Imipramine Inhibits LPS-induced NLRP3 Inflammasome Activation Through Cer/ASM Pathway In RAW264.7 Macrophages

Objective NLRP3 inflammasome is associated with a variety of inflammatory diseases.This study used LPS to induce RAW264.7 cells as an in vitro inflammation model.At the cellular level,the inhibitory effect of the acid sphingomyelinase(ASM)inhibitor imipramine on LPS-induced NLRP3 inflammasome activation in RAW264.7cells was studied,and explore the role of ASM/Ceramide(Cer)pathway in the activation mechanism of NLRP3 inflammasome,and further provide new targets and new ideas for the prevention and treatment of inflammatory related diseases.Methods1 Activation of NLRP3 inflammasome in RAW264.7 cells induced by LPS as an in vitro inflammation model.2 Treatment of cells with different concentrations of imipramine(0,20,40,60,80,100 μmol/L)or exogenous C2-ceramide(0,10,20,30,40,50 μmol/L),the effect of different concentrations of imipramine or C2-ceramide on the activity of RAW264.7cells was detected by CCK-8 method.3 RAW264.7 cells were pretreated with different concentrations of imipramine for 2hours and then treated with LPS for 8 hours.The protein expression of acid sphingomyelinase(ASM),NLRP3,caspase-1,IL-1β and IL-18 were determined by Western blot.The Elisa method was used to detect the release levels of inflammatory cytokines IL-1β and IL-18 in the supernatant of RAW264.7 cells.4 RAW264.7 cells were pretreated with different concentrations of imipramine for 2hours and then treated with LPS for 8 hours.The content of Cer was detected by the method of cell immunofluorescence.5 RAW264.7 cells were treated with different concentrations of C2-ceramide,The protein expression of NLRP3,caspase-1,IL-1β and IL-18 were determined by Western blot.Results1 The effect of different concentrations of imipramine or exogenous C2-ceramide on the activity of RAW264.7 cells by CCK-8 showed that the concentration of imipramine ranged from 0 to 60 μmol/L or C2-ceramide ranged from 0 to 30 μmol/L,there was no obvious toxicity to RAW264.7 cells,and the cell viability was not significantly different from that of the blank control group.2 Compared with the control group,LPS significantly up-regulated the protein expression levels of ASM,NLRP3,caspase-1,IL-1β and IL-18 in RAW264.7 cells(P<0.01),However,pretreatment with imipramine significantly inhibited the protein expression levels of ASM,NLRP3,caspase-1,IL-1β and IL-18 in RAW264.7 cells(P<0.01).3 Compared with the control group,LPS significantly increased the release levels of IL-1β and IL-18 in the supernatants(P<0.01),However,pretreated with imipramine significantly inhibited the release levels of IL-1β and IL-18 in the supernatants(P<0.01).4 Under fluorescence microscope,LPS significantly enhanced the fluorescence intensity in RAW264.7 cells.However,imipramine inhibits this fluorescence enhancement.Through quantitative analysis,LPS significantly increased the content of Cer in RAW264.7 cells(P<0.01),however,pretreated with imipramine significantly inhibited Cer changes induced by LPS(P<0.01).5 C2-ceramide significantly increased the protein expression levels of NLRP3,caspase-1,IL-1β and IL-18,and as the concentration of C2-ceramide increases,the expression of each protein gradually increases(P<0.01).Conclusions Imipramine as an inhibitor of ASM significantly inhibits protein expression of ASMin RAW264.7 cells induced by LPS and further inhibits Cer production.In combination with imipramine inhibits the activation of NLRP3 inflammasome in LPS-induced RAW264.7 cells and exogenous Cer activates NLRP3 inflammasome in RAW264.7 cells,indicates that the ASM/Cer pathway plays a key role in the formation and activation of NLRP3 inflammasome,Imipramine exerts anti-inflammatory activity by inhibiting the activation of NLRP3 inflammasome through the regulation of ASM/Cer pathway.