Sun Huiyuan, Ling Shukuan, Zhao Dingsheng, Li Jianwei, Li Yang, Qu Hua, Du Ruikai, Zhang Ying, Xu Feng, Li Yuheng, Liu Caizhi, Zhong Guohui, Liang Shuai, Liu Zizhong, Gao Xingcheng, Jin Xiaoyan, Li Yingxian, Shi Dazhuo
State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing, China.
Science and Technology Department, Beijing University of Chinese Medicine Third Affiliated Hospital, Beijing, China.
Front Pharmacol. 2020 Sep 9;11:532041. doi: 10.3389/fphar.2020.532041. eCollection 2020.
Previous studies have shown an attenuating effect of ginsenoside Re on myocardial injury induced by hypoxia/reoxygenation (H/R). However, the underlying mechanism remains unclear. This study was designed to determine the underlying mechanism by which ginsenoside Re protects from myocardial injury induced by H/R. HL-1 cells derived from AT-1 mouse atrial cardiomyocyte tumor line were divided into control, H/R, and H/R + ginsenoside Re groups. Cell viability was measured by CCK-8 assay. ATP levels were quantified by enzymatic assays. Signaling pathway was predicted by network pharmacology analyses and verified by luciferase assay and gene-silencing experiment. The relationship between ginsenoside Re and its target genes and proteins was analyzed by docking experiments, allosteric site analysis, real-time PCR, and ubiquitination and immunoprecipitation assays. Our results showed that ginsenoside Re treatment consistently increased HL-1 cell viability and significantly up-regulated ATP levels after H/R-induced injury. Network pharmacology analysis suggested that the effect of ginsenoside Re was associated with the regulation of the Hypoxia-inducing factor 1 (HIF-1) signaling pathway. Silencing of abrogated the effect of ginsenoside Re on HL-1 cell viability, which was restored by transfection with an HIF-1α-expressing plasmid. Results of the bioinformatics analysis suggested that ginsenoside Re docked at the binding interface between HIF-1α and the von Hippel-Lindau (VHL) E3 ubiquitin ligase, preventing VHL from binding HIF-1α, thereby inhibiting the ubiquitination of HIF-1α. To validate the results of the bioinformatics analysis, real-time PCR, ubiquitination and immunoprecipitation assays were performed. Compared with the mRNA expression levels of the H/R group, ginsenoside Re did not change expression of mRNA, while protein level of HIF-1α increased and that of HIF-1α[Ub]n decreased following ginsenoside Re treatment. Immunoprecipitation results showed that the amount of HIF-1α bound to VHL substantially decreased following ginsenoside Re treatment. In addition, ginsenoside Re treatment increased the expression of GLUT1 (glucose transporter 1) and REDD1 (regulated in development and DNA damage response 1), which are targets of HIF-1α and are critical for cell metabolism and viability. These results suggested that Ginsenoside Re treatment attenuated the myocardial injury induced by H/R, and the possible mechanism was associated with the inhibition of HIF-1α ubiquitination.
先前的研究表明人参皂苷Re对缺氧/复氧（H/R）诱导的心肌损伤具有减轻作用。然而，其潜在机制仍不清楚。本研究旨在确定人参皂苷Re保护心肌免受H/R诱导损伤的潜在机制。源自AT-1小鼠心房心肌细胞瘤系的HL-1细胞分为对照组、H/R组和H/R+人参皂苷Re组。通过CCK-8法检测细胞活力。通过酶法测定ATP水平。通过网络药理学分析预测信号通路，并通过荧光素酶测定和基因沉默实验进行验证。通过对接实验、变构位点分析、实时PCR以及泛素化和免疫沉淀实验分析人参皂苷Re与其靶基因和蛋白质之间的关系。我们的结果表明，人参皂苷Re处理可持续提高HL-1细胞活力，并在H/R诱导损伤后显著上调ATP水平。网络药理学分析表明，人参皂苷Re的作用与缺氧诱导因子1（HIF-1）信号通路的调节有关。敲除 可消除人参皂苷Re对HL-1细胞活力的影响，而转染表达HIF-1α的质粒可恢复该影响。生物信息学分析结果表明，人参皂苷Re对接于HIF-1α与冯·希佩尔-林道（VHL）E3泛素连接酶之间的结合界面，阻止VHL与HIF-1α结合，从而抑制HIF-1α的泛素化。为验证生物信息学分析结果，进行了实时PCR、泛素化和免疫沉淀实验。与人参皂苷Re处理后，与H/R组的mRNA表达水平相比，人参皂苷Re未改变 的mRNA表达，但HIF-1α蛋白水平升高，HIF-1α[Ub]n蛋白水平降低。免疫沉淀结果表明，人参皂苷Re处理后与VHL结合的HIF-1α量显著减少。此外，人参皂苷Re处理增加了葡萄糖转运蛋白1（GLUT1）和发育及DNA损伤反应调节蛋白1（REDD1）的表达，它们是HIF-1α的靶标，对细胞代谢和活力至关重要。这些结果表明，人参皂苷Re处理减轻了H/R诱导的心肌损伤，其可能机制与抑制HIF-1α泛素化有关。
