The Role And Mechanism Of NLRP3 Inflammasome Activation In Schistosoma Japonicum Induced Liver Fibrosis

Backgroud and aims Since granulomatous and fibrosing inflammation in the liver constitute the main pathology during S.japonicum infection,understanding the immunopathogenisis of granulomatous inflammation may elucidate novel therapeutic targets and approaches by which to treat schistosomiasis-associated liver fibrosis(SSLF).Our previous study has demonstrated that S.japonicum infection in vivo or soluble egg antigen(SEA)stimulation in vitro can activate the NLRP3 inflammasome in HSCs.In the present study,we aimed to further investigate the role of NLRP3 inflammasome activation in liver fibrogenesis during S.japonicum infection,and to discuss the activation mechanism of NLRP3 inflammasome in hepatic stellate cells(HSCs).Methods The mouse model of schistosomiasis-associated liver fibrosis was established by abdominal infection with S.japonicum cercariae,the NLRP3 deleted mouse model was established by tail vein injection with adeno-associated virus 8(AAV8).Fluorescent microscopy was used to detect the green fluorescence in liver tissue frozen sections,Western-blotting was used to detect NLRP3 in liver tissues and verify that the NLRP3 deleted mouse model was successfully established.H&E and Masson staining methods were used to detect liver tissue damage and determine whether the mouse model of schistosomiasis-associated liver fibrosis was successfully established,and to verify the effect of NLRP3 deletion on schistosomiasis-associated liver fibrosis.Confocal microscopy was used to detect the co-localization of NLRP3 and ASC,NLRP3 and caspase-1 in liver tissue frozen sections,and verify NLRP3 inflammasome formation in the liver.The co-localization of Desmin and NLRP3 in liver tissue frozen sections was detected to verify NLRP3 inflammasome formation in HSCs.Primary mouse HSCs were used to confirm the activation of NLRP3inflammasome induced by SEA.Immunoprecipitation,si RNA or drug inhibition were used to discuss the mechanism of NLRP3 inflammasome activation in HSCs.Results The mouse model of schistosomiasis-associated liver fibrosis and the NLRP3 deleted were successfully constructed by abdominal infection with S.japonicum cercaria and tail vein injection with AAV8.S.japonicum infection caused liver structural damage,infiltration of inflammatory cells and increased collagen deposition in the liver,which were inhibited by inhibition of NLRP3 inflammasome.The co-localization of NLRP3 and ASC,NLRP3 and caspase-1,NLRP3 and desmin in liver tissues of mice infected with S.japonicum were increased.HSCs were stimulated with SEA(50μg/ml),intracellular activated caspase-1 and IL-1β levels in supernatant increased over time.After transfection with si NLRP3 to inhibit NLRP3,the intracellular activated caspase-1 and IL-1β levels in supernatant were decreased.The protein levels of p-Syk,p-JNK and p-p38 in HSCs were increased after stimulated with SEA.After inhibiting Syk and JNK,the activation of NLRP3 inflammasome induced by SEA was decreased.It was also demonstrated that Syk was associated with the components of NLRP3 inflammasome.After blocking the dectin-1 receptor on the HSC surface,SEA induced p-syk,activated caspase-1 in HSC,and IL-1β in supernatant were significantly reduced.Conclusions S.japonicum infection can activate the NLRP3 inflammasome in HSCs,inhibiting NLRP3 inflammasome can reduce the fibrosis caused by S.japonicum infection.The activation of NLRP3 inflammasome in HSC is associated with Syk,dectin-1 receptor and JNK.