- Email: [email protected]
The effect of elafibranor and obeticholic acid on NAFLD Activity score and fibrosis stage in a novel metabolic rat model of diet-induced and biopsy-confirmed NASH
POSTER PRESENTATIONS Results: Massive lipid accumulation and increased ALT and AST levels were showed in HFHC rats compared with ICD and NC in all timepoints. High level of ammonia was found in ICD and HFCH compared with NC at 10 weeks, which was associated with progressive reduction in the activity of the urea cycle enzyme, OTC. HFHC-fed rats showed massive collagen accumulation and increase in inflammatory infiltrates at 16 weeks, whereas no signs of inflammation and fibrosis were found in ICD fed rats. Progressive neuropsychological dysfunction was observed in the HFHC animals (Table). Differences in depression and anxiety were found between ICD and HFHC groups at 16 weeks compared to NC. Moreover, differences in spatial working memory were found in HFHC group at 10 weeks and ICD at 4 weeks. Differences along the time have been found in depression, anxiety and cognitive functions. No differences were found between groups in motor activity and short-term social memory. Normal Chow Liver ALT (U/L) AST (U/L) + NH4 (µmol/L) Urea (mmol/L) OTC (nmol/min/ µgprotein)
4 weeks
ICD
10 weeks
4 weeks
HFHC 10 weeks
4 weeks
48 ± 3 50 ± 4 30 ± 4 47 ± 5 100 ± 11*** 77 ± 7 97 ± 4 75 ± 5 96 ± 5 130 ± 11*** 53 ± 4 75 ± 3 48 ± 3 140 ± 7** 47 ± 3 6.21 ± 0.7 4.03 ± 0.4 6.04 ± 0.3 5.23 ± 0.4* 5.5 ± 0.4* 0.67 ± 0.09 0.65 ± 0.05 0.38 ± 0.05** 0.32 ± 0.04** 0.27 ± 0.09***
10 weeks 155 ± 12*** 250 ± 21*** 138 ± 8** 4.01 ± 0.5 0.25 ± 0.05***
Conclusions: The results of this study show a temporal evolution of defect of the urea cycle with consequent hyperammonemia and progressive neuropsychological dysfunction in a clinically-relevant rodent model of NAFLD even prior to cirrhosis stages. The data provide an explanation for the poor quality of life of NAFLD patients and a potential therapeutic target. SAT-338 Interleukin-33/interleukin-33r signaling promotes liver inflammation and fibrogenesis in obesogenic model of nonalcoholic steatohepatitis in mice N. Jovicic1, I. Jeftic2, M.M. Kovacevic1, I. Tanaskovic1, N. Arsenijevic3, M.L. Lukic3, N. Pejnovic2. 1Institute of Histology; 2Institute of Pathophysiology; 3Center for Molecular Medicine, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Republic of Serbia E-mail: [email protected] Background and Aims: Cytokines have been shown to modulate liver fibrosis, but the role of IL-33/IL-33R(ST2) signaling pathway in obesity-associated liver pathology is only partially understood. In order to dissect the role of IL33/ST2 axis we used obesogenic model of nonalcoholic steatohepatitis (NASH) in C57BL/6 and BALB/c mice, the prototypical Th1 and Th2 strains. Methods: Male, 8-week old ST2 deficient (ST2-/-), wild-type (WT) BALB/c mice and C57Bl/6 mice were placed on high-fat diet (HFD; 60% kcal fat) or standard diet (10% kcal fat) for 24 weeks. After 10 weeks on HFD, one group of C57Bl/6 mice were injected intraperitoneally with murine recombinant IL-33 (0.5 μg) five times every
other day. Histological, immunophenotypic and gene expression analyses were performed. Results: HFD enhanced liver inflammation and fibrosis in C57Bl/6 mice, associated with more numerous pro-inflammatory CD11b+Ly6Chi monocytes, triple positive F4/80+CD11b+CD11c+ and IL1β-expressing F4/80+ macrophages (Mφ) and increased expression of procollagen α1, IL-33, IL-13 and TGF-β mRNA compared to mice on standard diet. IL-33 administration in vivo enhanced liver inflammation and fibrosis and increased the number of hepatic CD11b+ myeloid cells expressing ST2 and IL-13. In BALBc, deletion of ST2 markedly