Effect Of Tauroursodeoxycholic Acid On Autophagy Induced By Mechanical Injury Of Spinal Nerve Cells In Vitro

Objective: To investigate the effect of ursodeoxycholic acid(ursodeoxycholic acid)on the induction of autophagy in the spinal nerve cell injury model of SD rats by the scratch method of aseptic razor blades.Methods: The spinal cord neurons of SD rats were cultured in vitro by Liu Xiwei and Qi Jianguo.The spinal nerve cells in the spinal cord of SD rats were identified by Nissl staining of spinal nerve cells.Que Hai Ping et al reference method,according to the actual situation and their specific requirements to prepare the model of spinal cord nerve cells mechanical injury in SD rats,scratch intervals respectively in 1mm group,3mm group and 5mm group respectively;after injury,6h,12 h,24h,48 h,72h model of injury was observed by inverted microscope and MTT method to observe the cell inhibition rate to determination of SD rat spinal cord nerve cells after mechanical injury,and injury in time to determine the extent of drug intervention.Taurine ursodeoxycholic acid was prepared from 0.5mmol/L,1.0mmol/L,2.0mmol/L,4.0mmol/L,6.0mmol/L,10mmol/L,15mmol/L and 20mmol/L respectively,and the best drug concentration was determined by MTT method.The cultured cells were inoculated in 6-well plate after strict sterilization treatment,divided into biological characteristics of blank group,injury group,drug intervention group were observed with electron microscopy autophagy in spinal cord nerve cells in the formation and development processes of spinal nerve cells.The cultured cells were inoculated in 6-well plate after strict sterilization treatment,divided into control group,injury group,drug intervention group,were extracted from the protein,using Western blotting method,to detect the expression ofautophagy related factor Beclin-1 and LC3 II /I.The cultured cells were inoculated in 6-well plate after strict sterilization treatment,divided into control group,injury group,drug intervention group,by immunohistochemical method,to detect the expression of autophagy related factors Beclin-1 and LC3.Results: Identification results of spinal cord neurons in vitro: the cell morphology and Nissl staining of spinal nerve cells under inverted microscope proved that the cultured cells were spinal cord neurons,and their purity was above 90%.The results of mechanical injury model MTT analysis showed that the inhibition rate of cells was detected by MTT method.The inhibition rate was nearly 50% when the interval of injury was 3mm and the time was 24 h.Compared with each group,there were statistical differences(P < 0.05),so the degree and time of injury were selected as the observation points of drug intervention.The concentration of interfering drugs was determined by MTT.Results: the survival rate of cells was detected by MTT.When the drug concentration was 4mmol/L,the cell survival rate was close to 80%,which was statistically different from each group(P < 0.05).The application of electron microscope to observe the spinal nerve cell autophagy results:spinal cord nerve cells in blank group were almost normal morphology,nuclear membrane,membrane integrity,mitochondria,endoplasmic reticulum,no fragmentation changed,no swelling,no degeneration injury.The injury group can be found in large vacuolar changes in cells,mitochondria,endoplasmic reticulum damaged,can be found a small amount of autophagosomes and lysosomes with formation of autophagy lysosome monolayer structure,performance and lysosome preliminary combination,cytoplasmic components or cell fragments of the inner wrapping was gradually degraded.In the drug intervention group,a large number of newly formed autophagy precursors and autophagic vesicles combined with lysosomes formed asingle membrane structure of autophagy lysosomes,and their organelle damage and cell integrity damage were also lighter than those in the injury group.The expression of Beclin-1 and LC3 II / I was detected by Western blotting.Results: the expression of Beclin-1 in injury group was higher than that in blank group(P < 0.05).The expression of the drug intervention group Beclin-1 were higher than the injury group,there were significant differences(P < 0.05);the expression of Beclin-1 in the intervention group were higher than that in control group,there was significant difference(P < 0.05);the expression of LC3 I / II injury group were higher than those of control group,there was significant difference(P < 0.05);drug intervention group LC3 II / I.expression were higher than the injury group,there were significant differences(P < 0.05);drug intervention group expression of LC3 II / 1 were higher than that in control group,there was significant difference(P < 0.05).By the method of immunohistochemistry to detect the expression of LC3 and Beclin-1results: injury group LC3 and the expression of Beclin-1 was higher than that of blank group(P < 0.05);drug intervention group LC3 and Beclin-1 was higher than that in the injury group(P < 0.05);drug intervention group LC3 and Beclin-1 was higher than that of blank group(P< 0.05).Conclusion: The intervention of Taurodeoxycholic acid can improve the level of autophagy induced by the mechanical damage model of spinal nerve cells of SD rats constructed by the scratch method of aseptic razor.