Shikonin ameliorates renal fibrosis by targeting HSPA1A to regulate lipid metabolism and inhibit ferroptosis.

Abstract

BACKGROUND: Renal fibrosis (RF) is a critical step in the progression of chronic kidney disease to end-stage renal failure, but its treatment remains highly challenging, highlighting the urgent clinical need for novel therapeutic strategies. Shikonin, a naturally derived compound, shows promise in attenuating the progression of RF. A thorough understanding of its specific molecular targets and mechanism of action is essential. PURPOSE: This study elucidates the molecular mechanism through which Shikonin modulates HSPA1A to suppress ferroptosis and regulate lipid metabolism in RF. METHODS: A unilateral ureteral obstruction (UUO) mouse model was used to assess the antifibrotic effects of Shikonin. Efficacy was evaluated by analyzing renal pathomorphological changes and fibrosis indicators, and by using ELISA to detect the inflammation levels and kidney function. We integrated results from network pharmacology, transcriptomics, and non-targeted metabolomics to explore Shikonin's mechanism in renal fibrosis and verified it through experiments including qRT-PCR, immunofluorescence, western blot, transmission electron microscopy, ROS staining, and Bodipy staining. In vitro, human renal tubule epithelial cells (HK-2) were stimulated with TGF-β1 and then treated with Shikonin. Lipid-MS, CETSA, SPR, and molecular dynamics simulations were jointly employed to identify Shikonin's target. After knocking down this target in vivo using AAV, the effect of Shikonin on renal fibrosis was observed. RESULTS: Shikonin notably ameliorated histopathological lesions, renal dysfunction, pathological damage and fibrosis in UUO model. Transcriptomic and non-targeted metabolomics analyses indicated that lipid metabolism specifically the arachidonic acid (AA) pathway, is a key pathway through which Shikonin exerted its effects. Mechanistically, Shikonin directly bind to HSPA1A and, through this interaction, downregulated the expression of key AA-metabolizing enzymes ACSL4 and ALOX15, thereby suppressing lipid peroxidation and ferroptosis. This mechanism was confirmed both in vivo and in vitro. Crucially, the anti-fibrosis efficacy of Shikonin was abolished upon HSPA1A knockdown or inhibition, and could be functionally reversed by exogenous AA supplementation. CONCLUSION: This study has revealed a novel mechanism by which Shikonin alleviates renal fibrosis: by targeting HSPA1A to reprogram AA metabolism and inhibit ferroptosis. These findings display the HSPA1A-AA metabolism-ferroptosis axis as a critical therapeutic pathway and provide a compelling mechanistic foundation for developing Shikonin as a promising candidate drug.