Protective Effect And Mechanism Of Cordyceps Sinensis Extract On Cerebral Microvascular Endothelial Cell Ischemic Injury Model

Cordyceps sinensis(CS)is a valuable Chinese medicine that has been used for thousands of years.The previous study of the research group found that Cordyceps sinensis extract(CSE)has a significant protective effect on cerebral ischemic injury caused by middle cerebral artery occlusion(MCAO)in rats.Although the protective effect of CSE on cerebral ischemic injury was studied from the perspective of the whole animal model,it was not discussed from the perspective of cells and molecular biology.Cerebral ischemia is one of the causes of the destruction of the blood brain barrier(BBB).As an important component of BBB,brain microvascular endothelial cells(BMEC)play an important role in maintaining brain homeostasis and reducing brain permeability.Cerebral ischemia-induced apoptosis is one of the mechanisms of cerebral ischemia.Mitochondria supply energy to the brain and play an important role in apoptosis.Once it is damaged,it activates the mitochondrial apoptosis pathway.which intensifies cerebral ischemic injury.Therefore,mitochondrial apoptosis pathway can be used as a target for the treatment of cerebral ischemia.Therefore,this project established an oxygen-glucose-deprivation(OGD)model of BMEC.studied the protective effect of CSE on the OGD model of BMEC by the intervention of mitochondrial apoptosis pathway to explore the protective effect and molecular mechanism of CSE on cerebral ischemia injury.Objective:The effect of CSE on the OGD model of BMEC was preliminarily discussed based on cell morphology and cell activity.The protective effect of CSE on the OGD model of BMEC was explored based on inflammatory reaction,oxidative stress and apoptosis.The mechanism of CSE on BMEC pseudo ischemic damage model was deeply studied based on mitochondrial apoptosis pathway,so as to explore the protective effect and molecular mechanism of CSE on cerebral ischemia injury.Method:1.To establish an OGD model of BMEC:The rat BMEC was isolated and primary cultured,and the cells cultured in the third generation were used to establish the OGD model of BMEC.2.To study the effect of CSE on cell morphology and cell viability of the OGD model of BMEC:Ultrasonic extraction of CSE,preparation of CSE lyophilized powder and preparation of appropriate concentration,and administration after sterilization.The third generation of BMEC were isolated and cultured and divided into normal group,model group and CSE group(5,10,20,50,100,200,400 ?g·mL-1).The model group and CSE group were modeled by OGD.The inverted microscope and CCK-8 method were used to observe the changes of cell morphology and cell activity in each group of BMEC and determine the most appropriate concentration of CSE for the next experiment.3.To study the effect of CSE on oxidative stress of the OGD model of BMEC:The third generation of rat BMEC was isolated and cultured and divided into normal group,model group and CSE group(5.10,20 ?g·mL-1).The model group and CSE group were modeled by OGD.The release of lactate dehydrogenase(LDH)in the supernatant and intracellular Superoxide Dismutase(SOD),Malonydialdehyde(MDA),and Nitric Oxide(NO)of each group of BMEC were detected by biochemical method.4.To study the effect of CSE on inflammatory response of the OGD model of BMEC:The third generation of rat BMEC was isolated and cultured and divided into normal group,model group and CSE group(5,10,20 ?g·mL-1).The model group and CSE group were modeled by OGD.The levels of Tumor Necrosis Factor-alpha(TNF-?)and Interleukin-1 beta(IL-1?)in BMEC were detected by Enzyme linked immunosorbent assay(ELISA).5.To study the effect of CSE on the apoptosis of the OGD model of BMEC:The third generation of rat BMEC was isolated and cultured and divided into normal group,model group and CSE group(5,10,20 ?g·mL-1).The model group and CSE group were modeled by OGD.The apoptosis of BMEC cells in each group was detected by flow cytometry using Annexin V-FITC/PI assay.6.To study the effect of CSE on mitochondrial membrane potential(MMP)of the OGD model of BMEC:The third generation of rat BMEC were isolated and cultured and divided into normal group,model group and CSE group(5,10,20 ?g·mL-1).The model group and CSE group were modeled by OGD.Using flow cytometry,JC-1 fluorescent probes were used to detect the changes of MMP in each group of BMEC.7.To study the effect of CSE on the expression of Cytochrome C(Cyt C),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),caspase-3 proteins in the mitochondrial apoptosis pathway of the OGD model of BMEC:The third generation of rat BMEC was isolated and cultured and divided into normal group,model group,and The CSE group(5,10,20 ?g·mL-1).The model group and CSE group were modeled by OGD.The protein expression of Cyt C,Bcl-2,Bax and caspase-3 in BMEC was detected by Western blot.8.To study the effect of CSE on the contents of caspase-3,caspase-8.and caspase-9 in the mitochondrial apoptosis pathway of the OGD model of BMEC:The third generation of rat BMEC was isolated and cultured and divided into normal group,model group,and The CSE group(5,10,20 ?g·mL-1).The model group and CSE group were modeled by OGD.The contents of caspase-3,caspase-8 and caspase-9 in BMEC were detected by biochemical method.Results:1.The