基于内源性大麻素探讨康氏抗纤方治疗肝纤维化大鼠的作用机制

Study on the Mechanism of Kangshi Kangxian Recipe in Treatment of Liver Fibrosis Based on Endogenous Cannabinoid

目的研究康氏抗纤方对肝纤维化大鼠的药理效应及对内源性大麻素的作用机制。方法将40只Wistar雄性大鼠随机分为正常组、模型组、康氏抗纤方组和安络化纤丸组,每组10只。造模8周期间,正常组给予普通饲料喂养,模型组、康氏抗纤方组和安络化纤丸组给予四氯化碳(CCl_4)腹腔注射复制肝纤维化模型。从第5周开始,康氏抗纤方组、安络化纤丸组分别给予康氏抗纤方、安络化纤丸灌胃治疗4周。检测大鼠血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)活性;ELISA检测肝组织羟脯氨酸(Hyp)含量;HE染色法观察大鼠肝组织的病理变化;采用高效液相色谱-质谱联用法(LC/MS)检测肝组织内源性大麻素N-花生四烯酸氨基乙醇(AEA)、2-花生四烯酸甘油(2-AG)含量变化;通过real-time PCR法检测大鼠肝组织内源性大麻素水解酶脂肪酰胺水解酶(FAAH)和单酰基甘油酯酶(MGL)m-RNA表达水平变化。结果与正常组比较,模型组大鼠的肝组织内出现明显肝纤维化状态,血清ALT、AST活性及肝组织Hyp、AEA、2-AG含量升高(P<0.01),FAAH、MGL m-RNA表达降低(P<0.01)。治疗4周后,与模型组比较,康氏抗纤方组、安络化纤丸组肝细胞炎症坏死及胶原纤维增生明显减轻,血清ALT、AST活性、肝组织Hyp含量均显著降低(P<0.01),康氏抗纤方组、安络化纤丸组血清大麻素AEA、2-AG含量明显降低(P<0.01,P<0.05),FAAH、MGL的m-RNA表达显著升高(P<0.01,P<0.05)。结论康氏抗纤方对肝纤维化具有良好的治疗作用,其作用机制与抑制内源性大麻素密切相关。

Objective To study the pharmacological effect of Kangshi Kangxian Recipe(KSKXR)on hepatic fibrosis rats and its mechanism of action on endogenous cannabinoid.Methods Forty Wistar male rats were randomly divided into normal group,model group,KSKXR group,and Anluo Huaxian Pill(ALHXP),10 rats in each group.During the 8 weeks of modeling,rats in the normal group were fed with common diet,while those in the rest three groups were intraperitoneally injected with carbon tetrachloride(CCl4)to replicate the liver fibrosis model.Starting from the 5 th week,KSKXR and ALHXP were respectively administered to rats in the KSKXR group and ALHXP group for 4 successive weeks.Activities of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST),hepatic hydroxyproline(Hyp)were detected.Pathological changes of liver tissue were observed by HE staining.Contents of endogenous cannabinoid N-arachidonic acid aminoethanol(AEA)and 2-arachiodonoylglycerol(2-AG)in liver tissue were detected by LC/MS.m-RNA expressions of endogenous cannabinoid fatty acid amide hydrolase(FAAH)and monoacylglycerol lipase(MGL)were detected by Real-time PCR.Results Compared with the normal group,obvious liver fibrosis occurred,serum activities of ALT and AST significantly increased(P<0.01),the contents of Hyp in liver tissue increased(P<0.01),the contents of AEA and 2-AG in liver tissue significantly increased(P<0.01),and m-RNA expressions of FAAH and MGL decreased(P<0.01)in the model group.Compared with the model group after 4 weeks treatment,inflammation and necrosis of hepatocytes and the proliferation of collagen fibers were significantly alleviated,and serum activities of ALT and AST,hepatic Hyp content decreased significantly in the KSKXR group and the ALHXP group(P<0.01).Contents of AEA and 2-AG decreased obviously in the KSKXR group and the ALHXP group(P<0.01,P<0.05).m-RNA expressions of FAAH and MGL increased obviously(P<0.01,P<0.05).Conclusion KSKXR had good therapeutic effect on hepatic fibrosis,and its mechanism was closely related to inhibiting endogenous cannabinoid.