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1.
Am J Pathol ; 194(1): 71-84, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37925018

RESUMO

Alcohol-associated liver disease (ALD) is a serious public health problem with limited pharmacologic options. The goal of the current study was to investigate the efficacy of pharmacologic inhibition of soluble epoxide hydrolase (sEH), an enzyme involved in lipid metabolism, in experimental ALD, and to examine the underlying mechanisms. C57BL/6J male mice were subjected to acute-on-chronic ethanol (EtOH) feeding with or without the sEH inhibitor 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid (TUCB). Liver injury was assessed by multiple end points. Liver epoxy fatty acids and dihydroxy fatty acids were measured by targeted metabolomics. Whole-liver RNA sequencing was performed, and free modified RNA bases were measured by mass spectrometry. EtOH-induced liver injury was ameliorated by TUCB treatment as evidenced by reduced plasma alanine aminotransferase levels and was associated with attenuated alcohol-induced endoplasmic reticulum stress, reduced neutrophil infiltration, and increased numbers of hepatic M2 macrophages. TUCB altered liver epoxy and dihydroxy fatty acids and led to a unique hepatic transcriptional profile characterized by decreased expression of genes involved in apoptosis, inflammation, fibrosis, and carcinogenesis. Several modified RNA bases were robustly changed by TUCB, including N6-methyladenosine and 2-methylthio-N6-threonylcarbamoyladenosine. These findings show the beneficial effects of sEH inhibition by TUCB in experimental EtOH-induced liver injury, warranting further mechanistic studies to explore the underlying mechanisms, and highlighting the translational potential of sEH as a drug target for this disease.


Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Hepatopatias Alcoólicas , Camundongos , Animais , Masculino , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Transcriptoma , Camundongos Endogâmicos C57BL , Hepatopatias Alcoólicas/genética , Ácidos Graxos , Etanol , RNA
2.
Biology (Basel) ; 12(5)2023 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-37237453

RESUMO

Alcohol-associated liver disease (ALD) is the most common chronic liver disease and carries a significant healthcare burden. ALD has no long-term treatment options aside from abstinence, and the mechanisms that contribute to its pathogenesis are not fully understood. This study aimed to investigate the role of formyl peptide receptor 2 (FPR2), a receptor for immunomodulatory signals, in the pathogenesis of ALD. WT and Fpr2-/- mice were exposed to chronic-binge ethanol administration and subsequently assessed for liver injury, inflammation, and markers of regeneration. The differentiation capacity of liver macrophages and the oxidative burst activity of neutrophils were also examined. Compared to WT, Fpr2-/- mice developed more severe liver injury and inflammation and had compromised liver regeneration in response to ethanol administration. Fpr2-/- mice had fewer hepatic monocyte-derived restorative macrophages, and neutrophils isolated from Fpr2-/- mice had diminished oxidative burst capacity. Fpr2-/- MoMF differentiation was restored when co-cultured with WT neutrophils. Loss of FPR2 led to exacerbated liver damage via multiple mechanisms, including abnormal immune responses, indicating the crucial role of FPR2 in ALD pathogenesis.

3.
FASEB J ; 37(1): e22705, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36520060

RESUMO

Alcohol-associated liver disease (ALD) is a major health problem with limited effective treatment options. Alcohol-associated hepatitis (AH) is a subset of severe ALD with a high rate of mortality due to infection, severe inflammation, and ultimately multi-organ failure. There is an urgent need for novel therapeutic approaches to alleviate the human suffering associated with this condition. Resolvin D1 (RvD1) promotes the resolution of inflammation and regulates immune responses. The current study aimed to test the therapeutic efficacy and mechanisms of RvD1-mediated effects on liver injury and inflammation in an experimental animal model that mimics severe AH in humans. Our data demonstrated that mice treated with RvD1 had attenuated liver injury and inflammation caused by EtOH and LPS exposure by limiting hepatic neutrophil accumulation and decreasing hepatic levels of pro-inflammatory cytokines. In addition, RvD1 treatment attenuated hepatic pyroptosis, an inflammatory form of cell death, via downregulation of pyroptosis-related genes such as GTPase family member b10 and guanylate binding protein 2, and reducing cleavage of caspase 11 and gasdermin-D. In vitro experiments with primary mouse hepatocytes and bone marrow-derived macrophages confirmed the effectiveness of RvD1 in the attenuation of pyroptosis. In summary, our data demonstrated that RvD1 treatment provided beneficial effects against liver injury and inflammation in an experimental animal model recapitulating features of severe AH in humans. Our results suggest that RvD1 may be a novel adjunct strategy to traditional therapeutic options for AH patients.


