Protective Role of Klotho in Cardiomyocyte Injury Caused by Hypoxia-Reoxygenation via Regulation of the NF-κb...

This study aimed to investigate the protective role of klotho in cardiomyocytes subjected to hypoxia/reoxygenation (H/R) injury. An in vitro H/R model was established using H9C2 rat cardiomyoblasts, which were transfected with klotho-overexpressing lentiviral vectors. Cells were divided into six experimental groups: control, H/R, negative control (NC), klotho-overexpression, PDTC (an NF-κB inhibitor), and a combination group treated with klotho and lipopolysaccharide (LPS). Klotho expression levels were determined by quantitative real-time PCR and western blotting. Cell viability was measured via the MTT assay, while apoptosis was assessed using flow cytometry. The expression of proteins involved in the NF-κB signaling pathway was analyzed by western blotting, and the cellular expression of pro-inflammatory cytokines was evaluated through immunofluorescence staining. Klotho overexpression significantly elevated klotho levels in H9C2 cells and led to improved cell viability, reduced apoptosis, and decreased cellular expression of pro-inflammatory cytokines compared to the H/R group. Western blot analysis revealed that klotho overexpression, as well as PDTC treatment, suppressed NF-κB pathway activation, as indicated by a reduced p-P65/P65 ratio and increased p-IκBα/IκBα levels. These effects were partially reversed upon co-treatment with LPS. These results suggest that klotho confers cardioprotection against H/R-induced injury by modulating NF-κB signaling, reducing inflammation, and promoting cell survival. This protocol provides a reproducible method for studying the molecular mechanisms underlying myocardial ischemia-reperfusion injury and the therapeutic potential of klotho in cardiac inflammation.