reduced HFD-induced liver steatosis and inflammation, accompanied with lower expression of CD36, LXRα, PPAR-γ and decreased number of CD68+ Mφ and CD11c+ dendritic cells (DCs). Further, HFD-fed ST2-/mice had lower collagen deposition in livers associated with less numerous profibrotic CD11b+Ly6Clow monocytes and CD4+IL-17+ T cells and lower hepatic procollagen α1, IL-33 and IL-13 mRNA expression, and lower serum levels of IL-33 and IL-13, while the serum levels and hepatic TGF-β mRNA expression were similar between ST2-/- and WT mice. In an additional experiment in which we used HFD/fructosis diet, fibrosis exacerbated in WT mice, but diet had no effect on ST2-/- mice. In contrast, HFD-fed ST2-/- mice exhibited higher weight gain, amount of visceral adipose tissue (VAT), and enhanced VAT inflammation with higher percentages of VAT associated CD11c+ DCs, IFNγ and IL-17 expressing T cells, and CXCR3+ T cells compared to diet-matched WT mice. Conclusions: IL-33/ST2/IL-13 pathway has an important role in NASH associated fibrosis. Deletion of IL-33R has differential effects on obesity-associated VAT inflammation and liver pathology. IL-33/ST2 signaling is involved in obesity-associated liver pathology in Th1 and Th2 dominant strains. SAT-339 The effect of elafibranor and obeticholic acid on NAFLD Activity score and fibrosis stage in a novel metabolic rat model of dietinduced and biopsy-confirmed NASH N. Vrang1, K.S. Tølbøl1, M. Illemann1, S.S. Veidal1, L.D. Fensholdt1, M. Feigh1. 1Gubra, Horsholm, Denmark E-mail: [email protected] Background and Aims: To determine the effect of the PPAR α/δ agonist, elafibranor, and the FXR receptor agonist obeticholic acid (OCA), on hepatic histopathology in a diet-induced rat model of biopsy-confirmed nonalcoholic steatohepatitis (NASH). Methods: Male Wistar rats were fed a choline-deficient amino-acid defined diet high in fat, sucrose and cholesterol (CDAA-HFSC) or control diet. After 6 weeks, a liver biopsy was obtained under isoflurane anesthesia. Only histology confirmed steatotic and fibrotic rats (steatosis score 3; Fibrosis Stage ≥1) were randomized (n = 11– 12/group) to receive either vehicle (PO, BID), OCA (15 mg/kg, PO, BID) or elafibranor (15 mg/kg, PO, BID) for five weeks. Primary endpoints included a blinded histological assessment (pre- vs post-biopsy) of NAFLD Activity Score (NAS) (steatosis, inflammation, ballooning degeneration), Fibrosis Stage and image analysis for collagen 1a1 (Col1a1) immunoreactivity. Furthermore, metabolic and biochemical
Table: (abstract: SAT-337). Brain Cognition
Normal Chow 4 weeks
10 weeks
ICD 16 weeks
4 weeks
10 weeks
HFHC 16 weeks
4 weeks
10 weeks
16 weeks
Motor activity 104 ± 22.6 154.2 ± 15.2 163.7 ± 18.2 159.7 ± 13.7 123.2 ± 10.8 122.7 ± 12.9 134.2 ± 18.6 154 ± 18.8 141.8 ± 13 Depression 7.3 ± 3 6.3 ± 3 3.0 ± 3 10.8 ± 3 17.3 ± 3* 10.7 ± 3 9.2 ± 3 14.2 ± 3 18.0 ± 3* Anxiety 176.1 ± 23 164.9 ± 23 159.8 ± 23 211.4 ± 23 192.9 ± 23 249.4 ± 23* 215.6 ± 23 146.0 ± 23# 269.8 ± 23* Short-term social memory 48.3 ± 11 62.8 ± 11 45.3 ± 11 72.7 ± 11# 54.2 ± 11 30 ± 11 36 ± 11 57 ± 11# 11.5 ± 11 Working memory 39.2 ± 6.7 44.8 ± 9.7 93.1 ± 36.4 103 ± 31.5* 232 ± 49.8* 91.6 ± 25.7* 28.6 ± 8.5 74.8 ± 24.9* 90.2 ± 31.9* ANOVA with Tukey’s posthoc test. *P < 0.05, **P < 0.01, ***P 0.0001 compared with Normal chow at same timepoint. #P < 0.05 when compared evolution (4 weeks, 10 weeks and 16 weeks). S602
Journal of Hepatology 2017 vol. 66 | S543–S750
