Idebenone Regulates CD38-SIRT3-P53 Pathway To Counteract Oxidative Stress-induced Apoptosis Of Neuronal Cells

Objective:Oxidative stress can induce neuronal cell apoptosis,and the application of Idebenone can reduce the neuronal cell apoptosis induced by oxidative stress,but the specific mechanism remains unclear.Therefore,in this study,bioinformatics methods and in vitro experiments were used to explore the effects and specific mechanisms of Idebenone against oxidative stress.Methods:In this study,the oxidative stress model was constructed using mouse hippocampal neuron HT22 cell line,and the damage concentration of H₂O₂and the therapeutic concentration of Idebenone were determined by MTT method and LDH enzyme activity assay.After that,the apoptosis of HT22 cells before and after the addition of H₂O₂and Idebenone was detected by flow cytometry and TUNEL staining,respectively.The expression levels of apoptosis-related proteins P53 and Caspase3protein and m RNA were detected by Western Blot and RT-q PCR techniques.On this basis,bioinformatics was used to analyze the potential targets of Idebenone for anti-oxidative stress.Subsequently,the expression levels of Idebenone-related proteins CD38 and SIRT3,which were screened by bioinformatics,in protein and m RNA were detected by Western Blot,RT-q PCR,SIRT3 si RNA and other technologies,and the changes of NAD⁺were detected with NAD⁺detection kit,to investigate the role of CD38-SIRT3-P53in the anti-oxidative stress of Idebenone.Results:（1）MTT assay and LDH enzyme activity assay showed that 250μM H₂O₂could significantly cause the death of HT22 cells,and 20μM Idebenone could significantly reduce the death of HT22 cells.（2）The results of flow cytometry and TUNEL staining showed that H₂O₂could increase the apoptosis rate of HT22 cells,while Idebenone could decrease the high apoptosis rate caused by oxidative stress injury.（3）The results of Western Blot and RT-q PCR showed that P53 and Caspase3 protein and m RNA increased significantly in the process of H₂O₂oxidative stress damage,but the expression levels of P53 and Caspase3 protein and m RNA decreased after the application of Idebenone.（4）Bioinformatics analysis showed that CD38 was a potential drug target of Idebenone,and CD38 interacted with mitochondrial deacetylase SIRT3.（5）In vitro experiments showed that H₂O₂application could significantly increase the expression level of CD38,decrease the concentration of NAD⁺,decrease the expression level of SIRT3,and significantly increase the level of acetylated P53.The application of Idebenone can down-regulate the expression of CD38 and acetylated P53,and increase the levels of NAD⁺and SIRT3.After the knockdown of SIRT3,the expression of P53Ac showed a slight upward trend.Conclusions:Idebenone can reduce the apoptosis of HT22 cells damaged by oxidative stress by regulating CD38-SIRT3-P53 pathway.