Assuntos
Etanol , Lipopolissacarídeos , Humanos , Camundongos , Animais , Lipopolissacarídeos/toxicidade , Etanol/toxicidade , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Ácidos Docosa-Hexaenoicos/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Fígado/metabolismo
4.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557303

RESUMO

Chronic alcohol consumption leads to disturbances in intestinal function which can be exacerbated by inflammation and modulated by different factors, e.g., polyunsaturated fatty acids (PUFAs). The mechanisms underlying these alterations are not well understood. In this study, RNA-seq analysis was performed on ileum tissue from WT and fat-1 transgenic mice (which have elevated endogenous n-3 PUFAs). Mice were chronically fed ethanol (EtOH) and challenged with a single lipopolysaccharide (LPS) dose to induce acute systemic inflammation. Both WT and fat-1 mice exhibited significant ileum transcriptome changes following EtOH + LPS treatment. Compared to WT, fat-1 mice had upregulated expression of genes associated with cell cycle and xenobiotic metabolism, while the expression of pro-inflammatory cytokines and pro-fibrotic genes was decreased. In response to EtOH + LPS, fat-1 mice had an increased expression of genes related to antibacterial B cells (APRIL and IgA), as well as an elevation in markers of pro-restorative macrophages and γδ T cells that was not observed in WT mice. Our study significantly expands the knowledge of regulatory mechanisms underlying intestinal alterations due to EtOH consumption and inflammation and identifies the beneficial transcriptional effects of n-3 PUFAs, which may serve as a viable nutritional intervention for intestinal damage resulting from excessive alcohol consumption.


Assuntos
Etanol/toxicidade , Ácidos Graxos Dessaturases/fisiologia , Ácidos Graxos Ômega-3/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Íleo/metabolismo , Inflamação/metabolismo , Animais , Depressores do Sistema Nervoso Central/toxicidade , Perfilação da Expressão Gênica , Humanos , Íleo/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/genética , Masculino , Camundongos , Camundongos Transgênicos
5.
FASEB J ; 35(2): e21377, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33481293

RESUMO

Alcohol-associated liver disease (ALD) is a major human health issue for which there are limited treatment options. Experimental evidence suggests that nutrition plays an important role in ALD pathogenesis, and specific dietary fatty acids, for example, n6 or n3-PUFAs, may exacerbate or attenuate ALD, respectively. The purpose of the current study was to determine whether the beneficial effects of n3-PUFA enrichment in ALD were mediated, in part, by improvement in Wnt signaling. Wild-type (WT) and fat-1 transgenic mice (that endogenously convert n6-PUFAs to n3) were fed ethanol (EtOH) for 6 weeks followed by a single LPS challenge. fat-1 mice had less severe liver damage than WT littermates as evidenced by reduced plasma alanine aminotransferase, hepatic steatosis, liver tissue neutrophil infiltration, and pro-inflammatory cytokine expression. WT mice had a greater downregulation of Axin2, a key gene in the Wnt pathway, than fat-1 mice in response to EtOH and LPS. Further, there were significant differences between WT and fat-1 EtOH+LPS-challenged mice in the expression of five additional genes linked to the Wnt signaling pathway, including Apc, Fosl1/Fra-1, Mapk8/Jnk-1, Porcn, and Nkd1. Compared to WT, primary hepatocytes isolated from fat-1 mice exhibited more effective Wnt signaling and were more resistant to EtOH-, palmitic acid-, or TNFα-induced cell death. Further, we demonstrated that the n3-PUFA-derived lipid mediators, resolvins D1 and E1, can regulate hepatocyte expression of several Wnt-related genes that were differentially expressed between WT and fat-1 mice. These data demonstrate a novel mechanism by which n3-PUFAs can ameliorate ALD.


