| ObjectiveCerebral ischemia-reperfusion injury(CIRI)often results in blood-brain barrier(BBB)destruction,which leads to brain homeostasis imbalance and disease aggravation.Our previous study found that Pachymic acid(PA)can repair BBB,but the mechanism is not clear.Brain microvascular endothelium is an important component of BBB.Therefore,this study aims to duplicate set up oxygen glucose deprivation/reoxygenation(OGD/R)model of brain microvascular endothelial cells in vitro.Based on it clinic efficiency,Pharmmapper and Swiss database were used to predict possible targets of PA,which were then verified in vitro to explore its mechanism of how to protect BBB.Methods1.Pharmacodynamic evaluation of how PA alleviates OGD/R injury of bEnd.3 cells which is a mouse derived microvascular endothelial cells.OGD/R model of bEnd.3 cells was established in vitro.Then,CCK-8 method,LDH release method and the Trans-Epithelial Electrical Resistance(TEER)were used to assess the injury.A series of concentrations of PA were administrated to intervene the model,and mRNA transcription levels of OGD/R injury related genes were examined while TEER was also assessed to explore the pathways of how PA alleviated OGD/R injury of bEnd.3 cells.2.To predict possible targets of PA on bEnd.3 cells to alleviate OGD/R injury,3D structure of PA was obtained from Pub Chem Compound database while related target genes of PA were achieved from Swiss Target Prediction database.Pharm Mapper database and Uni Prot database which were then enriched by KEGG using David 6.8.Their outputs were visualized using"weishengxin"website to predict the action pathway of PA.;3D structure of PA was obtained by TCMSP database,key proteins of PI3K/Akt signal pathway were obtained from PDB whose molecular docking was performed by Auto Dock Vina software and the outputs were visualized by Py MOL software.3.In vitro experiments were carried out to verify the predicted targets of PA to clarify its mechanism of action.PI3K is a possible target for PA to alleviate OGD/R injury of bEnd.3cells.The expression of claudin-5 at bEnd.3 cells was detected by immunofluorescence,and the protein phosphorylation levels of PI3K,Akt,Fox O1 and the protein expression levels of claudin-5 and ZO-1 were assessed by Western blot.On the basis of exploring the positive targets LY294002,an inhibitor of PI3K,was used to intervene the model,and the effects of PI3K inhibitor on the possible targets of drugs were observed and compared,and the target of pachymic acid was verified in reverse,so as to clarify the mechanism of Pachymic acid in alleviating OGD/R injury of bend.3 endothelial cells.Results1.Establishment and examination of OGD/R injury model of bEnd.3 cells(1)The TEER values of 12 h,24 h,36 h,48 h and 60 h after bEnd.3 cells were seeded and cultured were measured by transcellular electrical resistance meter.The maximum Teer value(150.30Ω/cm~2)was observed at 36 h which was used as baseline culture time to examine the barrier effect of bEnd.3 cells in vitro.(2)CCK8 method,LDH release method and TEER were used to evaluate the damage of bEnd.3 cells at different OGD/R time.Compared with control group,the cell viability of OGD/R group decreased to 58.09%of baseline at OGD5h/R19h(deprivation for 5 hours then reperfusion for 19 hours),LDH activity increased to148%of baseline,and TEER value also decreased significantly(P<0.01).(3)The model was evaluated by CCK8,LDH release and TEER(P<0.01).In conclusion,the OGD/R injury model of bEnd.3 cells was established by 36 h culture with OGD 5h/R19h.2.Pharmacodynamic evaluation of PA in alleviating OGD/R injury of bEnd.3 endothelial cells2.1 PA attenuates OGD/R injury of bEnd.3 endothelial cellsCompared with control group,TEER value of OGD/R group decreased significantly(P<0.01).TEER values of PA 1.2μM,0.6μM and 0.3μM groups increased significantly cmpared with those in OGD/R group(P<0.05).2.2 PA up regulates claudin-5 and ZO-1 mRNA transcription levels in bEnd.3 cells with OGD/R injuryCompared with control group,the mRNA transcription levels of tight junction proteins Claudin-5 and ZO-1 in OGD/R group was significantly down regulated(P<0.05).The mRNA transcription levels of claudin-5 in PA 2.4μM group was significantly down regulated(P<0.05),PA 1.2μM