Yang Jia-Yao, Shi Zhao-Hong, Ma Wei, Tao Dong-Qing, Liu Song, Chen Lu, Zhou Xiao-Li
Department of Gastroenterology, Wuhan First Hospital, Wuhan 430022, China.
Department of Endocrinology, Hubei Third Renmin Hospital, Wuhan 430020, China.
Zhongguo Zhong Yao Za Zhi. 2018 Aug;43(15):3176-3183. doi: 10.19540/j.cnki.cjcmm.2018.0094.
To investigate the protective effect and relevant mechanism of Fuzi Lizhong decoction (FZLZD) on liver of rats with non-alcoholic 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-α and IL-6 in liver tissue. Real-time quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of fat metabolism-related factors SREBP-1c 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 (<0.01); compared with the model group, the indexes of serum lipid and liver function of rats in each treatment group were significantly decreased (<0.01), and those in FZLZD group were significantly decreased (<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-1c and FASN in the liver tissue of model group rats were significantly increased (<0.01); compared with model group, the secretion levels of TNF-α, IL-6 and the mRNA levels of SREBP-1c, FASN in liver tissue of rats in each treatment group were significantly decreased (<0.01); compared with YSF group, the secretion levels of TNF-α and IL-6 and the mRNA levels of SREBP-1c and FASN in FZLZD group were significantly different (<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 (<0.01), while the protein expression of p-NF-κBp65 was significantly decreased (<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-1c and FASN, and the inhibition of the NF-κBp65 pathway and thereby the reduction of the release of inflammatory factors.
为探讨附子理中汤（FZLZD）对非酒精性脂肪性肝病（NAFLD）大鼠肝脏的保护作用及相关机制，将32只雄性SPF级Wistar大鼠随机分为4组：对照组、模型组、益善复（YSF）组（200 mg·kg⁻¹·d⁻¹）和FZLZD组（10 g·kg⁻¹·d⁻¹），每组8只。通过灌胃给予脂肪乳剂4周制备NAFLD大鼠模型。造模成功后，各给药组大鼠连续给药4周。8周后，处死各组大鼠，采用HE染色检测肝组织病理变化。用自动生化分析仪检测各组大鼠空腹血脂水平（总胆固醇、甘油三酯、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇）和肝功能（谷丙转氨酶、总蛋白、白蛋白）。通过称重法计算大鼠肝脏指数。采用酶联免疫吸附测定（ELISA）法检测肝组织中炎症细胞因子肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的分泌水平。采用实时定量聚合酶链反应（qRT-PCR）法检测肝组织中脂肪代谢相关因子固醇调节元件结合蛋白-1c（SREBP-1c）和脂肪酸合酶（FASN）的mRNA表达水平。采用蛋白质免疫印迹法检测肝组织中磷酸化腺苷酸活化蛋白激酶（p-AMPK）和磷酸化核因子κB p65（p-NF-κBp65）蛋白表达水平。HE染色结果显示，与对照组相比，模型组大鼠肝组织病理变化明显；具体表现为肝小叶轮廓不清，肝细胞呈弥漫性脂肪变性，并伴有炎症浸润、核固缩、染色加深；与模型组相比，各治疗组肝脏病变均明显减轻；尤其FZLZD组缓解程度最为显著。血清检测结果显示，模型组大鼠血脂（总胆固醇、甘油三酯、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇）、肝功能（谷丙转氨酶、总蛋白、白蛋白）水平及肝脏指数均显著高于对照组（P<0.01）；与模型组相比，各治疗组大鼠血脂及肝功能指标均显著降低（P<0.01），FZLZD组显著降低（P<0.05），而YSF组无明显变化。ELISA和qRT-PCR结果显示，与对照组相比，模型组大鼠肝组织中TNF-α、IL-6分泌水平及SREBP-1c、FASN的mRNA水平均显著升高（P<0.01）；与模型组相比，各治疗组大鼠肝组织中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组相比，FZLZD组TNF-α、IL-6分泌水平及SREBP-1c、FASN的mRNA水平差异有统计学意义（P<0.01）。蛋白质免疫印迹法显示，与模型组相比，FZLZD组大鼠肝组织中p-AMPK蛋白表达显著升高（P<0.01），而p-NF-κBp65蛋白表达显著降低（P<0.01）。FZLZD可显著改善NAFLD大鼠肝脏病理变化，降低血脂水平，改善肝功能及肝脏指数，其机制可能与激活AMPK通路进而抑制SREBP-1c和FASN的表达，以及抑制NF-κBp65通路进而减少炎症因子释放有关。
