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Chronic pancreatitis increases the risk of developing pancreatic cancer through the upregulation of pathways favouring proliferation, fibrosis, and sustained inflammation. We established in previous studies that the ligand tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) signals through its cognate receptor fibroblast growth factor-inducible 14 (Fn14) to regulate these underlying cellular processes in the chronic liver injury niche. However, the role of the TWEAK/Fn14 signalling pathway in pancreatic disease is entirely unknown. An analysis of publicly available datasets demonstrated that the TWEAK receptor Fn14 is upregulated in pancreatitis and pancreatic adenocarcinoma, with single cell RNA sequencing revealing pancreatic ductal cells as the main Fn14 producers. We then used choline-deficient, ethionine-supplemented (CDE) diet feeding of wildtype C57BL/6J and Fn14 knockout littermates to (a) confirm CDE treatment as a suitable model of chronic pancreatitis and (b) to investigate the role of the TWEAK/Fn14 signalling pathway in pancreatic ductal proliferation, as well as fibrotic and inflammatory cell dynamics. Our time course data obtained at three days, three months, and six months of CDE treatment reveal that a lack of TWEAK/Fn14 signalling significantly inhibits the establishment and progression of the tissue microenvironment in CDE-induced chronic pancreatitis, thus proposing the TWEAK/Fn14 pathway as a novel therapeutic target.

Hepatic ischemia and reperfusion (IR) injury contributes to the morbidity and mortality associated with liver transplantation. microRNAs (miRNAs) constitute a family of noncoding RNAs that regulate gene expression at the posttranslational level through the repression of specific target genes. Here, we hypothesized that miRNAs could be targeted to enhance hepatic ischemia tolerance. A miRNA screen in a murine model of hepatic IR injury pointed us toward the liver-specific miRNA miR122. Subsequent studies in mice with hepatocyte-specific deletion of miR122 (miR122loxP/loxP Alb-Cre+ mice) during hepatic ischemia and reperfusion revealed exacerbated liver injury. Transcriptional studies implicated hypoxia-inducible factor-1α (HIF1α) in the induction of miR122 and identified the oxygen-sensing prolyl hydroxylase domain 1 (PHD1) as a miR122 target. Further studies indicated that HIF1α-dependent induction of miR122 participated in a feed-forward pathway for liver protection via the enhancement of hepatic HIF responses through PHD1 repression. Moreover, pharmacologic studies utilizing nanoparticle-mediated miR122 overexpression demonstrated attenuated liver injury. Finally, proof-of-principle studies in patients undergoing orthotopic liver transplantation showed elevated miR122 levels in conjunction with the repression of PHD1 in post-ischemic liver biopsies. Taken together, the present findings provide molecular insight into the functional role of miR122 in enhancing hepatic ischemia tolerance and suggest the potential utility of pharmacologic interventions targeting miR122 to dampen hepatic injury during liver transplantation. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:131 issue:7 ispartof: location:United States status: published

[EN]Objective Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD. Design RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or Ripk3-deficient (Ripk3(-/-)) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks. Results RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, Ripk3 deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in Ripk3(-/-) mice. Furthermore, Ripk3 deficiency hampered tumourigenesis. Intriguingly, Ripk3(-/-) mice displayed increased body weight gain, while lipidomics showed that deletion of Ripk3 shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor. (PPAR.) was increased in Ripk3(-/-) mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPAR. in controlling fat deposition and fibrosis. Conclusion Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression. Main funding is provided by FEDER funds through the COMPETE programme and by national funds through Fundacao para a Ciencia e a Tecnologia to CMPR (grants SAICTPAC/0019/2015-LISBOA-01-0145--FEDER-016405 and PTDC/MED-FAR/29097/2017 -LISBOA-01-0145-FEDER-029097). Additional funding comes from research grant APEF (Portuguese Association for the Study of Liver)/BAYER 2020 to MBA. JG is funded by the Fondation pour la Recherche Medicale (ARF20170938613), the Institute of Cardiometabolism and Nutrition (PAP17NECJG), the Societe Francophone du Diabete (R19114DD) and the Mairie de Paris (Emergences -R18139DD). MBA, PMR, MMP and ALS were investigators or students funded by Fundacao para a Ciencia e a Tecnologia.

