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JAK2/STAT3信号通路介导原花青素抗H9C2细胞缺氧/复氧损伤

俞辰斌, 赵国龙, 于立明, 俞世强, 段维勋, 张海锋

江苏省中医药研究院急诊科，南京 210028；第四军医大学教学实验中心，西安 710032；第四军医大学西京医院心血管外科，西安710032

摘要

本研究旨在探讨原花青素(proanthocyanidin, Pro)抗H9C2细胞缺氧/复氧(hypoxia/reoxygenation, H/R)损伤的机制，明确蛋白质酪氨酸激酶2/信号传导子与激活子3 (Janus kinase 2/signal transducer and activator of transcription 3, JAK2/STAT3)信号通路在此过程中的作用。取对数生长期的H9C2细胞株，随机分为5组：对照组(Con)、H/R损伤组(H/R)、Pro处理组(H/R+ Pro)、JAK2小干扰RNA处理组(H/R+Pro+JAK2 siRNA)和JAK2小干扰RNA对照组(H/R+JAK2 siRNA)。Pro (40 µmol/L)预处理8 h，H9C2细胞系缺氧2 h、复氧4 h后用MTT和TUNEL法分别检测各组细胞活力和凋亡率，用试剂盒检测超氧化物生成量，用Western blot法检测JAK2/STAT3通路相关分子，氧化应激指标及内质网应激相关蛋白的表达。结果显示，与H/R组相比，Pro处理可显著提高H/R处理的H9C2细胞活力，并降低细胞凋亡率，明显上调p-JAK2及p-STAT3水平，下调氧化应激指标超氧化物生成量及gp91phox蛋白表达量，下调内质网应激标志蛋白葡萄糖调节蛋白78 (glucose-regulated protein 78, GRP78)、CCAAT/增强子结合蛋白(CCAAT/enhancer binding protein homologous protein, CHOP)及caspase-12的表达，而Pro以上保护作用均被JAK2 siRNA所抑制。本研究结果表明，Pro可通过JAK2/STAT3信号通路减轻H/R引起的H9C2细胞氧化应激与内质网应激损伤。本研究为阐明Pro的心血管保护作用及相关药物的开发提供了实验依据。

关键词： 原花青素; 缺氧/复氧损伤; JAK2/STAT3; 氧化应激; 内质网应激

分类号：R331. 3

Proanthocyanidin protects H9C2 cells against hypoxia/reoxygenation injury via JAK2/STAT3 signaling pathway

YU Chen-Bin, ZHAO Guo-Long, YU Li-Ming, YU Shi-Qiang, DUAN Wei-Xun, ZHANG Hai-Feng

Emergency Department of Jiangsu Research Institute of Traditional Chinese Medicine, Jiangsu 210028； Experimental Teaching Center, The Fourth Military Medical University, Xi’an 710032, China； Department of Cardiovascular Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China

Abstract

The present study was aimed to investigate the underlying mechanisms of the protective effect of proanthocyanidin (Pro) against hypoxia/reoxygenation (H/R) injury in H9C2 cells with a focus on Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway. H9C2 cells were randomly assigned to 5 groups, including the control group (Con), the H/R-injured group (H/R), the Pro-treated group (H/R+Pro), the JAK2 siRNA-treated group (H/R+Pro+JAK2 siRNA) and the JAK2 siRNA control group (H/R+JAK2 siRNA). The cells were pretreated with Pro (40 µmol/L) for 8 h before 2 h of hypoxia and 4 h of reoxygenation. Cellular viability and apoptosis rate were detected by MTT and TUNEL methods, and superoxide generation was measured. JAK2/STAT3 signaling, oxidative stress markers and endoplasmic reticulum stress markers were also detected by Western blot. We found that Pro treatment significantly improved cellular viability and reduced apoptosis rate in H/R-treated H9C2 cells. In addition, Pro treatment significantly up-regulated the phosphorylation levels of JAK2 and STAT3, down-regulated the superoxide generation, gp91phox, glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP) and caspase-12 expression. However, these protective effects of Pro were all attenuated by JAK2 siRNA administration. Taken together, we demonstrated that Pro protects H9C2 cells against H/R-induced oxidative stress and endoplasmic reticulum stress injury via JAK2/STAT3 signaling pathway.

Key words: proanthocyanidin; hypoxia/reoxygenation injury; JAK2/STAT3; oxidative stress; endoplasmic reticulum stress

收稿日期：2016-02-25　　录用日期：2016-04-28

通讯作者：段维勋,张海锋　　E-mail: duanweixun@126.com,hfzhang@fmmu.edu.cn

引用本文：

俞辰斌, 赵国龙, 于立明, 俞世强, 段维勋, 张海锋. JAK2/STAT3信号通路介导原花青素抗H9C2细胞缺氧/复氧损伤[J]. 生理学报 2016; 68 (5): 568-574.

YU Chen-Bin, ZHAO Guo-Long, YU Li-Ming, YU Shi-Qiang, DUAN Wei-Xun, ZHANG Hai-Feng. Proanthocyanidin protects H9C2 cells against hypoxia/reoxygenation injury via JAK2/STAT3 signaling pathway. Acta Physiol Sin 2016; 68 (5): 568-574 (in Chinese with English abstract).