Exendin-4 improves neuron protection and functional recovery ...2020/11/09 · Nrf-2 / OH-1 is an antioxidant pathway that plays an important role in the body's 69 antioxidant response,

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1 Exendin-4 improves neuron protection and functional recovery in experimental

2 spinal cord injury in rats through regulating PCBP2 expression

3 Huaichao Luo 1*, Qingwei Wang 2, Lei Wang 3†

4

5 1. Department of Clinical Laboratory, Sichuan Cancer Hospital & Institute, Sichuan

6 Cancer Center, School of Medicine, University of Electronic Science and

7 Technology of China, Chengdu, 610041, China.

8 2. The Sichuan Provincial Key Laboratory for Human Disease Gene Study, Hospital

9 of the University of Electronic Science and Technology and Sichuan Provincial

10 People’s Hospital, Chengdu, 610072, China.

11 3. Department of Medical Ultrasound, Sichuan Academy of Medical Sciences &

12 Sichuan Provincial People's Hospital, Chinese Academy of Sciences Sichuan

13 Translational Medicine Research Hospital; Chengdu, 610072, China.

14

15 † Corresponding Author: Lei Wang, Department of Medical Ultrasound, Sichuan

16 Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chinese

17 Academy of Sciences Sichuan Translational Medicine Research Hospital, No.32,

18 West 2nd section, Yihuan road, Chengdu 610072, Sichuan, China, Tel:

19 +86-028-87393999; E-mail: [email protected].

20

21 Keywords: spinal cord injury; exendin-4; PCBP2; neuroprotective effect; cell

22 apoptosis; inflammation

.CC-BY 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

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https://doi.org/10.1101/2020.11.09.373993

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22

23 Abstract

24 Aims

25 In the present research, we assessed the therapeutic effects of Exendin-4 (Ex-4) on rat

26 models with spinal cord injury (SCI).

27 Materials and methods

28 36 male Sprague–Dawley rats were randomly allocated into three groups, including

29 sham operation group, SCI group and SCI+Ex-4 group (Ex-4 treatment (10 µg/rat)

30 after SCI, i.p.). In the SCI group, a laminectomy was performed at the T10 vertebrae,

31 followed by weight-drop contusion of the spinal cord. In the sham group, a

32 laminectomy was carried out without SCI contusion.

33 Key findings

34 Our results showed that Basso-Beattie-Bresnahan scale scores were significantly

35 decreased after SCI, and were obviously improved in SCI rats with Ex-4

36 administration. Additionally, the water content of spinal cord in SCI group was

37 dramatically increased than that in sham group, and after Ex-4 treatment, degree of

38 edema of spinal cord was remarkably reduced. And also, concentration levels of

39 inflammatory cytokines (IL-1α, IL-1β, IL-6 and TNF-α) in the spinal cord were

40 significantly elevated after SCI, and were remarkably reduced in SCI rats with Ex-4

41 administration. Subsequently, cell apoptosis rate in the injured spinal cord was

42 significantly increased, and after Ex-4 treatment, cell apoptosis rate was remarkably

43 decreased. We also revealed that levels of PCBP2 mRNA and protein were

44 significantly up-regulated after SCI, and were dramatically dropped in SCI rats with



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45 Ex-4 administration.

46 Significance

47 Take altogether, our findings disclosed that Ex-4 plays a role in promoting

48 neurological function recovery and inhibiting neuronal apoptosis through effecting

49 PCBP2 expression in SCI rat models.

50 Keywords: spinal cord injury; exendin-4; PCBP2; neuroprotective effect; cell

51 apoptosis; inflammation

52 Introduction

53 The pathophysiological changes of spinal cord injury (SCI), a highly devastating

54 pathology that seriously harms human health worldwide (1), is categorized into two

55 temporally-related mechanisms: the primary injury and the secondary injury. The

56 primary injury refers to a short-time impairment caused by the initial mechanical

57 trauma, which leads to irreversible damage of neurons. The secondary injury, induced

58 by multiple biological events including oxidative stress, immune dysfunction and

