hnRNPK knockdown alleviates NLRP3 inflammasome priming by repressing FLIP expression in Raw264.7 macrophages.

Inflammation is an important predisposing and progressive factor in chronic kidney disease (CKD). Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is associated with many fundamental cellular processes, but in chronic inflammatory pathologies remains unclear. An peripheral inflammation model was established using lipopolysaccharide (LPS)-stimulated mouse RAW264.7 macrophages, followed by inflammasome activation by ATP treatment. Knockdown of hnRNPK by sihnRNPK and FLICE-like inhibitory protein (FLIP) by siFLIP transfection were achieved in Raw264.7 macrophages. ELISA was used to determine the expression of IL-1β, IL-18 and TNF-α. Real time PCR was applied to detect the mRNA levels of hnRNPK, NOD-like receptors family pyrin domain-containing 3 (NLRP3), FLIP, Caspase-1, IL-1β and IL-18. Western blot and immunofluorescence were performed to detect relevant protein expressions. Co-immunoprecipitation (Co-IP) was used to assess the interaction of hnRNPK with FLIP. Results showed that LPS plus ATP activated NLRP3 inflammasome, which evidenced by the up-regulation of TNF-α, IL-1β and IL-18. Notably, hnRNPK and FLIP were significantly up-regulated in activated NLRP3 inflammasome of macrophages. HnRNPK or FLIP knockdown significantly suppressed the activation of NLRP3 inflammasome, as reflected by down-regulation of Caspase-1, IL-1β and IL-18. Importantly, hnRNPK could directly bind to FLIP in activated NLRP3 inflammasome. Our findings suggest that hnRNPK could promote the activation of NLRP3 inflammasome by directly binding FLIP, which might provide potential new therapeutic targets for CKD.