Study On The Effect And Mechanism Of Baicalin/gardenia Jasminoides On Endothelial Injury After Cerebral Ischemia Based On HIF-1α-VEGF/NLRP3-eNOS Pathwa

ObjectiveBaicalin(BC)and gardenia glycosides(GD)are one of the important anti-inflammatory components in Chinese traditional medicine Scutellaria baicalensis and Gardenia jasminoides,respectively,which have the effects of inhibiting inflammation,diastole,improving cerebral edema and microglia polarization status.Our group has been devoted to the research of BC/GD against cerebral ischemic injury.This study intends to further investigate the mechanism of BC/GD against cerebral ischemic injury by taking the postischemic vascular function as the entry point.Methods1.The vasodilatation and improvement of endothelial injury of BC/GD and its mechanism were initially investigated by using isolated vascular ring assay.The effect of BC/GD on resting vascular ring tone,the effect of cumulative concentration on vascular tone in normal rats,the effect of endothelium on its vasodilatory effect and its mechanism,the evaluation of endothelial function of blood vessels in MCAO rats by acetylcholine and the intervention effect of BC/GD were investigated respectively.2.By constructing middle cerebral artery obstruction(MCAO)model rats,using neurological function score;HE staining to observe brain histopathological condition;laser Doppler flow imaging perfusion instrument to monitor cerebral blood flow;TTC staining to determine cerebral infarct volume;WB to detect HIF-1α,VEGF,NLRP3,ASC,IL-1βprotein expression in cortical tissue on the ischemic side of the brain and brain basal e NOS and i NOS expression in vasculature;chemiluminescence detection of ROS level in brain tissue on the ischemic side;microplate assay kit to detect NO level in brain tissue on the ischemic side;Immunofluorescence staining with CD31(PECAM-1)was used to observe the integrity of cerebral basilar artery endothelium,while Hematopoietic promoter cell antigen CD34(Hematopoietic promoter cell antigen CD34)was used to observe vascular status and neovascularization.3.To explore the mechanism of BC/GD to ameliorate hypoxia-glucose deprivationinduced endothelial cell injury at the cellular level by constructing a human brain microvascular endothelial cell(Hc MEC)oxygen-glucose deprivation(OGD)model.After determining the optimal administration concentration by detecting cell viability and apoptosis,we observed the effects of BC/GD on apoptosis rate,ROS,NO and HIF-1α-VEGF/NLRP3-e NOS pathway protein expression content,and inflammatory factor levels in cell supernatant after OGD,respectively Results1.BC/GD had no significant effect on resting blood vessels;however,it could partially endothelium-dependent diastole U46619 precontracted cerebral basal vascular rings;the diastolic effect of BC/GD on blood vessels was significantly downregulated by L-NAME;the diastolic effect of cerebral basal vessels on Ach was significantly reduced in rats after MCAO,while 60 mg-kg-1 could effectively improve the diastolic effect of cerebral basal vessels on Ach after cerebral ischemia.The diastolic effect of 60 mg-kg-1 could effectively improve the diastolic effect of basal vessels on Ach after cerebral ischemia,and this effect was partially inhibited by PX-478.2.(1)After MCAO,the cortical sparing edema and pathological damage on the ischemic side of rats were more serious,with significantly higher neurological function scores,significantly lower cerebral blood flow,significantly larger infarct volume,significantly higher HIF-1α,VEGF,e NOS protein expression and lower i NOS protein expression;and PX-478 could further aggravate the damaged brain tissue;while after administration of DMOG,the above pathological damage of cerebral cortex,cerebral blood flow and infarction were significantly improved,and the expression of HIF-1α,VEGF,and e NOS proteins were significantly increased and i NOS protein expression was decreased after DMOG administration.It is suggested that the promotion of HIF-1α in a certain range at 24 h of cerebral ischemia is beneficial to increase NO expression,improve the adaptation of ischemic tissues during ischemia and hypoxia,and stabilize the vascular state.(2)Compared with sham surgery,the right cortex of MCAO group rats were severely damaged,and the score,cerebral blood flow,and cerebral infarct volume at 24 h were not different from 2h;the expression of e NOS was decreased and i NOS was increased in the basilar artery,and the endothelium was broken and structurally incomplete;the expression of HIF-1α,VEGF,CD34,and ROS was increased in the ischemic side cortex,and the expression of NLRP3 inflammatory vesicles and NO was decreased.After BC/GD treatment,it could improve the injury of MCAO group to different degrees,but PX-478 could partially antagonize the improvement effect of high dose of BC/GD.3.BC/GD intervention could differentially slow down the apoptosis of h CMEC cells induced by OGD,upregulate the expression of HIF-1α-VEGF-e NOS,reduce the activation of NLRP3 inflammatory vesicles,increase the content of NO,and reduce the level of ROS,and the above effects could be inhibited by PX-478.Conclusion1.BC/GD can partially endothelium-dependent diastolic U46619 precontracted cerebral basilar artery vascular ring and protect the endothelial condition after cerebral ischemia,and the mechanism may be related to HIF-1α expression.2.(1)Promoting HIF-1α expression in MCAO model rats within 24 h of cerebral ischemia improved brain histopathological conditions,increased cerebral blood flow and reduced infarct volume in the ischemic area,and the mechanism may be related to upregulation of VEGF and e NOS in the ischemic side of the cortex and downregulation of i NOS protein expression in the basilar artery.(2)BC/GD promotes microvascular formation in the ischemic area,reduces inflammatory factor adhesion,and improves blood supply to the ischemic area by upregulating HIF-1α and VEGF expression and downregulating NLRP3 inflammatory vesicle activation in post-ischemic brain tissue;upregulates e NOS and downregulates i NOS expression in the basilar artery to improve vascular endothelial function and diastolic blood vessels.3.By upregulating the expression of HIF-1α/VEGF/e NOS pathway and inhibiting NLRP3 inflammatory vesicle activation,we intervene in the pathology of vascular endothelial cell injury after cerebral ischemia,improve endothelial function and microcirculation in the ischemic area,thus reducing brain injury.