The Iridoid-rich Fraction From Valeriana Jatamansi Jones Activat The Nrf2/ARE Alleviating Oxidative Stress Reaction Of Spinal Cord Injury

Objective To investigate whether the iridoid-rich fraction from Valeriana jatamansi Jones?IRFV?has anti-oxidative protective effects on spinal cord injury and its regulation of oxidative genes related to Nrf2/ARE signal pathway by establishing a rat model of spinal cord injury?SCI?.In addition,the model of oxidative damage of PC12 cells induced by H₂O₂was selected to further reasearch the protective mechanism of IRFV regulating Nrf2/ARE signal pathway on PC12 cells in vitro.Method 1.The 60 SPF adult male Sprague-Dawley?SD?rats were randomly divided into3 groups?20 rats per group?:the sham-operated group,the model group,and the treatment group with 10 mg/kg IRFV?treatment group?.The model of SCI was established by a mini medical aneurysm clip?force 70g,clamp 10 s?,and the sham-operated group was only performed with the laminectomy without clipping the spinal cord,and the spinal cord of the rest rats were damaged by clamping.Two hours after model establishment successfully,the treatment group was given IRFV by intragastrical administration with 10 mg/kg,and the sham-operated group and the model group were given an equal volume of sodium carboxymethylcellulose?CMC-Na?solution with the same way.And each of group was given intragastrical administration once a day.The rats were sacrificed at the third days after administration,Hematoxylin/eosin?HE?staining was used to observe the pathological changes of the spinal cord tissue at the perilesional area;and Terminal-deoxynucleoitidyl transferase mediated nick end labeling?TUNEL?was used to observe the apoptosis of nerve cells of the spinal cord tissue at the perilesional area;and the contents of oxidative stress indexs superoxide dismutase?SOD?and malondialdehyde?MDA?in serum were detected by Enzyme linked immunosorbent assay?ELISA?;the related biochemical kits was used to test glutathione peroxidase?GSH-Px?and catalase?CAT?viability of the spinal cord tissue;the expression levels of proteins of nuclear NF-E2-related factor 2?Nrf2?and its downstream regulators heme oxygenase 1?HO-1?,NAD?P?H quinone oxidoreductase 1?NQO1?,glutamate cysteine ligase-catalytic subunit?GCLC?and glutamate-cysteine ligase modifier subunit?GCLM?were detected by Western blot.2.To further investigate the neuroprotective effects of IRFV regulating Nrf2/ARE signal pathway on PC12 cells from the cellular level.The oxidative damage model of PC12 cells was induced by different concentrations of hydrogen peroxide?H₂O₂??100,200,300,400,500,600,700??mol/L,and MTT assay was used to detect the effect of different concentrations of H₂O₂ on the survival rate of PC12cells to determine the optimal injury concentration.To study the protective effect of IRFV on oxidative damage of PC12 cells,the cells were divided into four groups:normal control group,H₂O₂ oxidative damaged group?model group?,IRFV+H₂O₂ group?IRFV group?,positive control drug vitamin C+H₂O₂ group?VC group?.The protective effect of IRFV on oxidative damage of PC12 cells was evaluated by MTT assay;the morphology of the cells was observed by inverted microscope;the cell leakage rate was measured by LDH;the cell apoptosis was detected by Annexin V FITC/PI double staining;the level of ROS in the cells was determined by fluorescein DCFH-DA probe method;the activity of MDA,SOD,GSH-Px and CAT was detected by related biochemical kits;the expression of nuclear Nrf2and its downstream regulators HO-1,NQO1,GCLC and GCLM were detected by Western blot.This further reveals the protective mechanism of IRFV on nerve cells,and provides experimental basis and theoretical basis for IRFV treating SCI.Result 1.?1?HE staining results showed that compared with the sham-operated group,the spinal cord structure was severely damaged in the spinal cord tissue of the model group,and the treatment group had protective effects against neuronal morphological changes and also the pathological score was lower than the model group?P<0.05?;?2?The results of the TUNEL method showed that no obvious brown-yellow nerve cells which represent