NLRC3 Alleviates Hypoxia/Reoxygenation Induced Inflammation In RAW264.7 Cells By Inhibiting K63-linked Ubiquitination Of TRAF6

Objective: To explore the effect and related mechanisms of NLR family CARD-containing 3(NLRC3)on hypoxia/reoxygenation(H/R)-induced pro-inflammatory outcome in RAW264.7 cells.Methods: A mouse model of hepatic ischemia/reperfusion(I/R)was constructed to detect the expression of NLRC3 protein in liver and content of IL-1β in serum respectively by Western blot and ELISA.H/R model in RAW264.7 cells was built in further study.Western blot method was used to detect the protein expression level of NLRC3、TRAF6、p-p65、p65、IκBα and K63-linked ubiquitination of TRAF6.Co-IP technology was used to detect the interaction between NLRC3 and TRAF6 molecules in cells.Immunofluorescence method was used to detect the nuclear level of NF-κB subunit p65.ELISA was used to detect the IL-1β content in cell culture supernatant.Results: In vivo,NLRC3 protein decreased with the prolongation of reperfusion time(P<0.05)in mouse model of hepatic I/R,meanwhile,the serum IL-1β level in mice increased(P<0.05).In vitro,the expression level of NLRC3 and IκBα decreased in cells subjected to hypoxia for 12 hours followed by reoxygenation for the indicated time point,while the expression level of p-p65/p65、TRAF6 and K63-linked ubiquitination level of TRAF6 increased(P<0.05).The physiological interaction betweenTRAF6 and NLRC3 was confirmed by Co-IP.Western blot analysis showed that overexpressing NLRC3 had no effect on protein expression of TRAF6 but significantly lowered the TRAF6-K63 linked ubiquitination level and protein expression of p-p65/p65 after H/R treatment,while increased the IκBα oppositely(P<0.05).The nuclear translocation of NF-κB p65 subunit in NLRC3-overexpressing group was significantly inhibited(P<0.05),and the inflammatory factor IL-1β in the cell supernatant decreased accordingly(P<0.05).Conclusion: NLRC3 expression decreases in I/R and H/R models with the prolongation of reperfusion/reoxygenation time.NLRC3 can inhibit the release of pro-inflammatory factors in RAW264.7 macrophages after H/R treatment,and its molecular mechanism may be through regulating the level of K63-ubiquitinated TRAF6 to further inhibit the activation of NF-κB,thereby reducing the inflammatory response.