黄芩苷通过调控人脑星形胶质细胞上TRPV4/AQP4改善OGD/R所致的细胞水肿

Baicalin improves cell edema caused by OGD/R by regulating TRPV4/AQP4 on human brain astrocytes

目的:研究黄芩苷(BA)对氧糖剥夺(OGD/R)诱导损伤的人脑星形胶质细胞SVGp12的影响,从抑制钙离子内流和细胞水肿的角度,调控离子通道瞬时受体电位香草素受体4型通道蛋白(TRPV4)和水通道蛋白4(AQP4)。方法:首先BA在SVGp12细胞上的安全用药浓度进行筛选,其次筛选OGD/R细胞模型,选用OGD R(4 h/2 h)的方法模拟细胞在脑中缺血缺氧的环境,将分组设定为正常组、Model组、BA组(5μmol/L),并加入TRPV4抑制剂HC067047和激动剂GSK1016790A进行对照。用吸光度法测定了细胞活力,ELISA法测定细胞乳酸脱氢酶(LDH)渗透率,Western Blot法观察TRPV4和AQP4的表达,流式细胞术测定细胞内Ca^(2+)浓度。结果:BA在12.5μmol/L及更低的浓度范围内对正常培养的SVGp12细胞活力不产生影响;在OGD/R(4 h/2 h)的条件下,SVGp12细胞的OD值降低约50%(P<0.01);OGD/R诱导损伤后,BA的浓度为5μmol/L时细胞活力值最高(P<0.01);BA能够改善GSK1016790A诱导后的SVGp12细胞的活力(P<0.05);BA能降低SVGp12细胞的LDH渗透率(P<0.05);BA能够抑制TRPV4和AQP4的表达(P<0.05);BA能够降低SVGp12细胞内Ca^(2+)浓度(P<0.01)。结论:BA对OGD/R诱导的SVGp12细胞具有保护作用,可能是通过抑制OGD/R后TRPV4和AQP4通道蛋白的上调,从而减少Ca^(2+)内流并抑制细胞肿胀。

Objective:To investigate the effects of Baicalin(BA)on human astrocyte SVGp12 with oxygen-glucose depriration/reperfusion(OGD/R)-induced injury,to regulate ion channel transient receptor potential vanilloid 4(TRPV4)and aquaporin 4(AQP4)from the perspective of inhibiting calcium influx and cell edema.Methods:The safe drug concentration of BA on SVGp12 cells and the OGD/R cell model were screened.OGD/R(4 h/2 h)method was used to simulate the environment of ischemia and hypoxia in the brain,and the groups were set into Control group,Model group,and BA group(5μmol/L).At the same time,TRPV4 inhibitor HC067047 and agonist GSK1016790A were added.The cell viability was determined by absorbance,the permeability of lactate dehydrogenase(LDH)by ELISA,the expression of TRPV4 and AQP4 by Western Blot and the concentration of Ca^(2+)by flow cytometry.Results:BA had no effect on the viability of SVGp12 cells in the concentration range of 12.5μmol/L and lower.Under the condition of OGD/R(4 h/2 h),the OD value of SVGp12 cells decreased by about 50%(P<0.01).After OGD/R induced injury,the cell viability was the highest when BA concentration was 5μmol/L(P<0.01).BA could improve the activity of SVGp12 cells induced by GSK1016790A(P<0.05).BA decreased LDH permeability of SVGp12 cells(P<0.05).BA could inhibit the expression of TRPV4 and AQP4(P<0.05).BA could decrease the Ca^(2+)concentration in SVGp12 cells(P<0.01).Conclusion:BA has a protective effect on OGD/R-induced SVGp12 cells,possibly by inhibiting the up-regulation of TRPV4 channel protein and AQP4 channel protein after OGD/R,thereby reducing Ca^(2+)influx and inhibiting cell swelling.