NLRP3 Inflammasomes Activation And Related Mechanisms Of Activation In Mouse Hepatic Stellate Cells During Schistosoma J.Infection

ObjectiveTo study the effects of Schistosoma J.infection on the activation of NLRP3 inflammasomes in primary hepatic stellate cells(HSCs).Method1)There are three groups:Control group,Infected group and Infected + YVAD group.In the Infected + YVAD group,infected mice were injected with YVAD(I.P.lmg/kg/24h),a caspase-1 inhibitor prior to Schistosoma J.infection for 1 week and after such infection for 6 weeks.HE and Masson staining were used to measure liver injuries and liver fibrosis.2)Immunohistochemistry(IHC)was used to detect the expression of IL-1β in liver tissues and compare the differences among Control group,Infected group and Infected+ YVAD group.3)Detect the co-localization of IL-1β and desmin,HSC marker,in liver tissues by confocal microscopy and compare the differences among these three groups.Results1)Mice were sacrificed to extract liver tissues for HE and Masson stain after 6 weeks of such infection.In Infected group,the results revealed damaged liver structure,eggs deposition into hepatic portal vein and the formation of egg granulomas with a large amount of collagens deposition and inflammatory cells infiltration around.However,these typical characteristics were not observed in the livers of control mice.Besides,caspase-1 inhibitor,YVAD,can obviously reduce liver injuries and hepatic fibrosis.2)IHC results showed that there were increased IL-1β expressions around egg granulomas in liver tissues in infected mice,which indicated that Schistosoma J.infection can activate NLRP3 inflammasomes in liver.However,YVAD can inhibit the increase in IL-1β levels.3)Detect the co-localization of IL-1β and desmin,HSCs marker,in liver tissues by confocal microscopy.The aggregation of these two molecules indicated the NLRP3 inflammasomes activation in HSCs,which could be inhibited by YVAD,caspase-1 inhibitor.ConclusionSchistosoma J.infection can activate NLRP3 inflammasome in HSCs.YVAD,caspase-1 inhibitor,can inhibit the NLRP3 inflammasome activation in HSCs and obviously reduce the liver injuries and liver fibrosis during Schistosoma J.infection.ObjectiveTo study the effects of soluble egg antigen(SEA)on NLRP3 inflammasome formation and activation in hepatic stellate cells(HSCs)in vitroMethod1)Extract primary mouse HSCs from the normal mice.2)Mouse HSCs were exposed to SEA stimulation at different time points(12,24 and 48 hours).Observe the co-localization of NLRP3 with ASC or Caspase-1 by confocal microscopy and the expressions of pro-caspase-1 and active caspase-1 in HSCs by western blotting.Besides,detect caspase-1 activity in HSCs and IL-1β production in the supernatant of cultured HSCs.3)Mouse NLRP3 gene in HSCs was silenced by Nlrp3 shRNA transfection.Detect the co-localization of NLRP3 with ASC or caspase-1,expressions of pro-caspase-1 and active caspase-1,caspase-1 activity in HSCs and IL-1β production in the supernatant of cultured HSCs after 24-hour treatment of SEA.4)HSCs were treated by both caspase-1 inhibitor,YVAD and SEA for 24h,then observe the co-localization of NLRP3 with ASC or caspase-1,expressions of pro-caspase-1 and active caspase-1,caspase-1 activity in HSCs and IL-1β production in the supernatant of cultured HSCs.Results1)The co-localization of NLRP3 with ASC or caspase-1 increased upon SEA stimulation at different time points(12,24,48 hours),indicating the aggregation of these inflammasome molecules.The maximum co-localization level was observed after 24-houur treatment of SEA.We also found that SEA significantly increased the level of active caspase-1.However,SEA has no effects on pro-caspase-1 level.Consistently,biochemical analysis showed that SEA enhanced caspase-1 activity and IL-1β release in HSCs.2)Knockdown of mouse NLRP3 gene by Nlrp3 shRNA in HSCs markedly inhibited SEA-induced co-localization of NLRP3 with ASC or caspase-1 and attenuated SEA-increased level of active caspase-1.Besides,NLRP3 gene silencing significantly blocked SEA-induced caspase-1 activity and IL-1β production in HSCs.3)Blockade of caspase-1 activity by caspase-1 inhibitor,YVAD almost