Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.448
Filtrar
1.
Eur J Pharmacol ; 921: 174866, 2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35231468

RESUMO

The proliferation of hepatic progenitor cells (HPCs) contributes to liver regeneration and fibrogenesis during chronic liver injury; however, the mechanism modulating HPC proliferation remains unknown. Y-box binding protein-1 (YB-1) is a transcription factor that regulates the transcription of several genes and is highly expressed in liver injury. We explored the role of YB-1 in HPC proliferation and liver fibrosis. We detected increased expansion of HPCs and elevated levels of YB-1 in HPCs from patients with hepatitis B virus-related fibrosis and choline-deficient ethionine-supplemented or 5-diethoxycarbonyl-1,4-dihydrocollidine diet-induced mice compared with those in control groups. HPC-specific deletion of YB-1 using YB-1flox/flox; Foxl1-Cre+/- mice led to reduced HPC expansion and less collagen deposition in the liver tissues compared with that in Cre-/- mice. In cultured primary HPCs, YB-1 knockdown inhibited HPC proliferation. Further experiments indicated YB-1 negatively regulated p53 expression, and silencing of p53 blocked YB-1 knockdown-mediated inhibition of HPC proliferation. Collectively, YB-1 negatively regulates HPC proliferation and alleviates liver fibrosis by p53.


Assuntos
Cirrose Hepática , Células-Tronco , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Animais , Proliferação de Células/genética , Etionina/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Fígado/metabolismo , Cirrose Hepática/patologia , Regeneração Hepática/genética , Camundongos , Células-Tronco/metabolismo
2.
Biochim Biophys Acta Mol Basis Dis ; 1868(1): 166290, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34662704

RESUMO

Hepatic fibrosis is characterized by excessive extracellular matrix deposition and ductular reactions, manifested as the expansion of hepatic progenitor cells (HPCs). We previously reported that the Y-box binding protein 1 (YB-1) in HPCs is involved in chronic liver injury. In this study, we constructed YB-1f/f Foxl1-Cre mice and investigated the role of YB-1 in HPC expansion in murine choline-deficient, ethionine-supplemented (CDE), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) models. Liver injury and fibrosis were measured using hematoxylin and eosin (HE), Masson, and Sirius Red staining. HPC proliferation was detected using EdU and immunofluorescence (IF). Autophagic flow was measured by mCherry-GFP-LC3B staining and transmission electron microscopy (TEM). YB-1 expression was measured by immunofluorescence and western blotting. CUT & Tag analysis, chromatin immunoprecipitation, and RT-PCR were performed to explore the regulation of autophagy-related protein 7 (Atg7) transcription by YB-1. Our results indicated that liver injury was accompanied by high expression of YB-1, proliferative HPCs, and activated autophagy in the CDE and DDC models. YB-1f/f Cre+/- mice displayed less liver injury and fibrosis than YB-1f/f Cre-/- mice in the CDE and DDC models. YB-1 promoted proliferation and autophagy of HPCs in vitro and in vivo. Transforming growth factor-ß (TGF-ß) induced YB-1 nuclear translocation and facilitated the proliferation and autophagy of HPCs. YB-1 nuclear translocation promoted the transcription of Atg7, which is essential for TGF-ß/YB-1 mediated HPCs expansion in vitro and in vivo. In summary, YB-1 nuclear translocation induced by TGF-ß in HPCs promotes the proliferation and autophagy of HPCs and Atg7 participates in YB-1-mediated HPC-expansion and liver fibrosis.


Assuntos
Proteína 7 Relacionada à Autofagia/genética , Doença Hepática Induzida por Substâncias e Drogas/genética , Cirrose Hepática/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Animais , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Deficiência de Colina/induzido quimicamente , Deficiência de Colina/genética , Deficiência de Colina/patologia , Modelos Animais de Doenças , Etionina/toxicidade , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Piridinas/toxicidade , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia
3.
Epigenetics Chromatin ; 14(1): 52, 2021 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-34863249

