YAP Expression Mediates Ketamine-evoked Apoptosis In SH-SY5Y Cells

Objective To investigate the effects of different concentrations of ketamine on the viability and proliferation of SH-SY5Y cells and clarify the neurotoxicity of ketamine;And to explore the role of YAP in promoting cell proliferation,thus suggests a potentially novel pharmacological target for the treatment of ketamine-evoked neurotoxicity.Methods SH-SY5Y cells were obtained from Ke zunji experimental research group of Neurodegenerative Diseases Research Group,Shanghai University of Traditional Chinese Medicine.The cells were cultured under standard cell culture conditions in Dulbecco^'s Modified Eagle Medium?DMEM?and the nutrient solution was changed every two days during subsequent experiments.Considering that various treatments has different effects on SH-SY5Y cells,cells passed from 2 to 4 generations were selected in all experiments.1.SH-SY5Y cells were cultured under standard cell culture conditions and treated with different concentrations of ketamine when cells growth adhering to the wall after 24hours.The cells were divided into six groups:H₂O₂-induced apoptosis as positive control group?group P?.In other words,H₂O₂ reagent was added into the cell solution at a concentration of 400?M.Blank control group?group B?.And depending on the different concentrations of ketamine,the experimental groups were set in four concentration gradients:the final concentration of ketamine was 400?M?group S1?,800?M?group S2?,1200?M?group S3?and 1600?M?group S4?.CCK-8 assay was applied after 12h and 24h ketamine treatment,and the number of cells was counted so we can observe the effects of ketamine on the activity and proliferation of SH-SY5Ycells.2.Annexin V-APC/7-AAD flow cytometry was used to detect apoptosis rate of SH-SY5Y cells under different treatment conditions.The expression level of apoptosis-related proteins was detected by immunofluorescence and western blotting,so we can explore the mechanism of ketamine-induced apoptosis of SH-SY5Ycells.3.As a downstream effector of Hippo signaling pathway,YAP can maintain self-renewal and differentiation of stem cells and regulate organ size as well as tumor formation.In this study,the expression level of YAP was increased by shRNA recombinant lentiviral assay and reduced by siRNA transfection with liposome 2000.Ketamine was administered to each group as described above,including the control group.Flow cytometry,immunofluorescent staining and western blotting were used to detect the expression level of apoptotic protein.Thus,we can speculate the effects of YAP on ketamine-evoked neurotoxicity.Results 1.Compared with group B,the activity and proliferation of SH-SY5Y cells under different treatment time?12h,24h?were decreased in S1,S2,S3 and S4 groups?P<0.05?.The effects of ketamine on the activity and proliferation of SH-SY5Y cells were also different with the concentration of ketamine.As the concentration of ketamine increases,the number of SH-SY5Ycells decreased to a greater extent,and the cell activity and proliferation capacity decreased to a greater extent,there were statistically significant differences among the groups?P<0.05?.The effect of 24h was more obvious than 12h,and ketamine reduced the activity and proliferation ability of SH-SY5Ycells in a dose-dependent manner.2.Compared with group P and group B,ketamine can induce apoptosis on SH-SY5Y cells in a dose-dependent manner.There were statistical differences between the groups at the concentration of 400,800,and 1600?M?P<0.05?.The difference between the experimental group and control group was statistically significant?P<0.05?.3.Compared with control group,SH-SY5Y cells treated with ketamine after YAP overexpression presented improved activity and proliferation compared with control group.On the one hand,the apoptosis rate decreased;on the other hand,the expression level of Cleaved Caspase-3 and Bax decreased as well as the expression level of Bcl-2increased,these results were statistically significant?P<0.05?.The increased level of YAP has a positive effect on cell activity and proliferation.Overexpression of YAP can promote cell proliferation and reduce ketamine-induced apoptosis of SH-SY5Y cells.The above results were statistically different?P<0.05?,and decreased expression of YAP would aggravate cell apoptosis induced by ketamine.Conclusion Ketamine can reduce viability and proliferation of SH-SY5Y cells and induce apoptosis in a dose-dependent manner.Overexpression of YAP can reduce apoptosis rate and the cytotoxicity of SH-SY5Y produced by ketamine,which has an antagonistic effect on ketamine induced apoptosis of SH-SY5Y cells.Decreased expression level of YAP can aggravate the apoptosis of SH-SY5Y cells induced by ketamine.It can be seen from the above results that YAP expression level has a certain regulatory effect on ketamine induced apoptosis of SH-SY5Y cells.