30], and induction of neuron apoptosis [31]. Absence of IL-1β inhibits lesion development and axonal plasticity and exerts positive effects on

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neurological outcome [32]. Blocking the IL-1 receptor could suppresses microglial activation and promotes ventral horn neuron survival [33], attenuates the severity of SCI [34], and promotes neurologic recovery after SCI [35]. IL-18 is another kind of important proinflammatory cytokine after SCI [36]. Inhibition of IL-18 reduces NF-κB phosphorylation in spinal astrocytes, and suppresses the induction of astroglial markers [37]. TNF-α can induce spinal cord motoneuron death [38] and exacerbate cell death after SCI [39] .Topical application of TNF-α antiserum can alleviate edema, microvascular permeability, and cell injury in rat SCI [40].Consistent with previous studies, we found that trauma to SCI causes an increase in pro-inflammatory cytokine

ACCEPTED MANUSCRIPT levels in the spinal cord [8]. Moreover, rutin administration markedly downregulated protein expression of IL-1β, IL-18, and TNF-α. Furthermore, a decrease in TNF-α levels may be related to reduced IL-1β production. Moreover, there was no significant difference on proinflammatory cytokine levels in the concentration of 50 mg/kg and

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100 mg/kg. Importantly, the NLRP3 inflammasome is increasingly recognized as an important proinflamatory mediator regulating the maturation and release of IL-1β and IL-18 [9]. Most recently, the NLRP3 inflammasome is recognized to play an

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important role in spinal cord tissue after SCI, and targeting of the NLRP3 inflammasome can inhibit neuroinflammation, improving functional recovery in SCI

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rats [8]. In this study, we found that SCI caused NLRP3 inflammasome activation, which is in accordance with previous studies [8].Furthermore, rutin administration significantly inhibited inflammasome activation, possibly via a decrease in ROS production. Moreover, there was no significant difference on NLRP3 inflammasome activation in the concentration of 50 mg/kg and 100 mg/kg.

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ROS also plays a key role in secondary oxidative stress, which contributes to spinal cord tissue damage [41]. A recent study has demonstrated that ROS can regulate NLRP3 inflammasome activation [13]. Inhibition of ROS production

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presumably suppresses inflammasome activation [42]. Another previous study showed that rutin can increase super oxide dismutase (SOD) activity, reduce MDA production, protect against Alzheimer’s disease (AD) through attenuating oxidative

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stress [43]. We demonstrated that SCI induction increases ROS and MDA levels; however, rutin obviously inhibited this process. Moreover, there was no significant difference on oxidative stress in the concentration of 50 mg/kg and 100 mg/kg

5. Conclusions Our data provide clear evidence that rutin attenuates tissue damage and improves locomotion recovery, and the mechanism may be related to the alleviation of inflammation and oxidative stress.

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Figure legends

Fig. 1 Time course of BBB score after SCI, and the effect of rutin on BBB score. P<0.001 SCI+Rutin100 vs. SCI. ##P<0.01 SCI+ Rutin50 vs. SCI. The data represent

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***

means± SEM.

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Fig. 2 Effects of rutin on histopathologic change of spinal cord tissue 72h after SCI. ***

P<0.001 SCI vs. Sham. ##p<0.01SCI+ Rutin50 vs. SCI.

###

p<0.001SCI+ Rutin100

vs. SCI. The data represent means± SEM. (A) sham group; (B)SCI group; (C) SCI+Rutin50 group; (D)SCI+Rutin100;(E) Histopathologic scores. Fig. 3 Effects of rutin on proinflammatory cytokines IL-1β (A),IL-18 (B) and TNF- α

#

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(C) protein levels in spinal cord tissue 72h after SCI.***P<0.001 SCI vs. Sham. p<0.05SCI+Rutin50 vs. SCI.

###

##

p<0.01SCI+ Rutin50 or SCI+ Rutin100 vs. SCI.

p<0.001SCI+ Rutin100 vs. SCI. The data represent means± SEM.

Fig. 4 Effects of rutin on NLRP3、ASC、active-caspase-1 protein levels in spinal cord

SCI.

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EP

tissue 72h after SCI.**p<0.01 and ***P<0.001 SCI vs.Sham. ##p<0.01 SCI+ Rutin50 vs. p<0.001 SCI+ Rutin50 or SCI+Rutin100 vs. SCI. The data represent means±

AC C

SEM.

Fig. 5 Effects of rutin on ROS (A) and MDA (B) productions in spinal cord tissue 72h after SCI.***p<0.01 SCI vs. Sham. #p<0.05SCI+ Rutin50 vs. SCI. Rutin100 vs. SCI. The data represent means± SEM.

##

p<0.01SCI+

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pro-caspase-1

active-caspase-1 β-actin

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