RESUMO

Neural tube defects (NTDs) remain one of the most life-threatening birth defects affecting infants. Most patients with NTDs eventually develop lifelong disability, which cause significant morbidity and mortality and seriously reduce the quality of life. Our previous study has found that ethionine inhibits cell viability by disrupting the balance between proliferation and apoptosis, and preventing neural stem cells from differentiating into neurons and astrocytes. However, how ethionine participates in the pathogenesis of neural tube development through N6-methyladenosine (m6A) modification remains unknown. This study aims to investigate METTL3- and ALKBH5-mediated m6A modification function and mechanism in NTDs. Herein, our results demonstrate that SAM play not only a compensatory role, it also leads to changes of m6A modification in neural tube development and regulation. Additionally, these data implicate that METTL3 is enriched in HT-22 cells, and METTL3 knockdown reduces cell proliferation and increases apoptosis through suppressing Wnt/ß-catenin signaling pathway. Significantly, overexpression of ALKBH5 can only inhibit cell proliferation, but cannot promote cell apoptosis. This research reveals an important role of SAM in development of NTDs, providing a good theoretical basis for further research on NTDs. This finding represents a novel epigenetic mechanism underlying that the m6A modification has profound and lasting implications for neural tube development.


Assuntos
Defeitos do Tubo Neural , Via de Sinalização Wnt , Animais , Etionina , Humanos , Camundongos , Defeitos do Tubo Neural/induzido quimicamente , Defeitos do Tubo Neural/genética , Qualidade de Vida , S-Adenosilmetionina
4.
Angew Chem Int Ed Engl ; 60(9): 4666-4672, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33935588

RESUMO

Radical S-adenosyl-l-methionine (SAM) enzymes initiate biological radical reactions with the 5'-deoxyadenosyl radical (5'-dAdo•). A [4Fe-4S]+ cluster reductively cleaves SAM to form the Ω organometallic intermediate in which the 5'-deoxyadenosyl moiety is directly bound to the unique iron of the [4Fe-4S] cluster, with subsequent liberation of 5'-dAdo•. Here we present synthesis of the SAM analog S-adenosyl-l-ethionine (SAE) and show SAE is a mechanistically-equivalent SAM-alternative for HydG, both supporting enzymatic turnover of substrate tyrosine and forming the organometallic intermediate Ω. Photolysis of SAE bound to HydG forms an ethyl radical trapped in the active site. The ethyl radical withstands prolonged storage at 77 K and its EPR signal is only partially lost upon annealing at 100 K, making it significantly less reactive than the methyl radical formed by SAM photolysis. Upon annealing above 77K, the ethyl radical adds to the [4Fe-4S]2+ cluster, generating an ethyl-[4Fe-4S]3+ organometallic species termed ΩE.


Assuntos
Proteínas de Escherichia coli/metabolismo , Etionina/metabolismo , Transativadores/metabolismo , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas de Escherichia coli/química , Etionina/análogos & derivados , Etionina/química , Radicais Livres/química , Radicais Livres/metabolismo , Modelos Moleculares , Estrutura Molecular , Transativadores/química
5.
Acta Biochim Biophys Sin (Shanghai) ; 52(10): 1156-1165, 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33083831

RESUMO

The central nervous system (CNS) diseases are still a major cause of morbidity and mortality throughout the world, which imposes heavy burden on the development of society. Ethionine is a non-proteinogenic amino acid having similar chemical structure and activity to that of methionine, with which it competes. Previous studies have confirmed that ethionine affects various cellular functions by inhibiting the biosynthesis of proteins, RNA, DNA, and phospholipids, or all of them. The relationship of ethionine with some CNS diseases, including neural tube defects, has been investigated recently. However, the detailed effects of ethionine on the nerve cell bioactivities and the underlying mechanisms have not been fully explored. Herein, we systematically investigated the influences of ethionine on the proliferation, differentiation, and apoptosis of neural stem cells (NSCs) and post-mitotic nerve cells. We demonstrated that ethionine inhibited cell viability by disrupting the balance between proliferation and apoptosis, prevented NSCs from differentiating into neurons and astrocytes, and blocked cell progression from G1 to S phase via reducing cyclin D1 function in nerve cells including NSCs, a mouse hippocampal neuron cell line (HT-22), and a mouse brain neuroma cell line (Neuro-2a). We speculated that the inhibitory effect of ethionine on cell viability and differentiation are associated with increased reactive oxygen species production. Our results also supported the concept that ethionine may be an underlying cause of abnormal folate metabolism-induced CNS diseases. Our findings may provide important direction for the application of abnormal folate metabolism-induced CNS diseases in future NSC-based therapies.


Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Etionina/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Astrócitos/metabolismo , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/metabolismo , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteína X Associada a bcl-2/metabolismo
6.
J Agric Food Chem ; 68(38): 10397-10402, 2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31825619

RESUMO

On the basis of the following data from the literature, we hypothesized the presence of ethionine in durian pulp: (1) the major odorants in terms of quantity as well as odor potency in durian pulp are ethanethiol and its derivatives; (2) genome analysis of durian assigned methionine γ-lyase (MGL), the enzyme that converts methionine to methanethiol, a key role for durian odor formation; and (3) MGL accepts not only methionine but also ethionine as a substrate. A targeted search by liquid chromatography-tandem mass spectrometry allowed us to confirm the presence of ethionine in durian pulp. Quantitation of ethionine in samples of different varieties (Monthong, Krathum, Chanee, and Kanyao) showed concentrations (621-9600 µg/kg) in the same range but below the methionine concentrations (16100-30200 µg/kg). During fruit ripening, the ethionine concentration increased as well as the ethanethiol concentration. Final evidence for the role of ethionine as an ethanethiol precursor was provided by demonstrating the formation of (2H5)ethanethiol after adding (2H5)ethionine to durian pulp.


Assuntos
Bombacaceae/química , Etionina/análise , Bombacaceae/classificação , Bombacaceae/crescimento & desenvolvimento , Bombacaceae/metabolismo , Cromatografia Líquida de Alta Pressão , Etionina/metabolismo , Frutas/química , Frutas/classificação , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Espectrometria de Massas , Metionina/análise , Metionina/metabolismo , Odorantes/análise
7.
Dig Dis Sci ; 65(1): 189-203, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31297627

RESUMO

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. METHODS: We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient-ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. RESULTS: CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. CONCLUSION: These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.


Assuntos
Carcinoma Hepatocelular/prevenção & controle , Antagonistas de Hormônios/farmacologia , Neoplasias Hepáticas/prevenção & controle , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Proglumida/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidores , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Deficiência de Colina/complicações , Modelos Animais de Doenças , Epigênese Genética , Etionina , Feminino , Regulação Neoplásica da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Receptor de Colecistocinina B/genética , Receptor de Colecistocinina B/metabolismo , Transdução de Sinais
8.
Spectrochim Acta A Mol Biomol Spectrosc ; 223: 117328, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31295632

RESUMO

Ethionine is an S-ethyl analog of methionine (Met) having a small change in structure. But it is a chemical carcinogen and an antagonist of Met, thus displaying a disparate biological profile. The oxidations of ethionine by biologically important oxidants have not been exploited. Oxidations of dl-ethionine by Pt(IV) anticancer model complexes trans-[PtX2(CN4)]2- (X = Cl or Br) were thus analyzed by time-resolved and stopped-flow spectral techniques. Overall second-order kinetics was established, being first-order in [Pt(IV)] and [Ethionine]tot (the total concentration of ethionine); the observed second-order rate constant k' versus pH profiles were obtained. A stoichiometry of Δ[Pt(IV)]:Δ[Ethionine]tot = 1:1 was unraveled, indicating that ethionine was oxidized to ethionine-sulfoxide which was confirmed by NMR spectroscopic and high-resolution mass spectral analyses. In the proposed reaction mechanism which is similar to that for the oxidation of Met by the same Pt(IV) compounds, the rate-determining steps are rationalized in terms of a bridge formation between one of the coordinated halides in [PtX2(CN4)]2- and the sulfur atom in ethionine, followed by an X+ transfer. Moreover, a large rate enhancement for the reaction of ethionine with [PtBr2(CN4)]2- compared with [PtCl2(CN4)]2- strongly supports an X+ transfer mechanism. Furthermore, a combined quantum-mechanical/molecular-mechanical (QM/MM) method was utilized to simulate a Cl+ transfer mechanism from trans-[PtCl2(CN)4]2- to ethionine. The simulations unraveled the energetically stable structures of reactants and products, which favor the Cl+ transfer process. Rate constants of the rate-determining steps have been derived. Ratios of k (ethionine)/k (Met) are between 2.2 and 2.6 obtained for the three protolytic species of ethionine and Met; the enhanced reactivity might be partially responsible for the disparate biological profiles.