59 neuronal apoptosis (2-5), often causes a great number of neurological, behavioral,

60 emotional, and cognitive deficits. Despite great achievements in the therapeutic

61 approaches, the prognosis of SCI patients still remains poor due to severe

62 neurological deficits. Accordingly, it is necessary for us to explore the main

63 mechanisms of neuronal apoptosis in SCI and identify a novel anti-apoptotic drug for

64 SCI treatment.

65 Oxidative stress is one of the important factors that cause the damage of SCI neurons.

66 Oxidative stress response can cause the levels of reactive oxygen species and



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67 inflammatory factors increase, thereby inducing neuronal damage and apoptosis (6)

68 (7). Nrf-2 / OH-1 is an antioxidant pathway that plays an important role in the body's

69 antioxidant response, inhibition of the Nrf-2 / OH-1 pathway will aggravate SCI, and

70 activate the Nrf-2 / OH-1 pathway play a protective effect on neurons (8, 9). It is

71 showed that Nrf-2 / OH-1 pathway can be activated by Exendin-4 (Ex-4) (10). Ex-4, a

72 39-amino acid peptide originally extracted from helodermasuspectum venom (11), is

73 extensively considered to be an effective drug for treating diabetes in the past decades

74 (12-14). In addition, more and more investigations showed that Ex-4 might serve a

75 neuro-protective role in several neurodegenerative diseases, including amyotrophic

76 lateral sclerosis (15), Huntington’s disease (16), Parkinson disease (17) and

77 Alzheimer’s disease (18). However, recent study demonstrated that Ex-4 could

78 prevent against SCI-induced mitochondrial apoptotic pathway (19). However, the

79 physiological or pathological functions of Ex-4 on SCI remained to be further

80 clarified.

81 PCBP2, a member of the poly(C)-binding protein (PCBP) family, exerts a crucial role

82 in posttranscriptional and translational regulation through interacting with

83 single-stranded poly(C) motifs in target mRNAs (20). Aberrant PCBP2 expression

84 was closely associated with a wide variety of human diseases, such as hepatic insulin

85 resistance (21), and cardiomyocyte hypertrophy (22). Roychoudhury et al. (23)

86 showed that PCBP2 was down-expressed in oral cancer cells, and up-regulation of

87 PCBP2 could contribute to a significant increase in the number of apoptotic cells.

88 Moreover, previous evidence indicated that PCBP2 might play important roles in



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89 neuronal apoptosis and astrocyte proliferation after SCI (24).

90 In the present article, we aimed to explore whether Ex-4 could exert a therapeutic

91 effect in SCI through regulating PCBP2 expression and Nrf-2 / OH-1 pathway. Our

92 research might provide a potential therapeutic strategy for patients with SCI.

93 Materials and methods

94 Animals and groups

95 A total of 36 adult male Sprague–Dawley (SD) rats weighing from 250 to 300 g were

96 purchased from Animal Center of Chinese Academy of Sciences, Shanghai, China.

97 Animals were housed in individual cages under standard laboratory conditions with a

98 12-hour light/dark cycle and were given ad libitum access to standard diet and water.

99 The rats were randomly divided into three groups (n=12 per group), including (1)

100 sham operation group, (2) SCI group and (3) SCI+Ex-4 group. The rats in sham

101 operation group were only received opening laminectomy operation and administrated

102 with normal saline, the rats in SCI group were received the process of SCI and

103 administrated with normal saline and the rats in SCI+Ex-4 group were administrated

104 intraperitoneally (i.p.) with Ex-4 (10 µg/rat) (Sigma-Aldrich, St. Louis, MO, USA) in

105 normal saline after SCI and dosing interval was 24h for three days. After 7 days, all

106 rats were sacrificed through cervical dislocation to collect samples for further

107 experiments. All procedure and handling techniques were approved by the Ethics

108 Committee of Sichuan Provincial People's Hospital and were in strict accordance with

109 the recommendations in the National Institutes of Health Guide for the Care and Use

110 of Laboratory Animals.



