The Mechanism Of Bile Acids And FXR/NLRP3 Inflammasome In Hepatic Fibrosis

Objective:Bile acids are produced in the liver and are metabolized in the intestine.Hepatic fibrosis often shows changes in the spectrum of bile acids,which may act as a signaling molecule to initiate inflammatory responses.Therefore,the aim of this study was to investigate the changes in bile acid metabolism in patients with hepatic fibrosis and its mechanism of action in the induction of inflammatory responses during hepatic fibrosis.To further investigate the mechanisms by which the Farnesoid X receptor（FXR）inhibits the inflammatory response and changes in hepatic bile acids and gut flora during hepatic fibrosis.The aim is to provide evidence for diagnosis and treatment of hepatic fibrosis.Methods:1.Changes of plasma bile acid spectrum and expression of IL-1βin patients with liver fibrosisPlasma samples from patients with different stages of liver fibrosis and normal controls were collected,and bile acids in plasma of patients were qualitatively and quantitatively analyzed by ultraperformance liquid chromatography coupled to mass spectrometry.The area under the curve of ROC was used to select bile acids with the most diagnostic value of liver fibrosis.Immunohistochemical staining was used to evaluate the expression ofα-SMA and IL-1βin fibrotic and normal liver tissue samples.2.GCDCA induces Liver Fibrosis through NLRP3 Inflammasome pathwayInterference with LPS with or without GCDCA in human hepatic stellate cell line LX2.Construction of NLRP3 knockdown lentivirus.Expression of NLRP3inflammasome-related proteins NLRP3,ASC,pro-Caspase-1,pro-IL-1β,liver fibrosis-associated protein Collagen 1 and bile acid receptor FXR were detected by western blot assay.The concentration of IL-1βin supernatant of cell culture was measured by enzyme-linked immunosorbent assay.The proliferation of LX2 was measured by CCK-8 assay.Wound healing and Transwell assays tested the migration of LX2.Twelve wild-type 8-week-old male C57BL/6J mice were randomly divided into three groups,and 8 NLRP3^-/-with C57BL/6J background male mice were randomly divided into two groups（n=4 in each group）:（1）vehicle group（ip with corn oil）;（2）CCl₄group（ip with 20%CCl₄–corn oil solution）;（3）the GCDCA gavage group;（4）the NLRP3^-/-+oil group;and（5）the NLRP3^-/-+CCl₄group.HE,Masson’s trichrome and Sirius red staining were used to observe the fibrosis and collagen expression.Immunohistochemical staining was used to evaluate the expressions of NLRP3,IL-1βandα-SMA.Western blot was used to detect the expression of NLRP3 inflammasome related proteins NLRP3,pro-Caspase-1,Cleaved caspase-1（20 kd）,pro-IL-1β,mature IL-1β（17 kd）,hepatic fibrosis proteinsα-SMA and Collagen 1 and FXR in liver tissue samples.3.Mechanism of FXR and NLRP3 in liver fibrosisConstruction of FXR overexpression lentivirus.LX2 was treated with the FXR agonist GW4064 or infected with FXR overexpressing lentiviral.The binding of FXR to NLRP3 at the protein level was measured by protein immunoprecipitation assay.The phosphorylation level of NLRP3 was detected by western blot with phospho-NLRP3（serine 295）antibody.4.Changes of bile acids in liver and intestinal flora in mice with liver fibrosis caused by NLRP3 knockout.Mice were divided into control group,CCl₄ group and NLRP3^-/-+CCl₄ group used in the second part of this study.Bile acids in the liver were characterized and quantified by ultraperformance liquid chromatography coupled to mass spectrometry（UPLC-MS/MS）.Bacterial sequencing of intestinal contents was performed with 16s RNA.Enzyme-linked immunosorbent assay（ELISA）was used to detect the concentration of LPS in mouse liver.Results:1.Changes of plasma bile acid spectrum and expression of IL-1βin patients with liver fibrosisA total of 15 bile acids were detected in plasma samples,and primary conjugated bile acids TCA,GCA,TCDCA,and GCDCA were increased at different stages of fibrosis compared to normal controls.GCDCA was increased at any stage of liver fibrosis classification,P<0.05.ROC curves were analyzed for the area under the curve（AUC）of differential bile acids,with the largest area under the curve of 0.758 for the four primary conjugated bile acids and the largest area under the curve of GCDCA for single bile acids.Immunohistochemistry staining showed thatα-SMA positive expression was significantly higher in fibrotic liver tissue samples than in control group,with increased expression of the inflammatory factor IL-1β.2.GCDCA induces Liver Fibrosis through NLRP3 Inflammasome