Notoginsenoside R1 interferes with PGAM5-NEK7 interaction to inhibit NLRP3 inflammasome activation in macrophages and...

Notoginsenoside R1 (NGR1), a primary bioactive constituent of P. notoginseng, has potent anti-inflammatory and immunomodulatory functions, but its role and mechanism in UC remain incompletely understood and warrant further exploration. The global prevalence of ulcerative colitis (UC) is rising steadily, threatening human health. Despite advances in modern therapies, UC treatments remain limited.

This work investigated whether NGR1 alleviates DSS-induced colitis by inhibiting the PGAM5-NEK7 interaction and NLRP3 inflammasome activation in macrophages, providing new mechanistic insights and therapeutic targets for UC.

UC model was established via 3% DSS administration to assess the protective efficacy of NGR1. Disease activity index (DAI), body weight and colon length were determined to assess disease severity and intestinal injury. HE staining, ELISA and immunofluorescence assays were employed to assess pathological changes in the colon and liver. Macrophages were depleted via tail vein injection of clodronate liposomes. RT-qPCR, ELISA, immunofluorescence and WB were performed to quantify macrophage markers, pro-inflammatory genes and NLRP3 inflammasome-related proteins. In vitro, LPS/ATP-stimulated bone marrow-derived macrophages (BMDMs) demonstrated that NGR1 exerts a potent inhibitory effect on NLRP3 inflammasome activation. After cell treatment, CCK-8, ELISA, RT-qPCR, and WB assays were performed to detect cell viability, inflammatory cytokine levels, and the expression of related genes and proteins. Meanwhile, molecular docking was utilized to verify the interactions among PGAM5, NEK7 and NLRP3. Co-IP in 293T cells overexpressing PGAM5, NEK7 and NLRP3 further verified their interplay.

Compared with the Model group, NGR1 intervention markedly reduced the DAI scores, ameliorated body weight loss and colon shortening, alleviated colonic and hepatic pathological damage, upregulated the expression of intestinal barrier tight junction proteins (ZO-1, Occludin and Claudin-1), and decreased LPS levels and the release of TNF-α, IL-1β and IL-6 in serum and tissues. Macrophage depletion was observed to markedly abolish protective effects of NGR1, and reduced PGAM5, NEK7, NLRP3, Caspase-1, C-Caspase-1 levels. In vitro, NGR1 inhibited inflammatory factor secretion and NLRP3 inflammasome activation in LPS/ATP-stimulated BMDMs, these effects were partially blocked after PGAM5 or NEK7 gene silencing. Co-IP assays confirmed endogenous interactions between PGAM5, NEK7 and NLRP3. And the molecular docking showed NGR1 could bind to PGAM5, and NGR1 intervention significantly suppressed the expression of the ternary complex formed by the three proteins.

This study revealed that NGR1 disrupted the PGAM5-NEK7 interaction, restrained NLRP3 inflammasome assembly in macrophages, and relieved colonic damage triggered by DSS, providing a novel target for UC treatment.