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111 Establishing SCI model

112 The rat models of SCI in this study were established based on the method by Allen as

113 described previously (25). All rats in this study were anesthetized with intraperitoneal

114 injection of 10% chloral hydrate (0.3 ml/kg, i.p.) and placed in a stereotactic frame.

115 The skin and muscle were incised and a laminectomy was carried out to explore the

116 thoracic vertebra level 10 (T10) of rats. Then a 10-g weight impactor was vertically

117 dropped from a 20-mm height onto the exposed cord. After the operation above, the

118 incision were closed with suture. In addition, the rats in sham operation group were

119 only received the laminectomy without SCI. All animals were then placed on a warm

120 pad until they recovered from anesthesia. Postoperative care included antibiotic

121 (crystalline penicillin 80000 units diluted in 5 ml NS) injections twice a day for 7

122 days and manual bladder expression twice a day until spontaneous voiding occurred

123 or till the end of the study.

124 Assessment of behavior

125 The motor functions of rats, including limb function, the degree of coordinated

126 stepping, stepping ability and trunk stability, were evaluated through the Basso,

127 Beattie and Bresnahan (BBB) rating scale (26). The BBB scores range from 0 points,

128 which indicates no observable hind limb movement, to 21 points, which indicates

129 normal hind movement. Each part was carried out for 10 minutes through two

130 individuals who were blind to treatment at pre-injury and 1, 3 and 7 days after

131 surgery.

132 Wet/dry weight ratio of spinal cord



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133 3-mm spinal cord sections from the center of the injury sites of three groups were

134 obtained and weighted for wet weight (WW). Then, all samples were dried under

135 110°C for 24 h and weighted for the dry weight (DW). [(WW − DW) × 100]/WW was

136 considered to be the water content of spinal cord.

137 Protein extraction and ELISA analysis

138 Following the instructions, the expression of inflammatory cytokines in spinal cord

139 tissue of rats, including IL-1α, IL-1β, IL-6 and TNF-α, were detected through

140 performing ELIS Aassay (Biotechnology Co., Ltd. Shanghai enzyme research,

141 Shanghai, China). With the instruction, total proteins were extracted from spinal cord

142 tissues of all groups by using protein extraction kit (Beyotime Biotechnology,

143 Shanghai, China), and the protein concentration was detected using the Bicinchoninic

144 Acid Protein Assay. Standard (50 μl) was set up and samples were added to the

145 ELISA plate (40 μl dilution+10 μl sample). OD values detected by MTP-800

146 Microplate reader (Corona Electric, Tokyo, Japan). The cytokine contents were

147 presented as pg/mg protein.

148 RNA extraction and RT-qPCR analysis

149 According to the instruction, total RNA was extracted from spinal cord samples

150 through using Trizol Reagents (Ke Min Biological Technology Co., Ltd., Shanghai,

151 China). For analysis of PCBP2 mRNA expression, total RNA was reverse transcribed

152 to cDNA using PrimeScript RT-PCR Kit (Takara, Dalian, China). All primers used in

153 this study were recorded in Table 1. qPCR analysis was performed using SYBR

154 Premix Ex Taq (Takara) on a 7900 Fast Real-Time PCR system (Applied Biosystems,



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155 Foster City, CA).The relative expression of PCBP2 mRNA was normalized to

156 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and calculated using

157 the 2−ΔΔCt method (ΔCt = Cttarget gene – Ctinternal control) (27).

158 Table 1 The sequences of primers.

Gene name Primer sequencesPCBP2Forward 5′-TGGACCCACTAATGCCATCT-3′Reverse 5′-CCTTGATCTTGCATCCACCT-3′GAPDHForward 5′-GGAAAGCTGTGGCGTGAT-3′Reverse 5′-AAGGTGGAAGAATGGGAGTT-3′

159 Western blot assay

160 Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, and

161 transferred onto a nitrocellulose membrane (Bio-Rad, Shanghai, China). After

162 blocking for 1 h, the membrane was incubated with primary antibodies (anti-PCBP2,

163 1:5000; Abcam, San Francisco, USA; anti-Nrf-2, 1:500; anti-OH-1, 1:500) for 4 h,

164 and then incubated with goat-anti rabbit HRP-conjugated secondary antibody for

165 another 2 hours. Immunoreactive protein bands were visualized using an ECL

166 detection system (Amersham, Little Chalfont, UK) and gray values were analyzed by

167 the Luminescent Image Analyzer (LAS-4000 mini, Fujifilm, Uppsala, Sweden).

