NLRP3 Inflammasome Activation Is Involved In Liver Fibrosis Of Schistosoma Japonicum Infected Mice Via NF-κB

Background and Aims:Liver fibrosis develops in chronic liver disease,leading to liver failure.Liver fibrosis is caused by Schistosoma japonicum(S.japonicum)infection.The schistosome eggs deposited in the liver induce inflammatory cells,such as kupffer cells(KCs),to produce cytokines.These progress in chronic inflammatory responses,which activate hepatic stellate cells(HSCs),resulting in schistosome-associated liver fibrosis(SSLF).The NOD-like receptor protein 3(NLRP3)inflammasome,as an important signaling molecule,involved in inflammatory responses in the liver.It has been found that NLRP3 inflammasome is expressed in SSLF,but the function and underlying mechanisms remain still unclear.In this study,we established a mouse model of S.japonicum infection,and investigated the roles and possible mechanisms of NLRP3 and its related signaling pathways in SSLF through intervening NLRP3 inflammasome at different stages of infection.Methods:Forty-five BALB/c mice(6-8 weeks old,female)were infected with 15 cercariae through the abdominal skin for 56 days to establish a model of S.japonicum infection.Fifteen mice were selected and received intraperitoneal(i.p.)injections of MCC950(10 mg/kg),an inhibitor of NLRP3 inflammasome,on the day of S.japonicum infection(the initial stage,M0 group)and on the day 22 post-infection(before production of the eggs,M4 group),followed by two consecutive days,and every two days thereafter,until day 56 after S.japonicum infection.Fifteen infected mice were used as the S.japonicum infection group(SJ group).Fifteen mice of the same age and sex were selected as the control group(C group).Six mice were used and were given intragastrically NF-κB inhibitor PDTC(100 mg/kg)every day from 7 days before S.japonicum infection until 56 days after the infection.The expressions of NLRP3,α-SMA and Collagen Ⅰ in liver tissues were detected by RT-qPCR.The expressions of NLRP3,pro-caspase-1,Caspase-1,pro-IL-1β,IL-1β,NF-κB p65,p-NF-κB p65,IκBα,and p-IκBα in liver tissues were determined by Western blot.The hydroxyproline(Hyp)contents in liver tissues were detected in Hyp kits.The serum aminotransferase was tested in ALT and AST kits.The serum IL-1β expression was examined by ELISA.The hepatic NLRP3 protein was stained by immunohistochemistry(ICH).HE and Masson staining was used to observe the areas of egg granulomas and collagen fibers,respectively.Immunofluorescence(IF)staining was performed for the co-localization area percentage analysis of NLRP3 or NF-κB p65 with F4/80 in KCs,and the co-localization area percentage analysis of NLRP3 with α-smooth muscle actin(α-SMA)in HSCs.In addition,the mice were weighed once a week and the weight curves were plotted.The livers and spleens were weighed,and the liver and spleen indexes were calculated.The length of the spleens was measured.The numbers of the male and female worms in the mesenteric veins,and the numbers of eggs in the livers were counted.The proportion and percentage of the male and female worms was calculatedThe human LX-2 cells were used for in vitro experiments.The experiments were divided into three groups.The first group experiment was established that the expressions of NLRP3,pro-IL-1β,IL-1β,α-SMA,and Collagen Ⅰ were determined in LX-2 cells after MCC950 treatment.The second group experiment was performed that the expressions ofα-SMA,and Collagen Ⅰ were measured in LX-2 cells after PDTC treatment.The third group experiment was administered that PDTC treatment affected on the expressions ofα-SMA,and Collagen Ⅰ after the LX-2 cells were transfected pLVX-NLRP3.The expressions of NLRP3,α-SMA,and Collagen Ⅰ were detected by RT-qPCR.The expressions of NLRP3,pro-IL-1β,IL-1β,IκBα,p-IκBα,α-SMA,and Collagen Ⅰ were determined by Western blot.The fluorescence intensity of NLRP3 in LX-2 cells was labeled in IF staining.The primary KCs and HSCs were isolated from the livers of the different group mice.The NLRP3 expression was examined in primary KCs and HSCs by IF staining.The expressions of α-SMA,and Collagen Ⅰ in the primary HSCs were detected by RT-qPCR.The expressions of NLRP3,pro-caspase-1,Caspase-1,pro-IL-1β,and IL-1βin primary KCs and HSCs,the expression of NF-κB p65,and p-NF-κB p65 in the primary KCs,and the expressions of α-SMA,and Collagen Ⅰ in the primary HSCs,were determined by Western blot.Results:Compared to blank control(C group),the expressions of NLRP3,pro-caspase-1,Caspase-1,pro-IL-1β,IL-1β,α-SMA,Collagen Ⅰ,NF-κB p65,p-NF-κB p65,p-IκBα and the contents of Hyp were increased,and the levels of serum IL-1β and ALT/AST were also increased in liver tissues of the mice infected with S.japonicum with 56 days(SJ group)Compared with simple infection