CJI (Traditional Chinese Medicine)
基于AMPK/mTOR/ERRα信号通路探讨慈菇消脂方影响非酒精性脂肪性肝炎小鼠肝细胞自噬作用机制
郭才,郑兰兰,何燕芳,谢娇娇,高芬,于春,马燕花
甘肃中医药大学第一临床医学院,甘肃 兰州 730000
摘要:目的 基于AMPK/mTOR/ERRα信号通路探讨慈菇消脂方对非酒精性脂肪性肝炎(NASH)小鼠肝细胞自噬的调控机制。方法 小鼠随机分为空白组、模型组、慈菇消脂方组和抑制剂组,每组10只。采用蛋氨酸胆碱缺乏饲料喂养构建NASH小鼠模型,造模成功后给药组予相应药物干预,连续4周。HE染色观察小鼠肝组织形态并进行非酒精性脂肪性肝病(NAFLD)活动度评分,电镜观察肝细胞超微结构,免疫组化检测肝组织 Beclin1、LC3、p62 蛋白表达,Western blot检测肝组织p-AMPK、p-mTOR、雌激素相关受体α (ERRα)蛋白表达。结果 与空白组比较,模型组小鼠肝组织出现明显脂肪变性并可见大量炎性细胞浸润,肝细胞可见大量形态较规则的单层膜脂质结构,NAFLD活动度评分升高(P<0.01),肝组织Beclin1、LC3、p-AMPK蛋白表达降低(P<0.01),p62、p-mTOR、ERRα蛋白表达升高(P<0.01);与模型组比较,各给药组小鼠肝组织脂肪变性减少,炎性细胞浸润改善,可见少量自噬体,NAFLD活动度评分降低,肝组织Beclin1、LC3、p-AMPK蛋白表达升高(P<0.01,P<0.05),p62、p-mTOR、ERRα蛋白表达降低(P<0.01)。结论 慈菇消脂方可能通过调控AMPK/mTOR/ERRα信号通路相关蛋白表达介导自噬,延缓NASH进展。关键词:非酒精性脂肪性肝炎;慈菇消脂方;AMPK/mTOR/ERRα信号通路;自噬;小鼠中图分类号:R285.5;R259.755 文献标识码:A文章编号:1005-5304(2024)05-0099-06 DOI:10.19879/j.cnki.1005-5304.202308560开放科学(资源服务)标识码(OSID): Discussion on the Mechanism of Cigu Xiaozhi Prescription on Hepatocytes Autophagy in Nonalcoholic Steatohepatitis Mice Based on AMPK/mTOR/ERRα Signaling Pathway
GUO Cai, ZHENG Lanlan, HE Yanfang, XIE Jiaojiao, GAO Fen, YU Chun, MA Yanhua
The First Clinical Medical College of Gansu University of Chinese Medicine, Lanzhou 730000, China Abstract: Objective To explore the mechanism of Cigu Xiaozhi Prescription on hepatocytes autophagy in nonalcoholic steatohepatitis (NASH) mice based on AMPK/mTOR/ERRα signaling pathway. Methods The mice were randomly divided into blank group, model group, Cigu Xiaozhi Prescription group and inhibitor group, with 10 mice in each group. A mouse model of NASH was constructed using methionine-choline-deficient chow, and drug interventions were administered after successful modelling for consecutive 4 weeks. Histological changes in liver tissue was observed by HE staining, and the NAFLD activity score was evaluated, electron microscopy was used to observe the ultrastructure of hepatocytes, immunohistochemistry was used to detect the relative expressions of Beclin1, LC3 and p62 proteins in liver tissue, Western blot was used to detect the expressions of p-AMPK, p-mTOR and ERRα proteins. Results Compared with the blank group, the liver tissue of mice in the model group showed obvious steatosis and a large number of inflammatory cells infiltration, a large number of regularly shaped monolayer lipid structures could be observed in liver cells, and the NAFLD activity score significantly increased (P<0.01), the expressions of Beclin1, LC3, and p-AMPK proteins in liver tissue significantly decreased (P<0.01), while the protein expressions of p62, p-mTOR, and ERRα significantly increased (P<0.01). Compared with the model group, the基金项目:国家自然科学基金(81860821);甘肃省优秀研究生创新之星项目(2023CXZX-754)通讯作者:马燕花,E-mail:617747928@qq.com
steatosis in liver tissue of mice in each administration group reduced, inflammatory cell infiltration was improved, a small amount of autophagosomes were visible, and the NAFLD activity score was reduced, the expressions of Beclin1, LC3, and p-AMPK proteins in liver tissue significantly increased (P<0.01, P<0.05), while the protein expressions of p62, p-mTOR, and ERRα significantly decreased (P<0.01). Conclusion Cigu Xiaozhi Prescription may regulate related protein expressions of AMPK/mTOR/ERRα signaling pathway to mediate autophagy and delay the progression of NASH.
