柴胡疏肝颗粒调控Keap1-Nrf2/HO-1信号通路改善四氯化碳诱导的小鼠急性肝损伤

Chaihu Shugan Granules Improve Tetrachloromethane-Induced Acute Liver Injury in Mice through Regulating the Keap1-Nrf2/HO-1 Signal Pathway

目的探讨柴胡疏肝颗粒对小鼠急性肝损伤的保护作用及可能机制。方法将柴胡疏肝颗粒按低、中、高(生药量:11.4、22.8、45.6 g·kg^(-1))3个剂量连续给药7 d,末次给药2 h后,除正常对照组外,用体积分数0.2%四氯化碳(tetrachloromethane,CCl4)溶液造模,12 h后,收集小鼠的血清和肝组织,制作肝组织病理切片。测定血清中丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)和肝组织中超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、白细胞介素6(interleukin 6,IL-6)、肿瘤坏死因子α(tumor necrosis factorα,TNF-α),以及活性氧自由基(reactive oxygen species,ROS)含量;转录组学获得肝脏组织差异表达mRNA并富集与差异表达基因显著相关的通路;代谢组学研究肝组织中内源性代谢物变化并进行通路富集分析;免疫组织化学法及Western blot法检测肝组织中c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MARK)、Kelch样ECH关联蛋白1(kelch-like ECH-associated protein 1,Keap1)、核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、血红素加氧酶-1(heme oxygenase-1,HO-1)表达及定位情况。结果与模型组比较,柴胡疏肝颗粒能显著降低小鼠血清ALT、AST和肝组织MDA、IL-6、TNF-α及ROS水平(P<0.05或P<0.01),显著升高肝组织中SOD水平(P<0.01),明显减轻肝细胞坏死及炎性浸润程度;上调Nqo1基因,上调醌氧化还原酶1[NAD(P)H:quinone oxidoreductase 1,NQO1]表达;下调Ccl2基因,下调单核细胞趋化蛋白-1(monocyte chemotactic protein 1,MCP-1)表达,且代谢物中有机酸类显著下调,糖类显著上调。肝组织中JNK、p38 MARK、Keap1表达均显著下调(P<0.05或P<0.01),Nrf2、HO-1表达显著上调(P<0.01)。结论柴胡疏肝颗粒可能通过激活Keap1-Nrf2/HO-1信号通路,对CCl4诱导的小鼠急性肝损伤具有保护作用。

OBJECTIVE To investigate the protective effect and possible mechanism of Chaihu Shugan Granules on acute liver injury in mice.METHODS Chaihu Shugan Granules was administered to mice at low,medium and high dosages(crude drug dose:11.4,22.8,45.6 g·kg^(-1))continuously for 7 d.Two hours after the last administration,the animal model was made with 0.2%tetrachloromethane(CCl4)solution except the control group.The serum and liver tissues were collected after 12 h.The levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and superoxide dismutase(SOD),malondialdehyde(MDA),interleukin 6(IL-6),tumor necrosis factorα(TNF-α)and reactive oxygen species(ROS)in liver tissues were measured by ELISA.HE staining was conducted to reveal the histopathological changes in liver.Transcriptomics was used to obtain differentially expressed mRNA in liver tissues and enrich differentially expressed pathways,while metabolomics was used to obtain changes in liver endogenous metabolites and enriches pathways of differential metabolites using KEGG database.The expression and location of c-Jun N-terminal kinase(JNK),p38 mitogen-activated protein kinase(p38 MARK),kelch-like ECH-associated protein 1(Keap1),nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)in liver tissues were measured by immunohistochemistry and Western blot.RESULTS Compared with the model group,the serum levels of ALT,AST and MDA,IL-6,TNF-αand ROS in liver tissues of mice in each group of Chaihu Shugan Granules decreased significantly(P<0.05 or P<0.01),while the level of SOD in liver tissues increased significantly(P<0.01).The degree of necrosis and inflammatory infiltration in liver cells decreased significantly.Nqo1 gene and NAD(P)H:quinone oxidoreductase 1(NQO1)expression were up-regulated while Ccl2 gene and monocyte chemotactic protein 1(MCP-1)expression were down-regulated.Organic acids were significantly down-regulated and carbohydrate was significantly up-regulated,the expressions of JNK,p38 MARK and Keap1 in liver tissues were significantly decreased(P<0.05 or P<0.01),while the expressions of Nrf2 and HO-1 were significantly increased(P<0.01).CONCLUSION Chaihu Shugan Granules might have protective effect on CCl4-induced acute liver injury in mice by activating the Keap1-Nrf2/HO-1 signal pathway.