Bilirubin preserves corpus cavernosum smooth muscle phenotype via inhibition of ferroptosis: Implications for oxidative stress-related erectile dysfunction

Emerging evidence highlights bilirubin as a potent endogenous antioxidant with protective roles in cardiovascular and metabolic diseases. However, its effects on penile vascular homeostasis and the underlying molecular mechanisms remain poorly understood. This study investigates whether exogenous bilirubin modulates the phenotype of corpus cavernosum smooth muscle cells (CCSMCs) and enhances erectile function, with a focus on the potential involvement of ferroptosis inhibition. Healthy Sprague-Dawley rats were randomly assigned to control (n = 10) or bilirubin-treated (10 mg/kg/day, intraperitoneal, 2 weeks; n = 10) groups. Erectile function was assessed by measuring intracavernosal pressure (ICP) in response to electrical stimulation of the cavernous nerve. Penile tissues were collected for histological, immunofluorescent, and molecular analyses. Collagen and smooth muscle content were evaluated by Masson’s trichrome staining and α-SMA/collagen I immunofluorescence. Protein expression of GPX4, SLC7A11, Nrf2, and pro-inflammatory cytokines (IL-6, TNF-α) was analyzed by Western blotting. Bilirubin treatment significantly enhanced erectile function, as indicated by increased ICP/mean arterial pressure (MAP) ratios. Histological analysis revealed a higher smooth muscle-to-collagen ratio in the corpus cavernosum, with upregulated α-SMA and downregulated collagen I expression—consistent with a shift toward a contractile phenotype. Mechanistically, bilirubin activated the SLC7A11/GPX4 axis and upregulated Nrf2, key regulators of ferroptosis suppression. Lipid peroxidation and inflammatory markers (IL-6, TNF-α) were significantly reduced in the penile tissue of treated animals. Bilirubin preserves the contractile phenotype of corpus cavernosum smooth muscle cells and supports erectile function in healthy rats by inhibiting ferroptosis and inflammation. These findings identify ferroptosis as a novel regulatory pathway in penile vascular biology and suggest that enhancing bilirubin signaling may protect against oxidative stress-related erectile dysfunction.