Zhang Linluo, Zhou Jie, Feng Zhe, Jiang Baoping, Li Changqing, Zhou Lingling, Zhou Xueping
The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, China.
Jiangsu Provincial Key Laboratory of Pharmacology and Safety Evaluation of Material Medical, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China.
Front Pharmacol. 2022 Jul 7;13:918466. doi: 10.3389/fphar.2022.918466. eCollection 2022.
Qingluo Tongbi Formula (QTF) is an empirical formula of Chinese medicine master Zhongying Zhou for the treatment of rheumatoid arthritis. Although including Hook. F. (TW), it has not shown liver toxicity in clinical application for many years. Our previous studies have shown that QTF can significantly reduce TW-caused hepatotoxicity, but the mechanism is still unclear. This study aimed to explore the important roles of mitophagy and endoplasmic reticulum stress (ERS) and the relationship between them in QTF in alleviating TW-induced hepatotoxicity. , C57BL/6J female mice were used to build a model of TW-induced liver toxicity; Then mice were randomly divided into control, TW, TW + RG, TW + PN, TW + SA, TW + BM, and QTF groups. After intragastric administration for 7 days, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in serum were detected; H and E staining, Oil Red O staining, transmission electron microscopy, Western blotting, and RT-qPCR were used to detect the pathological changes in liver tissue, the levels of ERS and mitophagy related proteins and genes, including GRP78, PERK, DRP1, LC3, etc., , triptolide (TP), catalpol (CAT), and panax notoginseng saponins (PNS), the main active ingredients of QTF, were selected. The mitophagy inhibitor, ERS inhibitor, and PERK inhibitor were used to further study the relationship between TW-induced ERS and mitophagy in HepaRG cells. The results showed that, QTF reduced excessive mitophagy and ERS in TW-induced hepatotoxicity in C57BL/6J mice, and the attenuating effects of RG and PN in QTF were most obvious, and they also significantly restrained the TW-induced ERS and mitophagy by the PERK-ATF4 pathway. Furthermore, PNS was superior to CAT in inhibiting the expression levels of GRP78, PERK, and ATF4, while CAT was superior to PNS in reversing the expression levels of DRP1, P62, and LC3. The combination of CAT and PNS had the best attenuating effect and the most significant regulation on ERS and mitophagy. In conclusion, QTF can alleviate TW-induced hepatotoxicity by differentially downregulating the PERK-ATF4 pathway and excessive mitophagy by different components.
清络通痹方（QTF）是中医大师周仲瑛治疗类风湿关节炎的经验方。虽含雷公藤（TW），但多年临床应用未显示肝毒性。我们前期研究表明QTF可显著减轻TW所致肝毒性，但其机制尚不清楚。本研究旨在探讨线粒体自噬和内质网应激（ERS）在QTF减轻TW诱导肝毒性中的重要作用及二者关系。采用C57BL/6J雌性小鼠建立TW诱导肝毒性模型；然后将小鼠随机分为对照组、TW组、TW+RG组、TW+PN组、TW+SA组、TW+BM组和QTF组。灌胃给药7天后，检测血清中丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）和乳酸脱氢酶（LDH）水平；采用苏木精-伊红染色、油红O染色、透射电镜、蛋白质免疫印迹法和实时定量聚合酶链反应检测肝组织病理变化、ERS和线粒体自噬相关蛋白及基因水平，包括GRP78、PERK、DRP1、LC3等。选取QTF主要活性成分雷公藤甲素（TP）、梓醇（CAT）和三七总皂苷（PNS），用线粒体自噬抑制剂、ERS抑制剂和PERK抑制剂进一步研究TW诱导的HepaRG细胞中ERS与线粒体自噬的关系。结果显示，QTF减轻C57BL/6J小鼠TW诱导肝毒性中的过度线粒体自噬和ERS，QTF中RG和PN的减轻作用最明显，它们还通过PERK-ATF4途径显著抑制TW诱导的ERS和线粒体自噬。此外，PNS在抑制GRP78、PERK和ATF4表达水平上优于CAT，而CAT在逆转DRP1、P62和LC3表达水平上优于PNS。CAT与PNS联合使用对ERS和线粒体自噬的减轻作用最佳且调控最显著。综上所述，QTF可通过不同成分差异性下调PERK-ATF4途径和过度线粒体自噬来减轻TW诱导的肝毒性。
