| Objective To investigate the effect of resveratrol(Res)on induced autophagy of spinal cord ischemic-reperfusion injury(SCII)in SD rats.Methods According to liu xiwei et al.'s mature method of cultured spinal cord nerve cells in vitro,the primary spinal cord nerve cells of SD rats were cultured in vitro for 1 week in this experiment..Primary nerve cells of SD rat spinal cord were identified by Nissl staining.Referring to Lin xiang et al.'s experience in establishing ischemia-reperfusion injury of spinal cord by using glutamic acid solution,the model of ischemia-reperfusion injury of spinal cord nerve cells was established based on the basic requirements of the experiment.The concentration of glutamic acid was 50umol/mL,100umol/mL,150umol/mL,200umol/mL and250umol/mL,respectively.After the treatment of spinal nerve cells with different concentrations of glutamic acid for 10 min,15min and 20 min,the cell morphology of spinal nerve cells with different concentrations of glutamic acid for different periods of time was observed under an inverted microscope.After successful modeling,MTT assay was used to determine the cell survival rate and inhibition rate,so as to determine the optimal concentration and time for glutamic acid to cause ischemia-reperfusion injury in spinal cord nerve cells.Resveratrol of 2mmol/L,4mmol/L,6mmol/L,8mmol/L,10mmol/L,12mmol/L and 14mmol/L was prepared,and the most suitable drug concentration was determined by MTT method.The cultured primary spinal cord cells were randomly divided into 3 groups:blank control group,model group and resveratrol experimental group.Each group had 64 Wells.The expressions of beclin-1 and LC3II/LC3 I in spinal cord nerve cells of each groupwere detected by Western blot and cell immunohistochemistry.The structures of autophagosomes in each group were observed by fluoroscopy electron microscopy.Results Identification results of primary spinal cord nerve cells of SD rats cultured in vitro: cell morphology and Nissl staining of spinal cord nerve cells were observed under an inverted microscope,and the cultured cells were confirmed to be spinal cord nerve cells with purity above 97%.MTT method was used to identify the ischemia-reperfusion injury model: when the concentration of glutamic acid was 150umol/mL and the cell mortality was close to 50% after the injury for 15 min,the difference between groups was statistically significant.Therefore,glutamate concentration of 150umol/L and action time of 15 min were selected as observation points for the intervention of the experimental drug resveratrol.MTT method was used to determine the optimal concentration of resveratrol: when the concentration of resveratrol was 8mmol/L,the cell survival rate was close to 80%,and the difference between groups was statistically significant(P < 0.05).Identification results of primary spinal cord nerve cells of SD rats cultured in vitro: cell morphology observed under an inverted microscope and nizhlet staining of spinal cord nerve cells proved that the cultured cells were spinal cord nerve cells with purity above 95%.Ischemia-reperfusion injury model determined by MTT method appraisal result: through the cell inhibitory rate determined by MTT method and pancreatic acid concentration is 150umol/L,injury time is 15 min cell inhibition rate of the most close to 50%,compared with group were statistically significant(P < 0.05),so choose 150 umol/L,pancreatic acid concentration effect 15 min as resveratrol intervention observation point.The autophagy of spinal cord nerve cells was observed by electron microscopy: in the blank group,the cell bodies of spinal cord nerve cells were normal,nuclear membrane,mitochondria and endoplasmic reticulum were normal.In the model group,the number of mitochondria was reduced and swollen,with a large number of vacuoles and looseendoplasmic reticulum.Autophagosomal lysosomes with double-layer membrane structure could be seen.In the resveratrol group,autophagosomal lysosomes with bilayer membrane structure were observed,and organelle and cell integrity were less damaged than in the model group.Using western blot detection Beclin 1 and LC3 ?/? expression results showed that the experimental group Beclin 1,LC3 ?/? expression is higher than the blank control group,there are statistical differences(P < 0.05);Experimental Beclin 1,LC3 ?/? expression is higher than the model group,statistically significant(P < 0.05);Model group Beclin 1,LC3 ?/? expression is higher than the blank control group,there are statistically significant(P < 0.05);The expressions of LC3 and beclin-1 in the model group were higher than those in the control group(P < 0.05).The expressions of LC3 and beclin-1 in the experimental group were higher than those in the model group(P < 0.05).The expressions of LC3 and beclin-1 in the experimental group were higher than those in the blank control group(P < 0.05).Conclusion The experimental drug resveratrol can alleviate the ischemia-reperfusion injury of rat spinal cord nerve cells by glutamic acid modeling,which is related to the promotion of autophagy of injured cells. |
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