POSTER PRESENTATIONS parameters including liver triglyceride (TG), total cholesterol (TC) and collagen content (hydroxyproline (HYP)) were measured. Results: CDAA-HFSC diet-induced rats developed histology confirmed hepatosteatosis, inflammation, ballooning degeneration (NAS score 6–7) and fibrosis (Stage F3-F4). In addition, liver TG/TC/HYP content were increased when compared to control rats. Importantly, CDAA-HFSC rats preserved the metabolic phenotype during the study period (10% body weight gain). OCA treatment induced a 5% weight loss, albeit with no effect on hepatomegaly, whereas elafibranor treatment increased liver weight. No effect of OCA or elafibranor was observed for liver TG/TC content. Elafibranor, but not OCA, significantly reduced liver HYP content when compared to vehicle-treated CDAA-HFSC rats, supported by a comparable change in Col1a1 deposition. In conjunction, 6/12 elafibranor treated rats demonstrated no worsening in Fibrosis Stage, as compared to 10/12 vehicle treated animals. Finally, only elafibranor treatment significantly reduced NAS by improving all components (steatosis, inflammation and ballooning degeneration). Conclusions: The novel CDAA-HFSC diet-induced rat model progressively developed key histological hallmarks of biopsy-confirmed NASH in conjunction with a preserved metabolic phenotype. Elafibranor treatment alleviated NAFLD Activity Score as reflected by improvements in steatosis, inflammation and ballooning degeneration. In addition, elafibranor treatment exerted anti-fibrotic effects in CDAA-HFSC diet-induced rats. SAT-340 Precision-cut liver slices as an ex-vivo model for human NAFLD show hepatic progenitor cell activation and fibrogenesis O. Govaere1, L.H. Reed1, H. Brown1, S.J. Cockell2, J.J. French3, S.A. White3, D.M. Manas4, A.K. Daly1, S.M. Robinson5, C. Griffiths1, L.A. Borthwick1, M.J. Drinnan1, D. Tiniakos1, Q.M. Anstee1, F. Oakley1 and On behalf of the EPoS consortium. 1Institute of Cellular Medicine; 2Bioinformatics Support Unit, Newcastle University; 3 Department of Hepatobiliary Surgery; 4Liver Unit; 5Department of Gastroenterology and Hepatology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom E-mail: [email protected] Background and Aims: Long-lasting damage to the liver results in fibrosis and inflammation, which is a complex interaction between different cell types. Although rodent models are widely used to investigate chronic liver disease, there are still questions about how representative such models are of human disease. The current study aimed to establish an ex-vivo human model to understand interactions between different cell types during liver damage. Methods: 250 µm thick precision-cut liver slices were cultured in a novel bioreactor and culture plate system that rocks the slices to mimic hepatic blood flow. Healthy rat liver tissue (n = 4) was used to optimise and test the novel system. Medium was collected daily to analyse protein secretion using ELISA and tissue was harvested on days 2, 4 and 6 for qPCR and histopathological analysis. Normal human liver tissue samples (n = 5), collected following surgical resection of colorectal cancer metastasis, were obtained after informed consent from patients at the Freeman Hospital, Newcastle-upon-Tyne, UK. After 24 h in culture human liver slides were loaded with a mixture of oleic, palmitic and linoleic acid for 48 h to induce lipid loading and processed for RNA sequencing and histochemistry. Results: Compared to a static system, the flow generated by the rocking bioreactor significantly increased cell viability and albumin production in the rat precision-cut liver slices and preserved tissue morphology as the bile ducts, the central venules and the sinusoids remained intact for up to 7 days in culture. After 72 h the human liver samples showed a loss of hepatocyte-related genes (e.g. GSTA1, ALDH2, GCDH, IGFBP3, IDO2), and a strong increase in extracellular matrix (e.g. COL1A1, MMP12, LOX, ITGB5, PDGFRB) and hepatic progenitor cell (e.g. EPCAM, CD24, KRT19, HEYL, LAMC2)-related