Assuntos
Ácidos Graxos Ômega-3/metabolismo , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/prevenção & controle , Substâncias Protetoras/metabolismo , Via de Sinalização Wnt , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Etanol/efeitos adversos , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Dessaturases/genética , Feminino , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Inflamação/genética , Lipopolissacarídeos/efeitos adversos , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
6.
Front Physiol ; 12: 812882, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35153819

RESUMO

Alcohol-associated liver disease (ALD) is a prevalent liver disorder and significant global healthcare burden with limited effective therapeutic options. The gut-liver axis is a critical factor contributing to susceptibility to liver injury due to alcohol consumption. In the current study, we tested whether human beta defensin-2 (hBD-2), a small anti-microbial peptide, attenuates experimental chronic ALD. Male C57Bl/6J mice were fed an ethanol (EtOH)-containing diet for 6 weeks with daily administration of hBD-2 (1.2 mg/kg) by oral gavage during the final week. Two independent cohorts of mice with distinct baseline gut microbiota were used. Oral hBD-2 administration attenuated liver injury in both cohorts as determined by decreased plasma ALT activity. Notably, the degree of hBD-2-mediated reduction of EtOH-associated liver steatosis, hepatocellular death, and inflammation was different between cohorts, suggesting microbiota-specific mechanisms underlying the beneficial effects of hBD-2. Indeed, we observed differential mechanisms of hBD-2 between cohorts, which included an induction of hepatic and small intestinal IL-17A and IL-22, as well as an increase in T regulatory cell abundance in the gut and mesenteric lymph nodes. Lastly, hBD-2 modulated the gut microbiota composition in EtOH-fed mice in both cohorts, with significant decreases in multiple genera including Barnesiella, Parabacteroides, Akkermansia, and Alistipes, as well as altered abundance of several bacteria within the family Ruminococcaceae. Collectively, our results demonstrated a protective effect of hBD-2 in experimental ALD associated with immunomodulation and microbiota alteration. These data suggest that while the beneficial effects of hBD-2 on liver injury are uniform, the specific mechanisms of action are associated with baseline microbiota.

7.
Sci Rep ; 10(1): 19930, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199802

RESUMO

The intestine interacts with many factors, including dietary components and ethanol (EtOH), which can impact intestinal health. Previous studies showed that different types of dietary fats can modulate EtOH-induced changes in the intestine; however, mechanisms underlying these effects are not completely understood. Here, we examined intestinal transcriptional responses to EtOH in WT and transgenic fat-1 mice (which endogenously convert n6 to n3 polyunsaturated fatty acids [PUFAs]) to identify novel genes and pathways involved in EtOH-associated gut pathology and discern the impact of n3 PUFA enrichment. WT and fat-1 mice were chronically fed EtOH, and ileum RNA-seq and bioinformatic analyses were performed. EtOH consumption led to a marked down-regulation of genes encoding digestive and xenobiotic-metabolizing enzymes, and transcription factors involved in developmental processes and tissue regeneration. Compared to WT, fat-1 mice exhibited a markedly plastic transcriptome response to EtOH. Cell death, inflammation, and tuft cell markers were downregulated in fat-1 mice in response to EtOH, while defense responses and PPAR signaling were upregulated. This transcriptional reprogramming may contribute to the beneficial effects of n3 PUFAs on EtOH-induced intestinal pathology. In summary, our study provides a reference dataset of the intestinal mucosa transcriptional responses to chronic EtOH exposure for future hypothesis-driven mechanistic studies.


Assuntos
Caderinas/fisiologia , Gorduras na Dieta/administração & dosagem , Etanol/farmacologia , Ácidos Graxos Ômega-3/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Animais , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
8.
Alcohol ; 83: 105-114, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31129175

RESUMO

The symposium "Mechanisms, Biomarkers and Targets for Therapy in Alcohol-associated Liver Injury: From Genetics to Nutrition" was held at the 19th Congress of International Society for Biomedical Research on Alcoholism on September 13th, 2018 in Kyoto, Japan. The goal of the symposium was to discuss the importance of genetics and nutrition in alcoholic liver disease (ALD) development from mechanistic and therapeutic perspectives. The following is a summary of this session addressing the gene polymorphisms in ALD, the role of zinc in gut-liver axis perturbations associated with ALD, highlighting the importance of dietary fat in ALD pathogenesis, the hepatic n6 and n3 PUFA oxylipin pattern associated with ethanol-induced liver injury, and finally deliberating on new biomarkers for alcoholic hepatitis and their implications for diagnosis and therapy. This summary of the symposium will benefit junior and senior faculty currently investigating alcohol-induced organ pathology as well as undergraduate, graduate, and post-graduate students and fellows.


