Metformin reduced NLRP3 inflammasome activity in Ox-LDL stimulated macrophages through adenosine monophosphate activated protein kinase and protein phosphatase 2A

Abstract

Metformin has been suggested to have cardiovascular protective effects. Previous researches showed that metformin activates Adenosine Monophosphate Activated Protein Kinase (AMPK) and Protein Phosphatase 2A (PP2A). This research aimed to elucidate whether and how metformin affects NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activity in oxidized low-density lipoprotein (ox-LDL) stimulated macrophages. Macrophages were treated with ox-LDL and metformin in a continuous manner, and NLRP3 inflammasome activation was evaluated by detecting IL-1β and caspase-1 p10 release by ELISA and western blot, respectively. AMPK α1 and α2 gene expression was silenced in macrophages by siRNA transduction. Expression of NLRP3 and pro-IL-1β was monitored by RT-qPCR and western blot. PP2A activity was inhibited by LB-100 treatment. Activation of NF-κB signaling was evaluated by detecting the nuclear accumulation of p65 and phosphorylation of IκBα by western blot. Activation of Tristetraprolin was evaluated by detecting its serine phosphorylation level by immunoprecipitation and western blot. In the results, upregulation of NLRP3 protein expression and NLRP3 inflammasome activation induced by ox-LDL treatment in macrophages were significantly attenuated by metformin treatment. AMPK gene silencing partially rescued NLRP3 inflammasome activation. Inhibition of PP2A significantly restored NLRP3 and pro-IL-1β protein expression level downregulated by metformin in ox-LDL-stimulated macrophages. PP2A catalytic activity was required for NF-κB inhibition and Tristetraprolin activation induced by metformin in ox-LDL-stimulated macrophages. Our data showed Metformin reduced NLRP3 protein expression and NLRP3 inflammasome activation in ox-LDL-stimulated macrophages through AMPK and PP2A.