The Protective Effect Of Apigenin On Cerebrovascular After Ischemic Cerebral Injury Via Caveolin-1 Pathway

Background and Objective:The dysfunction of cerebral microvascular affects the brain remodeling and repairing after cerebral ischemic injury.Cerebrovascular endothelial cell is an important part of cerebral microcirculation.The survival functions of cerebrovascular endothelial cells including proliferation,autophagy and apoptosis are changed after ischemic and hypoxic injury.Caveolin-1 is the marker protein of caveolae in the endothelial cell membrane.Researches have indicated that Caveolin-1 plays an important role in cell proliferation,autophagy and apoptosis.However,its role in ischemic cerebrovascular endothelial cells is still not clear.To study the regulation function of Caveolin-1 in cerebrovascular endothelial cells will provide a new target for the prevention and treatment of ischemic cerebral injury.Apigenin is a widely used plant flavonoid,which has neuroprotective effect on ischemic brain injury and promotes the regeneration of vascular endothelial cells.However,the protective effect of apigenin on ischemic cerebrovascular endothelial cells and the underlying mechanisms are need to be further researched.The aim of this study is to explore the effects of apigenin on the survival functions of cerebrovascular endothelial cells and angiogenesis after ischemic injury.Also,the regulatory role of Caveolin-1 in the protective effect of apigenin on the ischemic cerebrovascular endothelial cells is also explored.So as to provide a theoretical foundation for the relevant clinical application of apigenin.Methods:The model of cerebral ischemia and reperfusion injury in rat was established with insertion of thread embolish into middle cerebral artery,which was indicated as Middle Cerebral Artery Occlusion(MCAO).Apigenin or Caveolin-1 inhibitor(daidzin)was intraperitoneal injected.The neurological behavior scores of rats were evaluated by scoring of Zea longa.The coronal brain slices were stained with 2,3,5-triphenyl tetrazolium chloride(TTC)to observe the cerebral infarct volume.The new vessels density in the cerebral ischemic penumbra was observed under fluorescence microscope by vascular endothelial growth factor receptor2(VEGFR2)/CD34 double-staining immunofluorescence.The mRNA and protein expressions of Caveolin-1 in the ischemic penumbra were detected by RT-qPCR and Western Blot,respectively.The protein expressions of vsacular endothelial cell growth factor(VEGF)and endothelial nitric oxide synthase(eNOS)in the ischemic penumbra were detected by Western Blot.In order to validate the protective effect and the underlying mechanisms of apigenin on ischemic cerebrovascular endothelial cells via Caveolin-1 pathway.The oxygen-glucose deprivation/reoxygenation(OGD/R)model of human brain microvascular endothelial cells(HBMECs)was established.To investigate the related mechanisms,we constructed the lentiviral vector carrying RNAi sequence targeting the Caveolin-1 gene,to build the Caveolin-1 gene silencing model.The optimum interfering concentration of apigenin was used to interfere cell culture.The viability of HBMECs was measured by Cell Counting Kit-8(CCK-8)assay,the migration of HBMECs was analyzed by Transwell method,and the tube formation assay was performed using the branch point method.The autophagosome of HBMECs was observed by electron microscopy.The apoptosis of HBMECs was detected by flow cytometry.The mRNA and protein expressions of Caveolin-1 and related-genes of autophagy and apoptosis process in HBMECs after OGD/R were examined by RT-qPCR and Western Blot,respectively.Results:In vivo,the loss of Caveolin-1 effectively inhibited the angiogenesis,increased the cerebral infarct volume,and then aggravated the neurological deficits of rats after MCAO,as well as reduced the protein expressions of VEGF and eNOS. Apigenin promoted the protein expressions of Caveolin-1,VEGF and eNOS,and induced angiogenesis,then reduced the cerebral infarct volume,and improved the neurological deficits of rats after MCAO.In vitro,compared with the control group,the silencing of Caveolin-1 effectively inhibited the proliferation of HBMECs,and declined the expression of VEGF after OGD/R.And the silencing of Caveolin-1 contributed to aggravate autophagy of HBMECs after OGD/R,with the up-regulation of the expression of autophagy related gene of Beclin-1 and down-regulation of the protein expression of mammalian target of rapamycin(mTOR),as well as increasing the protein ratio of microtubule-associated protein light chain3(LC3)II to LC3I.Meanwhile,the loss of Caveolin-1 resulted in exacerbating apoptosis of HBMECs after OGD/R,with the down-regulation of the protein expression of B-cell lymphoma-2(Bcl-2).Apigenin had a good effect on promoting the proliferation,however,inhibiting the autophagy and apoptosis of HBMECs after OGD/R.Simultaneously,apigenin effectively increased the protein expressions of Caveolin-1,VEGF,mTOR and Bcl-2,and decreased the protein expression of Beclin-1,also reduced the protein ratio of LC3? to LC3? in HBMECs after OGD/R.We additionally demonstrated that the silencing of Caveolin-1 could drop off the regulatory effect of apigenin on the survival functions of HBMECs and the expressions of related-genes in HBMECs after OGD/R.Conclusion:1.Apigenin promoted the angiogenesis of ischemic penumbra and effecxtively improved the neurological deficits of rats after cerebral ischemic injury.2.Apigenin showed protective effect on the ischemic cerebrovascular endothelial cells,including increased the proliferation and inhibited the autophagy and apoptosis of cerebrovascular endothelial cells by up-regulating the expression of Caveolin-1 after ischemic injury.