168 Relative optical density was calculated relative to GAPDH.

169 TUNEL assay

170 Rats were anesthetized by intraperitoneal injection of about chloral hydrate (4%) and

171 the rats were sacrificed. Spinal cord tissue was collected and embedded in paraffin.

172 The sample was cut into 4 μm-thick slices, dewaxed with xylene, and incubated with



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173 gradient of ethanol. The samples were incubated with the proteinase K working

174 solution for 30 min at 37 °C. The reagents were added in the dark according to the

175 instructions of the In Situ Cell Death Detection Kit (Roche, US). Images were

176 acquired under a fluorescence microscope.

177 Statistical analysis

178 The data were presented as mean ± standard deviation (SD). All the data were

179 analyzed and calculated by using SPSS 19.0 software (SPSS, Chicago, IL, USA) and

180 GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). One-way ANOVA and

181 t-tests were used for the statistical analysis. The statistical tests were two-sided; a P

182 value <0.05 was considered to indicate a statistically significant result.

183 Results

184 Exendin-4 protects hindlimb locomotion function after SCI

185 As shown in Figure 1, prior to model establishment, there was no remarkable

186 difference in BBB locomotor rating scale scores in all groups. After SCI, the BBB

187 scores in SCI group were significantly dropped in comparison to those in sham

188 operation group, and the difference had statistically significant (all P<0.001).

189 Moreover, after Ex-4 treatment, the BBB scores were obviously increased at 3 and 7

190 days after SCI in SCI+Ex-4 group compared with those in SCI group, and the

191 difference had statistically significant (all P<0.05).

192 Exendin-4 alleviates edema of the injured spinal cord after SCI

193 To investigate the degree of edema of spinal cord in three groups, wet/dry weight

194 ratio test was performed to calculate the water content of spinal cord in this study. As



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195 shown in Figure 2, the water content of spinal cord in SCI group was significantly

196 elevated than that in sham group (P<0.01). After Ex-4 treatment, degree of edema of

197 spinal cord was remarkably reduced in SCI+Ex-4 group in comparison with that in

198 SCI group, and the difference also had statistically significant (P<0.05).

199 Exendin-4 suppresses inflammation of the injured spinal cord after SCI

200 Post-traumatic inflammatory reaction is a critical event in secondary damage after

201 SCI (28). As shown in Figure 3, concentration level of IL-1α in SCI group was

202 significantly increased than that in sham group (P<0.001). Moreover, compared with

203 that in SCI group, concentration level of IL-1α in SCI+Ex-4 group was remarkably

204 reduced after Ex-4 treatment (P<0.01). For the other cytokines (IL-1β, IL-6 and

205 TNF-α), the results of above comparisons were similar as those of IL-1α.

206 Exendin-4 inhibits cell apoptosis in the injured spinal cord after SCI

207 Apoptosis is a very important mechanism of secondary injury after SCI. As

208 demonstrated in Figure 4, the cell apoptosis rate in the injured spinal cord of SCI

209 group was significantly increased than that of sham group (P<0.001). Consistently,

210 after Ex-4 administration, the cell apoptosis rate was remarkably decreased in

211 comparison with that in SCI group, and the difference also had statistically significant

212 (P<0.01). These results demonstrated that Ex-4 treatment could alleviate neurological

213 deficits via inhibiting cell apoptosis in the injured spinal cord.

