疏肝健脾方药含药血清对LPS刺激下大鼠肝细胞炎症损伤及TLR4/p38MAPK信号通路影响的研究

Effects of soothing liver and invigorating spleen recipes on LPS-induced hepatocytes injury of rats and TLR4 / p38MAPK signal pathway

目的： 研究疏肝健脾方药含药血清对脂多糖（lipopolysaccharide，LPS）刺激下大鼠肝细胞炎症反应及TLR4（Toll样受体-4）/p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase，p38MAPK）信号通路的影响。 方法： 体外培养并鉴定SD大鼠肝细胞，制备疏肝健脾方药含药血清，对肝细胞进行疏肝健脾方药、p38MAPK抑制剂等药物干预，酶联免疫法（ELISA）检测细胞上清液中白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）含量的表达，Western blot检测 TLR4，p38MAPK，p-p38MAPK蛋白的表达。 结果： 可提取大鼠疏肝健脾方药含药或空白血清约2～3 mL，大鼠获得纯化后肝细胞1.5×10^8～2.0×10^8个，Typan blue染色测定细胞活力均在95%以上，免疫荧光法鉴定确定提取的为肝细胞。肝细胞培养上清的检测： LPS组较空白血清组IL-6，TNF-α水平均有显著升高（P〈0.01）。与LPS组比较，药物干预组IL-6，TNF-α表达水平呈显著降低（P〈0.01）。肝细胞的蛋白检测：与空白血清组比较，LPS组的p38MAPK，p-p38MAPK，TLR4蛋白表达均有显著升高（P〈0.01）；与LPS组比较，SB239063能显著下调p38MAPK的蛋白表达水平（P〈0.01），疏肝健脾组亦有下调，但无显著差异；疏肝健脾组，SB239063组的p-p38MAPK的蛋白表达水平均下调显著（P〈0.01）；TLR4蛋白表达水平以疏肝健脾组下调具有显著性差异（P〈0.01），SB239063组下调无显著统计学差异。 结论： 疏肝健脾方药可能通过TLR4/p38MAPK信号通路调控大鼠肝细胞炎症损伤，含药血清可能是其重要的作用途径之一。

Objective： To explore the effects of soothing liver and invigorating spleen recipes on lipopolysaccharide（LPS） induced hepatocyte inflammation of rats and TLR4/p38MAPK signal pathway. Method： The hepatocytes of SD rats were cultured and identified in vitro. The medicated serum of soothing liver and invigorating spleen recipes was prepared. The hepatocytes were treated with soothing liver and invigorating spleen recipes. Then Interleukin- 6 （IL-6） and tumor necrosis factor-α （TNF-α） expression in cultural supernatants were assayed by ELISA. The expressions of Toll-Like 4（TLR4）, p38 mitogen activated protein kinases （p38MAPK） and p-p38 mitogen-activated protein kinase （p-p38MAPK） were detected by Western blot. Result： The rat medicated serum of soothing liver and invigorating spleen recipes was extracted for 2-3 mL. The purified rat hepatocytes were 1.5×10^8-2.0×10^8. The cell viability was above 95% detected by Typan blue staining. The hepatocytes were identified by immumofluorescence assay. The detection of hepatocyte cultural supernatants： compared with that of the control group, IL-6 and TNF-α expression were increased in the LPS group （P〈0.01）. While compared with that of the LPS group, the expressions of IL-6 and TNF-α were decreased after soothing liver and invigorating spleen recipes intervention （P〈0.01）. The detection of hepatocyte proteins： compared with that of the control group, the protein expressions of p38MAPK, p-p38MAPK and TLR4 were all increased significantly in the LPS group （P〈0.01）. Compared with that of the LPS group, the protein expressions of p38MAPK was decreased significantly in SB239063 group and it was also decreased in the soothing liver and invigorating spleen recipes group, but with no significant difference. Compared with that of the LPS group, p38MAPK expression was reduced significantly in the soothing liver and invigorating spleen recipes group and the SB239063 （p38MAPK pathway inhibitor） group （P〈0.01）. TLR4 protein expression was decreased markedly in the soothing liver and invigorating spleen recipes group （P〈0.01） but had no difference between the SB239063 group and the LPS group. Conclusion： The soothing liver and invigorating spleen recipes may regulate hepatocyte inflammatory injury of rats through TLR4/p38MAPK signaling pathway.