附子理中汤通过激活AMPK通路及抑制NF-κBp65通路降低非酒精性脂肪肝大鼠肝脏损伤

Effect of Fuzi Lizhong decoction in reducing liver injury of rats with non-alcoholic fatty liver via activating AMPK and suppressing NF-κBp65 pathway

探究附子理中汤（Fuzi Lizhong decoction,FZLZD）对非酒精性脂肪肝（non-alcoholic fatty liver disease,NAFLD）大鼠肝脏的保护作用及相关机制。32只SPF级雄性Wistar大鼠随机分为4组,对照组,模型组,易善复（Yishanfu,YSF）组（200 mg·kg^-1·d-1）,FZLZD组（10 g·kg^-1·d^-1）,每组8只。采用脂肪乳持续4周灌胃制备大鼠非酒精性脂肪肝模型。确定造模成功后,各给药组大鼠持续给药4周。8周后处死各组大鼠,HE染色检测大鼠肝组织病理学变化;采用全自动生化分析仪检测各组大鼠空腹血清中血脂含量（总胆固醇,T-Chol;甘油三酯,TG;低密度脂蛋白胆固醇,LDL-C;高密度脂蛋白胆固醇,HDL-C）以及肝功能（丙氨酸氨基转移酶,ALT;总蛋白,TP;白蛋白,ALB）;称重法计算各组大鼠肝指数;酶联免疫吸附反应（ELISA）检测肝组织中炎症因子TNF-α,IL-6的分泌水平;实时荧光定量PCR（qRT-PCR）检测肝组织中脂肪代谢相关因子SREBP-1c,FASN的mRNA水平;Western blot法检测肝组织中p-AMPK,p-NF-κBp65蛋白表达量。结果显示,与对照组比较,模型组大鼠肝组织出现明显的病理学变化：肝小叶轮廓不清,肝细胞呈弥漫性脂肪样空泡病变,并呈炎性浸润,胞核固缩、着色深;与模型组比较,各治疗组大鼠肝组织病变均出现显著缓解,尤其以FZLZD组缓解程度最为显著。与对照组比较,模型组大鼠血清中血脂（T-Chol,TG,LDL-C,HDL-C）、肝功能（ALT,TP,ALB）及肝指数均显著升高（P〈0.01）;与模型组比较,各治疗组大鼠血脂及肝功能各指标均显著降低（P〈0.01）,FZLZD组大鼠肝指数显著降低（P〈0.05）,而YSF组大鼠肝指数无显著变化。与对照组比较,模型组大鼠肝组织中炎症因子TNF-α,IL-6分泌水平及SREBP-1c,FASN的mRNA水平均显著升高（P〈0.01）;与模型组比较,各治疗组大鼠肝组织中炎症因子TNF-α,IL-6分泌水平及SREBP-1c,FASN的mRNA水平均显著降低（P〈0.01）;与YSF组比较,FZLZD组TNF-α,IL-6分泌水平及SREBP-1c,FASN的mRNA水平差异显著（P〈0.01）。与模型组比较,FZLZD组大鼠肝组织中p-AMPK蛋白表达量显著升高（P〈0.01）,而p-NF-κBp65蛋白表达量显著降低（P〈0.01）。附子理中汤能显著改善NAFLD大鼠肝脏病理变化,降低血脂含量,改善肝功能,降低肝指数,这可能与其激活AMPK通路进而抑制SREBP-1c,FASN的表达以及抑制NF-κBp65通路进而降低炎症因子的释放密切相关。

To investigate the protective effect and relevant mechanism of Fuzi Lizhong decoction（FZLZD） on liver of rats with nonalcoholic fatty liver disease（NAFLD）,totally 32 male SPF Wistar rats were randomly divided into 4 groups： control group,model group,Yishanfu（YSF） group（200 mg·kg^-1·d^-1） and FZLZD group（10 g·kg^-1·d^-1）,with 8 rats in each group. Rat model of NAFLD was prepared through the intragastric administration with fat emulsion for 4 weeks. After the successful modeling,rats in each administration group were continuously administered for 4 weeks. After 8 weeks,the rats in each group were put to death,and the pathological changes in liver tissue were detected by HE staining. Automatic biochemical analyzer was used to detect fasting serum lipid levels（T-Chol,TG,LDL-C,HDL-C） and liver functions（ALT,TP,ALB） of rats in each group. The rat liver index was calculated by weighing method. Enzyme-linked immunosorbent assay（ELISA） was used to detect the secretion of inflammatory cytokines TNF-αand IL-6 in liver tissue. Real-time quantitative PCR（qRT-PCR） was used to detect the mRNA expressions of fat metabolism-related factors SREBP-1 c and FASN in liver tissue. Western blot was used to detect the p-AMPK and p-NF-κBp65 protein expressions in liver tissue. The results of HE staining showed that compared with the control group,the pathological changes in liver tissue in the model group rats were obvious; specifically,the outline of hepatic lobule was unclear,the hepatic cells showed diffuse steatosis of adipose tissue,and were accompanied by inflammatory infiltration,nuclear condensation,coloring deep; compared with the model group,liver lesions of all of the treatment groups were significantly alleviated; especially,the FZLZD group showed the most significant degree of remission. The results of serum test showed that the levels of serum lipids（T-Chol,TG,LDL-C,HDL-C）,liver functions（ALT,TP,ALB） and liver index in model group were significantly higher than those in control group（P〈0. 01）; compared with the model group,the indexes of serum lipid and liver function of rats in each treatment group were significantly decreased（P〈0. 01）,and those in FZLZD group were significantly decreased（P 0. 05）,while those in YSF group were not significantly changed. The results of ELISA and qRT-PCR showed that compared with the control group,the secretion levels of TNF-α,IL-6 and the mRNA levels of SREBP-1 c and FASN in the liver tissue of model group rats were significantly increased（P〈0. 01）; compared with model group,the secretion levels of TNF-α,IL-6 and the mRNA levels of SREBP-1 c,FASN in liver tissue of rats in each treatment group were significantly decreased（P〈0. 01）; compared with YSF group,the secretion levels of TNF-α and IL-6 and the mRNA levels of SREBP-1 c and FASN in FZLZD group were significantly different（P〈0. 01）. Western blotting showed that compared with the model group,the protein expression of p-AMPK in liver tissue of rats in FZLZD group was significantly increased（P〈0. 01）,while the protein expression of p-NF-κBp65 was significantly decreased（P〈0. 01）. FZLZD can significantly improve hepatic pathological changes,reduce serum lipid levels,promote liver function and liver index in NAFLD rats,which may be associated with the activation of the AMPK pathway and thereby the inhibition of the expressions of SREBP-1 c and FASN,and the inhibition of the NF-κBp65 pathway and thereby the reduction of the release of inflammatory factors.