primary BMEC has been successfully isolated and cultured,and the OGD model of BMEC has been successfully established.2.Cordyceps sinensis extract can alleviate the damage of cell morphology and cell activity caused by the OGD model of BMEC:By invert microscope observation and CCK-8 assay to detect BMEC,CSE can significantly reduce the damage and shrinkage of the cells caused by OGD,make cell volume normal,increases the number of viable cells,reduces the number of necrotic cells,and increases cell viability(P<0.05).These results indicated that CSE could protect the the OGD model of BMEC,which could improve the morphology of cells and increase the activity of cells.The appropriate concentration of CSE(5,10,20?g·mL-1)was selected for further experiments.3.Cordyceps sinensis extract has antioxidative effect on the OGD model of BMEC:Compared with normal group,the content of MDA,NO and leakage of LDH in the model group increased significantly(P<0.01),SOD activity in the model group decreased significantly(P<0.01).Compared with the model group,the CSE group(5,10,and 20?g·mL-1)significantly decreased intracellular MDA,NO,LDH leakage and significantly increased SOD activity(P<0.05).It was shown that CSE inhibited oxidative stress factors produced by the model of ischemic injury in BMEC and exerted antioxidative effects,which had a protective effect on cerebral ischemia.4.Cordyceps sinensis extract has anti-inflammatory effect on the OGD model of BMEC:Compared with the normal group,the levels of TNF-? and IL-1? in the model group were significantly increased(P<0.01).Compared with the model group,the CSE group(5,10,and 20 ?g·mL-1)significantly reduced intracellular TNF-? and IL-1? levels(P<0.05).These results indicated that CSE can inhibit the production of inflammatory factors in the OGD model of BMEC and exert anti-inflammatory effects,thus it has a protective effect on cerebral ischemic injury.5.Cordyceps sinensis extract has anti-apoptosis effect on the OGD model of BMEC:Compared with normal group,the apoptotic rate of the model group was significantly higher,the difference is statistically significant(P<0.01).Compared with the control group,the apoptosis rate of CSE 5,10,and 20 ?g·mL-1 cells was significantly decreased(P<0.01),and it was concentration-dependent.The results indicated that CSE can inhibit the apoptosis of BMEC induced by OGD,relieve the injury of the OGD model of BMEC,and protect cerebral ischemic injury.6.Cordyceps sinensis extract can increase the reduction of MMP induced by the OGD model of BMEC:Under normal conditions,the MMP of BMEC and the percentage of cells emitting P2 in red fluorescence were high,but the MMP was decreased after modeling.Compared with the normal group,the number of red fluorescent cells in the model group and the percentage of P2 was decreased(P<0.01).Compared with the model group,the CSE group(5,10,and 20 ?g·mL-1)increased the number of red fluorescent cells,increased the percentage of P2 and the MMP,which was statistically significant(P<0.01),and it was concentration-dependent.The results indicated that CSE can increase the reduction of MMP induced by the OGD model of BMEC,alleviate the injury of OGD model in BMEC,and protect cerebral ischemic injury.7.Cordyceps sinensis extract influenced the expression of related proteins in the mitochondrial pathway of the OGD model of BMEC,and thus inhibited the mitochondrial apoptosis pathway:Compared with the normal group,the expression of Cyt C,Bax and caspase-3 in the model group were up-regulated,and Bcl-2 expression was down-regulated,which were statistically significant(P<0.01).Compared with the model group,the expression of Cyt C,Bax and caspase-3 in CSE group were down-regulated,and the expression of Bcl-2 was up-regulated.The CSE 20 ?g·mL-1 were significantly different(P<0.05).The results showed that CSE could protect the injury caused by the OGD model of BMEC by inhibiting the mitochondrial apoptosis pathway,so as to exerted the protective effect on cerebral ischemic injury.8.Cordyceps sinensis extract inhibited caspase activity in the mitochondrial pathway of the OGD model of BMEC:Compared with the normal group,the caspase-3,caspase-8,and caspase-9 levels in the model group increased significantly(P<0.01).Compared with the model group,the CSE group(5,10,20 ?g·mL-1)significantly reduced the content of caspase-3,caspase-8,and caspase-9 in the cells(P<0.05).The results showed that CSE interfered with the mitochondrial apoptosis pathway and decreased the caspase content,thus alleviating the injury of the OGD model of BMEC and protecting cerebral ischemic injury.Conclusion:1.Cordyceps sinensis extract can improve cell morphology,increase cell activity,inhibit oxidative stress,reduce inflammatory response and apoptosis in the OGD model of BMEC.2.Cordyceps sinensis extract can increase MMP,alleviate mitochondrial membrane damage,down-regulate Cyt C,Bax,caspase-3 protein expression,up-regulate Bcl-2 protein expression,and decrease caspase-3,caspase-8,caspase-9 content in the OGD model of BMEC,which indicates that CSE has a protective effect on the OGD model of BMEC by inhibiting mitochondrial apoptosis pathway.3.To study the protective effect and mechanism of CSE on the OGD model of BMEC provides basis for exploring the indications of the traditional valuable Chinese medicine Cordyceps sinensis.