Jianpi Qushi Heluo Formula ameliorates podocytes injury related with ROS-mediated NLRP3 inflammasome activation in membranous nephropathy by promoting PINK1-dependent mitophagy

Ethnopharmacological relevance Jianpi Qushi Heluo formula (JQHF) is an evidence-based herbal formula based on "Fangji Huangqi Decoction" in the classic of traditional Chinese medicine (TCM) synopsis of the Golden Chamber. Its effectiveness in reducing idiopathic membranous nephropathy (IMN) proteinuria, edema and other clinical symptoms and improving kidney injury has been confirmed by clinical trials and animal experiments. These results demonstrate the critical significance of reducing podocyte injury by regulating mitophagy through integrating ethnopharmacology. Aim of the study This study aimed to explore the protective effects of JQHF on podocyte injury in IMN, focusing on its role in promoting PINK1-dependent mitophagy to inhibit reactive oxygen species (ROS) mediated activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. Materials and Methods The main components of JQHF were identified by ultra-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MSE). Passive Heymann nephritis (PHN) was induced in rats using anti-Fx1A antiserum, while podocyte injury in vitro was stimulated with sublytic complement C5b-9 (sC5b-9). The PHN rats were treated with JQHF, and autophagy inhibitor 3-methyladenine (3-MA) combined with JQHF was used for pathway verification, and Benazepril served as a positive control. In vitro, podocytes were exposed to palmitic acid (PA, a ROS inducer) and N-acetylcysteine (NAC, a ROS scavenger). Further, we silenced the expression of PINK1 in podocytes model and intervened with JQHF-containing serum. Renal function was assessed through biochemical analyses and histopathology. Mitochondrial function was measured by detecting mitochondrial membrane potential (MMP), ROS levels as well as mitochondrial ultrastructure. The expression of podocyte structural proteins (desmin, nephrin, podocin) and inflammasome-related markers (NLRP3, caspase-1, IL-1β, IL-6) was analyzed to assess podocyte injury and inflammasome activation. Results (1) In vitro, in contrast to the control group, PA intervention caused increased ROS accumulation, reduced MMP, upregulated NLRP3 and caspase-1 expression, as well as elevated expression of inflammatory factors IL-1β and IL-6. The expression of nephrin and podocin was notably reduced. By contrast, NAC reversed these effects. (2) In vivo, JQHF effectively ameliorated mitochondrial damage, reduced NLRP3 expression and mitigated podocyte injury in PHN rats. The protective effects of JQHF were diminished by 3-MA, confirming the involvement of autophagy. (3) In vitro, JQHF-containing serum attenuated C5b-9-induced podocyte injury, improved mitochondrial dysfunction, and inhibited ROS-mediated activation of NLRP3 inflammasome. Silencing PINK1 significantly reversed these protective effects. Conclusion JQHF can alleviate podocyte damage, that through inhibiting ROS-mediated NLRP3 inflammasome activation by improving PINK1-mediated mitophagy.