214 Exendin-4 inhibits PCBP2 and promotes Nrf-2 and OH-1 expression at mRNA

215 and protein levels in the injured spinal cord after SCI

216 Actually, it still remains elusive how Ex-4 protects neurological function and inhibit



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217 cell apoptosis. In the present study, RT-qPCR analysis was performed to investigate

218 whether mRNA expression of PCBP2, Nrf-2 and OH-1 had an association with SCI.

219 As shown in Figure 5A and 6A, compared with that in sham group, the mRNA

220 expression level of PCBP2 was significantly up-regulated and the levels of Nrf-2 and

221 OH-1 were down-regulated after SCI (P<0.001). The mRNA expression level of

222 PCBP2 was dramatically decreased in SCI+Ex-4 group than that in SCI group, and

223 Nrf-2 and OH-1 were increased in SCI+EX-4 group (P<0.01).

224 Moreover, western blot assay was performed to detect the protein expression of

225 PCBP2, Nrf-2 and OH-1 in SCI. As shown in Figure 5B and 6B, compared with that

226 in sham group, the protein expression level of PCBP2 was significantly up-regulated

227 and Nrf-2 and OH-1 were down-regulated after SCI (P<0.001). The protein

228 expression level of PCBP2 was obviously decreased and Nrf-2 and OH-1 were

229 increased in SCI+Ex-4 group than that in SCI group (P<0.01). There results indicated

230 that Ex-4 might serve a neuro-protective role in SCI through inhibiting the expression

231 of PCBP2 and promoting Nrf-2 / OH-1 pathway.

232 Discussion

233 As we all know, SCI is a severe and complicated medical condition leads to lifelong

234 neurological disabilities in both motor and sensory systems. To our knowledge, it was

235 the first time to reveal that Ex-4 plays a role in promoting neurological function

236 recovery and inhibiting neuronal apoptosis through effecting PCBP2 expression in

237 SCI rat models.

238 Neurological impairment in SCI is mainly caused by two different mechanisms,



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239 including primary injury and secondary damage. The primary injury refers to

240 neurological impairment caused by trauma, including neurons necrosis and axotomy,

241 and the secondary damage occurs with a series of changes in cell metabolism and

242 gene expression after primary injury, including edema, inflammation, ischemia, which

243 eventually leads to neuronal apoptosis (29, 30). Therefore, neuroprotection and

244 neurorecovery still remain the major strategies for SCI treatment.

245 An ideal neuroprotectant for treating SCI should be limited-toxic, easily delivered,

246 and provide protection at all stages of injury. Ex-4, as a glucagon-like peptide-1(GLP)

247 receptor agonist, can effectively down-regulate blood glucose level, stimulate

248 pancreatic β-cell regeneration, induce transcription of pro-insulin gene and promote

249 maturation and secretion of insulin (31). Ex-4 can also stimulate the growth and

250 proliferation of human β-cells, and suppress cytokine-induced apoptosis (32). Fanet

251 al. (33) demonstrated that Ex-4 could inhibit the apoptosis of retinal cell, balance the

252 ratios of Bcl-2/Bax and Bcl-xL/Bax and decrease retinal reactive gliosis in diabetic

253 Goto-Kakizaki rats. In addition, it was reported that Ex-4 serves a cardioprotective

254 role, which could inhibit apoptosis of cardiomyocytes (34). Consistently, Gupta et al.

255 (35) found that Ex-4 could inhibit cell apoptosis and promote the initiation of

256 lipolysis, which played a protective role in ischemic injury of fatty liver. Moreover,

257 increasing reports have demonstrated that Ex-4 could cross the blood-brain barrier

258 (BBB) and act as neurotrophic factors in spinal cord and brain tissues through

259 intraperitoneal administration (15, 36). Wang et al. (37) indicated that the ratio of

260 cortical neuron apoptosis under oxygen/glucose deprivation (OGD) was obviously



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261 reduced after the treatment of Ex-4. Li et al. (15) found that Ex-4 treatment could

262 reduce apoptosis of neuronal cell and ameliorate degeneration of motor neuron in

263 amyotrophic lateral sclerosis. Of note, there are several similar conclusions about the

