吴茱萸碱对四氯化碳诱导肝纤维化小鼠肠道菌群的影响

Effects of evodiamine on carbon tetrachloride-induced liver fibrosis mice based on modulating gut microbiota

目的研究吴茱萸碱对四氯化碳(CCl4)诱导肝纤维化小鼠的改善作用及其对肠道菌群的影响。方法于2019年8月,将30只SPF级雄性C57BL/6小鼠平均分为正常组、模型组及吴茱萸碱组。正常组小鼠腹腔注射橄榄油(2 ml/kg),模型组和吴茱萸碱组小鼠腹腔注射20%的CCl4(2 ml/kg),每周2次,连续6周;成功建立肝纤维化模型后,吴茱萸碱组小鼠每天灌胃吴茱萸碱18 mg/kg,连续4周。检测各组小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、白蛋白(ALB)及总蛋白(TP)水平,观察小鼠肝组织病理学改变,测定吴茱萸碱对肝纤维化小鼠肠道菌群丰度及多样性的影响,检测各组小鼠肝脏组织中炎症因子白细胞介素6(IL-6)、白细胞介素1β(IL-1β)及肿瘤坏死因子α(TNF-α)的mRNA及蛋白表达水平。结果与正常组相比,模型组小鼠体重和血清中ALB、TP水平降低,肝指数和ALT、AST水平升高,肠道菌群Shannon和Simpson指数降低(P<0.01);与正常组相比,模型组小鼠粪便中乳酸菌属(Lactobacillus)、阿克曼氏菌(Akkermansia)、拟杆菌属(Bacteroides)丰度降低,肠球菌属(Enterococcus)、腔隙杆菌属(Lachnoclostridium)丰度升高(P<0.01);与正常组相比,模型组小鼠肝组织中IL-6、IL-1β、TNF-αmRNA和蛋白表达明显上调(P<0.01)。与模型组相比,吴茱萸碱能降低肝纤维化小鼠肝指数和血清中ALT、AST水平,提高ALB及TP水平(P<0.05),改善肝组织炎细胞浸润及纤维化程度,上调肝纤维化小鼠肠道菌群Shannon和Simpson指数(P<0.05);与模型组相比,吴茱萸碱可提高乳酸菌属、阿克曼氏菌、拟杆菌属丰度,降低肠球菌属、腔隙杆菌属丰度(P<0.05);与模型组相比,吴茱萸碱可降低肝纤维化小鼠肝组织中IL-6、IL-1β及TNF-αmRNA和蛋白表达水平(P<0.05)。结论吴茱萸碱对肝纤维化具有改善作用,其机制可能与改善肠道菌群、抑制肝脏炎症反应有关。

Objective To access the effects of evodiamine on carbon tetrachloride(CCl4)-induced liver fibrosis mice and study the mechanism based on modulating gut microbiota.Methods From August 2019,30 SPF male C57BL/6 mice were randomly divided into normal,model and evodiamine groups.Mice in control group received intraperitoneal injection of olive oil(2 ml/kg,twice per week)for 6 weeks.Mice in model and evodiamine groups received intraperitoneal injection of 20%CCl4(2 ml/kg,twice per week)for 6 weeks to induce liver fibrosis mice.Then,mice in evodiamine group received orally of evodiamine(18 mg/kg)for 4 weeks.The levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),albumin(ALB)and total protein(TP)were detected.The pathological changes of liver tissue were observed.The effects of evodiamine on the abundance and diversity of intestinal microflora in liver fibrosis mice were determined.The mRNA and protein expression levels of inflammatory factors[interleukin-6(IL-6),interleukin-1β(IL-1β),and tumor necrosis factorα(TNF-α)]in liver tissue were measured.Results Compared with the normal group,the body weight,serum ALB and TP levels of the model group were decreased,the liver index,ALT and AST levels were increased,and the intestinal flora Shannon and Simpson indexes were decreased(P<0.01).Compared with the normal group,the abundance of Lactobacillus,Akkermansia and Bacteroides in the feces of the model group decreased,while the abundance of Enterococcus and Lachnoclostridium increased(P<0.01).Compared with the normal group,the mRNA and protein expressions levels of IL-6,IL-1β,and TNF-αin the liver tissue of the model group were significantly increased(P<0.01).Compared with the model group,evodiamine could reduce liver index and serum ALT and AST levels,increase ALB and TP levels(P<0.05),improve inflammatory cell infiltration and fibrosis degree in liver tissue,and up regulate intestinal flora Shannon and Simpson indexes in liver fibrosis mice(P<0.05).Compared with the model group,evodiamine could increase the abundance of Lactobacillus,Akkermansia,Bacteroides,and reduce the abundance of Enterococcus and Lachnoclostridiun(P<0.05).Compared with the model group,evodiamine could reduce the mRNA and protein levels of IL-6,IL-1βand TNF-αin liver tissue of liver fibrosis mice(P<0.05).Conclusion Evodiamine can ameliorate CCl4-induced liver fibrosis through modulating gut microbiota and inhibiting the inflammatory response in liver.