markers. Histopathogical assessment confirmed early fibrosis and collagen deposition in the human samples after 72 h in vitro while none was detected at time zero. In addition an expansion of the hepatic progenitor cell compartment and parenchymal congestion was observed. Fat loading aggravated the process of bridging fibrosis, and focally induced steatosis and ballooning of remaining hepatocytes. Conclusions: Human precision-cut liver slices can be used to model features of non-alcoholic fatty liver disease in vitro. SAT-341 miR-21 ablation and obeticholic acid ameliorate NASH in mice P.M. Rodrigues1, M.B. Afonso1, A.L. Simão1, C.C. Carvalho2, A. Trindade2,3, A. Duarte2,3, P.M. Borralho1, M.V. Machado4, H. Cortez-Pinto4, C.M.P. Rodrigues1, R.E. Castro1. 1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa; 2 Reproduction and Development, Interdisciplinary Centre of Research in Animal Health (CIISA), Faculty of Veterinary Medicine, Universidade de Lisboa, Lisbon; 3Instituto Gulbenkian de Ciência (IGC), Oeiras; 4 Gastrenterology, Hospital Santa Maria, Lisbon, Portugal E-mail: [email protected] Background and Aims: microRNAs were recently suggested to participate in non-alcoholic fatty liver disease (NAFLD) pathogenesis. Moreover, nuclear receptors, namely peroxisome proliferator-activated receptors (PPARs) and the farnesoid X receptor (FXR) are currently under scrutiny as modulators of lipid/glucose metabolism in non-alcoholic steatohepatitis (NASH). We aimed to elucidate the role of the miR-21/PPARα pathway in liver and muscle tissues of murine NASH models and ascertain the therapeutic potential of miR21 abrogation alone or in combination with FXR agonist obeticholic acid (OCA). Methods: Wild-type (WT) and miR-21 KO mice were fed with chow (n = 10) or methionine and choline-deficient (MCD; n = 10) diets for 2 and 8 weeks. Alternatively, mice were fed either chow (n = 12) or fast food diet (FF; n = 12) for 25 weeks. Six animals from each group had their diet supplemented with OCA 10 mg/kg/day (Intercept Pharmaceuticals, Inc.). Human biopsies were obtained from morbid obese NAFLD patients (n = 28). Liver/muscle samples were processed for histological analysis and assessment of miR-21, pro-inflammatory/pro-fibrogenic cytokines, PPARα and metabolic relevant genes and for insulin resistance mediators, by qRT-PCR and immunoblotting. A Taqman® Array was performed to evaluated the expression of lipid regulated genes. Results: WT mice fed with the MCD diet developed steatohepatitis and fibrosis, displaying increased levels of lipoapoptosis and serum ALT. In contrast, miR-21 KO mice displayed a significant decrease in steatosis severity, cell death and liver damage, inflammation and did not develop fibrosis. Also, WT FF-fed mice developed hepatomegaly, macrovesicular steatosis, inflammatory infiltrates and increased oxidative stress. miR-21 levels were increased in WT FF-fed mice, in both liver and muscle, concomitantly with decreased expression of PPARα. Importantly, miR-21 ablation in combination with OCA stoutly reduced steatosis severity and inflammation, preventing oxidative stress and restoring metabolic pathways, reinstated insulin sensitivity in the liver and muscle. Finally, miR-21/PPARa axis was found increased in liver and muscle samples of NAFLD patients, as well as in serum samples. Conclusions: In conclusion, miR-21 abrogation, together with FXR activation by OCA, significantly improves metabolic parameters in NASH, highlighting the therapeutic potential of multitargeting therapies for NAFLD and the metabolic syndrome. (Supported by PTDC/BIM-MEC/0895/2014[Office1], SFRH/BD/ 88212/2012, FCT, PT).
