RESUMO
BACKGROUND/PURPOSE OF THE STUDY: Vitamin D3-deficiency is common in patients with chronic liver-disease and may promote disease progression. Vitamin D3-administration has thus been proposed as a therapeutic approach. Vitamin D3 has immunomodulatory effects and may modulate autoimmune liver-disease such as primary sclerosing cholangitis. Although various mechanisms of action have been proposed, experimental evidence is limited. Here we test the hypothesis that active 1,25-(OH)2-vitamin D3 inhibits activation of hepatic stellate cells (HSC) in vitro and modulates liver-injury in vivo. METHODS: Proliferation and activation of primary murine HSC were assessed by BrdU- and PicoGreen(®)-assays, immunoblotting, immunofluorescence-microscopy, quantitative-PCR, and zymography following calcitriol-treatment. Wild-type and ATP-binding cassette transporter b4(-/-) (Abcb4(-/-))-mice received calcitriol for 4 weeks. Liver-damage, inflammation, and fibrosis were assessed by serum liver-tests, Sirius-red staining, quantitative-PCR, immunoblotting, immunohistochemistry and hydroxyproline quantification. RESULTS: In vitro, calcitriol inhibited activation and proliferation of murine HSC as shown by reduced α-smooth muscle actin and platelet-derived growth factor-receptor-ß-protein-levels, BrdU and PicoGreen®-assays. Furthermore, mRNA-levels and activity of matrix metalloproteinase 13 were profoundly increased. In vivo, calcitriol ameliorated inflammatory liver-injury reflected by reduced levels of alanine aminotransferase in Abcb4(-/-)-mice. In accordance, their livers had lower mRNA-levels of F4/80, tumor necrosis factor-receptor 1 and a lower count of portal CD11b positive cells. In contrast, no effect on overall fibrosis was observed. CONCLUSION: Calcitriol inhibits activation and proliferation of HSCs in vitro. In Abcb4(-/-)-mice, administration of calcitriol ameliorates inflammatory liver-damage but has no effect on biliary fibrosis after 4 weeks of treatment.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Calcitriol/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Hepatite Animal/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/patologia , Hepatite Animal/imunologia , Hepatite Animal/patologia , Fatores Imunológicos/farmacologia , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Western lifestyle-associated malnutrition causes steatosis that may progress to liver inflammation and mitochondrial dysfunction has been suggested as a key factor in promoting this disease. Here we have molecularly, biochemically and biophysically analyzed mitochondria from steatotic wild type and immune-compromised mice fed a Western diet (WD) - enriched in saturated fatty acids (SFAs). WD-mitochondria demonstrated lipidomic changes, a decreased mitochondrial ATP production capacity and a significant sensitivity to calcium. These changes preceded hepatocyte damage and were not associated with enhanced ROS production. Thus, WD-mitochondria do not promote steatohepatitis per se, but demonstrate bioenergetic deficits and increased sensitivity to stress signals.
Assuntos
Fígado Gorduroso/patologia , Hepatócitos/patologia , Mitocôndrias/fisiologia , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Dieta/métodos , Modelos Animais de Doenças , Ácidos Graxos/administração & dosagem , Metabolismo dos Lipídeos , Camundongos , Mitocôndrias/metabolismoRESUMO
The data presented in this article describe the fatty acid composition of chow, liver tissue and isolated liver mitochondria from mice fed for 6-24 weeks with a high caloric western diet (WD) in comparison to control diet (normal diet, ND). The fatty acid composition was measured via gas chromatography flame ionization detection (GC-FID). Moreover, WD-induced mitochondrial protein changes are presented in this work and were analyzed by mass spectrometry (LC-MS/MS). For further interpretation and discussion of the presented data please refer to the research article entitled "Mitochondrial adaptation in steatotic mice" (Einer et al., 2017) [1].
RESUMO
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 (qPCR)]. For in vivo experiments, murine hepatic stellate cells (HSCs) were isolated via pronase-collagenase perfusion followed by density gradient centrifugation using female mice. Murine HSCs were stimulated with up to 1 ng/mL IL-1ß with or without 2.5 µg/mL Anakinra, an IL-1 receptor antagonist, respectively. The proliferation of murine HSCs was assessed via the BrdU 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, qPCR, 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; Mann-Whitney U-test) and lower mRNA 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 mRNA 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-1ß mRNA 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-1ß 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-1ß concentration of 0.1 ng/mL and 1.18 ± 0.73 A.U. at an IL-1ß concentration of 1 ng/mL 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/mL Anakinra (0.81 ± 0.60 A.U. in untreated controls, 0.92 ± 0.68 A.U. at an IL-1ß concentration of 0.1 ng/mL, and 0.91 ± 0.69 A.U. at an IL-1ß concentration of 1 ng/mL; 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 hydroxyproline content, liver serum tests (ALT and AP) and pro-fibrotic (collagen 1α1, collagen 1α2, transforming growth factor-ß, and TIMP-1) and anti-fibrotic [matrix metalloproteinase 2 (MMP2), MMP9 and MMP13] gene expression after 4 wk of treatment. Furthermore, the hepatic IL-1ß and F4/80 mRNA expression levels were unaffected by Anakinra treatment. CONCLUSION: IL-1ß 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.