Assuntos
Biomarcadores/análise , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/fisiopatologia , Fenômenos Fisiológicos da Nutrição/fisiologia , Animais , Dieta , Gorduras na Dieta , Hepatite Alcoólica , Humanos , Metabolismo dos Lipídeos/genética , Fígado/química , Fígado/metabolismo , Hepatopatias Alcoólicas/terapia , Camundongos , Oxilipinas/análise , Zinco
9.
J Lipid Res ; 60(12): 2034-2049, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31586017

RESUMO

Ethanol (EtOH)-induced alterations in intestinal homeostasis lead to multi-system pathologies, including liver injury. ω-6 PUFAs exert pro-inflammatory activity, while ω-3 PUFAs promote anti-inflammatory activity that is mediated, in part, through specialized pro-resolving mediators [e.g., resolvin D1 (RvD1)]. We tested the hypothesis that a decrease in the ω-6:ω-3 PUFA ratio would attenuate EtOH-mediated alterations in the gut-liver axis. ω-3 FA desaturase-1 (fat-1) mice, which endogenously increase ω-3 PUFA levels, were protected against EtOH-mediated downregulation of intestinal tight junction proteins in organoid cultures and in vivo. EtOH- and lipopolysaccharide-induced expression of INF-γ, Il-6, and Cxcl1 was attenuated in fat-1 and WT RvD1-treated mice. RNA-seq of ileum tissue revealed upregulation of several genes involved in cell proliferation, stem cell renewal, and antimicrobial defense (including Alpi and Leap2) in fat-1 versus WT mice fed EtOH. fat-1 mice were also resistant to EtOH-mediated downregulation of genes important for xenobiotic/bile acid detoxification. Further, gut microbiome and plasma metabolomics revealed several changes in fat-1 versus WT mice that may contribute to a reduced inflammatory response. Finally, these data correlated with a significant reduction in liver injury. Our study suggests that ω-3 PUFA enrichment or treatment with resolvins can attenuate the disruption in intestinal homeostasis caused by EtOH consumption and systemic inflammation with a concomitant reduction in liver injury.


Assuntos
Etanol/efeitos adversos , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Animais , Ácidos e Sais Biliares/metabolismo , Fezes/química , Feminino , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL
10.
PLoS One ; 13(9): e0204119, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30256818

RESUMO

Alcoholic liver disease (ALD), a significant health problem, progresses through the course of several pathologies including steatosis, steatohepatitis, fibrosis, and cirrhosis. There are no effective FDA-approved medications to prevent or treat any stages of ALD, and the mechanisms involved in ALD pathogenesis are not well understood. Bioactive lipid metabolites play a crucial role in numerous pathological conditions, as well as in the induction and resolution of inflammation. Herein, a hepatic lipidomic analysis was performed on a mouse model of ALD with the objective of identifying novel metabolic pathways and lipid mediators associated with alcoholic steatohepatitis, which might be potential novel biomarkers and therapeutic targets for the disease. We found that ethanol and dietary unsaturated, but not saturated, fat caused elevated plasma ALT levels, hepatic steatosis and inflammation. These pathologies were associated with increased levels of bioactive lipid metabolites generally involved in pro-inflammatory responses, including 13-hydroxy-octadecadienoic acid, 9,10- and 12,13-dihydroxy-octadecenoic acids, 5-, 8-, 9-, 11-, 15-hydroxy-eicosatetraenoic acids, and 8,9- and 11,12-dihydroxy-eicosatrienoic acids, in parallel with an increase in pro-resolving mediators, such as lipoxin A4, 18-hydroxy-eicosapentaenoic acid, and 10S,17S-dihydroxy-docosahexaenoic acid. Elucidation of alterations in these lipid metabolites may shed new light into the molecular mechanisms underlying ALD development/progression, and be potential novel therapeutic targets.


