Acacetin alleviates pyroptosis, inflammatory response, and oxidative stress in renal tubular epithelial cells caused by kidney stone via targeting GPR35

Nephrolithiasis, particularly calcium oxalate (CaOx) stones, remains a global health burden with limited pharmacological options for recurrence prevention. In this study, we investigated the therapeutic mechanism of acacetin (ACA) using both in vitro (COM-stimulated HK-2 cells) and in vivo (glyoxylate-induced mice) models of CaOx kidney stone. Our results demonstrated that ACA treatment dose-dependently attenuated renal injury, reduced the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), and activated the Nrf2/HO-1/NQO1 antioxidant pathway. Moreover, ACA significantly suppressed the NLRP3/caspase-1/GSDMD-mediated pyroptosis pathway. Through network pharmacology analysis, molecular docking, and molecular dynamics simulations, we identified GPR35 as a high-affinity direct binding target of ACA. The cellular thermal shift assay (CETSA) further confirmed that ACA enhanced the thermal stability of GPR35, indicative of direct interaction. Functional validation revealed that GPR35 overexpression recapitulated the protective effects of ACA by inhibiting NLRP3-related pyroptosis, whereas GPR35 knockdown significantly abrogated ACA-mediated pyroptosis suppression. Collectively, these findings establish the GPR35/NLRP3/pyroptosis axis as a critical pathway in CaOx kidney stone pathogenesis and identify ACA as a novel GPR35 agonist that concurrently suppresses pyroptosis and activates antioxidant defenses. Thus, ACA represents a promising therapeutic candidate for the prevention and treatment of kidney stone disease.