Protective Effect Of Mitochondria-targeted Antioxidants On Subarachnoid Hemorrhage Cell Model

BackgroundAneurysmal Subarachnoid Hemorrhage(aSAH)is a common fatal disease,accounting for about 5.7% of all strokes,with an annual incidence of 0.7 to 23.9 per 100,000,but the incidence in different regions A little different.After the first bleeding,30% of patients died within 1 day,and despite the new treatment,the total mortality rate exceeded 50%.In addition,69% of patients with aSAH have a poor quality of life.Recently,more and more studies indicate that Early Brain Injury(EBI)is the main cause of poor prognosis in patients with aSAH.Therefore,EBI treatment is considered to be the main target for early attention in SAH patients.Mitochondria not only provide energy to cells,but also participate in the regulation of calcium ions and the apoptotic pathway of cells.Maintaining mitochondrial function is vital for nerve cells.Recent studies have suggested that mitochondrial targeted antioxidants(Elamipretide TFA,MTP-131)have good results for diseases such as inflammation and ischemia-reperfusion injury.However,the mitochondrial dysfunction and mitochondrial apoptotic pathways after SAH are rarely described.In this study,MTP-131 was applied to the cell model of EBI after SAH,and the protective effect of MTP-131 on EBI after SAH was explained through mitochondrial function and mitochondrial apoptosis pathway.ObjectiveTo explore the protective effect of MTP-131 on nerve cells in the EBI stage after SAH,and provide theoretical basis for clinical treatment.MethodsPC12 cells and oxyhaemoglobin(OxyHb)were used to establish an SAH model in vitro,and MTP-131 intervention was used to divide them into control group,SAH group,and MTP-131 + SAH group.The observation point is 48 h after model making.MTT method was used to detect the viability of PC12 cells.The mitochondrial membrane potential(Δψm)after JC-1 fluorescent probe staining was observed with a fluorescence microscope,and the changes in Δψm were detected by flow cytometry.Western blot was used to detect the cytoplasm,Expression of cytochrome c(Cyt C),B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)in mitochondria.Results1.MTT assay for PC12 cell viability: Compared with the control group,the cell viability of the SAH group was significantly increased.The viability of PC12 cells decreased in the MTP-131-treated groups,and the vitality of the 0.5 μM MTP-131 group decreased significantly(P <0.01).2.Fluorescence microscope and flow cytometry were used to detect the effect of MTP-131 on mitochondrial membrane potential of PC12 cells induced by OxyHb: fluorescence microscope observation showed that the green-red fluorescence of the control group was relatively low;the green-red fluorescence ratio was significantly increased after the SAH model was established(P <0.05);After MTP-131 intervention,compared with the SAH group,the green-red fluorescence ratio was significantly reduced(P <0.05).Quantitative analysis of green-red fluorescence intensity by flow cytometry.Compared with the control group,the green-red fluorescence ratio of SAH was significantly increased(P <0.05).After the intervention of MTP-131,the green-red fluorescence was significantly reduced compared with that before(P <0.05).3.Western Blot detected Cyt C expression in cytoplasm and mitochondria of PC12 cells: Cyt C in mitochondria of SAH cells was significantly lower than that of control group(P <0.05).The expression of Cyt C protein in mitochondria of PC12 cells in MTP-131 group was significantly increased(P <0.05);Cyt C in cytoplasm of SAH cells was significantly higher than that in the control group(P <0.05);Compared with SAH group,Cyt C protein expression in PC12 cells in SAH + MTP-131 group was significantly reduced(P <0.05).4.Western Blot detected the expression of apoptotic proteins in PC12 cells: Compared with the control group,the expression of Bcl-2 associated X protein(Bax)in the SAH group increased significantly(P <0.05);Bcl-2 Compared with SAH group,Bax expression in PC12 cells of SAH + MTP-131 group was significantly reduced(P <0.05);Bcl-2 expression was significantly increased(P <0.05).ConclusionAfter SAH,MTP-131 plays a neuroprotective role by protecting mitochondrial function and inhibiting cell apoptosis.