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

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.