AIM:To study the interleukin-1(IL-1)pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4^(-/-)(Abcb4^(-/-))mouse model.METHODS:Female and male Abcb4^(-/...AIM:To study the interleukin-1(IL-1)pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4^(-/-)(Abcb4^(-/-))mouse model.METHODS:Female and male Abcb4^(-/-)mice from 6 to 13 mo of age were analysed for the degree of cholestasis(liver serum tests),extent of liver fibrosis(hydroxyproline content and Sirius red staining)and tissue-specific activation of signalling pathways such as the IL-1 pathway[quantitative polymerase chain reaction(q PCR)].For in vivo experiments,murine hepatic stellate cells(HSCs)were isolated via pronasecollagenase perfusion followed by density gradient centrifugation using female mice.Murine HSCs were stimulated with up to 1 ng/m L IL-1βwith or without 2.5μg/m L Anakinra,an IL-1 receptor antagonist,respectively.The proliferation of murine HSCs was assessed via the Brd U assay.The toxicity of Anakinra was evaluated via the fluorescein diacetate hydrolysis(FDH)assay.In vivo 8-wk-old Abcb4^(-/-)mice with an already fully established hepatic phenotype were treated with Anakinra(1 mg/kg body-weight daily intraperitoneally)or vehicle and liver injury and liver fibrosis were evaluated via serum tests,q PCR,hydroxyproline content and Sirius red staining.RESULTS:Liver fibrosis was less pronounced in males than in female Abcb4^(-/-)animals as defined by a lower hydroxyproline content(274±64μg/g vs 436±80μg/g liver,respectively;n=13-15;P<0.001;MannWhitney U-test)and lower m RNA expression of the profibrogenic tissue inhibitor of metalloproteinase-1(TIMP)(1±0.41 vs 0.66±0.33 fold,respectively;n=13-15;P<0.05;Mann-Whitney U-test).Reduced liver fibrosis was associated with significantly lower levels of F4/80 m RNA expression(1±0.28 vs 0.71±0.41 fold,respectively;n=12-15;P<0.05;Mann-Whitney U-test)and significantly lower IL-1βm RNA expression levels(1±0.38 vs 0.44±0.26 fold,respectively;n=13-15;P<0.001;Mann-Whitney U-test).No gender differences in the serum liver parameters[bilirubin;alanine aminotransferase(ALT);aspartate aminotransferase and alkaline phosphatase(AP)]were found.In vitro,the administration of IL-1βresulted in a significant increase in HSC proliferation[0.94±0.72 arbitrary units(A.U.)in untreated controls,1.12±0.80 A.U.at an IL-1βconcentration of 0.1 ng/m L and 1.18±0.73 A.U.at an IL-1βconcentration of 1 ng/m L in samples from n=6 donor animals;P<0.001;analyses of variance(ANOVA)].Proliferation was reduced significantly by the addition of 2.5μg/m L Anakinra(0.81±0.60 A.U.in untreated controls,0.92±0.68 A.U.at an IL-1βconcentration of 0.1 ng/m L,and 0.91±0.69 A.U.at an IL-1βconcentration of 1 ng/m L;in samples from n=6 donor animals;P<0.001;ANOVA)suggesting an anti-proliferative effect of this clinically approved IL-1 receptor antagonist.The FDH assay showed this dose to be non-toxic in HSCs.In vivo,Anakinra had no effect on the hepatic hydroxyprolinecontent,liver serum tests(ALT and AP)and profibrotic(collagen 1α1,collagen 1α2,transforming growth factor-β,and TIMP-1)and anti-fibrotic[matrix metalloproteinase 2(MMP2),MMP9 and MMP13]gene expression after 4 wk of treatment.Furthermore,the hepatic IL-1βand F4/80 m RNA expression levels were unaffected by Anakinra treatment.CONCLUSION:IL-1βexpression is associated with the degree of liver fibrosis in Abcb4^(-/-)mice and promotes HSC proliferation.IL-1 antagonism shows antifibrotic effects in vitro but not in Abcb4^(-/-)mice.展开更多