Keywords: nonalcoholic steatohepatitis; Cigu Xiaozhi Prescription; AMPK/mTOR/ERRα signaling pathway; autophagy; mice
非酒精性脂肪性肝病( non-alcoholic fatty liver disease,NAFLD)已成为全球最常见的慢性肝病之一。作为NAFLD的一种病理类型,非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)的组织变化表现为脂肪变性、炎性细胞浸润和肝细胞气球样变,可能伴纤维化,且随着纤维化的发展,肝硬化、肝细胞癌等终末期肝病的发生风险随之增加。包括遗传、胰岛素抵抗、氧化应激、自噬受损在内的多种因素相互作用,共同促进NASH的发生发展。研究发现,自噬能调节NAFLD的主要病理变化,包括肝脏脂质代谢、炎症和纤维化[1]。
慈菇消脂方是甘肃中医药大学附属医院院内制剂,由山慈菇、泽泻、净山楂、丹参、土鳖虫、柴胡、黄芩、法半夏、决明子等中药组成,临床应用多年,治疗NAFLD疗效显著[2]。前期实验研究显示,该方能明显改善NASH大鼠肝功能及血脂水平、减轻脂质过氧化和炎症[3],并从细胞层面验证慈菇消脂方含药血清可通过自噬发挥改善肝细胞脂肪变性的作用[4]。本研究通过动物模型进一步探讨其改善NASH的作用机制。
1 实验材料
1.1 动物
SPF级C57BL/6J雄性小鼠40只,8周龄,体质量(20±2)g,购于北京斯贝福生物技术有限公司,动物生产许可证号SCXK(京)2019-0010。饲养于甘肃中医药大学SPF级动物房,温度( 23±2)℃ ,湿度55%±5%,光照12 h。动物使用许可证号SYXK(甘) 2023-568。本研究经甘肃中医药大学动物伦理委员会审批[SYXK(甘)2023-568]。
1.2 药物及制备
慈菇消脂方由山慈菇10 g、柴胡12 g、丹参20 g、土鳖虫20 g、薏苡仁30 g、黄芩10 g、泽泻15 g、枸杞子20 g、净山楂20 g、法半夏15 g、茯苓20 g、何首乌20 g、决明子20 g、炙甘草10 g组成,饮片购于甘肃中
医药大学附属医院,煎煮方法参考课题组前期实验[5],
制成浓度为3.276 g/mL慈菇消脂方药液。
1.3 主要试剂与仪器雷帕霉素(货号 HY-10219 ),美国 MedChem Express公司;DAB显色试剂盒(货号K193328E)、兔SP试剂盒(货号SP-9001),北京中杉金桥公司;4%组织细胞固定液(货号P1110)、Gluta固定液(货号P1126)、DMSO(货号D8370)、BCA蛋白定量试剂盒(货号PC0020)、苏木素染色液(货号G1080)、伊红染色液(货号G1100),北京索莱宝公司;Beclin-1抗体(货号 3738S ),美国 CST 公司; LC3抗体(货号GTX127375),美国 GeneTex公司;p62抗体(货号
β
5114S ),美国 CST 公司; -actin 抗体(货号YM3028),美国Immunoway公司;腺苷酸活化蛋白激酶(AMPK)抗体(货号T55326)、p-AMPK抗体(货号TA3423)、哺乳动物雷帕霉素靶蛋白(mTOR)抗体(货号T55306)、p-mTOR抗体(货号T56571),中国
α(ERRα)抗体(货号Abmart公司;雌激素相关受体bs-6998R ),北京博奥森公司。切片机(型号RM2016),德国徕卡公司;显微镜(型号Ⅸ51),日本Olympus 公司;Western blot电源(型号DYY-6D)、Western blot电泳仪(型号DYCZ-25D)、Western blot转膜仪(型号DYCZ-40 G),北京六一生物科技公司;透射电镜(型号TECNAI G2 20 TWIN),美国 FEI公司。
2 实验方法
2.1 分组、造模及给药
40只小鼠适应性喂养1周,采用随机数字表法分为空白组、模型组、慈菇消脂方组和抑制剂组,每组
10只。参照文献[6]造模方法,空白组予正常饲料,其
余组予蛋氨酸和胆碱缺乏饲料(北京斯贝福生物技术有限公司),造模6周后,根据人与小鼠体表面积换算给药剂量,慈菇消脂方组予慈菇消脂方药液65.52 g/kg灌胃,抑制剂组予雷帕霉素溶液(将雷帕霉素粉末溶于2%DMSO中,依次加入40%聚乙二醇、5%吐温-80和53%生理盐水促溶)4.0 g/kg灌胃,给药体积0.2 mL/kg,
1