Journal of Hepatology 2017 vol. 66 | S543–S750
S603
4 weeks
ICD
10 weeks
4 weeks
HFHC 10 weeks
4 weeks
48 ± 3 50 ± 4 30 ± 4 47 ± 5 100 ± 11*** 77 ± 7 97 ± 4 75 ± 5 96 ± 5 130 ± 11*** 53 ± 4 75 ± 3 48 ± 3 140 ± 7** 47 ± 3 6.21 ± 0.7 4.03 ± 0.4 6.04 ± 0.3 5.23 ± 0.4* 5.5 ± 0.4* 0.67 ± 0.09 0.65 ± 0.05 0.38 ± 0.05** 0.32 ± 0.04** 0.27 ± 0.09***
10 weeks 155 ± 12*** 250 ± 21*** 138 ± 8** 4.01 ± 0.5 0.25 ± 0.05***
Conclusions: The results of this study show a temporal evolution of defect of the urea cycle with consequent hyperammonemia and progressive neuropsychological dysfunction in a clinically-relevant rodent model of NAFLD even prior to cirrhosis stages. The data provide an explanation for the poor quality of life of NAFLD patients and a potential therapeutic target. SAT-338 Interleukin-33/interleukin-33r signaling promotes liver inflammation and fibrogenesis in obesogenic model of nonalcoholic steatohepatitis in mice N. Jovicic1, I. Jeftic2, M.M. Kovacevic1, I. Tanaskovic1, N. Arsenijevic3, M.L. Lukic3, N. Pejnovic2. 1Institute of Histology; 2Institute of Pathophysiology; 3Center for Molecular Medicine, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Republic of Serbia E-mail: [email protected] Background and Aims: Cytokines have been shown to modulate liver fibrosis, but the role of IL-33/IL-33R(ST2) signaling pathway in obesity-associated liver pathology is only partially understood. In order to dissect the role of IL33/ST2 axis we used obesogenic model of nonalcoholic steatohepatitis (NASH) in C57BL/6 and BALB/c mice, the prototypical Th1 and Th2 strains. Methods: Male, 8-week old ST2 deficient (ST2-/-), wild-type (WT) BALB/c mice and C57Bl/6 mice were placed on high-fat diet (HFD; 60% kcal fat) or standard diet (10% kcal fat) for 24 weeks. After 10 weeks on HFD, one group of C57Bl/6 mice were injected intraperitoneally with murine recombinant IL-33 (0.5 μg) five times every
other day. Histological, immunophenotypic and gene expression analyses were performed. Results: HFD enhanced liver inflammation and fibrosis in C57Bl/6 mice, associated with more numerous pro-inflammatory CD11b+Ly6Chi monocytes, triple positive F4/80+CD11b+CD11c+ and IL1β-expressing F4/80+ macrophages (Mφ) and increased expression of procollagen α1, IL-33, IL-13 and TGF-β mRNA compared to mice on standard diet. IL-33 administration in vivo enhanced liver inflammation and fibrosis and increased the number of hepatic CD11b+ myeloid cells expressing ST2 and IL-13. In BALBc, deletion of ST2 markedly reduced HFD-induced liver steatosis and inflammation, accompanied with lower expression of CD36, LXRα, PPAR-γ and decreased number of CD68+ Mφ and CD11c+ dendritic cells (DCs). Further, HFD-fed ST2-/mice had lower collagen deposition in livers associated with less numerous profibrotic CD11b+Ly6Clow monocytes and CD4+IL-17+ T cells and lower hepatic procollagen α1, IL-33 and IL-13 mRNA expression, and lower serum levels of IL-33 and IL-13, while the serum levels and hepatic TGF-β mRNA expression were similar between ST2-/- and WT mice. In an additional experiment in which we used HFD/fructosis diet, fibrosis exacerbated in WT mice, but diet had no effect on ST2-/- mice. In contrast, HFD-fed ST2-/- mice exhibited higher weight gain, amount of visceral adipose tissue (VAT), and enhanced VAT inflammation with higher percentages of VAT associated CD11c+ DCs, IFNγ and IL-17 expressing T cells, and CXCR3+ T cells compared