Assuntos
Gorduras na Dieta/efeitos adversos , Etanol/efeitos adversos , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Hepatopatias Alcoólicas/metabolismo , Fígado/metabolismo , Oxilipinas/metabolismo , Animais , Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Gorduras na Dieta/administração & dosagem , Modelos Animais de Doenças , Etanol/administração & dosagem , Regulação da Expressão Gênica , Metabolismo dos Lipídeos/genética , Fígado/lesões , Fígado/patologia , Hepatopatias Alcoólicas/patologia , Masculino , Metaboloma , Camundongos Endogâmicos C57BL , Modelos Biológicos , Oxirredução
11.
Biomolecules ; 8(2)2018 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29587455

RESUMO

The symposium, "Role of Nutrition in Alcoholic Liver Disease", was held at the European Society for Biomedical Research on Alcoholism Congress on 9 October 2017 in Crete, Greece. The goal of the symposium was to highlight recent advances and developments in the field of alcohol and nutrition. The symposium was focused on experimental and clinical aspects in relation to the role of different types of dietary nutrients and malnutrition in the pathogenesis of alcoholic liver disease (ALD). The following is a summary of key research presented at this session. The speakers discussed the role of dietary fats and carbohydrates in the development and progression of alcohol-induced multi-organ pathology in animal models of ALD, analyzed novel nutrition-related therapeutics (specifically, betaine and zinc) in the treatment of ALD, and addressed clinical relevance of malnutrition and nutrition support in ALD. This summary of the symposium will benefit junior and senior faculty currently investigating alcohol-induced organ pathology as well as undergraduate, graduate, and post-graduate students and fellows.


Assuntos
Alcoolismo , Hepatopatias Alcoólicas , Desnutrição , Gorduras na Dieta/metabolismo , Etanol/metabolismo , Humanos , Hepatopatias Alcoólicas/dietoterapia , Hepatopatias Alcoólicas/tratamento farmacológico , Hepatopatias Alcoólicas/metabolismo
12.
Biomolecules ; 8(1)2018 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-29342874

RESUMO

Both chronic and acute (binge) alcohol drinking are important health and economic concerns worldwide and prominent risk factors for the development of alcoholic liver disease (ALD). There are no FDA-approved medications to prevent or to treat any stage of ALD. Therefore, discovery of novel therapeutic strategies remains a critical need for patients with ALD. Relevant experimental animal models that simulate human drinking patterns and mimic the spectrum and severity of alcohol-induced liver pathology in humans are critical to our ability to identify new mechanisms and therapeutic targets. There are several animal models currently in use, including the most widely utilized chronic ad libitum ethanol (EtOH) feeding (Lieber-DeCarli liquid diet model), chronic intragastric EtOH administration (Tsukamoto-French model), and chronic-plus-binge EtOH challenge (Bin Gao-National Institute on Alcohol Abuse and Alcoholism (NIAAA) model). This review provides an overview of recent advances in rodent models of binge EtOH administration which help to recapitulate different features and etiologies of progressive ALD. These models include EtOH binge alone, and EtOH binge coupled with chronic EtOH intake, a high fat diet, or endotoxin challenge. We analyze the strengths, limitations, and translational relevance of these models, as well as summarize the liver injury outcomes and mechanistic insights. We further discuss the application(s) of binge EtOH models in examining alcohol-induced multi-organ pathology, sex- and age-related differences, as well as circadian rhythm disruption.


Assuntos
Consumo Excessivo de Bebidas Alcoólicas/complicações , Modelos Animais de Doenças , Etanol/administração & dosagem , Hepatopatias Alcoólicas/etiologia , Animais , Consumo Excessivo de Bebidas Alcoólicas/patologia , Etanol/toxicidade , Hepatopatias Alcoólicas/patologia , Camundongos , Ratos
13.
Am J Pathol ; 187(10): 2232-2245, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28923202

