补中益气汤对刀豆蛋白A致小鼠急性肝衰竭的保护作用

目的:研究补中益气汤对刀豆蛋白A(Con A)致急性肝衰竭小鼠的保护作用及机制。方法:80只小鼠随机分为正常组,模型组,环孢素A(Cs A)组,补中益气汤低、高剂量(10.5,21 g·kg^-1)组,每组16只。除正常组外,其余各组静脉注射15 mg·kg^-1Con A诱导小鼠急性肝衰竭模型。治疗组于Con A注射30 min后分别静脉注射50 mg·kg^-1Cs A,口服灌胃补中益气汤,正常组和模型组采用同时间、同体积双蒸水灌胃对照。造模3,10 h后取眼球血、肝脏、脾脏,采用流式微球技术检测血清肿瘤坏死因子-α(TNF-α),白细胞介素(IL)-6,IL-12,γ-干扰素(IFN-γ)和单核细胞趋化蛋白-1(MCP-1)水平,采用全自动生化分析仪检测血清丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST)活性,苏木素-伊红(HE)染色观察肝组织病理学变化,流式细胞术分析脾脏CD4+T淋巴细胞活化程度,蛋白免疫印迹法(Western blot)检测肝脏细胞外调节蛋白激酶1/2(ERK1/2),p38丝裂原活化蛋白激酶(p38 MAPK)的表达及磷酸化水平。结果:与正常组比较,模型组小鼠血清ALT和AST水平均显著升高(P<0.01),肝组织病理损伤明显,血清炎性细胞因子TNF-α,IL-6,IL-12,IFN-γ和MCP-1水平显著升高(P<0.01),脾脏CD4+T淋巴细胞中IL-2,IFN-γ和IL-4表达显著上调(P<0.01),肝脏ERK1/2和p38 MAPK磷酸化水平显著升高(P<0.01);与模型组比较,补中益气汤高剂量组血清ALT和AST水平明显降低(P<0.05,P<0.01),肝组织病理损伤程度减轻,血清炎性细胞因子TNF-α,IL-6,IL-12,IFN-γ和MCP-1水平明显降低(P<0.05,P<0.01),脾脏CD4+T淋巴细胞中IL-2和IFN-γ表达明显下调(P<0.05,P<0.01),肝脏ERK1/2和p38 MAPK磷酸化水平明显降低(P<0.05,P<0.01)。结论:补中益气汤对Con A诱导的急性肝衰竭小鼠具有明显的保护作用,其保护作用机制可能通过抑制ERK1/2和p38 MAPK信号通路,从而降低T淋巴细胞活化和炎性细胞因子分泌。

Protective Effect and Mechanism of Buzhong Yiqitang on Concanavalin A-induced Acute Liver Failure in Mice

Objective:To explore the hepatoprotective effect and the mechanism of Buzhong Yiqitang(BZYQT)on mice with acute liver failure induced by Concanavalin A(ConA).Method:A total of 80 mice were randomly divided into normal group,model group,Cyclosporine A(CsA)group,BZYQT low and high dose group(10.5,21 g·kg^-1),16 mice per group.All the mice except for normal group were injected intravenously with15 mg·kg^-1 ConA.The treatment group mice were orally administrated with BZYQT,or intravenously administrated with 50 mg·kg^-1 CsA 30 min post ConA injection,normal and model group mice were orally administrated with dd H2O at the same time.Blood,liver and spleen were collected 3 and 10 h post ConA injection.Cytokine levels of tumor necrosis factor alpha(TNF-α),interleukin-6(IL-6),interleukin-12(IL-12),interferon-gamma(IFN-γ)and monocyte chemoattractant protein-1(MCP-1)in the serum were detected with cytometric bead array.The alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels in the serum were analyzed with fully automatic biochemical analyzer.The pathological changes of liver tissues were observed by hematoxylin-eosin(HE)staining.The activation of splenic CD4+T lymphocytes was analyzed by flow cytometry.The expression and phosphorylation of extracellular regulated protein kinase 1/2(ERK1/2)and p38 mitogen-activated protein kinase(p38 MAPK)was analyzed by Western blot.Result:Compared with normal group,model group showed higher levels of ALT and AST in the serum(P<0.01),obvious pathological damage of liver tissue,higher levels of TNF-α,IL-6,IL-12,IFN-γand MCP-1 in the serum(P<0.01),higher expression of IL-2,IFN-γand IL-4 CD4+T lymphocytes in the spleen(P<0.01),and elevated levels of phosphorylation of ERK1/2 and p38 MAPK(P<0.01).Compared with the model group,BZYQT high dose group showed decreased levels of ALT and AST(P<0.05,P<0.01),reduced liver injury,decreased levels of TNF-α,IL-6,IL-12,IFN-γand MCP-1 in the serum(P<0.05,P<0.01),reduced level of IL-2 and IFN-γCD4+T lymphocytes in the spleen(P<0.05,P<0.01),and reduced levels of phosphorylation of ERK1/2 and p38 MAPK(P<0.05,P<0.01).Conclusion:BZYQT has a protective effect on mice with acute liver failure induced by ConA.The mechanism may be through inhibiting ERK1/2 and p38 MAPK signaling pathways,thereby reducing T lymphocyte activation and inflammatory cytokine secretion.