Investigate The Ameliorative Effect And Mechanism Of Icariin On Cerebral Ischemia-Reperfusion Injury From Regulating MPTP Over-Opening

Background In the past 30 years,with the change of diet structure and the continuous progress of population aging,the incidence and mortality of stroke in China have been rising continuously,among which ischemic stroke is the majority.At present,the main treatment is to restore blood flow through thrombolytic therapy,but the increase of reactive oxygen species（ROS）during reperfusion will further exacerbate brain damage.Objective The aim of this study was to investigate the protective effect of Icariin（ICA）on cerebral ischemia-reperfusion injury in SD rats,and further explore its mechanism through mitochondrial permeability transition pore（mPTP）-mediated oxidative damage pathway.Methods In vitro experiments:PC-12 cells were stimulated with H₂O₂ 200μM for 8h to establish a model of cell damage.Different concentrations of ICT and combined use of mPTP inhibitor Cyclosporin A（Cs A）and mPTP activator Lonidamine（LND）were used to pre-protect the cells for 12h.Cell viability was measured by MTT.Flow cytometry and immunefluorescence were used to detect the level of reactive oxygen species（ROS）.The opening state of mPTP and the level of mitochondrial membrane potential were detected by immunofluorescence.Cell apoptosis was detected by flow cytometry.The expression levels of VDAC,Cy PD,Bcl2 and Bax were detected by Western blot.In vivo experiment:SD rats were randomly divided into sham operation group（Sham）,ischemic-reperfusion group（I/R）,ICA low dose group（10mg/kg/Bid）,ICA high dose group（30mg/kg/Bid）,and Cs A group（15mg/kg,once,30min before operation）.The middle cerebral artery occlusion（MCAO）model was established in the rats except the Sham group（2 hours of ischemia and 22 hours of reperfusion）.The neurological function was evaluated by double-blind Zea Longa and MNSS scores.The effect of ICA on infarct volume was evaluated by TTC staining,and the pathological status of ischemic cortex was detected by HE staining.Nissl staining was applied to observe the changes of Nissl bodies in ischemic cortex.The expression level of VDAC protein was estimated by Western blot.Results In vitro experiments:After H₂O₂ stimulation,the viability of PC-12 cells decreased significantly,the cell volume increased,and the cell membrane boundary was unclear.Furthermore,Ros level increased significantly,mPTP was in an abnormal open state,MMP decreased dramaticly,and apoptosis increased.After ICT intervention,cell viability was significantly increased,ROS level decreased.Meanwhile,mPTP opening was inhibited,MMP increased,ROS production decreased,and cell apoptosis decreased.The protective effect of ICT was not further improved when ICT was co-treated with Cs A.On the contrary,the improvement effect of ICT was reversed,when it was co-treated with mPTP activator LND.In vivo experiment:Compared with the Sham group,the rats in the I/R group had a severe infarction,with a percentage of infarct volume of 32%to 40%.In parallel,the rats in the I/R group had severe neurological impairment,manifested by a significant increase in Zea and m NSS scores.Furthermore,in the ischemic cortex,the number of Nissl bodies decreased,the staining color became lighter,cell morphology and structure are severely disrupted,and mPTP opening is increased.ICA intervention significantly reduced infarct volume,improved morphological changes in cortical cells on the ischemic side,increased the number of Nissl vesicles and remarkably inhibited the over-opening of mPTP in rats with ischemia-reperfusion injury.Cs A has a comparable role to ICA.Conclusion ICT,the main metabolite of ICA,can effectively ameliorate H₂O₂-induced oxidative damage in vitro.ICA pre-protection can significantly reduce the infarct size of MCAO model in vivo.These protective effects are closely related to the high efficiency of ICT in inhibiting the opening of mPTP,reducing ROS levels,and improving mitochondrial membrane potential.