茵栀黄联合二甲双胍对非酒精性脂肪肝大鼠的保护作用和机制研究

Protective Effect and Mechanism of Yinzhihuang Combined with Metformin on Nonalcoholic Fatty Liver Disease in Rats

目的:探讨茵栀黄联合二甲双胍对非酒精性脂肪肝(NAFLD)大鼠的保护作用及机制。方法:将54只雄性SD大鼠随机分为2组,其中正常对照组(12只)和高脂饲料组(42只),进行NAFLD建模。8 w后每组随机各抽取2只,经病理检查确定NAFLD建模成功后,再将高脂饲料组随机分为模型对照组、茵栀黄9 g/kg组、二甲双胍0.2 g/kg组、茵栀黄联合二甲双胍(4.5 g/kg+0.1 g/kg)组,每组10只。除了正常对照组及模型对照组以外,其他各组同时给予相应治疗药物,每天1次,连续5 w,末次给药后禁食不禁水16 h,取血液和肝脏。采用生化法检测血清中门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)以及肝组织中丙二醛(MDA)含量、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力;酶联免疫吸附法(ELISA)检测肝组织中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)以及转化生长因子-β(TGF-β)的水平;蛋白质印迹法(Western blot)检测大鼠肝组织中过氧化物酶增殖物激活受体-α(PPAR-α)蛋白的表达情况。苏木精-伊红(HE)染色观察肝组织病变程度。结果:与正常对照组比较,模型对照组大鼠血清中ALT、AST活性、TC、TG、LDL-C含量以及肝组织中MDA的含量显著升高(P<0.01);血清中HDL-C以及肝组织中SOD、GSH-Px的活力显著降低(P<0.01);肝组织中TNF-α、IL-6以及TGF-β含量均显著升高(P<0.01);肝组织中PPAR-α蛋白表达水平显著下调(P<0.01)。与模型对照组比较,各给药组大鼠以上指标均呈现不同程度的改善(P<0.05或P<0.01),其中茵栀黄联合二甲双胍组(4.5 g/kg+0.1 g/kg)改善程度最为明显。结论:茵栀黄和二甲双胍对NAFLD大鼠均具有治疗作用,并且联合应用二者的疗效明显优于单独应用茵栀黄或二甲双胍,其机制很可能与调节PPAR-α信号通路有关。

Objective: To investigate the protective effect and mechanism of Yinzhihuang combined with metformin on non-alcoholic fatty liver(NAFLD) in rats. Methods: 54 male SD rats were randomly divided into 2 groups, including the control group(12 rats) and the high-fat diet group(42 rats) for the establishment of NAFLD model. After 8 weeks, 2 rats were randomly selected from each group. After the establishment of NAFLD model was proved successful by pathological examination, the rats in high-fat diet groups were randomly divided into the model group, 9 g/kg Yinzhihuang group, 0.2 g/kg metformin group, Yinzhihuang combined with metformin(4.5 g/kg+0.1 g/kg) group, 10 rats in each group. Except for the control group and the model group, the rats in the other groups were given corresponding drugs at the same time, once a day for 5 weeks, after the last administration, rats were fasted for 16 hours. Blood and liver tissues were collected. The contents or activities of serum aspartate aminotransferase(AST), alanine aminotransferase(ALT), total cholesterol(TC), triglyceride(TG), low-density lipoprotein(LDL-C), high-density lipoprotein(HDL-C) and liver tissue malondialdehyde(MDA), superoxide dismutase(SOD) and glutathione Peroxidase(GSH-Px) were detected by biochemical methods. The contents of tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and transforming growth factor-β(TGF-β) in liver tissues were determined by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of peroxidase proliferator-activated receptor-α(PPAR-α) protein in rat liver tissue. Hematoxylin-Eosin(HE) staining was used to observe the pathological changes of liver tissues. Results: Compared with the control group, the serum contents of ALT, AST, TC, TG, LDL-C and MDA in the model group were significantly increased(P<0.01), the serum contents of HDL-C,SOD and GSH-Px were significantly decreased(P<0.01), the liver tissue contents of TNF-α, IL-6 and TGF-β were significantly increased(P<0.01), the PPAR-α protein expression level in the liver tissue was significantly decreased(P<0.01). Compared with the model group, the above indicators of rats in each administration group showed different degrees of improvement(P<0.05 or P<0.01), and Yinzhihuang combined with metformin(4.5 g/kg+0.1 g/kg) group showed the most obvious improvement. Conclusion: Both Yinzhihuang and metformin have therapeutic effects on NAFLD rats, and the effects of Yinzhihuang combined with metformin are significantly better than that of Yinzhihuang or metformin alone. The mechanism is probably related to the regulation of PPAR-α signaling pathway.