Luhong formula attenuates cardiomyocyte hypoxia/reoxygenation injury in vitro through SLC7A11-mediated ferroptosis.

This study investigates whether Luhong Formula (LHF) inhibits hypoxia/reoxygenation (H/R)-induced ferroptosis in cardiomyocytes and the underlying mechanism. H9c2 cells were subjected to H/R injury and treated with LHF extract, diltiazem (positive control), or Fer-1 (ferroptosis inhibitor). CCK-8, colorimetric assays, and transmission electron microscopy showed that LHF significantly increased cell viability, reduced LDH release, restored SOD activity and GSH content, and alleviated mitochondrial damage. FerOrange staining and C11-BODIPY flow cytometry revealed that LHF decreased intracellular Fe2+ levels and lipid ROS accumulation. Immunofluorescence and Western blot demonstrated that LHF up-regulated GPX4 expression, showing effects comparable to those of Fer-1 and diltiazem in this in vitro system. Using SLC7A11-overexpressing and -silencing stable cell lines, we found that SLC7A11 overexpression mimicked LHF's protective effects, while SLC7A11 silencing partially reversed them. Co-immunoprecipitation confirmed that H/R increased SLC7A11 ubiquitination, whereas LHF reduced this modification, thereby stabilizing SLC7A11 protein. Collectively, LHF is associated with reduced SLC7A11 ubiquitination, which correlates with up-regulated GPX4 and blocked ferroptosis, alleviating H/R injury in cardiomyocytes. These findings provide preliminary evidence for a potential regulatory effect of LHF on SLC7A11 protein stability, although direct measurements of protein half-life and detailed ubiquitin topology require further investigation. These findings suggest that LHF inhibits H/R-induced ferroptosis in H9c2 cells, at least in part, through the SLC7A11/GPX4 pathway, which may contribute to its cardioprotective effect.