JAK2/STAT3信号通路介导原花青素抗H9C2细胞缺氧/复氧损伤

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

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 si RNA)和JAK2小干扰RNA对照组(H/R+JAK2 si RNA)。Pro(40μmol/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 si RNA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。

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.