疏肝健脾方对脂多糖诱导的大鼠肝细胞损伤及TLR4/p38丝裂原活化蛋白激酶信号通路的影响

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

作者信息

Gong Xiang-Wen, Yang Qin-He, Yan Hai-Zhen, Zhang Yu-Pei, Liang Yin-Ji, Liu Yi-Zhen, Lin Chun-Mei, Li Yuan-Yuan

出版信息

Zhongguo Zhong Yao Za Zhi. 2014 Oct;39(20):4027-33.

PMID:25751957

Abstract

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 x 10(8)-2.0 x 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.

摘要

目的

探讨疏肝健脾方对脂多糖（LPS）诱导的大鼠肝细胞炎症及TLR4/p38MAPK信号通路的影响。

方法

体外培养并鉴定SD大鼠肝细胞。制备疏肝健脾方含药血清。用疏肝健脾方处理肝细胞。然后采用酶联免疫吸附测定法（ELISA）检测培养上清液中白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的表达。采用蛋白质免疫印迹法检测Toll样受体4（TLR4）、p38丝裂原活化蛋白激酶（p38MAPK）和磷酸化p38丝裂原活化蛋白激酶（p-p38MAPK）的表达。

结果

提取疏肝健脾方大鼠含药血清2 - 3 mL。纯化的大鼠肝细胞为1.5×10⁸ - 2.0×10⁸个。经台盼蓝染色检测细胞活力在95%以上。通过免疫荧光法鉴定肝细胞。肝细胞培养上清液检测：与对照组比较，LPS组IL-6和TNF-α表达升高（P < 0.01）。与LPS组比较，疏肝健脾方干预后IL-6和TNF-α表达降低（P < 0.01）。肝细胞蛋白检测：与对照组比较，LPS组p38MAPK、p-p38MAPK和TLR4蛋白表达均显著升高（P < 0.01）。与LPS组比较，SB239063组p38MAPK蛋白表达显著降低，疏肝健脾方组也降低，但差异无统计学意义。与LPS组比较，疏肝健脾方组和SB239063（p38MAPK通路抑制剂）组p38MAPK表达显著降低（P < 0.01）。疏肝健脾方组TLR4蛋白表达明显降低（P < 0.01），但SB239063组与LPS组比较无差异。