Capsaicin ameliorates diabetic nephropathy by targeted activation of GPX4 and inhibition of pyroptosis.

Abstract

Purpose: Capsaicin (CAP) is the primary pungent alkaloid found in chili peppers. The aim of this study is to screen for candidate therapeutic agents targeting the mitochondrial dysfunction-pyroptosis axis in diabetic nephropathy (DN) and to validate the efficacy and mechanism of action of capsaicin.

Methods: Bioinformatics analysis was conducted to identify candidate genes and drugs. The protective effects and underlying mechanisms of CAP were investigated using a high-glucose (HG)-induced HK-2 cell model and a streptozotocin-induced T1DM DN rat model. Experimental techniques included CCK8 assay, flow cytometry (pyroptosis and ROS), JC-1 staining, Western blot analysis, and histopathological staining. GPX4-IN-3 was applied in vivo and in vitro to determine whether it reversed the protective effects of capsaicin. The data were analyzed using the Shapiro-Wilk test, one-way analysis of variance (ANOVA) with corresponding post-hoc tests, and correlation analysis.

Results: Bioinformatic analysis identified 12 intersecting genes and highlighted CAP as a potential GPX4-targeting compound. Molecular docking demonstrated a high binding affinity of CAP for GPX4 and caspase-9. In vitro, CAP dose-dependently restored cell viability and suppressed HG-induced pyroptosis in HK-2 cells. It significantly alleviated mitochondrial damage by reducing ROS production, restoring mitochondrial membrane potential, and improving mitochondrial ultrastructure. Notably, these protective effects were reversed by GPX4-IN-3. Western blot analysis revealed that CAP upregulated GPX4 expression and inhibited activation of the NLRP3/caspase-1/GSDMD-N pyroptosis pathway. In vivo, CAP treatment markedly improved renal histopathology and renal function in DN rats; however, these effects were attenuated by co-administration of GPX4-IN-3.

Conclusion: This study demonstrates that CAP ameliorates diabetic renal injury by targeting GPX4, thereby mitigating mitochondrial dysfunction and subsequent NLRP3-mediated pyroptosis. These findings validate the bioinformatic prediction and reveal a novel mechanism of action for CAP, positioning it as a promising therapeutic candidate for DN.