摘要
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-1β with or without 2.5 μg/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 ± 64 μg/g vs 436 ± 80 μg/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-1β m 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-1β resulted 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-1β concentration of 0.1 ng/m L and 1.18 ± 0.73 A.U. at an IL-1β concentration 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 2.5 μg/m L Anakinra(0.81 ± 0.60 A.U. in untreated controls, 0.92 ± 0.68 A.U. at an IL-1β concentration of 0.1 ng/m L, and 0.91 ± 0.69 A.U. at an IL-1β concentration 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-1β and F4/80 m RNA expression levels were unaffected by Anakinra treatment.CONCLUSION: IL-1β expression 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.
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-1β with or without 2.5 μg/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 ± 64 μg/g vs 436 ± 80 μg/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-1β m 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-1β resulted 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-1β concentration of 0.1 ng/m L and 1.18 ± 0.73 A.U. at an IL-1β concentration 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 2.5 μg/m L Anakinra(0.81 ± 0.60 A.U. in untreated controls, 0.92 ± 0.68 A.U. at an IL-1β concentration of 0.1 ng/m L, and 0.91 ± 0.69 A.U. at an IL-1β concentration 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-1β and F4/80 m RNA expression levels were unaffected by Anakinra treatment.CONCLUSION: IL-1β expression 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.
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)