control(SJ group),the expressions of hepatic NLRP3,Caspase-1,IL-1β,α-SMA,Collagen I,NF-κB p65,p-NF-κB p65,and the contents of hepatic Hyp,and serum IL-1β and ALT/AST were reduced;the areas of hepatic collagen fibers and the egg granulomas were smaller in mice treated with MCC950 on the day of S.japonicum infection(M0 group).However,the expressions of hepatic NLRP3,Caspase-1,IL-1β,α-SMA,Collagen I,NF-κB p65,p-NF-κB p65,the contents of hepatic Hyp,and serum IL-1β and ALT/AST were increased;the areas of hepatic collagen fibers and egg granulomas were larger in mice given with MCC950 at day 22 post-infection(M4 group)compared to simple infection control(SJ group).When compared to simple infection control(SJ group),the expressions of hepatic α-SMA,Collagen I,p-IκBα,and serum ALT/AST were reduced;the areas of hepatic collagen fibers were smaller in mice treated with PDTC 7 days before S.japonicum infection(SJ+PDTC group).The results of the IF double staining showed that the co-localization area percentage of NLRP3 or NF-κB p65 with F4/80 in KCs,and the co-localization area percentage of NLRP3 with α-SMA in HSCs,had both a rise,in S.japonicum infection group,compared to C group.Compared with SJ group,the co-localization area percentage of NLRP3 or NF-κB p65 with F4/80 in KCs,and NLRP3 with α-SMA in HSCs,was decreased after MCC950 was administered on the day of infection;while it was increased after MCC950 was given at day 22 post-infection Moreover,compared with the results in the SJ group,the liver and spleen indexes were increased in the M4 group,whereas the spleen index was only decreased in the M0 group The spleens were enlarged at 56 days after infection,but there were no evident differences among the three groups(SJ,M0 and M4 groups).It was similar in the numbers of female worms,the ratio and percentage of female and male worms in the mesenteric veins,and the numbers of eggs in liver tissues.But the numbers of male worms in the M4 group were less than the SJ group.Additionally,the weight of SJ,M0,and M4 group mice showed a sharp drop from the 35th to 42nd day,followed by more gradual weight loss,while control mice showed a steady gain in weight until the end of the experiment.There were no significant weight differences among SJ,M0 and M4 groups at the 56th day,but a higher weight was observed at the 7th day in the M0 group,and a lower weight was observed at the 35th day in the M4 group.In vitro,soluble egg antigens(SEA)-lipopolysaccharide(LPS)-adenosine triphosphate(ATP)could induce the expressions of NLRP3,pro-IL-1β,IL-1β,p-IκBα,α-SMA,and Collagen Ⅰ in LX-2 cells.MCC950 treatment could down-regulate the expressions of NLRP3,IL-1β,α-SMA,and Collagen Ⅰ induced by SEA-LPS-ATP in LX-2 cells.PDTC treatment could reduce the expressions of p-IκBα,α-SMA,and Collagen Ⅰ induced by SEA-LPS-ATP in LX-2 cells.The LX-2 cells were transfected with pLVX-NLRP3,and then treated with SEA-LPS-ATP.The expressions of p-IκBα,α-SMA and Collagen Ⅰ were increased in LX-2 cells.PDTC treatment could decrease NLRP3-mediated expressions of p-IκBα,α-SMA,and Collagen Ⅰ.In addition,the protein expressions of NLRP3,pro-caspase-1,Caspase-1,pro-IL-1β,IL-1β,NF-κB p65 and p-NF-κB p65 were up-regulated in the primary KCs;the protein expressions of NLRP3,pro-caspase-1,Caspase-1,pro-IL-1β,IL-1β were increased,and the protein and mRNA expressions ofα-SMA,and Collagen Ⅰ were also increased in the primary HSCs isolated from the SJ group mice,compared with C group mice.However,the expressions of NLRP3,Caspase-1,IL-1β,NF-κB p65 and p-NF-κB p65 protein in the primary KCs were decreased;the expressions of NLRP3,Caspase-1,and IL-1β protein were reduced,and α-SMA and Collagen Ⅰ protein and mRNA in the primary HSCs were also reduced in mice treated with MCC950 on the day of infection compared to SJ group.Contrary to the aforementioned results in SJ group,the expressions of NLRP3,Caspase-1,IL-1β,and NF-κB p65 and p-NF-κB p65 protein in the primary KCs had a rise;the expressions of NLRP3,Caspase-1,and IL-1β protein were raised,and α-SMA and Collagen Ⅰ protein and mRNA in the primary HSCs were also raised in mice treated with MCC950 at day 22 post-infectionConclusion:NLRP3 inflammasome activation plays an important role in the liver fibrosis of S.japonicum infected mice.Block the activation of NLRP3 inflammasome by reducing the expressions of NLRP3/Caspase-1/IL-1β,and improve SSLF,after MCC950 is injected at the initial stage of S.japonicum infection.In addition,the NF-κB expression and activation are reduced after MCC950 is administered at the initial stage of S.japonicum infection.NF-κB inhibited mice are protected from SSLF with PDTC is administered 7 days before S.japonicum infection.Furthermore,PDTC could inhibit NLRP3-mediated liver fibrosis in LX-2 cells.