to diet-matched WT mice. Conclusions: IL-33/ST2/IL-13 pathway has an important role in NASH associated fibrosis. Deletion of IL-33R has differential effects on obesity-associated VAT inflammation and liver pathology. IL-33/ST2 signaling is involved in obesity-associated liver pathology in Th1 and Th2 dominant strains. SAT-339 The effect of elafibranor and obeticholic acid on NAFLD Activity score and fibrosis stage in a novel metabolic rat model of dietinduced and biopsy-confirmed NASH N. Vrang1, K.S. Tølbøl1, M. Illemann1, S.S. Veidal1, L.D. Fensholdt1, M. Feigh1. 1Gubra, Horsholm, Denmark E-mail: [email protected] Background and Aims: To determine the effect of the PPAR α/δ agonist, elafibranor, and the FXR receptor agonist obeticholic acid (OCA), on hepatic histopathology in a diet-induced rat model of biopsy-confirmed nonalcoholic steatohepatitis (NASH). Methods: Male Wistar rats were fed a choline-deficient amino-acid defined diet high in fat, sucrose and cholesterol (CDAA-HFSC) or control diet. After 6 weeks, a liver biopsy was obtained under isoflurane anesthesia. Only histology confirmed steatotic and fibrotic rats (steatosis score 3; Fibrosis Stage ≥1) were randomized (n = 11– 12/group) to receive either vehicle (PO, BID), OCA (15 mg/kg, PO, BID) or elafibranor (15 mg/kg, PO, BID) for five weeks. Primary endpoints included a blinded histological assessment (pre- vs post-biopsy) of NAFLD Activity Score (NAS) (steatosis, inflammation, ballooning degeneration), Fibrosis Stage and image analysis for collagen 1a1 (Col1a1) immunoreactivity. Furthermore, metabolic and biochemical
Table: (abstract: SAT-337). Brain Cognition
Normal Chow 4 weeks
10 weeks
ICD 16 weeks
4 weeks
10 weeks
HFHC 16 weeks
4 weeks
10 weeks
16 weeks
Motor activity 104 ± 22.6 154.2 ± 15.2 163.7 ± 18.2 159.7 ± 13.7 123.2 ± 10.8 122.7 ± 12.9 134.2 ± 18.6 154 ± 18.8 141.8 ± 13 Depression 7.3 ± 3 6.3 ± 3 3.0 ± 3 10.8 ± 3 17.3 ± 3* 10.7 ± 3 9.2 ± 3 14.2 ± 3 18.0 ± 3* Anxiety 176.1 ± 23 164.9 ± 23 159.8 ± 23 211.4 ± 23 192.9 ± 23 249.4 ± 23* 215.6 ± 23 146.0 ± 23# 269.8 ± 23* Short-term social memory 48.3 ± 11 62.8 ± 11 45.3 ± 11 72.7 ± 11# 54.2 ± 11 30 ± 11 36 ± 11 57 ± 11# 11.5 ± 11 Working memory 39.2 ± 6.7 44.8 ± 9.7 93.1 ± 36.4 103 ± 31.5* 232 ± 49.8* 91.6 ± 25.7* 28.6 ± 8.5 74.8 ± 24.9* 90.2 ± 31.9* ANOVA with Tukey’s posthoc test. *P < 0.05, **P < 0.01, ***P 0.0001 compared with Normal chow at same timepoint. #P < 0.05 when compared evolution (4 weeks, 10 weeks and 16 weeks). S602
Journal of Hepatology 2017 vol. 66 | S543–S750
POSTER PRESENTATIONS parameters including liver triglyceride (TG), total cholesterol (TC) and collagen content (hydroxyproline (HYP)) were measured. Results: CDAA-HFSC diet-induced rats developed histology confirmed hepatosteatosis, inflammation, ballooning degeneration (NAS score 6–7) and fibrosis (Stage F3-F4). In addition, liver TG/TC/HYP content were increased when compared to control rats. Importantly, CDAA-HFSC rats preserved the metabolic phenotype during the study period (10% body weight gain). OCA treatment induced a 5% weight loss, albeit with no effect on hepatomegaly, whereas elafibranor treatment increased liver weight. No effect of OCA or elafibranor was observed for liver TG/TC content. Elafibranor, but not OCA, significantly reduced liver HYP content when compared to vehicle-treated CDAA-HFSC rats, supported by a comparable change in Col1a1 deposition. In conjunction, 6/12 