pathwayIn the LPS-primed LX2 cells,with the increase of GCDCA stimulation concentration and the extension of time,the protein expressions of NLRP3-related proteins and collagen 1 increased,as well as IL-1βconcentration in supernatant of cell culture.While FXR expression decreased.GCDCA stimulation increased LX2 proliferation and migration.The knock-down of NLRP3 obviously weakened the trend of increasing NLRP3-related proteins and Collagen 1 caused by GCDCA stimulating in LX2.In liver fibrosis mouse models,both GCDCA and CCl₄induced fibrosis,while NLRP3 knockout significantly weakened the increasing expression of liver fibrosis and collagen 1 caused by CCl₄intervention.Immunohistochemical staining results showed that the positive expressions of inflammatory proteins NLRP3 and IL-1βand fibrosis marker proteinα-SMA in the liver tissues of GCDCA and CCl₄groups were higher than those in the control group,while NLRP3 knockout weakened these changes caused by CCl₄.3.Mechanism of FXR and NLRP3In LPS-primed LX2 cells,FXR agonist GW4064 or FXR overexpression reduced the expression of collagen 1 and secreted IL-1βin supernatant of cell culture induced by GCDCA.Immunocoprecipitation showed that FXR and NLRP3 protein combined with each other.Western blot showed that GCDCA could increase the phosphorylation of NLRP3 serine 295（ser 295）,while the activation of FXR by GW4064 weakened this trend.Moreover overexpression of FXR weakened the increasing trend of phosphorylation of NLRP3（ser 295）induced by LPS.4.Changes in liver bile acids and intestinal microbiota induced by NLRP3knockoutComparing the relative abundances of primary and secondary bile acids and 7α-hydroxy-4-cholesterol-3-one（C4）in control group,CCl₄group and NLRP3^-/-+CCl₄group,the C4 concentration was significantly different（P=0.0107）.In multivariate analysis,14 secondary bile acids decreased and 3 bile acids increased in CCl₄group compared to control group.In contrast,16 bile acids of the NLRP3^-/-+CCl₄group had increased compared to CCl₄groups,and 8 of them had decreased in the CCl₄group compared to control group（57%,8/14）.In univariate analysis,C4 in CCl₄group was higher,while the levels of apo CA,TDCA and UDCA-7s were lower compared with the control group.The levels of b MCA,TCA,UDCA-7S and apo CA increased in NLRP3^-/-+CCl₄group compared with CCl₄group.Potential biomarkers were screened by the intersection of multivariate and univariate analysis.The biomarkers in the CCl₄group included secondary bile acid apo CA（P=0.001,FC=0.16）,TDCA（P=0.026,FC=0.15）,UDCA-7S（P=0.029,FC=0.027）and C4（P=0.029,Fold Change=21.95）compared to control group.The The biomarkers in the NLRP3^-/-+CCl₄group included primary bile acid b MCA（P=0.017,FC=2.23）and TCA（P=0.045,FC=3.68）and secondary bile acid UDCA-7S（P=0.029,FC=5.90）and apo CA（P=0.031,FC=2.55）compared to CCl₄group.However,these two kinds of secondary bile acids were decreased in CCl₄group compared with the control group.Bacteroides and Parabacteroides were more abundant in the CCl₄group than in the other groups at the genus level.Prevotella,Turicibacter,Ruminococcus,and Clostridium were most abundant in the NLRP3^-/-+CCl₄group,while Allobaculum was most abundant in the control group.Spearman correlation of gut flora（genus level）with liver differentially metabolized bile acids showed that Parabacteroides were significantly negatively correlated with TDCA,apo CA,and UDCA-7S,and significantly positively correlated with C4,whereas Allobaculum was positively correlated with TDCA,apo CA and UDCA-7S,and negatively correlated with C4.In addition,Bacteroides was negatively correlated with TDCA and positively correlated with C4.Compared with the control group,the concentration of LPS in liver in CCl₄group increased,while NLRP3^-/-weakened this increasing trend.Spearman correlation analysis showed that LPS concentration in liver was positively correlated with C4 concentration in liver,Bacteroides and Parabacteroides in intestine,but negatively correlated with Allobaculum.Conclusions:（1）The levels of serum primary conjugated bile acids in patients with hepatic fibrosis are increased.（2）BAs induce hepatic fibrosis via the NLRP3 inflammasome signaling pathway.（3）FXR inhibits liver fibrosis by inhibiting the phosphorylation of NLRP3（ser 295）.（4）NLRP3 gene knockout could partially reverse the changes of bile acid in liver of mice with liver fibrosis,cause changes of intestinal flora and reduce the occurrence of intestinal bacterial translocation.（6）Allobaculum is a potential protective bacteria.