The Effect Of NLRP3 Inflammasome On NaAsO₂-mediated Hepatic Stellate Cell Activation And Liver Fibrosis

Objective:Related studies have shown that chronic inorganic arsenic exposure can cause liver damage and liver fibrosis in humans and rats.Hepatic stellate cells are the main source of collagen fibers,and their activation is a key factor in the formation of liver fibrosis.NOD-like receptor protein 3（NLRP3）inflammasome is the most classic member of the inflammasome family,which has been extensively studied,and is closely related to liver fibrosis and hepatic stellate cell activation.Our previous researches have confirmed that inorganic arsenic exposure can induce the occurrence of non-alcoholic fatty liver disease and the activation of hepatic stellate cells in mice,but liver fibrosis is not obvious.In addition,previous research by our group also showed that exposure to inorganic arsenic can activate NLRP3 inflammasome in the livers of mice,and stimulate liver autophagy at the same time.However,in the process of inorganic arsenic-induced liver fibrosis,the effects of changes in autophagy levels and activation of NLRP3inflammasome on hepatic stellate cells still not clear.Therefore,the purpose of this study is to investigate the role of NLRP3 inflammasome in the activation of hepatic stellate cells during NaAsO₂-induced liver fibrosis in Sprague-Dawley rats,and the regulating effect of autophagy in the process.Methods:In vivo,10-week-old male Sprague-Dawley rats were used as research objects and randomly divided into 3 groups,including a control group,2.5 mg/kg BW treatment group and 5 mg/kg BW treatment group.The exposure method was gavage with NaAsO₂ for every 12 h,and the exposure time was 9 months.During the exposure period,rats could drink and eat freely.After exposure,rats were sacrificed and liver tissues were collected and paraffin sections were made for experimental studies.Hematoxylin-eosin staining（H&E staining）was used to detect liver inflammation in rats;Sirius red staining and Masson staining were used to detect liver fibrosis in rats;serum alanine aminotransferase（ALT）detection kit was used to detect serum glutamate aminotransferase content;serum aspartate aminotransferase（AST）content was detected by the AST assay kit;tissue immunofluorescence was used to detect the expression level of hepatic stellate cell activation marker protein-smooth muscle actin（a-SMA）;Western blot detection of hepatic stellate cell activation-related marker protein expression levels in rat liver tissues:collagenⅠ（Collagen-1）,a-SMA,matrix metalloproteinase-1（MMP-1）and metalloproteinases tissue inhibitor-1（TIMP-1）,NLRP3 inflammasome and downstream related protein expression levels:NLRP3,caspase-1 precursor（pro-caspase-1）,apoptosis-associated speckle protein（ASC）containing caspase recruitment domain,caspase-1,interleukin-1bprecursor（pro-IL-1b）and interleukin-1b（IL-1b）,expression levels of autophagy-related proteins:microtubule-associated protein light chain 3-Ⅱ（LC3-Ⅱ）and p62,and cathepsin B in the cytoplasm（CTSB）expression level.In vitro,rat hepatic stellate cell line HSC-t6 and rat primary hepatic stellate cells were used as research objects.MTT assay was used to detect the survival rate of HSC-t6cells treated with NaAsO₂ at different concentrations;HSC-t6 cells were treated with 0,1,2,4mM NaAsO₂ for 24 h.The expression levels of autophagy-related proteins in HSC-t6 cells were detected by Western blot:LC3-Ⅱand p62,cytoplasmic CTSB levels;HSC-t6 cells were pretreated with lipopolysaccharide（LPS）and then treated with 0,1,2,4mM NaAsO₂ for 24 h,and the expression levels of NLRP3 inflammasome and relevant downstream proteins were detected by Western blot:NLRP3,caspase-1 p20 and IL-1b;hepatic stellate cell activation-related marker protein expression were detected by Western blot:a-SMA,Collage-1,MMP-1 and TIMP-1;specific inhibitors of NLRP3（MCC950）,CTSB release inhibitors（CA-074 me）,and autophagy specific inhibitor（3-MA）were used in HSC-t6 cells and rat primary hepatic stellate cells,and then treated with 4mM NaAsO₂ for 24 h.Western blot was used to detect the activation of HSC-t6cells,NLRP3 