基于CYP酶/Caspase-3途径探讨何首乌及其主要成分致药物性肝损伤的作用机制  

Mechanistic Study on the Mechanism of Drug-Induced Liver Injury Induced by Polygoni Multiflori Radix and Its Main Components Based on the CYP Enzyme/Caspase-3 Pathway

作　　者：刘思雨 张依娜 李敏[1] 雷一鸣 王丽平 卫培峰[2] 欧莉[1] 白杨[2] 董泰玮 高峰[1] LIU Siyu;ZHANG Yina;LI Min;LEI Yiming;WANG Liping;WEI Peifeng;OU Li;BAI Yang;DONGTaiwei;GAO Feng(Shaanxi University of Chinese Medicine,Xianyang 712046 Shaanxi,China;The Second Affiliated Hospital of Shaanxi University of Chinese Medicine,Xianyang 712000 Shaanxi,China)

机构地区：[1]陕西中医药大学,陕西咸阳712046 [2]陕西中医药大学第二附属医院,陕西咸阳712000

出　　处：《中药新药与临床药理》2025年第4期506-514,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：陕西省科技厅重点项目(2021JQ-728);中医药“双链融合”中青年科研创新团队建设项目(2022-SLRH-YQ-008);陕西省“特支计划”人才项目。

摘　　要：目的基于CYP酶/Caspase-3途径探讨何首乌及其主要成分致药物性肝损伤的作用机制。方法(1)体外实验:取HepG2细胞,分为空白组,大黄素低、中、高剂量组(50、100、150μmol·L^(-1)),大黄酸低、中、高剂量组(100、150、200μmol·L^(-1)),没食子酸低、中、高剂量组(100、150、200μmol·L^(-1)),以及二苯乙烯苷低、中、高剂量组(200、500、1000μmol·L^(-1)),给予相应药物干预24 h。采用CCK-8法检测细胞存活率;流式细胞术检测细胞凋亡率;qRT-PCR法检测肝细胞中CYP1A1、CYP3A4、Caspase-3 mRNA表达水平;Western Blot法检测肝细胞中CYP1A1、CYP3A4、Cleaved Caspase-3蛋白表达水平。(2)体内实验:将昆明种小鼠随机分为空白组、生首乌组(8 g·kg^(-1))、制首乌组(8 g·kg^(-1))、大黄素组(37.8 mg·kg^(-1))、大黄酸组(312μg·kg^(-1))、没食子酸组(4.58 g·kg^(-1)),每组10只。每天灌胃给药1次,连续给药4周。采用全自动酶标仪检测肝功能指标谷草转氨酶(AST)、谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、总胆红素(TBIL)、间接胆红素(IBIL);HE染色法观察肝组织病理变化;qRT-PCR法检测肝组织中CYP1A1、CYP3A4、Caspase-3 mRNA表达水平;Western Blot法检测肝组织中CYP1A1、CYP3A4、Cleaved Caspase-3蛋白表达水平。结果(1)体外实验:与空白组比较,中、高剂量大黄素及不同浓度大黄酸、没食子酸均对HepG2细胞有显著抑制作用(P<0.01),且呈浓度依赖性。二苯乙烯苷各浓度对HepG2细胞均无明显抑制作用(P>0.05)。与空白组比较,各给药组的细胞随着给药浓度升高,细胞形态开始发生变化,呈现皱缩、变圆、颜色加深,以及部分坏死、脱落,大黄素中、高剂量组及没食子酸中、高剂量组细胞坏死严重;大黄素、大黄酸及没食子酸中、高剂量组的细胞凋亡率均明显升高(P<0.05,P<0.01);大黄素、大黄酸及没食子酸各剂量组细胞的CYP1A1 mRNA表达显著上调(P<0.05,P<0.01);大黄素高剂量组、大黄�Objective To explore the mechanism of drug-induced liver injury(DILI)induced by Polygoni Multiflori Radix and its main components based on CYP enzyme/Caspase-3 pathway.Methods(1)In vitro experiment:HepG2 cells were divided into blank group,emodin low-,medium-and high-dose groups(50,100,150μmol·L^(-1)),rhein low-,medium-and high-dose groups(100,150,200μmol·L^(-1)),gallic acid low-,medium-and highdose groups(100,150,200μmol·L^(-1)),and stilbene glycoside low-,medium-and high-dose groups(200,500,1000μmol·L^(-1)).Cell viability was detected by CCK-8 method.The apoptosis rate was detected by flow cytometry.The mRNA expression levels of CYP1A1,CYP3A4 and Caspase-3 in hepatocytes were detected by qRT-PCR.The expression levels of CYP1A1,CYP3A4 and Cleaved Caspase-3 in hepatocytes were detected by Western Blot.(2)In vivo experiment:Kunming mice were randomly divided into blank group,Polygoni Multiflori Radix group(8 g·kg^(-1)),prepared Polygoni Multiflori Radix group(8 g·kg^(-1)),emodin group(37.8 mg·kg^(-1)),rhein group(312μg·kg^(-1))and gallic acid group(4.58 g·kg^(-1)),with 10 mice in each group.Intragastric administration was performed once a day for 4 weeks.The liver function indexes of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),total bilirubin(TBIL)and indirect bilirubin(IBIL)were detected by automatic microplate reader.The pathological changes of liver tissue were observed by HE staining.The mRNA expression levels of CYP1A1,CYP3A4 and Caspase-3 m in liver tissue were detected by qRT-PCR.The expression levels of CYP1A1,CYP3A4 and Cleaved Caspase-3 in liver tissue were detected by Western Blot.Results(1)In vitro experiments:Compared with the blank group,medium-and high-doses of emodin,as well as different concentrations of rhein and gallic acid,significantly inhibited HepG2 cells(P<0.01),showing concentration dependence.Stilbene glycoside at all concentrations showed no significant inhibitory effect on HepG2 cells(P>0.05).Compared with the blank group,the mor

关 键 词：何首乌 制首乌 大黄素 大黄酸 没食子酸 药物性肝损伤 CYP酶/Caspase-3途径 HEPG2细胞 小鼠 

分 类 号：R285.5[医药卫生—中药学]