RESUMO

Alcoholic liver disease is a major human health problem leading to significant morbidity and mortality in the United States and worldwide. Dietary fat plays an important role in alcoholic liver disease pathogenesis. Herein, we tested the hypothesis that a combination of ethanol and a diet rich in linoleic acid (LA) leads to the increased production of oxidized LA metabolites (OXLAMs), specifically 9- and 13-hydroxyoctadecadienoic acids (HODEs), which contribute to a hepatic proinflammatory response exacerbating liver injury. Mice were fed unsaturated (with a high LA content) or saturated fat diets (USF and SF, respectively) with or without ethanol for 10 days, followed by a single binge of ethanol. Compared to SF+ethanol, mice fed USF+ethanol had elevated plasma alanine transaminase levels, enhanced hepatic steatosis, oxidative stress, and inflammation. Plasma and liver levels of 9- and 13-HODEs were increased in response to USF+ethanol feeding. We demonstrated that primarily 9-HODE, but not 13-HODE, induced the expression of several proinflammatory cytokines in vitro in RAW264.7 macrophages. Finally, deficiency of arachidonate 15-lipoxygenase, a major enzyme involved in LA oxidation and OXLAM production, attenuated liver injury and inflammation caused by USF+ethanol feeding but had no effect on hepatic steatosis. This study demonstrates that OXLAM-mediated induction of a proinflammatory response in macrophages is one of the potential mechanisms underlying the progression from alcohol-induced steatosis to alcoholic steatohepatitis.


Assuntos
Gorduras na Dieta/efeitos adversos , Inflamação/patologia , Ácido Linoleico/efeitos adversos , Fígado/metabolismo , Fígado/patologia , Animais , Araquidonato 15-Lipoxigenase/metabolismo , Consumo Excessivo de Bebidas Alcoólicas , Composição Corporal , Citocinas/metabolismo , Modelos Animais de Doenças , Etanol , Ácidos Linoleicos/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Macrófagos/metabolismo , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Estresse Oxidativo , Células RAW 264.7
14.
Reprod Toxicol ; 67: 85-99, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27915011

RESUMO

In this study, we identify gene targets and cellular events mediating the teratogenic action(s) of 5-Aza-2'-deoxycytidine (AzaD), an inhibitor of DNA methylation, on secondary palate development. Exposure of pregnant mice (on gestation day (GD) 9.5) to AzaD for 12h resulted in the complete penetrance of cleft palate (CP) in fetuses. Analysis of cells of the embryonic first branchial arch (1-BA), in fetuses exposed to AzaD, revealed: 1) significant alteration in expression of genes encoding several morphogenetic factors, cell cycle inhibitors and regulators of apoptosis; 2) a decrease in cell proliferation; and, 3) an increase in apoptosis. Pyrosequencing of selected genes, displaying pronounced differential expression in AzaD-exposed 1-BAs, failed to reveal significant alterations in CpG methylation levels in their putative promoters or gene bodies. CpG methylation analysis suggested that the effects of AzaD on gene expression were likely indirect.


Assuntos
Azacitidina/análogos & derivados , Região Branquial/efeitos dos fármacos , Fissura Palatina/induzido quimicamente , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Azacitidina/toxicidade , Região Branquial/embriologia , Região Branquial/patologia , Proliferação de Células/efeitos dos fármacos , Fissura Palatina/embriologia , Fissura Palatina/genética , Fissura Palatina/patologia , Metilação de DNA/efeitos dos fármacos , Decitabina , Desenvolvimento Embrionário/genética , Feminino , Perfilação da Expressão Gênica , Idade Gestacional , Camundongos Endogâmicos ICR , Gravidez
15.
Reprod Toxicol ; 67: 100-110, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27923600

RESUMO

Defects in development of the secondary palate, which arise from the embryonic first branchial arch (1-BA), can cause cleft palate (CP). Administration of 5-Aza-2'-deoxycytidine (AzaD), a demethylating agent, to pregnant mice on gestational day 9.5 resulted in complete penetrance of CP in fetuses. Several genes critical for normal palatogenesis were found to be upregulated in 1-BA, 12h after AzaD exposure. MethylCap-Seq (MCS) analysis identified several differentially methylated regions (DMRs) in DNA extracted from AzaD-exposed 1-BAs. Hypomethylated DMRs did not correlate with the upregulation of genes in AzaD-exposed 1-BAs. However, most DMRs were associated with endogenous retroviral elements. Expression analyses suggested that interferon signaling was activated in AzaD-exposed 1-BAs. Our data, thus, suggest that a 12-h in utero AzaD exposure demethylates and activates endogenous retroviral elements in the 1-BA, thereby triggering an interferon-mediated response. This may result in the dysregulation of key signaling pathways during palatogenesis, causing CP.


