Novel natural inhibitor Rubimaillin targets NLRP3 R167/Y381 to ameliorate inflammatory and neurodegenerative diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubia cordifolia L. is traditionally used in Chinese medicine for treating arthritic and inflammatory conditions by cooling blood and activating circulation. Gouty arthritis, one of the disease models investigated in this study, falls within this traditional anti-arthritic application. This study investigates whether rubimaillin (Rub), a naphthoquinone from Rubia cordifolia, selectively inhibits NLRP3 inflammasome activation and exerts therapeutic effects in relevant disease models. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome is a key driver of various inflammatory, metabolic, and neurodegenerative disorders; yet, no clinically approved inhibitor is currently available. Rubia cordifolia L. is a traditional medicinal herb, and Rubimaillin (Rub), a naphthoquinone isolated from this herb, has potential anti-inflammatory properties, but its role and mechanism in regulating NLRP3 activation remain unclear. AIM OF STUDY: This study aimed to determine whether Rubimaillin (Rub), a naphthoquinone isolated from Rubia cordifolia L., can selectively inhibit NLRP3 inflammasome activation and yield therapeutic effects in relevant NLRP3-driven disease models. MATERIALS AND METHODS: Mouse primary microglia, bone marrow-derived macrophages (BMDMs), and the human macrophage cell line THP-1 were primed with lipopolysaccharide (LPS) for 3 h, then stimulated with Nigericin or ATP to induce NLRP3 inflammasome assembly and pyroptosis. Caspase-1 activation, apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, interleukin-1β (IL-1β) release, and lactate dehydrogenase (LDH) secretion were quantified. Mutagenesis studies were conducted to identify critical residues required for Rub's binding to NLRP3. The in vivo therapeutic potential of Rub was assessed in three murine models of NLRP3-driven inflammation: LPS-induced sepsis, monosodium urate crystal (MSU)-induced gouty arthritis, and the APP/PS1 double-transgenic Alzheimer's disease (AD) mouse model. Mice received intraperitoneal Rub or vehicle, and disease severity was evaluated by histopathology, cytokine profiling, and behavioral tests. RESULTS: Rub significantly reduced pyroptosis and IL-1β release in mouse primary microglia, BMDMs, and THP-1 cells in a dose-dependent manner, without affecting Absent in melanoma 2(AIM2) or NLR family CARD domain containing 4 (NLRC4) pathways. Mechanistically, Rub directly bound to NLRP3, thereby blocking the oligomerization of both NLRP3 and ASC, as well as preventing caspase-1 activation and gasdermin D (GSDMD) cleavage. Furthermore, mutagenesis studies identified arginine 167 and tyrosine 381 as critical residues for Rub's binding to NLRP3. In vivo, Rub treatment significantly prolonged survival and attenuated lung injury in the sepsis model, reduced paw swelling and bone erosion in the gout model, and ameliorated cognitive deficits and neuroinflammation in the AD model, respectively. CONCLUSION: Collectively, these findings demonstrate that Rub selectively targets the NLRP3 inflammasome and exerts therapeutic effects on NLRP3-driven diseases including sepsis, gout, and AD. This study provides a molecular basis for the traditional application of Rubia cordifolia L. and highlights Rub as a promising natural lead compound for the treatment of NLRP3-driven disorders.