Abstract：
This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1β, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1β in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1β, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1β in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.
摘要：
本研究观察二陈汤(ECD)对小鼠非酒精性脂肪性肝炎(NASH)的预防作用,并探讨其可能的作用机制。从而为临床应用ECD预防NASH提供科学依据。将C57BL/6雄性小鼠随机分为正常组(补充蛋氨酸和胆碱、MCS),模型组(蛋氨酸和胆碱缺乏，MCD),低剂量ECD组(ECD__L，6g·kg~(-1))，中剂量ECD组(ECD__M，12g·kg~(-1)),和高剂量ECD组(ECD__H，24g·kg~(-1))，每组8只小鼠。MCS组饲喂MCS饮食，和其他组喂食MCD饮食。各组小鼠给予相应的饮食,但是药物干预组的剂量低，medium-,和大剂量ECD（10mL·kg〜（-1）·d〜（-1）），在MCD饮食喂养的基础上，灌胃6周，在整个实验过程中，老鼠可以自由地进食和饮水。实验结束时,小鼠禁食过夜(12小时)并用20%尿烷麻醉。此后,收集血液和肝脏组织。血清检测丙氨酸氨基转移酶(ALT)水平,谷草转氨酶(AST),白细胞介素-1β（IL-1β），白细胞介素-6（IL-6），白细胞介素-10（IL-10），和肿瘤坏死因子-α(TNF-α)。采用苏木精-伊红(HE)染色对肝组织进行肝脏组织学分析,通过实时定量逆转录酶-聚合酶链反应(RT-qPCR)和Westernblot分析检测核因子-2相关因子2/谷胱甘肽过氧化物酶4(Nrf2/GPX4)通路相关基因和蛋白的表达水平,分别。结果表明,与MCS组相比,MCD组血清ALT和AST水平较高;HE染色显示肝组织脂肪空泡和明显的炎性细胞浸润;血清IL-1β,IL-6和TNF-α水平显著升高,血清IL-10水平明显降低。脂肪酸合成酶(FASN)的mRNA表达,单核细胞趋化蛋白-1（MCP-1），肝组织中IL-1β显著上调,而GPX4，Nrf2和NAD（P）H：奎宁氧化还原酶（NQO1）的表达显着下调。与MCD组相比，ECD_M和ECD_H组的血清ALT和AST水平显著降低,ECD__L组AST水平明显下降。肝组织中脂肪空泡数量和炎症细胞浸润程度均有改善;血清IL-1β,IL-6和TNF-α水平显著降低,但血清IL-10水平仅在ECD_H组显著升高。FASN的mRNA表达,肝组织中MCP-1和IL-1β显著下调,GPX4和NQO1表达显著上调。ECD_M组和ECD_H组Nrf2mRNA表达明显上调。Westernblot结果显示,与MCD组相比,ECD给药后,各组Nrf2和GPX4蛋白表达水平均显著升高,FASN蛋白表达水平明显降低；ECD_M组和ECD_H组NQO1蛋白表达升高。总之,ECD可以减少肝脏脂质积累，氧化应激，肝脏炎症,和NASH小鼠的肝损伤，这可能与Nrf2/GPX4通路的激活有关。
