健肝降脂丸通过激活NRF2通路改善非酒精性脂肪性肝炎小鼠脂代谢及炎症机制研究

Jiangan Jiangzhi Pill improved the lipid metabolism and inflammatory mechanism of nonalcoholic steatohepatitis mice by activating the NRF2 pathway

关键词:  健肝降脂丸  NRF2通路  非酒精性脂肪肝性肝炎  抗氧化  脂质代谢  炎症机制

Key words:  Jiangan Jiangzhi Pill  NRF2 pathway  nonalcoholic steatohepatitis  antioxidation  lipid metabolism  inflammatory mechanism

摘要:

[目的] 观察健肝降脂丸对蛋氨酸胆碱缺乏饲料（MCD）饮食诱导的非酒精性脂肪性肝炎（NASH）小鼠的脂质代谢及炎症作用机制。[方法] 小鼠随机分为对照组、模型组、阳性药组，健肝降脂丸低、中、高剂量组，每组10只。对照组正常饮食，其余实验组MCD饮食。阳性药组腹腔注射多烯磷脂酰胆碱 150 mg/kg，健肝降脂丸低、中、高剂量组分别灌胃健肝降脂丸100、200、400 mg/kg，每日1次，对照组、模型组平行给与等体积的生理盐水。每7天称质量并记录。共持续6周。苏木精-伊红（HE）染色观察肝组织病理学变化，生化试剂盒检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）浓度，BCA蛋白质定量试剂盒检测肝组织匀浆中甘总胆固醇（TC）、三酰甘油（TG）、超氧化物歧化酶（SOD）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）水平，聚合酶链反应（PCR）检测小鼠肝组织中核转录因子红系2相关因子2（NRF2）、血红素加氧酶1（HO-1）、谷氨酸半胱氨酸连接酶催化亚基（GCLC）重组蛋白、醌氧化还原酶1（NQO1）的信使核糖核酸（mRNA）表达水平，蛋白免疫印迹法（Western blot）检测小鼠肝组织中NRF2通路相关蛋白表达水平。[结果] 与正常组相比，结果显示，与正常组相比，模型组小鼠肝脏病理损伤严重，肝细胞结构破坏、细胞间隔增宽塌陷且大量炎细胞浸润，血清ALT、AST、TG、TC水平显著升高（P＜0.01），抗氧化指标SOD、GSH-Px活性降低，MDA水平升高，肝组织中NRF2、HO-1、GCLC、NQO1的mRNA及蛋白表达量均显著降低（P＜0.01）；与模型组相比，阳性药组及健肝降脂丸低、中、高剂量组小鼠肝脏细胞结构趋于清晰，细胞间隔缩短，炎性细胞浸润减少，体质量增加、肝指数降低，血清ALT、AST、TG、TC水平显著下降，SOD、GSH-Px活性升高，MDA水平降低，同时肝组织中NRF2通路相关mRNA及蛋白表达水平显著上调。[结论] 本研究首次揭示健肝降脂丸对MCD诱导的NASH小鼠具有多维度改善作用，其治疗效应与调控NRF2信号通路、增强抗氧化防御系统的功能密切关联，通过上调HO-1、GCLC、NQO1等下游靶基因表达，增强肝脏抗氧化能力，改善MCD诱导的NASH小鼠脂质代谢紊乱和炎症损伤。综合实验结果，NRF2通路活化可能是健肝降脂丸改善NASH的重要作用途径之一。

Abstract:

[Objective] To observe the lipid metabolism and inflammatory mechanism of Jiangan Jiangzhi Pills on nonalcoholic steatohepatitis（NASH） mice induced by methionine choline deficient（MCD）. [Methods] The mice were randomly divided into a control group，model group，positive drug group，and Jiangan Jiangzhi Pills low-dose，medium-dose，high-dose groups，with 10 mice in each group. The control group was fed a normal diet，while the other experimental groups received an MCD diet. The positive drug group was intraperitoneally injected with polyene phosphatidylcholine（150 mg/kg），and the Jiangan Jiangzhi Pill low-dose，medium-dose，and high-dose groups were orally administered the pill at doses of 100，200 and 400 mg/kg，respectively，once daily. The control and model groups were given an equal volume of saline in parallel. Body weight was measured and recorded every 7 days for a total duration of 6 weeks. Hematoxylin-eosin（HE） staining was used to observe histopathological changes in liver tissue. Serum concentrations of alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were measured using biochemical assay kits. The levels of total cholesterol（TC），triglycerides（TG），superoxide dismutase（SOD），malondialdehyde（MDA），and glutathione peroxidase（GSH-Px） in liver tissue homogenates were quantified using a BCA protein assay kit. PCR was performed to detect the mRNA expression levels of nuclear factor erythroid 2-related factor 2（NRF2），heme oxygenase-1（HO-1），glutamate-cysteine ligase catalytic subunit（GCLC），and NAD（P）H quinone oxidoreductase 1（NQO1） in mouse liver tissue. Western blot analysis was conducted to measure the expression levels of NRF2 pathway-related proteins in mouse liver tissue. [Results] Compared with the normal group，the results showed that compared with the normal group，the model group had severe liver pathological damage，hepatocyte structure destruction，widening and collapse of cell septum，and a large number of inflammatory cells infiltrated，serum ALT，AST，TG，TC levels were significantly increased（P＜0.01）. The activities of antioxidant indexes SOD and GSH-Px decreased，the levels of MDA increased，and the mRNA and protein expressions of NRF2，HO-1，GCLC and NQO1 in liver tissue were significantly decreased（P＜0.01）；compared with the model group，the liver cell structure of mice in the positive drug group and the low-dose，medium-dose and high-dose groups of Jiangan Jiangzhi Pills tended to be clear，the cell interval was shortened，the inflammatory cell infiltration was reduced，the body weight increased，the liver index decreased，the serum levels of ALT，AST，TG and TC decreased significantly，the activities of SOD and GSH-Px increased，the MDA level decreased，and the expression levels of NRF2 pathway-related mRNA and protein in liver tissue were significantly up-regulated. [Conclusion] This study revealed for the first time that Jiangan Jiangzhi Pill has a multi-dimensional improvement effect on MCD-induced NASH mice，and its therapeutic effect is closely related to the function of regulating NRF2 signaling pathway and enhancing the antioxidant defense system，and enhancing the liver’s antioxidant capacity by up-regulating the expression of downstream target genes such as HO-1，GCLC，and NQO1，and improving the lipid metabolism disorder and inflammatory damage induced by MCD. Based on the experimental results，the activation of NRF2 pathway may be one of the important ways for Jiangan Jiangzhi Pills to improve NASH.