264 association between Ex-4 and SCI. Li et al. (38) demonstrated that Ex-4 had a

265 protective role in SCI rat models via inhibiting neuronal apoptosis and promoting the

266 function recovery of motor nerve. Our present results extended previous findings by

267 showing that SCI-induced hindlimb locomotion deficits were obviously ameliorated

268 after Ex-4 treatment, indicating that Ex-4 could promote the recovery of locomotion

269 recovery. Spinal cord water content was remarkably reduced and cell apoptosis in the

270 spinal cord tissues was obviously inhibited by Ex-4 administration in SCI rats. All the

271 results corroborate the conclusions of previous relevant studies. Morever, Ex-4

272 intervention could significantly promote the Nrf-2 / OH-1 pathway, which suggested

273 that Ex-4 may alleviate oxidative stress and inflammatory responses by upregulating

274 the Nrf-2 / OH-1 pathway, thereby inhibiting neuronal apoptosis .

275 PCBP2 is involved in regulation of gene expression at multiple levels, including

276 transcription, processing, stability and translational regulation via regulating the

277 binding capacity of poly (C). Mao et al. (24) found that PCBP2 plays an important

278 role in neuronal apoptosis and astrocyte proliferation and PCBP2 knockdown

279 obviously suppresses neuronal apoptosis after SCI. In our study, the levels of PCBP2

280 mRNA and protein were significantly up-regulated after Ex-4 treatment in SCI rat

281 models, indicating a close association between Ex-4 administration and PCBP2

282 expression.



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283 Taken together, in the present research we provided evidence that Ex-4 plays a crucial

284 role in promoting the recovery of neurological function, alleviating the degree of

285 edema of spinal cord and inhibiting cell apoptosis in the injured spinal cord after SCI,

286 and PCBP2 might be a potential functional target of Ex-4 in SCI treatment.

287 Acknowledgements

288 Not applicable.

289 Ethics approval

290 The animal experiment was carried out according to the National Institute of Health’s

291 Guidelines for the Care and Use of Laboratory Animals and was given permission by

292 Animal Care and Research Committee of Sichuan Provincial People's Hospital

293 Conflict of interest

294 All authors declare that there are no conflicts of interest in this study.

295 Funding

296 This study was supported by Young talents fund of Hospital of the University of

297 Electronic Science and Technology and Sichuan Provincial People's Hospital (grant

298 2016QN05)

299 Authors' contributions

300 Huaichao Luo, Qingwei Wang and Lei Wang designed experiments, analyzed data

301 and interpreted results. Huaichao Luo and Qingwei Wang data acquisition. Huaichao

302 Luo, Qingwei Wang and Lei Wang wrote the manuscript and prepared the figures.

303 Huaichao Luo and Qingwei Wang reviewed and edited the manuscript. Lei Wang

304 coordinated and directed the project. All authors approved the final version of the



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305 manuscript.

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413 Inhibition of the MAP3 kinase Tpl2 protects rodent and human beta-cells from

414 apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory



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415 actions of exendin-4. Cell death & disease. 2016;7:e2065.

416 33. Fan Y, Liu K, Wang Q, Ruan Y, Zhang Y, Ye W. Exendin-4 protects retinal cells

417 from early diabetes in Goto-Kakizaki rats by increasing the Bcl-2/Bax and

418 Bcl-xL/Bax ratios and reducing reactive gliosis. Molecular vision. 2014;20:1557-68.

419 34. Younce CW, Niu J, Ayala J, Burmeister MA, Smith LH, Kolattukudy P, et al.

420 Exendin-4 improves cardiac function in mice overexpressing monocyte

421 chemoattractant protein-1 in cardiomyocytes. Journal of molecular and cellular

422 cardiology. 2014;76:172-6.

423 35. Gupta NA, Kolachala VL, Jiang R, Abramowsky C, Romero R, Fifadara N, et al.

