Mfn2-mediated Preservation Of Mitochondrial Function Contributes To The Protective Effects Of BHAPI In Response To Ischemia

Secondary brain injury after stroke is one of the main factors determining prognosis of the stroke,and oxidative damage is one of the main pathological mechanisms of brain injury in stroke.According to WHO statistics,there were 15million patients with new stroke worldwide each year,of which 1/3 patients died and1/3 patients were permanently disabled.In China,there were about 2 million patients with new stroke each year,of which 1.2 million patients died,and the annual growth rate was 8.7%.As a result,stroke was the second fatal disease after ischemic heart disease.With the improvement of medical and nursing technology,the global mortality of stroke has declined,but its incidence and disability rate keep increasing.The high disability rate of stroke seriously affects the life quality of patients,and obviously aggravates the economic burden of patients and society.Ischemic stroke is the main type of stroke disease,accounting for 75%.At present,the pathophysiological mechanism of ischemic stroke is still not fully elucidated,and its clinical treatment is also short of effective therapeutic drugs and methods.After a lot of studies in vivo and in vitro,more and more evidences indicated that iron participated in and aggravated the pathological process of ischemic stroke.After the stroke,the intracellular free iron ions were increased,which cause long-term damage to the cells,also known as ferroptosis induced by iron accumulation.Therefore,iron chelators,which could specific chelate and eliminate intracellular iron ions,may be a new neuroprotective agents.Studies in animal model have found that iron chelators have a protective effect on ischemic stroke,and methyl tirilazad?one of the iron chelators?can reduce the mortality,decrease symptoms and signs of nervous system,and improve the clinical prognosis in the clinical observation of patients with ischemic stroke,it has certain clinical value in the treatment of ischemic stroke.However,since traditional iron chelators can non-selectively interfere with the iron metabolism process,resulting in intracellular iron deficiency and death,which limits its clinical application.In recent years,the researchers have found?E?-N'-?1-?2-??4-?4,4,5,5-tetramethyl-1,2,3-dioxoborolan-2-yl?benzyl?oxy?phenyl?eth ylidene?isonicotinohydrazide?BHAPI?,which is improved from salicylaldehyde isonicotinic acid hydrazide?SIH?,is a new type of selective iron chelators.BHAPI has well stability and antioxidant ability,and only plays a specific role for the damaged area,does not affect the iron metabolism in normal cells,thus it has great potential protective effect.In the experiments of oxidative damage of strong oxidants of cardiomyocytes,it was confirmed that BHAPI has a good effect on the protection and maintenance of oxidative stress damage.But whether BHAPI has a protective effect on ischemic stroke is unclear.Therefore,we explored the protective effect of BHAPI on HT22 cell line and explored its possible mechanism through establishing ischemia-reperfusion OGD/R cell model in vitro.Experiment 1 Changes of BHAPI in OGD/R cell model and its effect on cells Objective:To detect whether BHAPI can be converted to HAPI in the ischemia-reperfusion OGD/R cell model,and investigate the neuroprotective effect of BHAPI on the OGD/R cell model.Methods:The ischemia-reperfusion OGD/R cell model in vitro was established using HT22 hippocampal neuron cell line,and BHAPI with different concentrations were added into the cell model.The cell viability were detected using CCK-8 assay.The optimal concentration of BHAPI was detected using HPLC.The methods including Flow cytometry and Western blot respectively were used to detect apoptosis of HT22 neurons and activation of caspase-3,and to evaluate effect of BHAPI on the ischemia-reperfusion OGD/R cell model.Results:?1?BHAPI is almost completely converted to HAPI after 24 h in the OGD/R cell model;?2?The cell viability in OGD/R group was significantly reduced.The reduction of cell viability in the OGD/R group treated with BHAPI was significantly lower than that in the OGD/R group?P<0.05?;?3?In contrast,the LDH content in the supernatant in the OGD/R group was significantly increased,and the LDH content in the supernatant in the OGD/R group treated with BHAPI was significantly lower than that in the OGD/R group?P<0.05?;?4?Flow cytometry showed that the degree of apoptosis in the OGD/R group treated with BHAPI was significantly lower than that in the OGD/R group;?5?The activation of caspase-3was significantly reduced in the OGD/R group treated with BHAPI.Conclusion:?1?BHAPI was activated and further converted to HAPI in the ischemia-reperfusion OGD/R cell model.?2?BHAPI can improve the damage and apoptosis of OGD/R cells induced by glucose oxygen deprivation.In the case of ischemia and hypoxia,BHAPI may have protective effect on HT22 hippocampal neuron,it may be potential protective treatment for neuron damage caused by ischemia and hypoxia.Experiment 2 Effect of BHAPI on intracellular mitochondria in OGD/R cells Objective:To explore the protective effect of BHAPI on mitochondria in OGD/R cells.Methods:After staining the cells with tetramethylrhodamine methyl ester?TMRM?,the mitochondrial membrane potential?MMP?was detected by FV10i laser confocal scanning microscopy.Mitochondria were extracted to detect the absorbance of mitochondria and the buffering capacity of calcium ions.The oxygen consumption of cells and the rate of extracellular acidification were detected using Seahorse Extracellular Flux Analyzers.Results:?1?The mitochondrial membrane potential in the OGD/R model was significantly lower than that in the normal control group?P<0.05?.The decrease of mitochondrial membrane potential in the OGD/R group treated with BHAPI was significantly lower than that in the OGD/R group?P<0.05?.?2?Compared with control group,mitochondrial Ca²⁺buffering capacity in the OGD/R group was reduced about 60%.The Ca²⁺-induced mitochondrial swelling in the OGD/R group treated with BHAPI was significantly lower than that in the OGD/R group?P<0.05?.?3?Compared with OGD/R group,BHAPI treatment group can partially improve OGD/R-induced oxygen consumption and extracellular acidification rate?P<0.05?.Conclusion:BHAPI can improve mitochondrial dysfunction in the OGD/R model induced by glucose oxygen deprivation.Experiment 3 To validate the effect of BHAPI on OGD/R cell model via regulating Mfn2Objective:To explore the regulation and mechanism of BHAPI on mitochondrial dynamics.Methods:The morphological changes of mitochondria were examined by Mito Tracker staining.The expressions of mitochondrial fusion and fission proteins?Mfn1,Mfn2,Drp1,Opa1?in OGD/R group treated with BHAPI and OGD/R group were detected by Western blot.The downstream targets were interfere with si-Mfn2transient transfection technique and the Gateway overexpression technique,and then the CCK-8 assay and flow cytometry were repeatedly operated to perform verification.Results:?1?The mitochondrial morphology in the OGD/R group was significantly changed.Compared with OGD/R group,changes of mitochondrial morphology in the BHAPI treatment group were partially inhibited;?2?In the OGD/R group,only the expression of Mfn2 protein was significantly decreased.In the BHAPI-treated group,only the expression of Mfn2 protein was partially increased.But there were no effect on the expressions of Mfn1,Drp1 and Opa1;?3?Si-Mfn2 partially reduced the expression of Mfn2 protein,and it had a significant inhibitory effect on protective effect of cell viability,LDH release,apoptosis and mitochondrial MMP level induced by 100?M BHAPI;?4?Gateway system significantly increased expression of Mfn2protein,and it had a significant improved effect on protective effect of cell viability,LDH release,apoptosis and mitochondrial MMP level induced by 10?M BHAPI.Conclusion:BHAPI may promote mitochondrial fusion through regulating Mfn2,and maintain mitochondrial function,thereby protecting HT22 cells.