| Objective:To investigate the protective effect of Alisol A 24-Acetate on Oxygen-glucose deprivation/reperfusion(OGD/R)injury in the mouse brain microvascular endothelial cells(bEnd.3)and its relationship with miR-92a-3p.Methods:1.The protective effect of Alisol A 24-Acetate on OGD/R injury in bEnd.3 cells(1)The OGD/R model was established in the hypoxia incubator,and the cell viability at different hypoxia times was detected by cck8 assay,with the relative viability of 50%-60% as the modeling condition.(2)The effects of Alisol A 24-Acetate on the morphology and viability of bEnd.3 cells were investigated.Nitric oxide(NO)kit and lactate dehydrogenase(LDH)kit were used to detect NO and LDH levels respectively.The expressions of TNF-? and IL-1? were detected by ELISA Kit.The expression of tight junction proteins(claudin-5,occludin,ZO-1)were detected by western bolt.(3)The expression of miR-92a-3p and miR-92a-1-5p in bEnd.3 cells were detected by qPCR.Possible target genes of miR-92a-3p were searched through Targetscan7.2 and miRDB databases.To further investigate the protective mechanism of Alisol A 24-Acetate on OGD/R-induced cells injury and its correlation with miR-92a-3p.2.The regulation of miR-92a-3p on OGD/R injury in bEnd.3 cellsBEnd.3 cells were transfected with miR-92a-3p mimics and miR-92a-3p inhibitor,and then treated with OGD/R injury.The cell viability was detected by cck8 assay.The levels of NO and LDH were detected by the kit.The expression of tight junction proteins(claudin-5,occludin and ZO-1)were detected by western bolt.To investigate the influence of miR-92a-3p on OGD/R-induced cells and tight junction.Further,the target genes of miR-92a-3p were verified by dual luciferase reporter gene methods.Results:1.Alisol A 24-Acetate had protective effects on OGD/R injury in bEnd.3 cells(1)The cell viability of OGD8h/R16 h was(57.29±5.87)%,as the modeling condition for subsequent experiments.(2)1mg/L Alisol A 24-Acetate alleviated the release of LDH(P<0.05),inhibited the expression of TNF-?(P<0.01)and up-regulated the expression of tight junction proteins(claudin-5 and ZO-1)(P<0.05).However,it had no obvious effect on the cell viability,NO level,IL-1? and occludin protein expression.10mg/L and 20mg/L Alisol A 24-Acetate obviously improved the cell viability(P < 0.01)and NO level(P < 0.01 or P < 0.05),reduced the release of LDH(P < 0.01),inhibited the expression of TNF-? and IL-1?(P < 0.01),increased tight junction proteins(claudin-5 and ZO-1)expression(P < 0.05).And,10mg/L Alisol A 24-acetate treatment reversed the low-expression of occludin induced by OGD/R injury(P<0.05).(3)The expressions of miR-92a-3p and miR-92a-1-5p in bEnd.3 cells was induced by OGD/R injury(P<0.01).10mg/L and 20mg/L Alisol A 24-Acetate specifically down-regulated the expression of miR-92a-3p(P<0.01),but it had no obvious effect on the expression of miR-92a-1-5p.The target genes of miR-92a-3p were found to be associated with the tight junction by Targetscan7.2 and miRDB databases.Using the principle of base pairing,the binding sites of the 3'UTR sequences of claudin-5,occludin and ZO-1 of miR-92a-3p were manually compared,and it was found that miR-92a-3p could bind to the 3'UTR sequences of occludin and ZO-1,suggesting that miR-92a-3p may directly target the expression of occludin and ZO-1.2.Alisol A 24-Acetate ameliorated OGD/R injury in bEnd.3 cells via inhibiting the expression of miR-92a-3p(1)After OGD/R injury,the cell viability,LDH and NO levels,the expression of tight junction proteins of mimics NC group and inhibitor NC group were similar to those of OGD group.It was indicated that transfection had no side effect on OGD/R injury in bEnd.3 cells.(2)MiR-92a-3p mimics(50nmol/L)further up-regulated miR-92a-3p expression in bEnd.3 cells,reduced cell viability and NO level(P <0.05),down-regulated tight junction proteins(claudin-5,occludin and ZO-1)expression(P<0.05),indicating that miR-92a-3p mimics further increased the damage of bEnd.3 cells.Compared with the transfection of miR-92a-3p mimics alone,the co-treatment of miR-92a-3p mimics and 10mg/L of Alisol A 24-Acetate significantly improved the cell viability,NO and LDH levels,and the expression of tight junction proteins(P<0.01).It was suggested that Alisol A 24-Acetate alleviated the cell damage induced by miR-92a-3p mimics.(3)After transfection miR-92a-3p inhibitor,the cell viability increased significantly(P<0.01),NO level increased(P<0.01),LDH overflow decreased(P<0.05),the expression of tight junction(claudin-5,occludin,and ZO-1)proteins increased(P<0.01 or P<0.05).It was further demonstrated that miR-92a-3p inhibitor had a protective effect on OGD/R injury in bEnd.3 cells,which was similar to Alisol A 24-Acetate.(4)The dual luciferase reporter gene assay showed that luciferase activity in occludin 3'UTR(WT)and ZO-1 3'UTR(WT)transfected cells were lower than those in control groups(P<0.01).However,there was less effect of miR-92a-3p on the luciferase activity in cells containing mutant occludin 3'UTR(MUT)and ZO-1 3'UTR(MUT),confirming that occludin and ZO-1 were the target genes of miR-92a-3p.It indicated that Alisol A 24-Acetate exerted the protective effect of OGD/R injury in endothelial cells through inhibiting the expression of miR-92a-3p via targeting occludin and ZO-1.Conclusions:1.Alisol A 24-Acetate improved cell morphology and cell viability,promoted the release of NO,reduced the expression of inflammatory cytokines(TNF-? and IL-1?)and protected tight junction proteins(claudin-5,occludin and ZO-1),and alleviated OGD/R injury in bEnd.3 cells.2.OGD/R induced the high expression of miR-92a-3p in bEnd.3 cells,and Alisol A 24-Acetate specifically inhibited the expression of miR-92a-3p.3.MiR-92a-3p mimics further increased OGD/R injury in bEnd.3 cells.MiR-92a-3p inhibitor improved the OGD/R injury in bEnd.3 cells by improving cell viability and NO release,promoting tight junction proteins expression.It indicated that miR-92a-3p inhibitor had a protective effect on OGD/R injury in bEnd.3 cells.4.MiR-92a-3p directly targeted to occludin and ZO-1,indicating that Alisol A 24-acetate improved OGD/R-induced bEnd.3 cells injury via miR-92a-3p targeting tight junctions. |
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