completely abolished SEA-induced increases in active caspase-1 level by western blot analysis and substantially suppressed SEA-enhanced caspase-1 activity and IL-1(3 release in HSCs.ConclusionSEA can induce the formation and activation of NLRP3 inflammasomes in HSCs in vitro.ObjectiveTo study the molecular mechanisms of SEA-induced NLRP3 inflammasomes formation and activation in hepatic stellate cells(HSCs).Method1)HSCs were stimulated without or with SEA(50 μg/ml)in the presence of PBS,cathepsin B inhibitor Ca-074Me(5 μM),potassium channel blocker glibenclamide(Glib,10μM)or ROS scavenger N-acetyl-L-cysteine(NAC,10μM).Then observe the co-localization of NLRP3 with ASC or caspase-1,expressions of po-caspase-1 and active caspase-1,caspase-1 activity in HSCs and IL-1β productions in the supernatant of cultured HSCs.2)HSCs were stimulated without or with SEA in the presence of PBS,NADPH oxidase inhibitor gp91 pep(20 μM).Then observe the expressions of pro-caspase-1 and active caspase-1 in HSCs and IL-1β productions in the supernatant of cultured HSCs.3)Cathepsin B gene in HSCs was silenced by cathepsin B siRNA transfection.Detect the changes of SEA-induced NLRP3 inflammasomes formation and activation in HSCs after 24-hour treatment of SEA.4)Observe the effects of silencing cathepsin B gene by cathsi RNA on SEA-induced lysosome membrane permeability,subsequent cathepsin B release and its activity in HSCs.Besides,detect the changes of SEA-induced cathepsin B activity after using cathepsin B inhibitor Ca-074Me.5)Observe the effects of lysosome membrane stabilizer,dexamethasone(Dex,100 μM)or chloroquine(CQ,20μM),on SEA-induced NLRP3 inflammasomes activation,including the expressions of pro-caspase-1 and active caspase-1 in HSCs and IL-1βproductions in the supernatant of cultured HSCs.Results1)It was found that inhibition of both cathepsin B activity and ROS release in HSCs markedly attenuated SEA-induced colocalization of NLRP3 with ASC or caspase-1.Consistently,inhibition of cathepsin B activity and scavenging of ROS release by using cathepsin B inhibitor,Ca-074Me and N-acetyl-L-cysteine(NAC)almostly completely blocked SEA-induced increases in active caspase-1 level,caspase-1 activity and IL-1βproduction in HSCs.However,glibenclamide(Glib),a K channel blocker had no significant effect on SEA-induced NLRP3 inflammasome formation and activation.2)We also tested whether inhibition of NADPH oxidase attenuates SEA-induced caspase-1 activation and IL-1β production in HSCs.It was found that inhibition of NADPH oxidase by gp-91pep significantly attenuated the SEA-induced increases in active caspase-1 levels and IL-1β production in HSCs,implicating a role of NADPH oxidase in NLRP3 infammasomes activation.3)We demonstrated that silencing of cathepsin B gene markedly inhibited SEA-induced co-localization of NLRP3 with ASC or caspase-1 and reduced SEA-enhanced cleavage of pro-caspase-1,caspase-1 activity and production of IL-1β.These results further confirmed that cathepsin B is indeed involved in SEA-induced NLRP3 inflammasomes formation and activation in HSCs.4)Our results demonstrated that SEA stimulation leaded to decreased fluorescence intensity of red puncta,and silencing cathepsin B genes decreased fluorescence intensity of cathepsin B substrate in lysosomes,and SEA stimulation lead to decrease in fluorescence intensity of cathepsin B substrate in these lysosomes,which is an indicator for the leakage of cathepsin B into cytoplasm.Silencing cathepsin B genes or cathepsin B inhibitor Ca-074Me also reduce cathepsin B activity,while SEA stimulation enhanced cathepsin B activity in control HSCs,but not in HSCs treated with cathepsin B siRNA or Ca-074Me.5)We also tested the effect of lysosome membrane stabilizer on SEA-induced caspase-1 activation and IL-1β production.It was found that lysosome membrane stabilizer dexamethasone or chloroquine significantly abolished the SEA-induced increases in active caspase-1 levels and IL-1β production in HSCs.ConclusionSEA induced the formation and activation of NLRP3 inflammasomes,which was associated with both redox regulation and lysosomal dysfunction,but not with potassium channel activation.