基金Supported by The Münchener Medizinische Wochenschrift(MMW)B.Braun-Stiftung(to Reiter FP)the Deutsche Forschungsgemeinschaft(HO 4460/2-1 to Hohenester S and RU 742/6-1 to Rust C)
文摘AIM:To study the interleukin-1(IL-1)pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4^(-/-)(Abcb4^(-/-))mouse model.METHODS:Female and male Abcb4^(-/-)mice from 6 to 13 mo of age were analysed for the degree of cholestasis(liver serum tests),extent of liver fibrosis(hydroxyproline content and Sirius red staining)and tissue-specific activation of signalling pathways such as the IL-1 pathway[quantitative polymerase chain reaction(q PCR)].For in vivo experiments,murine hepatic stellate cells(HSCs)were isolated via pronasecollagenase perfusion followed by density gradient centrifugation using female mice.Murine HSCs were stimulated with up to 1 ng/m L IL-1βwith or without 2.5μg/m L Anakinra,an IL-1 receptor antagonist,respectively.The proliferation of murine HSCs was assessed via the Brd U assay.The toxicity of Anakinra was evaluated via the fluorescein diacetate hydrolysis(FDH)assay.In vivo 8-wk-old Abcb4^(-/-)mice with an already fully established hepatic phenotype were treated with Anakinra(1 mg/kg body-weight daily intraperitoneally)or vehicle and liver injury and liver fibrosis were evaluated via serum tests,q PCR,hydroxyproline content and Sirius red staining.RESULTS:Liver fibrosis was less pronounced in males than in female Abcb4^(-/-)animals as defined by a lower hydroxyproline content(274±64μg/g vs 436±80μg/g liver,respectively;n=13-15;P<0.001;MannWhitney U-test)and lower m RNA expression of the profibrogenic tissue inhibitor of metalloproteinase-1(TIMP)(1±0.41 vs 0.66±0.33 fold,respectively;n=13-15;P<0.05;Mann-Whitney U-test).Reduced liver fibrosis was associated with significantly lower levels of F4/80 m RNA expression(1±0.28 vs 0.71±0.41 fold,respectively;n=12-15;P<0.05;Mann-Whitney U-test)and significantly lower IL-1βm RNA expression levels(1±0.38 vs 0.44±0.26 fold,respectively;n=13-15;P<0.001;Mann-Whitney U-test).No gender differences in the serum liver parameters[bilirubin;alanine aminotransferase(ALT);aspartate aminotransferase and alkaline phosphatase(AP)]were found.In vitro,the administration of IL-1βresulted in a significant increase in HSC proliferation[0.94±0.72 arbitrary units(A.U.)in untreated controls,1.12±0.80 A.U.at an IL-1βconcentration of 0.1 ng/m L and 1.18±0.73 A.U.at an IL-1βconcentration of 1 ng/m L in samples from n=6 donor animals;P<0.001;analyses of variance(ANOVA)].Proliferation was reduced significantly by the addition of 2.5μg/m L Anakinra(0.81±0.60 A.U.in untreated controls,0.92±0.68 A.U.at an IL-1βconcentration of 0.1 ng/m L,and 0.91±0.69 A.U.at an IL-1βconcentration of 1 ng/m L;in samples from n=6 donor animals;P<0.001;ANOVA)suggesting an anti-proliferative effect of this clinically approved IL-1 receptor antagonist.The FDH assay showed this dose to be non-toxic in HSCs.In vivo,Anakinra had no effect on the hepatic hydroxyprolinecontent,liver serum tests(ALT and AP)and profibrotic(collagen 1α1,collagen 1α2,transforming growth factor-β,and TIMP-1)and anti-fibrotic[matrix metalloproteinase 2(MMP2),MMP9 and MMP13]gene expression after 4 wk of treatment.Furthermore,the hepatic IL-1βand F4/80 m RNA expression levels were unaffected by Anakinra treatment.CONCLUSION:IL-1βexpression is associated with the degree of liver fibrosis in Abcb4^(-/-)mice and promotes HSC proliferation.IL-1 antagonism shows antifibrotic effects in vitro but not in Abcb4^(-/-)mice.
