Effect Of Tap63 On The Expression Of Apoptotic Factors In Parkinson’s Cell Model

Aim:To observe the effect of Tap63 on the expression of Bcl-2（B cell lymphoma-2protein）,Bax（Bcl-2-associated x protein）and Mcl-1（myeloid leukemia-1 protein）in Parkinson’s cell model induced by 1-methyl-4-phenylpyridine（MPP⁺）,and to explore the role of Tap63 in the mechanism of apoptosis in Parkinson’s disease.Method:Mouse neuroblastoma cells（SH-SY5Y cell line）were divided into 4 groups according to different treatment methods:（1）Blank control group:without any intervention.（2）MPP⁺treatment group:10μmol·L^-1MPP⁺was added to SH-SY5Y cells in a culture medium for 24 hours.（3）In Tap63-NC+MPP⁺treatment group,lentivirus particles carrying nonsense sequences were continuously transfected for 48 hours,followed by 10μmol·L^-1MPP⁺for 24 hours.（4）In the Tap63 overexpression+MPP⁺treatment group,lentiviral particles carrying Tap63 overexpression gene were continuously transfected for 48 hours,and 10μmol·L^-1MPP⁺continued to act for 24 hours after changing solution.The protein was extracted from the four groups of cells and the expression of Bcl-2,Bax,Mcl-1 apoptotic factors and the proportion of apoptotic cells in each group were detected by western-blot test and flow cytometry experiment.The grayscale of protein bands was measured by Image J and the data were counted by Graphpad prism 9.Results:The results of Western blotting showed that the expression of Tap63 protein in MPP⁺treated group（0.871±0.084,t=4.951,p<0.01）was lower than that in control group（1.171±0.123）,and the difference was statistically significant.Compared with Tap63-NC+MPP⁺group（0.85±0.097）,the expression of Tap63 protein in Tap63 overexpression+MPP⁺treatment group（1.073±0.135,t=3.301,p<0.01）increased significantly.Compared with the control group（Bax:0.968±0.051,Bcl-2:1.156±0.114,Mcl-1:2.065±0.102）,the MPP⁺treatment group（Bax:1.543±0.013,t=9.684,P<0.01;Bcl-2:0.949±0.075,t=3.69,P<0.01;Mcl-1:1.717±0.157,t=4.543,P<0.01）increased the level of Bax expression,while the expression of Bcl-2 and Mcl-1 decreased,which was statistically significant.Compared with the Tap63-NC+MPP⁺group（Bax:1.549±0.115,Bcl-2:0.961±0.087,Mcl-1:1.678±0.149）,the Bax expression level of the Tap63 overexpression+MPP⁺treatment group（Bax:0.913±0.049,t=12.48,P<0.01;Bcl-2:1.086±0.098,t=2.34,P<0.05;Mcl-1:1.918±0.094,t=3.331,P<0.01）decreased,while the expression of Bcl-2 and Mcl-1increased,and the difference was statistically significant.Flow cytometry analysis showed that the number of apoptotic cells in MPP⁺treated group（9.522±0.796,t=16.74,p<0.01）was significantly higher than that in control group（3.59±0.345）.Compared with Tap63-NC+MPP⁺group（10.75±0.64）,the number of apoptotic cells decreased significantly in Tap63 overexpression+MPP⁺treatment group（5.903±0.566,t=13.90,p<0.01）.Conclusion:After Western-blot and flow cytometry experiments on the four groups of cells,we found that the expression of Tap63 and anti-apoptotic factors decreased and pro-apoptotic factors increased in the cell model treated with MPP⁺.Overexpression of Tap63 could down-regulate pro-apoptotic factor Bax,up-regulate anti-apoptotic proteins Bcl-2 and Mcl-1,and inhibit apoptosis,indicating that Tap63 may be involved in the pathogenesis of Parkinson’s disease and protect Parkinson’s cell model.