Background Nonalcoholic steatohepatitis (NASH) is a form of liver disease characterized by steatosis, necroinflammation, and fibrosis, resulting in cirrhosis and cancer. Efforts have focused on reducing the intake of trans fatty acids (TFAs) because of potential hazards to human health and the increased risk for NASH. However, the health benefits of reducing dietary TFAs have not been fully elucidated. Here, the effects of TFAs vs. a substitute on NASH induced in mice by feeding a choline-deficient, methionine-lowered, L-amino acid-defined, high-fat diet (CDAA-HF) were investigated. Methods Mice were fed CDAA-HF containing shortening with TFAs (CDAA-HF-T(+)), CDAA-HF containing shortening without TFAs (CDAA-HF-T(−)), or a control chow for 13 or 26 weeks. Results At week 13, NASH was induced in mice by feeding CDAA-HF-T(+) containing TFAs or CDAA-HF-T(−) containing no TFAs, but rather mostly saturated fatty acids (FAs), as evidenced by elevated serum transaminase activity and liver changes, including steatosis, inflammation, and fibrosis. CDAA-HF-T(−) induced a greater extent of hepatocellular apoptosis at week 13. At week 26, proliferative (preneoplastic and non-neoplastic) nodular lesions were more pronounced in mice fed CDAA-HF-T(−) than CDAA-HF-T(+). Conclusions Replacement of dietary TFAs with a substitute promoted the development of proliferation lesions in the liver of a mouse NASH model, at least under the present conditions. Attention should be paid regarding use of TFA substitutes in foods for human consumption, and a balance of FAs is likely more important than the particular types of FAs.

Introdução: A doença hepática gordurosa não-alcoólica (DHGNA) é uma doença hepática progressiva que é considerada um fator de risco para o desenvolvimento de doença cardiovascular (DCV). Transtornos do eixo fígado-intestino, incluindo a disbiose intestinal, têm sido associados à DHGNA e DCV. Objetivos: Desenvolver um modelo experimental de DHGNA de origem metabólica que permita avaliar, na sequência, fatores relacionados à DCV, como índices aterogênicos, inflamação hepática e sistêmica e disfunção endotelial, bem como a associação entre a composição e a diversidade da microbiota intestinal e sua relação com o risco de desenvolvimento de DVC. Métodos: Ratos Sprague Dawley, adultos e machos, foram randomizados em dois grupos (n=10): controle, alimentado com dieta padrão, e intervenção, que recebeu dieta hiperlipídica deficiente em colina. O experimento teve duração de 16 semanas e após os animais foram eutanasiados. Os materiais biológicos foram coletados e armazenados até a realização das avaliações bioquímicas, inflamatórias, histológicas e da microbiota fecal. Resultados: Os animais do grupo intervenção apresentaram significativo aumento no delta de índice de Lee (p = 0,017), circunferência abdominal (p < 0,001), acúmulo de tecido adiposo abdominal (p < 0,001), bem como elevação dos níveis séricos de alanina aminotransferase (p = 0,010), glicemia (p = 0,013), colesterol total (p = 0,033), lipoproteína de baixa densidade (p = 0,011) e triglicerídeos (p = 0,011) e diminuição dos níveis séricos de lipoproteína de alta densidade (p = 0,006), similares às observadas na DHGNA em humanos. Todos os animais do grupo intervenção desenvolveram DHGNA, com ou sem a presença de esteato-hepatite, enquanto no grupo controle não houve qualquer alteração histológica. Houve aumento significativo no grupo intervenção dos índices aterogênicos, coeficiente aterogênico (p < 0,001), índice de risco Castelli-I (p < 0,001) e índice de risco Castelli-II (p < 0,001), além de aumento significativo na concentração dos marcadores de inflamação e disfunção endotelial sistêmica, interleucina (IL)-1β (p < 0,001), proteína quimiotática de monócitos-1 (p < 0,001), inibidor tecidual de metaloproteinases-1 (p < 0,001) e inibidor do ativador de plasminogênio-1 (p < 0,019). Houve um aumento na expressão gênica hepática do receptor toll like (TLR)-4 (p = 0,041), TLR-9 (p = 0,033), fator de diferenciação mielóide-88 (p = 0,001), receptor do tipo NOD-proteína-3 (p = 0,019), capase-1 (p < 0,001), fator de necrose tumoral-α (p = 0,028), IL-6 (p = 0,013) e IL-18 (p = 0,007), além da diminuição das expressão do receptor ativado por proliferadores de peroxissoma-α (p = 0,008) em relação ao grupo controle. Observamos aumento significativo de miR-122 (p = 0,041) e miR-33a (p = 0,001) no grupo intervenção, e o inverso foi observado para