Nepetin limits NLRP3 inflammasome activation and alleviates NLRP3-driven inflammatory diseases via PINK1-dependent mitophagy

The NLRP3 inflammasome plays a pivotal role in the progression of inflammatory diseases. Mitochondrial damage, oxidative stress and mitochondrial DNA (mtDNA) leak are the key upstream factors for NLRP3 inflammasome activation. Nepetin (Nep), a naturally occurring flavonoid found with anti-inflammatory properties; however, whether it can affect the NLRP3 inflammasome activation and its precise anti-inflammatory mechanism remains unclear. In this study, we demonstrated that Nep enhances PINK1-mediated ubiquitin phosphorylation, which promotes Mitophagy and subsequently inhibits NLRP3 inflammasome activation and Pyroptosis in macrophages. The administration of Nep to macrophages alleviated of mitochondrial damage, reduced mitochondrial superoxide production, restored mitochondrial membrane potential and prevented the mtDNA leakage. These findings provide compelling evidence for the antioxidant effect of Nep. Furthermore, the pivotal function of Mitophagy in the NLRP3 inflammasome inhibitory impact of Nep was substantiated through the utilisation of Mitophagy inhibitors and siRNA techniques. Notably, Nep increased survival and reduced organ damage in mice with systemic inflammation by inhibiting NLRP3 inflammasome activation. In addition, Nep suppressed NLRP3 inflammasome activation in obese mice, which led to reduced white adipose and liver inflammation, thereby ameliorating Insulin resistance. In conclusion, our findings suggest that Nep is a potent NLRP3 inflammasome inhibitor and a promising candidate for the development of anti-inflammatory therapies.