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、150 μmol·L-1 ），大黄酸低、中、 高剂量组（100、150、200 μmol·L-1 ），没食子酸低、中、高剂量组（100、150、200 μmol·L-1 ），以及二苯乙烯苷 低、中、高剂量组（200、500、1 000 μmol·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（312 μg·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、大黄酸中/高剂量组及没食子酸各剂量组细胞的 CYP3A4 mRNA 表达显著上调 （P＜0.01）；大黄素中/高剂量组、大黄酸高剂量组、没食子酸中/高剂量组细胞的 Caspase-3 mRNA 表达显著上 调（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、CYP3A4、Cleaved Caspase-3 蛋 白表达均显著上调（P＜0.05，P＜0.01）。（2）体内实验：从肝组织病理损伤程度看，大致为没食子酸组＞大黄酸 组＞大黄素组＞生首乌组＞制首乌组，制首乌组的肝组织病理损伤最低。与空白组比较，生首乌组、大黄素 组、没食子酸组小鼠的血清 IBIL、TBIL、AST、ALP 水平均显著升高（P＜0.05，P＜0.01），大黄酸组的血清 AST、ALP 水平显著升高（P＜0.05，P＜0.01），没食子酸组的血清 ALT 水平明显升高（P＜0.05）；生首乌组、 制首乌组、大黄素组、没食子酸组小鼠的肝组织 CYP1A1、CYP3A4、Caspase-3 mRNA 表达均显著上调（P＜ 0.05，P＜0.01），大黄酸组的肝组织 CYP3A4 mRNA 表达明显上调（P＜0.05）；生首乌组、制首乌组及没食子酸 组小鼠的肝组织 CYP1A1、CYP3A4 蛋白表达均显著上调（P＜0.05，P＜0.01），生首乌组、大黄酸组及没食子 酸组小鼠的肝组织 Cleaved Caspase-3 蛋白表达均显著上调（P＜0.05，P＜0.01）。结论 何首乌及其主要成分致 肝损伤的作用机制可能与通过激活 CYP1A1、CYP3A4 通路，促进 Caspase-3 凋亡因子表达有关，其中没食子 酸的肝损伤作用最强。

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， 150 μmol·L-1 ）， rhein low-，medium- and high- dose groups （100，150，200 μmol·L-1 ），gallic acid low-，medium- and highdose groups （100，150，200 μmol·L-1 ），and stilbene glycoside low-，medium- and high- dose groups （200，500， 1 000 μmol·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 （312 μg·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 morphology of cells in all treatment groups changed with increasing drug concentration，showing shrinkage，rounding， darkening，and partial necrosis and detachment. Severe necrosis was observed in the medium- and high-dose emodin groups and the medium- and high-dose gallic acid groups. The apoptosis rate significantly increased in the medium- and high-dose emodin， rhein， and gallic acid groups （P＜0.05， P＜0.01）. The mRNA expression of CYP1A1 was significantly upregulated in all dose groups of emodin， rhein， and gallic acid （P＜0.05， P＜0.01）. The mRNA expression of CYP3A4 was significantly upregulated in the high-dose emodin group， medium- and high-dose rhein groups，and all dose groups of gallic acid （P＜0.01）. The mRNA expression of Caspase-3 was significantly upregulated in the medium- and high-dose emodin groups，high-dose rhein group，and medium- and high-dose gallic acid groups （P＜0.05， P＜0.01）. The protein expressions of CYP1A1， CYP3A4， and Cleaved Caspase-3 were significantly upregulated in all dose groups of emodin， rhein， and gallic acid （P＜0.05， P＜0.01）.（2）In vivo experiments： Based on the degree of liver tissue pathological damage，the order was roughly gallic acid group＞rhein group＞emodin group＞Polygoni Multiflori Radix group＞prepared Polygoni Multiflori Radix group， with the prepared Polygoni Multiflori Radix group showing the least liver tissue damage. Compared with the blank group，the serum levels of IBIL， TBIL，AST，and ALP were significantly increased in the Polygoni Multiflori Radix group，emodin group，and gallic acid group （P＜0.05，P＜0.01）. The serum levels of AST and ALP were significantly increased in the rhein group （P＜ 0.05，P＜0.01），and the serum ALT level was significantly increased in the gallic acid group （P＜0.05）. The mRNA expressions of CYP1A1， CYP3A4， and Caspase-3 in liver tissues were significantly upregulated in the Polygoni Multiflori Radix group，prepared Polygoni Multiflori Radix group，emodin group，and gallic acid group （P＜0.05， P＜0.01）， and the mRNA expression of CYP3A4 was significantly upregulated in the rhein group （P＜0.05）. The protein expressions of CYP1A1 and CYP3A4 wrere significantly upregulated in the Polygoni Multiflori Radixgroup， prepared Polygoni Multiflori Radix group，and gallic acid group （P＜0.05，P＜0.01），and the protein expression of Cleaved Caspase-3 was significantly upregulated in the Polygoni Multiflori Radix group，rhein group，and gallic acid group（P＜0.05，P＜0.01）. Conclusion The mechanism of liver injury induced by Polygoni Multiflori Radix and its main components may be related to the activation of CYP1A1 and CYP3A4 pathways and the promotion of Caspase-3 apoptosis factor expression，among which gallic acid has the strongest liver injury effect.

R285.5

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。