20-羟基蜕皮甾酮通过Akt/NF-κB通路调节小胶质细胞活化

20-Hydroxyecdysone Inhibits Akt-Mediated NF-κB Activation in LPS-treated Microglial Cells

目的观察20-羟基蜕皮甾酮对脂多糖诱导的体外原代培养小胶质细胞活化的影响,并探讨其相关机制。方法脂多糖处理原代培养的SD大鼠小胶质细胞,构建其活化模型。实验分为正常组,脂多糖处理组和脂多糖+20-羟基蜕皮甾酮组。酶联免疫法测定各组细胞培养基中IL-1β和TNF-α的浓度,Western blot法检测各组细胞胞质内p-IκBα、细胞核内pNF-κB以及细胞内p-Akt水平。结果 10ng/ml LPS孵育小胶质细胞8h,细胞培养基中IL-1β和TNF-α浓度分别从33. 73±6. 42pg/ml和43. 67±7. 17pg/ml上升至87. 16±12. 78pg/ml和96. 55±13. 76pg/ml(P <0. 01)。50μmol/L 20-羟基蜕皮甾酮和LPS一起处理细胞8h后,笔者观察到细胞上清中IL-1β和TNF-α浓度分别降低至59. 37±9. 24和72. 81±12. 69pg/ml(P <0. 05)。进一步增加20-羟基蜕皮甾酮浓度至100μmol/L,细胞上清中IL-1β和TNF-α浓度进一步降低至48. 11±8. 42pg/ml和61. 44±9. 38pg/ml(P <0. 01)。此外,脂多糖导致小胶质细胞胞质内p-IκBα、细胞核内p-NF-κB和细胞内pAkt水平明显升高(P <0. 01),20-羟基蜕皮甾酮抑制上述蛋白水平的升高。结论 20-羟基蜕皮甾酮经Akt信号通路,失活NF-κB,抑制LPS诱导的小胶质细胞IL-1β和TNF-α分泌,最终改善小胶质细胞介导的炎症。

Objective To investigate the effects of 20-hydroxyecdysone on microglial activation and the related mechanisms in lipopolysaccharide(LPS)-stimulated microglial cells.Methods A model of activation of microglia was established by LPS in the primary cultured SD rat microglia.The experiment was divided into control group,LPS-treated group and LPS+20-hydroxyecdysone treated group.The concentration of IL-1βand TNF-αin the culture supernatant of microglia cells were determined by enzyme-linked immunosorbent assay in the experimental group.The levels of p-IκBαin the cytoplasm,p-NF-κB in the nucleus and p-Akt in cell were detected by Western blot.Results The concentration of IL-1βand TNF-αin the culture supernatant of microglia cells were increased from 33.73±6.42pg/ml and 43.67±7.17pg/ml up to 87.16±12.78pg/ml and 96.55±13.76pg/ml respectively after the microglia cells were treated by 10ng/ml LPS for 8 hours(P<0.01).We observed that the concentration of IL-1βand TNF-αin the culture supernatant of microglia cells were reduced to 59.37±9.24 and 72.81±12.69pg/ml after the microglia cells were treated by 50μmol/L 20-hydroxyecdysone and LPS for 8 hours(P<0.05).The concentration of IL-1βand TNF-αin the culture supernatant of microglia cells were further decreased to 48.11±8.42pg/ml and 61.44±9.38pg/ml respectively when The concentration of 20-hydroxyecdysone was increased to 100μmol/L(P<0.01).In addition,the levels of p-IκBαin the cytoplasm,p-NF-κB in the nucleus and p-Akt in cell were increased significantly in LPS-treated microglia cells,which were inhibited by 20-hydroxyecdysone.Conclusion 20-hydroxyecdysone caused inactivation of NF-κB via Akt signaling pathway,further inhibited the secretion of IL-1βand TNF-αof microglia and finally ameliorated the inflammatory responses of microglia induced by LPS.