miR-145 (p = 0,010) e miR-126 (p < 0,001). O grupo intervenção, apresentou diminuição significativa da diversidade bacteriana fecal e menor abundância relativa do filo Firmicutes (p = 0,040), o que se correlacionou com a gravidade histológica da DHGNA. Conclusão: O grupo intervenção apresentou alterações bioquímicas, inflamatórias e histopatológicas similares àquelas observadas na DHGNA em humanos. Houve neste grupo, um aumento da obesidade visceral, dislipidemia, hiperglicemia, inflamação hepática e sistêmica, disfunção endotelial e um maior risco de desenvolvimento de DCV. Este modelo experimental, poderia ser utilizado no entendimento da fisiopatologia da DHGNA e sua evolução, bem como para estudos terapêuticos pré-clínicos que visem à cardioproteção na DHGNA, um desfecho habitualmente não avaliado em ensaios clínicos em humanos. Introduction: Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease that is considered a risk factor for the development of cardiovascular disease (CVD). Liver-gut axis disorders, including intestinal dysbiosis, have been associated with NAFLD and CVD. Objective: To develop an experimental model of NAFLD of metabolic origin that would allow to evaluate, in the sequence, factors related to CVD, such as atherogenic indices, hepatic and systemic inflammation and endothelial dysfunction, as well as the association between the composition and diversity of the gut microbiota and its relationship with the risk of developing CVD. Methods: Adult male Sprague Dawley rats were randomized into two groups (n = 10): control group that was fed standard diet and intervention group that received a high-fat and choline-deficient (HFCD) diet. The experiment had duration of 16 weeks and after the animals were euthanized. Biological materials were collected and stored until the biochemical, inflammatory, histological and fecal microbiota evaluations were performed. Results: The animals in the intervention group, showed a significantly greater delta Lee index (p = 0.017), abdominal circumference (p < 0.001), abdominal adipose tissue accumulation (p < 0.001), as well as elevation of serum levels of alanine aminotransferase (p = 0.010), glycemic (p = 0.013), total cholesterol (p = 0.033), low-density lipoprotein (p = 0.011), triglycerides (p = 0.011) and decreased serum levels of high-density lipoprotein (p = 0.006), corresponding to those observed in NAFLD in humans. All animals in the intervention group developed NAFLD, with or without the presence of steatohepatitis, while no histological changes were found in the control group. There was a significant increase in the intervention group, of atherogenic indices, atherogenic coefficient (p < 0.001), Castelli-I risk index (p < 0.001) and Castelli-II risk index (p < 0.001), increased markers of systemic inflammation and endothelial dysfunction, interleukin (IL)-1β (p < 0.001), monocyte chemotactic protein-1 (p < 0.001), tissue inhibitor of metalloproteinases-1 (p < 0.001) and inhibitor of plasminogen activator-1 (p < 0.019). There was an increase in hepatic gene expression of toll-like receptor (TLR)-4 (p = 0.041), TLR-9 (p = 0.033), myeloid differentiation primary response-88 (p = 0.001), nod-like receptor protein-3 (p = 0.019) caspase-1 (p < 0.001), tumor necrosis factor-α (p = 0.028), IL-6 (p = 0.013) and IL-18 (p = 0.007), besides the diminution of the expression of peroxisome proliferator activated receptor-α (p = 0.008) in relation to the control group. We observed a significant increase of miR-122 (p = 0.041) and miR-33a (p = 0.001) in the intervention group, and the inverse was observed for miR-145 (p = 0.010) and miR-126 (p < 0.001). The intervention group showed a significant decrease in the fecal bacterial diversity and lower relative abundance of the Firmicutes phylum (p = 0.040), which correlated with the histological severity of NAFLD. Conclusion: The intervention group presented biochemical, inflammatory and histopathological alterations, similar to those observed in NAFLD in humans. There were in this group, an increase in visceral obesity, dyslipidemia, hyperglycemia, systemic and hepatic inflammation, endothelial dysfunction and an increased risk of developing CVD. This experimental model could be used to understand the pathophysiology of NAFLD and its evolution, as well as for preclinical therapeutic studies that aim at cardioprotection in NAFLD, an outcome not usually evaluated in clinical trials in humans.