group(P<0.05)and 0.6μM groups(P<0.01)were all significantly up regulated.The transcription levels of ZO-1 mRNA in 1.2μM,0.6μM and 0.3μM groups were all up-regulated(P<0.05).3.Prediction of PA target at OGD/R injury model at bEnd.3 cells3.1 PI3K/Akt signaling pathway is a possible pathway of PA to alleviate OGD/R injury of bEnd.3 cellsThe Swiss Target Prediction and Pharm Mapper database revealed 143 key targets of PA to alleviate OGD/R injury at bEnd.3 cells.KEGG enrichment analysis(top 20)showed PI3K/Akt signaling pathway,cAMP signaling pathway,calcium signaling pathway etc.,and the key protein of PI3K/Akt signaling pathway with the most significant difference was selected to screen interactors using molecular docking.3.2 Molecular docking technology predicts the target of PA in alleviating OGD/R injury of bEnd.3 endothelial cellsThe key proteins of PI3K Akt signaling pathway were"Pachymic acid protein"docking by molecular docking technology:(1)3D structure of Pachymic acid was obtained by TCMSP database while PI3K and Akt were obtained by PDB database.(2)Autodock Vina software was used to dock Pachymic acid with PI3K and Akt,and Py MOL was used to visualize the docking results.(3)The binding affinity between PA and protein Akt was-4.0/kcal·mol-1 while the binding affinity between PA and protein PI3K was-9.5/kcal·mol-1.Binding affinity more than the cut-off value of-5.0/kcal·mol-1 indicate the potential target to be further screened.4.Verification of predictive pathway and target of PA in alleviating OGD/R injury of bEnd.3 endothelial cells4.1 PA attenuates claudin-5 loss in OGD/R injury model of bEnd.3 endothelial cellsIn the control group,the fluorescence that stained claudin-5 was evenly distributed around the nucleus of b End3 cells with good continuity while the gray value of fluorescence in the OGD/R group was significantly lower(P<0.01)than that in control group,with discountinued perineucleus staining and scattered signal in cytoplasm.The gray value of fluorescence in PA1.2μM and 0.6μM groups was increased significantly compared with that in OGD/R group(P<0.05),with evenly distributed signal and good continuity around the nucleus no scattered fluorescence could be appreciated.4.2 Mechanism of PA in alleviating OGD/R injury of bEnd.3 endothelial cells(1)Compared with control group,the phosphorylation levels of PI3K,Akt and Fox O1 and the expression levels of claudin-5 and ZO-1 in OGD/R group were significantly down-regulated(p<0.05).Compared with OGD/R group,the phosphorylation levels of PI3K,Akt and Fox O1 and the protein expression levels of claudin-5 and ZO-1 were significantly up-regulated in PA 1.2μM and 0.6μM groups(P<0.05).(2)Based on(1),PI3K/Akt Fox O1 may regulate claudin-5 and ZO-1 protein,so LY294002,a PI3K inhibitor,was used to intervene.And the results show that,Compared with control group,phosphorylation levels of PI3K,Akt and Fox O1 and the expression levels of claudin-5and ZO-1 in OGD/R group were significantly down regulated(P<0.05);Compared with OGD/R group,the phosphorylation levels of PI3K,Fox O1 and Akt and the expression levels of Claudin-5 and ZO-1 were significantly up-regulated(p<0.05)in PA 1.2μM group.Compared with PA 1.2μM+LY294002group,the phosphorylation levels of PI3K,Akt,Fox O1 and the expression levels of claudin-5 and ZO-1 in PA 1.2μM group were significantly up-regulated(P<0.05).Compared with PA1.2μM group,the phosphorylation level of PI3K in LY294002+LY294002 group was significantly increased(P<0.01),but the phosphorylation levels of Akt and Fox O1 protein and the expressions of ZO-1 and claudin-5 protein were not significantly different.Conclusion1.Oxygen glucose deprivation for 5h and reperfusion 19h at 36 hours of culture was applied to establish OGD/R injury model of bEnd.3 cells in vitro.PA reduce the damage of b BEnd.3 cells by up regulating expression of tight junction associated proteins.2.Finally,we draw the conclusion that"regulating PI3K/Akt/Fox O1 signaling pathway"is the potential mechanism for PA to alleviate the damage of bEnd.3 cells induced by OGD/R as shown by the outcome from pharmmapper and Swiss target database screening,molecular docking prediction,and in vitro experiment verification. |
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