甘草次酸通过抑制p38丝裂原活化蛋白激酶信号通路促进脂多糖诱导的小胶质细胞M2极化

Glycyrrhetinic acid promotes LPS-induced M2 polarization of microglia by inhibiting p38 mitogen-activated protein kinase signaling pathway

目的探究甘草次酸(GA)通过抑制p38丝裂原活化蛋白激酶(p38 MAPK)信号通路促进脂多糖(LPS)诱导的小胶质细胞M2极化。方法将小鼠BV2小胶质细胞分为阴性对照(NC)组、LPS组(100μg/ml LPS)、GA组(100μg/ml LPS+10μmol/L GA)、GA+抑制剂组(100μg/ml LPS+10μmol/L GA+10μmol/ml p38 MAPK通路抑制剂)、抑制剂组(100μg/ml LPS+10μmol/ml p38 MAPK通路抑制剂)和GA+激活剂组(100μg/ml LPS+10μmol/L GA+300 ng/ml p38 MAPK通路激活剂)。用细胞计数试剂盒8法测定细胞活力,酶联免疫吸附测定炎性因子水平,倒置显微镜观察细胞形态,Western blot检测精氨酸酶1(Arg-1)、诱导型一氧化氮合酶(iNOS)及p38 MAPK通路相关蛋白表达水平。结果与LPS组比较,GA组和抑制剂组细胞足突增多、胞体变大、细胞间隔缩小,白细胞介素(IL)1β、IL-6、iNOS、P53蛋白、磷酸化p38 MAPK(p-p38 MAPK)表达显著降低(22.53±0.53、24.13±1.00 vs 29.56±1.46,10.37±0.59、10.16±0.21 vs 15.13±1.00,0.39±0.01、0.38±0.01 vs 1.12±0.01,0.34±0.01、0.31±0.01 vs 0.89±0.01,0.40±0.01、0.40±0.02 vs 0.88±0.01,P<0.05),IL-10、Arg-1表达显著升高(177.33±7.57、187.21±6.87 vs 64.67±11.50,0.41±0.01、0.44±0.03 vs 0.10±0.01,P<0.05)。与GA组比较,GA+抑制剂组细胞足突增多、胞体变大,IL-1β、IL-6、iNOS、P53蛋白、p-p38 MAPK表达显著降低,IL-10、Arg-1表达显著升高(P<0.05);GA+激活剂组细胞足突减少、胞体变小,IL-1β、IL-6、iNOS、P53蛋白、p-p38 MAPK表达显著升高,IL-10、Arg-1表达显著降低(P<0.05)。结论GA可通过阻断p38 MAPK信号通路促进LPS诱导的小鼠BV2小胶质细胞M2极化。

Objective To explore the effect of glycyrrhetinic acid(GA)on lipopolysaccharide(LPS)-induced M2 polarization of microglia by inhibiting p38 mitogen-activated protein kinase(p38 MAPK)signaling pathway.Methods Murine microglial BV2 cells were divided into negative control(NC)group,LPS group(100μg/ml),GA group(100μg/ml LPS+10μmol/L GA),GA+inhibitor group(100μg/ml LPS+10μmol/L GA+10μmol/ml p38 MAPK pathway inhibitor),inhibitor group(100μg/ml LPS+10μmol/ml p38 MAPK pathway inhibitor)and GA+activator group(100μg/mL LPS+10μmol/L GA+300 ng/ml P38 MAPK pathway activator).CCK-8 assay was used to measure cell viability,ELISA to determine inflammatory factor levels,inverted microscopy to observe cell morphology,and Western blotting to detect the protein levels of arginase 1(Arg-1),inducible nitric oxide synthase(iNOS)and p38-MAPK pathway-related proteins.Results Compared with the LPS group,more microglial processes,enlarged cell bodies and reduced intercellular space,decreased levels of IL-1βand IL-6,and reduced protein levels of iNOS,p53 and p-p38 MAPK(22.53±0.53 and 24.13±1.00 vs 29.56±1.46,10.37±0.59 and 10.16±0.21 vs 15.13±1.00,0.39±0.01 and 0.38±0.01 vs 1.12±0.01,0.34±0.01 and 0.31±0.01 vs 0.89±0.01,0.40±0.01 and 0.40±0.02 vs 0.88±0.01,P<0.05),while increased IL-10 level and Arg-1 expression(177.33±7.57 and 187.21±6.87 vs 64.67±11.50,0.41±0.01 and 0.44±0.03 vs 0.10±0.01,P<0.05)were observed in the GA group and the inhibitor group.The GA+inhibitor group showed more microglial processes,enlarged cell bodies,reduced IL-1βand IL-6 levels and iNOS,p53 and p-p38 MAPK levels,and increased IL-10 level and Arg-1 expression when compared with the GA group(P<0.05).Moreover,opposite phenomena were observed in the GA+activator group(P<0.05).Conclusion GA can promote LPS-induced M2 polarization of murine microglial BV2 cells by blocking the p38 MAPK signaling pathway.