The aim of this work was to investigate the potential protective effects of fish oil on the basis of kidney transcriptomic data on a nutritional experimental model. Methods Male weanling Wistar rats were divided into four groups and fed choline-deficient (CD) and choline-supplemented (CS) diets with vegetable oil (VO) and menhaden oil (MO): CSVO, CDVO, CSMO and CDMO. Animals were killed after receiving the diets for 6 days. Total RNA was purified from the right kidney and hybridized to Affymetrix GeneChip Rat Gene 1.0 ST Array. Differentially expressed genes were analyzed. Results All CSVO, CSMO and CDMO rats showed no renal alterations, while all CDVO rats showed renal cortical necrosis. A thorough analysis of the differential expression between groups CSMO and CDMO was carried out. There were no differential genes for p < 0.01. The analysis of the differential expression between groups CSVO and CSMO revealed 32 genes, 11 were over-expressed and 21 were under-expressed in CSMO rats. Conclusions This work was part of a large set of experiments and was used in a hypothesis-generating manner. The comprehensive analysis of genetic expression allowed confirming that menhaden oil has a protective effect on this nutritional experimental model and identifying 32 genes that could be responsible for that protection, including Gstp1. These results reveal that gene changes could play a role in renal injury. Fil: Denninghoff, Valeria Cecilia. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología. Centro de Patología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ossani, Georgina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología. Centro de Patología Experimental; Argentina Fil: Uceda, Ana Margarita. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología. Centro de Patología Experimental; Argentina Fil: Rugnone, Matias Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Fernandez, Elmer Andres. Area de Ciencias Agrarias, Ingeniería, Ciencias Biológicas y de la Salud de la Universidad Católica de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina Fil: Fresno Rodríguez, Cristóbal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Area de Ciencias Agrarias, Ingeniería, Ciencias Biológicas y de la Salud de la Universidad Católica de Córdoba; Argentina Fil: González, German. Area de Ciencias Agrarias, Ingeniería, Ciencias Biológicas y de la Salud de la Universidad Católica de Córdoba; Argentina Fil: Díaz, Maria Luisa. Hospital Británico de Buenos Aires; Argentina Fil: Avagnina, Alejandra. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; Argentina Fil: Elsner, Boris. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; Argentina Fil: Monserrat, Alberto Juan. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología. Centro de Patología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The present study was performed to assess the roles of hepatocellular oxidative damage to DNA and constituents other than DNA in rat liver carcinogenesis caused by a choline-deficient, L-amino acid-defined (CDAA) diet by examining the effects of the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD). The parameters used for cellular oxidative damage were the level of 8-hydroxy-guanine (8-OHGua) for DNA and that of 2-thiobarbituric acid-reacting substance (TBARS) for constituents other than DNA. A total of 40 male Fischer 344 rats, 6 weeks old, were fed the CDAA diet for 12 weeks with or without DPPD (0.05, 0.10 or 0.20%) or butylated hydroxytoluene (BHT, 0.25%). In the livers of the rats, the numbers and sizes of glutathione S-transferase (EC 2.5.1.18) placental form (GSTP)- and/or gamma-glutamyltransferase (GGT, EC 2.3.2.2)-positive lesions and levels of 8-OHGua and TBARS were determined. The GSTP-positive lesions of 0.08 mm2 or larger were all stained positively for GGT as well in cross-sectional area, whereas the smaller lesions were generally negative for GGT. DPPD and BHT reduced the size of the GSTP-positive lesions without affecting their total numbers. At the same time, they reduced TBARS generation without affecting 8-OHGua formation in DNA. The present results indicate that oxidative DNA damage (represented by 8-OHGua formation) and damage to constituents other than DNA (represented by TBARS generation) may play different roles in rat liver carcinogenesis caused by the CDAA diet; the former appears to be involved in the induction of phenotypically altered hepatocyte populations while the latter may be related to the growth of such populations.