Assuntos
Azacitidina/análogos & derivados , Região Branquial/efeitos dos fármacos , Fissura Palatina/induzido quimicamente , Metilação de DNA/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Animais , Azacitidina/toxicidade , Região Branquial/embriologia , Fissura Palatina/embriologia , Fissura Palatina/genética , Decitabina , Desenvolvimento Embrionário/genética , Feminino , Perfilação da Expressão Gênica , Idade Gestacional , Camundongos Endogâmicos ICR , Gravidez
16.
Exp Cell Res ; 342(1): 32-8, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26921506

RESUMO

p300 is a multifunctional transcriptional coactivator that interacts with numerous transcription factors and exhibits protein/histone acetyltransferase activity. Loss of p300 function in humans and in mice leads to craniofacial defects. In this study, we demonstrated that inhibition of p300 histone acetyltransferase activity with the compound, C646, altered the expression of several genes, including Cdh1 (E-cadherin) in mouse maxillary mesenchyme cells, which are the cells that give rise to the secondary palate. The increased expression of plasma membrane-bound E-cadherin was associated with reduced cytosolic ß-catenin, that led to attenuated signaling through the canonical Wnt pathway. Furthermore, C646 reduced both cell proliferation and the migratory ability of these cells. These results suggest that p300 histone acetyltransferase activity is critical for Wnt-dependent palate mesenchymal cell proliferation and migration, both processes that play a significant role in morphogenesis of the palate.


Assuntos
Caderinas/metabolismo , Proteína p300 Associada a E1A/fisiologia , Via de Sinalização Wnt , Animais , Benzoatos/farmacologia , Caderinas/genética , Movimento Celular , Células Cultivadas , Proteína p300 Associada a E1A/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Camundongos Endogâmicos ICR , Morfogênese , Nitrobenzenos , Palato/citologia , Palato/embriologia , Palato/metabolismo , Pirazóis/farmacologia , Pirazolonas , beta Catenina/metabolismo
17.
Dev Dyn ; 243(12): 1536-43, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25104574

RESUMO

BACKGROUND: Transforming growth factor-ß3 (TGF-ß3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF-ß3 functions during secondary palate fusion are still poorly understood. RESULTS: We found that mouse cytokeratin 6α and 17 mRNAs were expressed exclusively in the palate medial edge epithelium on embryonic day 14.5, and this expression was completely abolished in Tgf-ß3 mutant embryos. In contrast, we found that Jagged2 was initially expressed throughout the palate epithelium, but was specifically down-regulated in the medial edge epithelium during palatal fusion. Jagged2 down-regulation was regulated by TGF-ß3, since Jagged2 was persistently expressed in palatal medial edge epithelium in Tgf-ß3 null mutant embryos. Moreover, addition of DAPT, a specific inhibitor of Notch signaling, partially rescued the fusion defects in Tgf-ß3 null mutant palatal shelves. CONCLUSIONS: Based on these results, together with the previous study indicating that the loss of Jagged2 function promotes embryonic oral epithelial fusion, we concluded that TGF-ß3 mediates palate fusion in part by down-regulating Jagged2 expression in palatal medial edge epithelium. In addition, cytokeratin 6α and 17 are two TGF-ß3 downstream target genes in palate medial edge epithelium differentiation.


Assuntos
Embrião de Mamíferos/embriologia , Mucosa Bucal/embriologia , Palato/embriologia , Fator de Crescimento Transformador beta3/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Embrião de Mamíferos/citologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratina-6/biossíntese , Queratina-6/genética , Queratinas/biossíntese , Queratinas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Palato/citologia , Proteínas Serrate-Jagged , Fator de Crescimento Transformador beta3/genética
18.
Dev Growth Differ ; 56(6): 434-47, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24849136