inflammasome activation,and CTSB levels in the cytoplasm;immunofluorescence was used to detect the activation level of primary hepatic stellate cells and CTSB release levels in HSC-t6 cells;acridine orange（AO）fluorescent probe was used to detect the degree of lysosomal damage in HSC-t6 cells.Results:In vivo,rats were exposed to 0 mg/kg BW,2.5 mg/kg BW and 5 mg/kg BW NaAsO₂ for 9 months.The results of H&E staining,serum ALT and AST showed that compared with the control group,rats with high concentration of NaAsO₂appeared obvious inflammation and liver damage;Sirius red staining and Masson staining showed that compared with the control group,rats with NaAsO₂ exposure occurred a large amount of collagen deposition,suggesting liver fibrosis formation;tissue immunofluorescence results showed that compared with the control group,the expression level of hepatic stellate cell activation marker proteina-SMA was increased in the NaAsO₂-exposed group;compared with the control group,Western blot results showed that the expression level of autophagy-related protein LC3-Ⅱwas increased in the NaAsO₂-exposed group,and the expression level of p62 was decreased;the level of cytoplasmic CTSB was increased;NLRP3 inflammasome and downstream related protein:pro-caspase-1,caspase-1,pro-IL-1b,and IL-1bexpression levels were increased;hepatic stellate cell activation-related marker protein:Collagen-1,a-SMA,and TIMP-1 expression levels were increased,MMP-1 expression levels were decreased.In vitro,MTT assay result showed that:HSC-t6 cells were treated with different concentrations of NaAsO₂ for 24 hours,and the cell survival rate gradually decreased with the increase of the concentration of NaAsO₂;HSC-t6 cells were treated with 0,1,2,4mM NaAsO₂ for 24 hours.Compared with the control group,Western blot results showed that LC3-Ⅱ,cytoplasmic CTSB,NLRP3,caspase-1 p20,IL-1b,Collagen-1,a-SMA and TIMP-1 expression were increased,p62 and MMP-1 expression were decreased.After pretreatment with NLRP3 specific inhibitor MCC950,Western blot results of HSC-t6 cells showed that the inhibitor pretreatment group,compared with the exposed group（4mM NaAsO₂）,the expression levels of caspase-1 p20,IL-b,Collagen-1,a-SMA and TIMP-1 were decreased,and the expression level of MMP-1 was increased.For rat primary hepatic stellate cells,immunofluorescence results showed that compared with the high-dose exposure group（5 mg/kg BW）,the expression level ofa-SMA in the inhibitor pretreatment group was decreased.After pretreatment with CTSB release inhibitor CA-074 me,the results of Western blot of HSC-t6 cells showed that the inhibitor pretreatment group,compared with the exposed group,the expression levels of NLRP3,caspase-1 p20,IL-1b,Collagen-1,a-SMA and TIMP-1 were decreased,and the expression level of MMP-1 was increased;the results of cellular immunofluorescence showed that the release of CTSB in the inhibitor pretreatment group was reduced compared with the exposure group;for rat primary hepatic stellate cells,immunofluorescence results showed that compared with the high-dose exposure group,the expression level ofa-SMA in the inhibitor pretreatment group was reduced.After pretreatment with 3-MA,an autophagy-specific inhibitor,Western blot results on HSC-t6cells showed that the inhibitor pretreatment group,compared with the exposed group,the expression levels of NLRP3,caspae-1 p20,IL-1band cytoplasm CTSB,Collagen-1,a-SMA and TIMP-1 were decreased,and the expression level of MMP-1 was increased.Cellular immunofluorescence results showed that CTSB release was reduced in the inhibitor pretreatment group compared with the exposure group;AO fluorescent probe results showed that the degree of lysosomal damage was reduced in the inhibitor pretreated group compared with the exposed group;for rat primary hepatic stellate cells,immunofluorescence results showed that compared with the high-dose exposure group,the expression level ofa-SMA in the inhibitor pretreatment group was reduced.Conclusion:NaAsO₂ induce hepatic stellate cell activation through the autophagy-CTSB-NLRP3 inflammasome pathway,leading to the formation of liver fibrosis.