Rg1 activates the AMPK/mTOR-autophagy axis and inhibits the NOD-like receptor 3 inflammasome to alleviate pyroptosis in periodontal ligament fibroblasts

This study explored the mechanism of ginsenoside Rg1 (Rg1) alleviating lipopolysaccharide (LPS)-induced Pyroptosis in human periodontal ligament fibroblasts (HPDLFs). HPDLFs were treated with LPS to induce Pyroptosis, followed by the Rg1 intervention. Cell viability and Lactate Dehydrogenase (LDH) release were assessed by CCK-8 and kits. Levels of pyroptosis-related cytokines, NOD-like Receptor 3 (NLRP3) mRNA expression, and protein levels of NLRP3 inflammasome-related markers, pyroptosis-related markers, autophagy-related markers, and the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) pathway were determined by ELISA, RT-qPCR, and western blot. Autophagosome changes were also observed under transmission electron microscopy. HPDLFs were transfected with NLRP3-overexpression plasmids, or co-treated with LPS + Rg1 and the Autophagy inhibitor (3-MA), AMPK Inhibitor (BML-275), or mTOR Activator (MHY1485). LPS increased Pyroptosis (decreased cell viability, increased LDH release, increased GSDMD-N-terminal domain protein and IL-1β and IL-18 levels), activated the NLRP3 inflammasome (increased NLRP3, apoptosis-associated speck-like protein containing a CARD, cleaved Caspase-1 protein levels), induced Autophagy (increased Beclin1 protein, LC3-II/LC3-I levels, and autophagosome number), impaired autophagic flux, and inhibited p62 degradation. Partial AMPK activation and mTOR inhibition were observed. LPS + Rg1 further activated AMPK/mTOR signaling, mitigated impaired autophagic flux, inhibited the NLRP3 inflammasome, and alleviated HPDLF Pyroptosis. Rg1 promoted Autophagy by regulating the AMPK/mTOR pathway to inhibit NLRP3 inflammasome activation, thereby inhibiting HPDLF Pyroptosis. AMPK inhibition or mTOR activation partially averted Rg1's regulatory effects. Rg1 activates AMPK/mTOR-autophagy axis and inhibits NLRP3 inflammasome, thereby alleviating LPS-induced HPDLF Pyroptosis.

AMP-activated protein kinase; Autophagy; Ginsenoside Rg1; Human periodontal ligament fibroblasts; Mechanistic target of rapamycin; NLRP3 inflammasome; Periodontitis; Pyroptosis.