elafibranor treated rats demonstrated no worsening in Fibrosis Stage, as compared to 10/12 vehicle treated animals. Finally, only elafibranor treatment significantly reduced NAS by improving all components (steatosis, inflammation and ballooning degeneration). Conclusions: The novel CDAA-HFSC diet-induced rat model progressively developed key histological hallmarks of biopsy-confirmed NASH in conjunction with a preserved metabolic phenotype. Elafibranor treatment alleviated NAFLD Activity Score as reflected by improvements in steatosis, inflammation and ballooning degeneration. In addition, elafibranor treatment exerted anti-fibrotic effects in CDAA-HFSC diet-induced rats. SAT-340 Precision-cut liver slices as an ex-vivo model for human NAFLD show hepatic progenitor cell activation and fibrogenesis O. Govaere1, L.H. Reed1, H. Brown1, S.J. Cockell2, J.J. French3, S.A. White3, D.M. Manas4, A.K. Daly1, S.M. Robinson5, C. Griffiths1, L.A. Borthwick1, M.J. Drinnan1, D. Tiniakos1, Q.M. Anstee1, F. Oakley1 and On behalf of the EPoS consortium. 1Institute of Cellular Medicine; 2Bioinformatics Support Unit, Newcastle University; 3 Department of Hepatobiliary Surgery; 4Liver Unit; 5Department of Gastroenterology and Hepatology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom E-mail: [email protected] Background and Aims: Long-lasting damage to the liver results in fibrosis and inflammation, which is a complex interaction between different cell types. Although rodent models are widely used to investigate chronic liver disease, there are still questions about how representative such models are of human disease. The current study aimed to establish an ex-vivo human model to understand interactions between different cell types during liver damage. Methods: 250 µm thick precision-cut liver slices were cultured in a novel bioreactor and culture plate system that rocks the slices to mimic hepatic blood flow. Healthy rat liver tissue (n = 4) was used to optimise and test the novel system. Medium was collected daily to analyse protein secretion using ELISA and tissue was harvested on days 2, 4 and 6 for qPCR and histopathological analysis. Normal human liver tissue samples (n = 5), collected following surgical resection of colorectal cancer metastasis, were obtained after informed consent from patients at the Freeman Hospital, Newcastle-upon-Tyne, UK. After 24 h in culture human liver slides were loaded with a mixture of oleic, palmitic and linoleic acid for 48 h to induce lipid loading and processed for RNA sequencing and histochemistry. Results: Compared to a static system, the flow generated by the rocking bioreactor significantly increased cell viability and albumin production in the rat precision-cut liver slices and preserved tissue morphology as the bile ducts, the central venules and the sinusoids remained intact for up to 7 days in culture. After 72 h the human liver samples showed a loss of hepatocyte-related genes (e.g. GSTA1, ALDH2, GCDH, IGFBP3, IDO2), and a strong increase in extracellular matrix (e.g. COL1A1, MMP12, LOX, ITGB5, PDGFRB) and hepatic progenitor cell (e.g. EPCAM, CD24, KRT19, HEYL, LAMC2)-related
