NLRP3 Inflammasome/IL-1?/Smad Signal Activation Mediated By Aldosterone Induced Reactive Oxygen Species Contributes To Hepatic Fibrosis

BackgroundAldosterone acts as a key risk factor in hepatic fibrosis by activation Hepatic stellate cell(HSC).Unfortunately,its specific mechanism is still not fully understated.Some researchers show that aldosterone promoted the production of excessive reactive oxygen species(ROS)to induce hepatic fibrosis.There are numerous downstream molecules involved in oxidative stress,among which inflammasome has been identified as a novel mechanism of liver injury and fibrosis.NLRP3 inflammasome forms a complex comprised of adaptor proteins such as the apoptosis associated speck like protein(ASC),and the serine protease caspase-1(Caspl).The NI..RP3 inflammasome complex finally results in caspase-1 activation and processing of pro-IL-1? into IL-1?,which mediates the activation of HSC and amplifies chronic liver fibrosis.In this study we used NLRP3 knockout mice to prove the hypothesize that:ROS-mediated activation of NLRP3 inflammasome contributes to aldosterone-induced liver fibrosis in HSC both in vivo and in vitro.Methods1.In vivo,wild type and NLRP3 knockout mice were used to establish CC14 induced liver fibrosis model.In wild type mice group,the RNA level of aldosterone synthesis gene,H2O2 content and the protein levels of NLRP3,ASC,caspase-1,IL-1?,Smad2/3 were measured.In wild type and NLRP3 knockout mice group,all of the methods:HE staining,hepatic fibrosis score,Masson staining,western blot and immunohistochemistry staining were used to explore the levels of hepatic fibrosis.2.In vitro,the protein levels of NLRP3,ASC,caspase-1,IL-1?,type I collagen and ?-SMA were measured by western blot analysis in wild type HSC group and NLRP3 knockout HSC group treated with aldosterone and its inhibitor.H2O2 content was measured in wild type HSC group treated by aldosterone and its inhibitor.The above protein levels were measured in wild type HSC group treated by aldosterone,ROS inhibitor and IL-1? inhibitor(The protein levels of P-smad2/3 and Smad2/3 were also measured in IL-1? inhibitor group).Wild type and NLRP3 knockout HSC group were stimulated by IL-1?,the above protein levels were also tested by western blot.Results1.The RNA level of aldosterone synthesis gene,H2O2 content and protein levels of NLRP3,ASC,caspase-1,IL-1? and smad2/3 were upregulated in wild type CC14 group compared with oil group in vivo.NLRP3 gene knockout mice were also used to establish CC14 induced liver fibrosis model.The level of hepatic fibrosis decreased in NLRP3 knockout CC14 group compared with wild type CC14 group.The protein levels of IL-1?,Smad2/3,?-SMA and type I collagen were also decreased in NLRP3 knockout CC14 group compared with wild type CC14 group.2.To further investigate the role of aldosterone in liver fibrosis,the protein levels of NLRP3,ASC,caspase-1,IL-1?,type I collagen and ?-SMA were measured in vitro as well as H2O2 content and all of them increased in aldosterone stimulation group compared with control group in wild type HSC and decreased in aldosterone inhibitor group compared with aldosterone group.3.All of the above protein levels were decreased in wild type ROS inhibitor stimulation group compared with aldosterone group.The above protein levels were downregulated in NLRP3 knock HSC group treated by aldosterone compared with wild type group treated by aldosterone.4.To investigate the roles of IL-1? in aldosterone induced liver fibrosis,western blot analysis was used to test the above protein levels and the protein levels of P-smad2/3 and smad2/3,all of which increased in aldosterone stimulated group compared with control group in wild type HSC and IL-1? inhibitor reversed the results.The protein levels of above were increased in IL-1?stimulated group compared with control group in wild type HSC and decreased in NLRP3 knockout IL-1? stimulated group compared with wild type IL-1? stimulated group.ConclusionsThe activation of NLRP3 inflammasome/IL-1?/Smad via aldosterone induced ROS promoted the development of liver fibrosis.