H2 protects H9c2 cells from hypoxia/reoxygenation injury by inhibiting the Wnt/CX3CR1 signaling pathway

Jingsheng Wang, Bin Ma, Xue Jiang, Chao Li, Zhaochen Lin, Yumei Wang, Jingfei Shi, Gang Wang, Chao Cui


1Department of Pharmacy, Qilu Hospital of Shandong University Dezhou Hospital, Dezhou, Shandong Province, China2Department of Cardiovascular Medicine, The Second Affiliated Hospital of Shandong First Medical University, Taian, Shandong Province, China3Department of Cardiovascular Medicine, Taian City Taishan District People’s Hospital, Taian, Shandong Province, China4Hydrogen Medical Research Center, The Affiliated Taian City Central Hospital of Qingdao University, Taian, Shandong Province, China5Shandong First Medical University, Jinan, Shandong Province, China

Abstract

Myocardial ischemia‒reperfusion injury is a severe cardiovascular disease, and its treatment and prevention are crucial for improving patient prognosis and reducing the economic burden. This study aimed to explore the impact of hydrogen (H2) on hypoxia/reoxygenation (H/R) injury in H9c2 cells (derived from rat embryonic heart tissue) induced by hydrogen peroxide (H2O2) and to elucidate its underlying mechanism. An H/R injury model was established in H9c2 cells via exposure to 15 μM H2O2 for 3 hours, followed by incubation in a 5% CO2 atmosphere at 37°C for 24 hours. Then, the cells were treated with H2 (50%) for 6, 12 or 24 hours. The results demonstrated that H9c2 cells exposed to H2O2 and subjected to H/R injury presented a marked decrease in the cell survival rate, accompanied by severe morphological alterations, such as curling and wrinkling, and elevated lactate dehydrogenase levels. Notably, H2 mitigated H/R injury induced by H2O2 in a time-dependent manner, improving the morphological damage observed in H9c2 cells and decreasing lactate dehydrogenase levels. Compared with the model group, treatment with H2 increased the activities of antioxidant enzymes, including catalase, superoxide dismutase, and glutathione peroxidase, while concurrently reducing the level of malondialdehyde, an indicator of cellular damage. Furthermore, H2 treatment downregulated the expression of inflammatory cytokines and inflammatory-related factors, specifically interleukin-6, high-mobility group box 1, tumor necrosis factor-alpha, and Toll-like receptor 4, in H9c2 cells post-H/R injury. Furthermore, H2 treatment resulted in a marked decrease in the expression levels of proteins associated with the Wnt/C-X3-C-motif receptor 1 signaling pathway, such as β-catenin, glycogen synthase kinase-3 beta, adenomatous polyposis coli, and Wnt and C-X3-C-motif receptor 1. This observation suggests a potential mechanism for its protective effects against H/R injury. Therefore, H2 exerts a protective effect against H/R injury in H9c2 cells induced by H2O2, potentially by inhibiting the activated Wnt/C-X3-C-motif receptor 1 signaling pathway. This inhibition, in turn, prevents the generation of oxidative stress, inflammatory cytokines, and inflammation-associated factors.


摘要：心肌缺血再灌注损伤是一种严重的心血管疾病，其治疗和预防对于改善患者预后和减轻经济负担至关重要。本研究旨在探讨氢（H2）对过氧化氢（H2O2）诱导的大鼠胚胎心脏组织H9c2细胞缺氧/再氧化（H/R）损伤的影响，并阐明其机制。将H9c2细胞暴露于15 μM H2O2中3 H，然后在37℃5% CO2环境中孵育24 H，建立H/R损伤模型。然后，用H2（50%）处理细胞6、12或24小时。结果表明，H9c2细胞暴露于H2O2和H/R损伤后，细胞存活率明显下降，并伴有严重的形态改变，如卷曲和起皱，乳酸脱氢酶水平升高。值得注意的是，H2以时间依赖性的方式减轻H2O2诱导的H/R损伤，改善H9c2细胞的形态学损伤，降低乳酸脱氢酶水平。与模型组相比，H2处理增加了过氧化氢酶、超氧化物歧化酶和谷胱甘肽过氧化物酶等抗氧化酶的活性，同时降低了细胞损伤指标丙二醛的水平。此外，H2处理可下调h /R损伤后H9c2细胞中炎症因子和炎症相关因子的表达，特别是白细胞介素-6、高迁移率组盒1、肿瘤坏死因子- α和toll样受体4。此外，H2处理导致Wnt/C-X3-C-motif受体1信号通路相关蛋白的表达水平显著降低，如β-catenin、糖原合成酶激酶3 β、腺瘤性大肠息肉病、Wnt和C-X3-C-motif受体1。这一观察结果提示了其对H/R损伤的保护作用的潜在机制。因此，H2对H2O2诱导的H9c2细胞H/R损伤具有保护作用，可能是通过抑制激活的Wnt/C-X3-C-motif受体1信号通路。这种抑制反过来又阻止了氧化应激、炎症细胞因子和炎症相关因子的产生。