Yiqi Huoxue Formula ameliorates endometriosis by suppressing NF-κB p65/NLRP3 inflammasome-associated pyroptotic signaling.

Yiqi Huoxue Formula (YQHXF) is an 11-herb hospital-based traditional Chinese medicinal formula developed according to the therapeutic principle of tonifying qi and activating blood circulation. Previous clinical application has suggested its relevance to postoperative ovarian endometriosis with qi deficiency and blood stasis; however, its pharmacological basis remains incompletely understood. This study investigated whether YQHXF attenuates endometriotic lesion progression by modulating NF-κB p65/NLRP3 inflammasome-associated pyroptotic signaling. A rat model of endometriosis was established by autologous endometrial transplantation. LC-HRMS was used to characterize the chemical profile of YQHXF, and RNA sequencing was performed on ectopic lesions. Primary eutopic and ectopic endometrial stromal cells (ESCs) were isolated from patients with endometriosis. p65 overexpression, siRNA-mediated p65 knockdown, the NLRP3 inhibitor MCC950, and the NLRP3 agonist BMS-986299 were used to evaluate pathway involvement. Cell viability, inflammatory cytokines, inflammasome- and pyroptosis-associated markers, transmission electron microscopy, PI/Hoechst staining, and LDH release were assessed. LC-HRMS profiling yielded 287 putatively annotated records and showed recurrent chemical features across three independent YQHXF batches. YQHXF reduced ectopic lesion volume, alleviated histopathological injury, and downregulated Ki-67 and PCNA in vivo. Transcriptomic analysis linked its effects to immune-inflammatory responses, TNF/NF-κB signaling, NOD-like receptor signaling, and cytoskeletal/adhesion remodeling. In EM tissues and ectopic ESCs, NF-κB p65/NLRP3 inflammasome-associated signaling was activated, accompanied by increased IL-1β and IL-18 release, elevated cleaved caspase-1 and GSDMD-N, reduced E-cadherin, and pyroptosis-associated membrane injury. YQHXF suppressed these changes, whereas NLRP3 activation or p65 overexpression partially weakened its protective effects. YQHXF attenuates endometriosis progression, at least in part, by suppressing NF-κB p65/NLRP3 inflammasome-associated pyroptotic signaling and inflammatory injury. These findings provide preclinical mechanistic evidence supporting further investigation of YQHXF as a potential non-hormonal therapeutic candidate for endometriosis.