竹节参总皂苷对脂多糖诱导的BV2细胞炎症反应的影响及机制研究

Study on Effects of Total Saponins of Panacis Japonici Rhizoma on Lipopolysaccharideinduced Inflammation in BV2 Cells and Its Mechanism

目的观察竹节参总皂苷(TSPJ)对脂多糖诱导的小鼠小胶质细胞BV2炎症反应的影响,探讨其改善炎症的作用机制。方法采用脂多糖诱导BV2细胞炎症模型,将细胞分为正常组、模型组和TSPJ低、中、高浓度组,分别进行干预后,ELISA检测细胞上清液一氧化氮(NO)、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-4和IL-10含量,免疫荧光染色检测小胶质细胞标志物CD86、Arginase-1及自噬蛋白LC3表达,Western blot检测自噬相关蛋白p62、Beclin1、LC3及Akt、p-Akt、mTOR、p-mTOR蛋白表达。结果与正常组比较,模型组细胞上清液NO、TNF-α、IL-1β含量明显增加,IL-4、IL-10含量明显减少,细胞CD86表达明显升高,Arginase-1、LC3表达明显降低,Beclin1、LC3Ⅱ蛋白表达明显降低,p62、p-Akt、p-mTOR蛋白表达明显升高,差异均有统计学意义(P<0.01);与模型组比较,TSPJ高浓度组细胞上清液NO、TNF-α、IL-1β含量明显减少,IL-4和IL-10含量明显增加,细胞CD86表达明显降低,Arginase-1、LC3表达明显升高,Beclin1、LC3Ⅱ蛋白表达明显升高,p62、p-Akt、p-mTOR蛋白表达明显降低,差异均有统计学意义(P<0.05,P<0.01)。与TSPJ组比较,TSPJ和自噬抑制剂3-甲基腺嘌呤联用后细胞Beclin1、LC3Ⅱ蛋白表达明显降低(P<0.01),p62蛋白表达明显升高(P<0.01);TSPJ和Akt抑制剂GSK690693联用后细胞p-Akt、p-mTOR蛋白表达明显降低(P<0.01)。结论TSPJ能抑制脂多糖诱导的BV2细胞炎症反应,通过调节促炎因子和抗炎因子分泌,促进BV2细胞由M1型向M2转化,维持M1/M2平衡,其抗炎作用与提高细胞自噬水平、抑制Akt/mTOR通路磷酸化有关。

Objective To observe the effects of total saponins of Panax japonici Rhizoma(TSPJ)on lipopolysaccharide-induced BV2 inflammation in mouse microglia BV2 cells;To explore its mechanism of improving inflammation.Methods Lipopolysaccharide was used to induced BV2 cells inflammation model,the cells were divided into normal group,model group and TSPJ low-,medium-and high-concentration groups.After the intervention,the contents of nitric oxide(NO),tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-4 and IL-10 in cell supernatant were detected by ELISA,immunofluorescence staining was used to detect the expressions of microglial marker CD86,Arginase-1 and autophagy protein LC3,Western blot was used to detect the expressions of autophagy-related proteins p62,Beclin1,LC3,Akt,p-Akt,mTOR,p-mTOR proteins.Results Compared with the normal group,the contents of NO,TNF-α,and IL-1βin supernatant increased significantly in the model group,the contents of IL-4 and IL-10 significantly decreased,the expression of CD86 significantly increased,the expressions of Arginase-1 and LC3 significantly decreased,the expressions of Beclin1 and LC3Ⅱprotein significantly decreased,and the expressions of p62,p-Akt,p-mTOR protein significantly increased,with statistical significance(P<0.01).Compared with the model group,the contents of NO,TNF-α,and IL-1βin supernatant of TSPJ high-concentration group decreased significantly,the contents of IL-4 and IL-10 increased significantly,and the expression of CD86 decreased significantly,the expressions of Arginase-1 and LC3 significantly increased,the expressions of Beclin1 and LC3Ⅱprotein significantly increased,and the expressions of p62,p-Akt and p-mTOR protein significantly decreased,with statistical significance(P<0.05,P<0.01).Compared with the TSPJ group,the expressions of Beclin1 and LC3Ⅱprotein significantly decreased(P<0.01)after the combination of TSPJ and autophagy inhibitor 3-MA,and the expression of p62 protein significantly increased(P<0.01);the expressions of p-Akt and p-mTOR protein decreased significantly(P<0.01)after the combination of TSPJ and Akt inhibitor GSK690693.Conclusion TSPJ can inhibit lipopolysaccharide-induced inflammation of BV2 cells,promote the transformation of BV2 cells from M1 to M2 by regulating the secretion of inflammatory factors,and maintain the balance of M1/M2.The anti-inflammatory effect of TSPJ is related to the increase of autophagy level and inhibition of Akt/mTOR pathway phosphorylation.