424 The glucagon-like peptide-1 receptor agonist Exendin 4 has a protective role in

425 ischemic injury of lean and steatotic liver by inhibiting cell death and stimulating

426 lipolysis. The American journal of pathology. 2012;181(5):1693-701.

427 36. Yamamoto K, Amako M, Yamamoto Y, Tsuchihara T, Nukada H, Yoshihara Y,

428 et al. Therapeutic effect of exendin-4, a long-acting analogue of glucagon-like

429 peptide-1 receptor agonist, on nerve regeneration after the crush nerve injury. BioMed

430 research international. 2013;2013:315848.

431 37. Wang MD, Huang Y, Zhang GP, Mao L, Xia YP, Mei YW, et al. Exendin-4

432 improved rat cortical neuron survival under oxygen/glucose deprivation through PKA

433 pathway. Neuroscience. 2012;226:388-96.

434 38. Li HT, Zhao XZ, Zhang XR, Li G, Jia ZQ, Sun P, et al. Exendin-4 Enhances

435 Motor Function Recovery via Promotion of Autophagy and Inhibition of Neuronal

436 Apoptosis After Spinal Cord Injury in Rats. Molecular neurobiology.



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437 2016;53(6):4073-82.

438

439

440 Figure 1 BBB locomotor rating scale in the three groups of rats, including a sham

441 group, a SCI group and a SCI+Ex-4 group (n=12 per group). The BBB scores were

442 obviously reduced in SCI group and were up-regulated after Ex-4 treatment.

443 Figure 2 Water content of spinal cord in the three groups of rats, including a sham

444 group, a SCI group and a SCI+Ex-4 group (n=12 per group) by wet/dry weight

445 method. Ex-4treatment obviously alleviated edema of spinal cord in SCI rats.

446 **P<0.01 vs sham group, #P<0.05 vs SCI group.

447 Figure 3 Concentrations of inflammatory cytokines in the three groups of rats,

448 including a sham group, a SCI group and a SCI+Ex-4 group (n=12 per group). (A)

449 Concentration levels of IL-1α; (B) Concentration levels of IL-1β; (C) Concentration

450 levels of IL-6; (D) Concentration levels of TNF-α. ***P<0.001 vs sham group;

451 ##P<0.01 vs SCI group; #P<0.05 vs SCI group.

452 Figure 4 Cell apoptosis rates were detected through TUNEL assay in the three groups

453 of rats, including a sham group, a SCI group and a SCI+Ex-4 group (n=12 per group).

454 Ex-4 treatment obviously decreased the number of apoptotic cells after SCI.

455 ***P<0.001 vs sham group; ###P<0.001 vs SCI group.

456 Figure 5 mRNA and protein levels of Nrf-2 / OH-1 were investigated in the three

457 groups of rats, including a sham group, a SCI group and a SCI+Ex-4 group (n=12 per

458 group). (A) mRNA level of Nrf-2 and OH-1 were significantly up-regulated after SCI,



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459 and they were down-regulated under the treatment of Ex-4. GAPDH mRNA was used

460 as an internal control. (B) Protein level of Nrf-2 and OH-1 were significantly

461 up-regulated after SCI and were down-regulated under the treatment of Ex-4. GAPDH

462 protein was used as an internal control. ***P<0.001 vs sham group; ##P<0.01 vs SCI

463 group.

464 Figure 6 mRNA and protein levels of PCBP2 were investigated in the three groups of

465 rats, including a sham group, a SCI group and a SCI+Ex-4 group (n=12 per group).

466 (A) mRNA level of PCBP2 was significantly up-regulated after SCI and was

467 down-regulated under the treatment of Ex-4. GAPDH mRNA was used as an internal

468 control. (B) Protein level of PCBP2 was significantly up-regulated after SCI and was

469 down-regulated under the treatment of Ex-4. GAPDH protein was used as an internal

470 control. ***P<0.001 vs sham group; ##P<0.01 vs SCI group.

471



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Exendin-4 improves neuron protection and functional recovery ...2020/11/09 · Nrf-2 / OH-1 is an antioxidant pathway that plays an important role in the body's 69 antioxidant response,