apoptotic were observed in the spinal cord tissue of the sham-operated group,and,however,there were obvious brown-yellow apoptotic nerve cells in the spinal cord tissue at the perilesional area of the model group;compared with the model group,the treatment group apparently attenuated the number of brown-yellow apoptotic nerve cells?P<0.05?;?3?The results of ELISA showed that compared with the sham-operated group,the SOD content decreased and the MDA content increased in the serum of the model group;compared with the model group,the SOD content in the serum of the treatment group increased?P<0.05?,and the MDA content decreased significantly?P<0.01?;?4?The results of the biochemical kits tests showed that the activity of GSH-Px and CAT antioxidant enzymes in the spinal cord of the model group was lower than that in the sham-operated group;compared with the model group,the treatment group significantly increased the activity of the antioxidant enzymes of GSH-Px?P<0.05?and CAT?P<0.01?of the spinal cord tissue;?5?The Western blot results showed that the NQO1 in the model group was higher than that in the sham-operated group?P<0.05?and there was no significant difference in other proteins expression;compared with the model group,the expression levels of nuclear Nrf2,NQO1,GCLC and GCLM proteins in the treatment group were Significantly increased?P<0.05?,and the protein expression of HO-1 increased,but not statistically significant.2.?1?Compared with the normal control group,after being cultured with final concentration of 500 mol/L H₂O₂after 2 h,the cell survival rate was significantly decreased in PC12 cells?P<0.01?,suggesting the model establistment successfully;and the intervention of 80?g/mL IRFV perfom the best protection effect;?2?Compared with the normal control group,the cell oxidative damaged by H₂O₂ significantly decreased the cell viability?P<0.01?;the cell viability of the IRFV group was significantly increased after IRFV intervention?P<0.05?;?3?Compared with the normal control cells,the synapses become shorter or the pseudopods turn into disappearing,and some cells shrink and fall off,and the number of cells decreases on the cell oxidative damaged by H₂O₂;however,the pseudopods of the cells become re-extension and the growth state of the cells is obviously improved after IRFV intervention;?4?Compared with the normal control group,the cell oxidative damaged by H₂O₂ increased LDH leakage and apoptosis rate?P<0.01?;the LDH leakage and apoptosis rate decreased significantly after IRFV intervention?P<0.01?;?5?Compared with the normal control group,the cell oxidative damaged by H₂O₂ increased the expression of Reactive Oxygen Species?ROS?and MDA,and the intervention of IRFV could reverse its effect?P<0.05?;?6?Compared with the normal control group,the cell oxidative damaged by H₂O₂ decreased the activity of antioxidant enzymes SOD,GSH-Px and CAT in PC12 cells?P<0.01?,and all the antioxidant enzyme activity increased significantly after IRFV intervention?P<0.05?;?7?Compared with the normal control group,there was no significant difference in the expression levels of nuclear Nrf2,HO-1,NQO1,GCLC and GCLM proteins after H₂O₂oxidative damage;the expression level of the Nrf2,HO-1,NQO1,GCLC and GCLM protein was significantly increased after IRFV intervention?P<0.05?.Conclusion?1?IRFV plays a neuroprotective role to spinal cord injury by improving the pathological changes and decreasing the apoptosis of nerve cells in rats with SCI;in addition,IRFV can promote the proliferation of PC12 cells with H₂O₂ oxidative damage,reduce the leakage of LDH and apoptosis rate,and improve the morphology changes induced by oxidative damage,protecting the cell membrane structure integrity.?2?IRFV can counteract oxidative stress by reducing the lipid peroxidation product MDA of SCI model rats and H₂O₂oxidatively damaged PC12 cells and increasing the activity of anti-oxidation proteases such as CAT,SOD and GSH-Px.?3?IRFV can reverse oxidative stress by up-regulating Nrf2 and downstream anti-oxidases?NQO1,GCLC and GCLM?in SCI model rats and H₂O₂oxidatively damaged PC12 cells,thereby exerting the protective effect of anti-oxidation.