| Objective:To investigate the protective effect of Alisol A on oxygen-glucose deprivation/reoxygenation(OGD/R)injurey in the mouse brain microvascular endothelial cells(b End.3)and its regulation of apoptosis through PI3K-AKT signaling pathway.Methods:CCK8 assay was used to detect the effects of 2 μmol/L、20 μmol/L、40 μmol/L、80 μmol/L、160 μmol/L、240 μmol/L and 320 μmol/L Alisol A on the viability of b End.3 cells to determine drug toxicity and IC50 value of Alisol A;OGD/R was applied to culture b End.3 to mimic cell injury and CCK8 assay was used to determine the hypoxic intervention time by detect the changes of cell viability after different hypoxia time and different concentration of Alisol A intervened in b End.3 cells;Lactate dehydrogenase(LDH)detection kit reflected the activity of LDH;Reactive oxide species(ROS)kit was used to detect intracellular ROS;the expression of interleukin 1β(IL-1β)and tumor necrosis factor α(TNF-α)were detected by ELISA;b End.3 cell permeability was tested by permeability experiment;the ability of cell migration and invasion were detected by wound healing test and cell invasion assay;flow cytometry was used to detect apoptosis;RT-q PCR was used to detect the expression level of Bcl-2 mRNA and Bax mRNA;the level of p-PI3 K,PI3K,p-AKT,AKT,Bcl-2 and Bax proteins were determined by western blot analysis.Results:1.Protective effect of Alisol A on OGD/R-induced bEnd.3(1)Cell viability: 2 μmol/L,20 μmol/L,40 μmol/L Alisol A had no obvious toxic effect on cells,while 80 μmol/L,160 μmol/L,240 μmol/L and 320 μmol/L Alisol A inhibited cell viability and the cytotoxicity of 160 μmol/L 240 μmol/L and320 μmol/L Alisol A was obvious;compared with the control group,the cell activity of OGD/R group was significantly reduced(P < 0.01)and LDH release up-regulated(P < 0.01);compared with OGD/R group,the cell activity of the Alisol A intervention groups were improved(P < 0.01 or P < 0.05)and LDH release alleviated(P < 0.01 or P < 0.05).(2)Inflammation: compared with the control group,intracellular ROS expression increased in OGD / R group(P < 0.01);compared with OGD/R group,intracellular ROS expression decreased in Alisol A intervention group(P < 0.01 or P< 0.05).The levels of IL-1β and TNF-α in the supernatant of OGD/R group were higher than those in the control group(P < 0.01);the inflammatory factors in low,middle and high concentrations Alisol A intervention groups were lower than those in OGD/R group(P < 0.01 or P < 0.05).(3)Cell permeability: the cell permeability of OGD/R group was higher than that of control group(P < 0.01),and Alisol A treatment reversed the high-permeability of b End.3 induced by OGD/R injury(P < 0.01 or P < 0.05).(4)Migration and invasion capacity: compared with the control group,the migration and invasion capacitites of cells in OGD/R group were reductive(P < 0.01);compared with OGD/R group,the migration and invasion abilities of cells under Alisol A intervention were heightened(P < 0.01).(5)Apoptosis: the apoptosis rate of OGD/R group was significantly elevated by OGD/R injury(P < 0.01),and Alisol A significantly inhibited the cell apoptpsis induced by OGD/R(P < 0.01 or P < 0.05).(6)Bcl-2 mRNA and Bax mRNA expression: compared with the control group,Bax mRNA expression was significantly up-regulated and Bcl-2 mRNA expression was significantly down-regulated(P < 0.01)in OGD/R group;compared with OGD/R group,Bcl-2 mRNA expression ehanced and Bax mRNA expression receded in low,medium and high concentrations Alisol A intervention groups(P < 0.01 or P < 0.05).(7)The expression of p-PI3 K,PI3K,p-AKT,AKT,Bcl-2 and Bax proteins:compared with the control group,the ratios of p-PI3K/PI3 K and p-AKT/AKT declined and the expression of Bcl-2 protein descended in OG /R group(P < 0.01),on the contrary,the expression of Bax protein increased in OGD/R group(P < 0.01);compared with ODG/R group,the ratios of p-PI3K/PI3 K and p-AKT/AKT as well as the expression of Bcl-2 protein enhanced after Alisol A treament while the expression of Bax protein decreased in low,medium and high dose Alisol A intervention groups(P<0.01 or P < 0.05).2.Alisol A protect BMECs against OGD/R injury through PI3K-AKT signaling pathway.The results of cell permeability test,wound healing test,cell invasion assay,flow cytometry,RT-q PCR and western blot in control group,OGD/R group and OGD+MA group(MA: 20 μmol/L Alisol A)were consistent with Result 1.However,inhibition of PI3 K protein expression led to alteration of effect of Alisol A in mediun concentration(MA).Compared with OGD+MA group and OGD+MA+DMSO group,the cell permeability of OGD+MA+LY group(LY: 10 μmol/L LY294002)increased,the migration and invasion abilities of OGD+MA+LY group weakened,meanwhile,OGD+MA+LY group cell apoptosis and Bax protein and mRNA expression increased,but Bcl-2 protein and mRNA expression OGD+MA+LY group showed a declining trend and the phosphorylation of PI3 K and AKT protein was significantly reduced(P< 0.01 or P < 0.05).Conclusion:1.Alisol A significantly improved cell viability and reduced LDH release induced by OGD/R injury;2.Alisol A decreased intracellular ROS expresseion and reduce the expression of inflammatory factors IL-1β and TNF-α induced by OGD/R injury.At the same time,Alisol A reduce cell permeability to alleviate OGD/R injury;3.Alisol A enhanced cell migration and invasion capacities,promoted the phosphorylation of PI3 K and AKT proteins,and facilitated the expression of Bcl-2protein and mRNA,but reduced the expression of Bax protein and mRNA.Alisol A plays a critical role to resist cell apoptosis and protect end 3 cells;4.Inhibition of PI3K protein increased cell permeability and weakened cell migration and invasion capacities.Inhibition of PI3 K protein decreased PI3 K and AKT protein phosphorylation and down-regulated anti-apoptotic protein Bcl-2 and Bcl-2 mRNA expression,but increased pro-apoptotic protein Bax and Bax mRNA expression.Inhibition of PI3 K protein weakened the inhibitory effect of Alisol A on apoptosis;5.Alisol A can protect BMECs against OGD/R injury by regulating apoptosis through PI3K-AKT signaling pathway. |
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