markers. Histopathogical assessment confirmed early fibrosis and collagen deposition in the human samples after 72 h in vitro while none was detected at time zero. In addition an expansion of the hepatic progenitor cell compartment and parenchymal congestion was observed. Fat loading aggravated the process of bridging fibrosis, and focally induced steatosis and ballooning of remaining hepatocytes. Conclusions: Human precision-cut liver slices can be used to model features of non-alcoholic fatty liver disease in vitro. SAT-341 miR-21 ablation and obeticholic acid ameliorate NASH in mice P.M. Rodrigues1, M.B. Afonso1, A.L. Simão1, C.C. Carvalho2, A. Trindade2,3, A. Duarte2,3, P.M. Borralho1, M.V. Machado4, H. Cortez-Pinto4, C.M.P. Rodrigues1, R.E. Castro1. 1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa; 2 Reproduction and Development, Interdisciplinary Centre of Research in Animal Health (CIISA), Faculty of Veterinary Medicine, Universidade de Lisboa, Lisbon; 3Instituto Gulbenkian de Ciência (IGC), Oeiras; 4 Gastrenterology, Hospital Santa Maria, Lisbon, Portugal E-mail: [email protected] Background and Aims: microRNAs were recently suggested to participate in non-alcoholic fatty liver disease (NAFLD) pathogenesis. Moreover, nuclear receptors, namely peroxisome proliferator-activated receptors (PPARs) and the farnesoid X receptor (FXR) are currently under scrutiny as modulators of lipid/glucose metabolism in non-alcoholic steatohepatitis (NASH). We aimed to elucidate the role of the miR-21/PPARα pathway in liver and muscle tissues of murine NASH models and ascertain the therapeutic potential of miR21 abrogation alone or in combination with FXR agonist obeticholic acid (OCA). Methods: Wild-type (WT) and miR-21 KO mice were fed with chow (n = 10) or methionine and choline-deficient (MCD; n = 10) diets for 2 and 8 weeks. Alternatively, mice were fed either chow (n = 12) or fast food diet (FF; n = 12) for 25 weeks. Six animals from each group had their diet supplemented with OCA 10 mg/kg/day (Intercept Pharmaceuticals, Inc.). Human biopsies were obtained from morbid obese NAFLD patients (n = 28). Liver/muscle samples were processed for histological analysis and assessment of miR-21, pro-inflammatory/pro-fibrogenic cytokines, PPARα and metabolic relevant genes and for insulin resistance mediators, by qRT-PCR and immunoblotting. A Taqman® Array was performed to evaluated the expression of lipid regulated genes. Results: WT mice fed with the MCD diet developed steatohepatitis and fibrosis, displaying increased levels of lipoapoptosis and serum ALT. In contrast, miR-21 KO mice displayed a significant decrease in steatosis severity, cell death and liver damage, inflammation and did not develop fibrosis. Also, WT FF-fed mice developed hepatomegaly, macrovesicular steatosis, inflammatory infiltrates and increased oxidative stress. miR-21 levels were increased in WT FF-fed mice, in both liver and muscle, concomitantly with decreased expression of PPARα. Importantly, miR-21 ablation in combination with OCA stoutly reduced steatosis severity and inflammation, preventing oxidative stress and restoring metabolic pathways, reinstated insulin sensitivity in the liver and muscle. Finally, miR-21/PPARa axis was found increased in liver and muscle samples of NAFLD patients, as well as in serum samples. Conclusions: In conclusion, miR-21 abrogation, together with FXR activation by OCA, significantly improves metabolic parameters in NASH, highlighting the therapeutic potential of multitargeting therapies for NAFLD and the metabolic syndrome. (Supported by PTDC/BIM-MEC/0895/2014[Office1], SFRH/BD/ 88212/2012, FCT, PT).
Journal of Hepatology 2017 vol. 66 | S543–S750
S603