RESUMO

Clefts of the lip and palate are thought to be caused by genetic and environmental insults but the role of epigenetic mechanisms underlying this common birth defect are unknown. We analyzed the expression of over 600 microRNAs in the murine medial nasal and maxillary processes isolated on GD10.0-GD11.5 to identify those expressed during development of the upper lip and analyzed spatial expression of a subset. A total of 142 microRNAs were differentially expressed across gestation days 10.0-11.5 in the medial nasal processes, and 66 in the maxillary processes of the first branchial arch with 45 common to both. Of the microRNAs exhibiting the largest percent increase in both facial processes were five members of the Let-7 family. Among those with the greatest decrease in expression from GD10.0 to GD11.5 were members of the microRNA-302/367 family that have been implicated in cellular reprogramming. The distribution of expression of microRNA-199a-3p and Let-7i was determined by in situ hybridization and revealed widespread expression in both medial nasal and maxillary facial process, while that for microRNA-203 was much more limited. MicroRNAs are dynamically expressed in the tissues that form the upper lip and several were identified that target mRNAs known to be important for its development, including those that regulate the two main isoforms of p63 (microRNA-203 and microRNA-302/367 family). Integration of these data with corresponding proteomic datasets will lead to a greater appreciation of epigenetic regulation of lip development and provide a better understanding of potential causes of cleft lip.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Lábio/embriologia , MicroRNAs/genética , Animais , Feminino , Perfilação da Expressão Gênica , Hibridização In Situ , Camundongos , Fosfoproteínas/genética , Gravidez , Transativadores/genética
19.
Int J Dev Biol ; 58(9): 713-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25896208

RESUMO

Recent studies have shown that mouse palatal mesenchymal cells undergo regional specification along the anterior-posterior (A-P) axis defined by anterior Shox2 and Msx1 expression and posterior Meox2 expression. A-P regional specification of the medial edge epithelium, which is directly responsible for palate fusion, has long been proposed, but it has not yet been demonstrated due to the lack of regional specific markers. In this study, we have demonstrated that the palate medial edge epithelium is regionalized along the A-P axis, similar to that for the underlying mesenchyme. Mmp13, a medial edge epithelium specific marker, was uniformly expressed from anterior to posterior in wild-type mouse palatal shelves. Previous studies demonstrated that medial edge epithelium expression of Mmp13 was regulated by TGF-beta3. We have found that the changes in Mmp13 expression in TGF-beta3 knockouts varied along the A-P axis, and can be broken down into three distinct regions. These regions correlated with regional specification of the underlying medial edge mesenchymal cells and timing of palate fusion. Mouse palate medial edge epithelium along the A-P axis can be divided into different regions according to the differential response to the loss of TGF-beta3.


Assuntos
Embrião de Mamíferos/citologia , Epitélio/embriologia , Mesoderma/embriologia , Mucosa Bucal/embriologia , Palato/embriologia , Animais , Embrião de Mamíferos/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Metaloproteinase 13 da Matriz/genética , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Palato/metabolismo , Fator de Crescimento Transformador beta3/fisiologia
20.
Microrna ; 3(3): 160-73, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25642850

RESUMO

Environmental factors contribute to the etiology of cleft palate (CP). Environmental factors can also affect gene expression via alterations in DNA methylation suggesting a possible mechanism for the induction of CP. Identification of genes methylated during development of the secondary palate provides the basis for examination of the means by which environmental factors may adversely influence palatal ontogeny. We previously characterized the methylome of the developing murine secondary palate focusing primarily on protein- encoding genes. We now extend this study to include methylated microRNA (miRNA) genes. A total of 42 miRNA genes were found to be stably methylated in developing murine palatal tissue. Twenty eight of these were localized within host genes. Gene methylation was confirmed by pyrosequencing of selected miRNA genes. Integration of methylated miRNA gene and expression datasets identified 62 miRNAs, 69% of which were non-expressed. For a majority of genes (83%), upstream CpG islands (CGIs) were highly methylated suggesting down-regulation of CGI-associated promoters. DAVID and IPA analyses indicated that both expressed and non-expressed miRNAs target identical signaling pathways and biological processes associated with palatogenesis. Furthermore, these analyses also identified novel signaling pathways whose roles in palatogenesis remain to be elucidated. In summary, we identify methylated miRNA genes in the developing murine secondary palate, correlate miRNA gene methylation with expression of their cognate miRNA transcripts, and identify pathways and biological processes potentially mediated by these miRNAs.


Assuntos
Metilação de DNA , MicroRNAs/genética , Palato/embriologia , Palato/metabolismo , Animais , Ilhas de CpG , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos ICR , Regiões Promotoras Genéticas
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