Assuntos
Antineoplásicos/farmacologia , Etionina/química , Modelos Teóricos , Platina/farmacologia , Análise Espectral , Concentração de Íons de Hidrogênio , Cinética , Conformação Molecular , Oxirredução , Espectroscopia de Prótons por Ressonância Magnética , Fatores de Tempo
9.
Cell Death Dis ; 10(6): 449, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31209199

RESUMO

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis. Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH, based on several murine models. However, the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear, because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH. In the present study, our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver. By establishing a simple method of discriminating between apoptosis and necrosis in the liver, we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model. To further investigate what type of necrosis is involved in the initial necrotic cell death, we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model. In addition, necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice, which is lacking in a terminal executor of necroptosis. Consequently, necroptosis inhibition failed to block the onset of necrotic cell death, while ferroptosis inhibition protected hepatocytes from necrotic death almost completely, and suppressed the subsequent infiltration of immune cells and inflammatory reaction. Furthermore, the amount of oxidized phosphatidylethanolamine, which is involved in ferroptosis pathway, was increased in the liver sample of the CDE diet-fed mice. These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis.


Assuntos
Ferroptose , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Apoptose/efeitos dos fármacos , Tetracloreto de Carbono/toxicidade , Cromanos/farmacologia , Citocinas/metabolismo , Dieta , Etionina , Ferroptose/efeitos dos fármacos , Hepatite/imunologia , Hepatite/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Quelantes de Ferro/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necroptose/efeitos dos fármacos , Necrose , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismo
10.
Methods Mol Biol ; 1866: 83-94, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30725410

RESUMO

The elevated methionine (MET) requirement for the growth of tumors, first observed by Sugimura in 1959, termed MET dependence, is a potentially highly effective therapeutic target. Proof of this principle is that when MET restriction (MR) was initially established in co-cultures of cancer and normal cells, MET dependence could be exploited to selectively kill cancer cells without killing co-cultured normal cells. MET-dependent cells become reversibly blocked in the late S/G2 phase of the cell cycle under MR enabling selective and effective S-phase chemotherapy against these blocked cancer cells. Subsequent MET repletion with an anti-mitotic drug was totally effective at selectively eliminating the MET-dependent cancer cells enabling the normal MET-dependent cells to take over the culture. We have also observed that the MET analog ethionine (ETH) is synergistic with MR in arresting the growth of the Yoshida sarcoma both in vitro and eliminating metastasis when transplanted to nude mice. MR increased the efficacy of cisplatinum (CDDP) against the MX-1 human breast carcinoma cell line when grown in nude mice. MR increased 5-fluorouracil (5-FU) efficacy on a human gastric cancer xenograft, SC-1-NU, in nude mice. MET-restricted total parenteral nutrition (MR TPN) was effective in Yoshida sarcoma-bearing rats. MR TPN with doxorubicin (DOX) and vincristine (VCR) resulted in significant tumor suppression and prolonged survival of Yoshida-sarcoma-bearing rats. These results were the basis of subsequent studies that used methioninase to effect MR for effective cancer therapy.


Assuntos
Dieta , Metionina/deficiência , Neoplasias/tratamento farmacológico , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Técnicas de Cocultura , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Etionina/administração & dosagem , Etionina/farmacologia , Etionina/uso terapêutico , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Masculino , Camundongos Nus , Metástase Neoplásica , Neoplasias/patologia , Nutrição Parenteral , Ratos , Sarcoma de Yoshida/patologia , Vincristina/farmacologia , Vincristina/uso terapêutico
11.
FASEB J ; 33(3): 3536-3548, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30452879

RESUMO

Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1-carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2-carbon-transporting folate in the form of 5,10-ethenyl-tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2-carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP-1 cells with ethionine, their transcription of TNF-α was inhibited and cell proliferation reduced. Mass spectrometry of THP-1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone-methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2-carbon transporting variant of the folate cycle and modify human chromatin via ethylation.-Röth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.


Assuntos
Carbono/metabolismo , Etionina/metabolismo , Ácido Fólico/metabolismo , Histonas/metabolismo , Imunomodulação/fisiologia , Lactobacillus reuteri/metabolismo , Aminoácidos/metabolismo , Proliferação de Células/fisiologia , Células Cultivadas , Meios de Cultura/metabolismo , Homocisteína/metabolismo , Humanos , Metionina/metabolismo , Metilação , Microbiota/fisiologia , Probióticos/metabolismo , Células THP-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
12.
J Gastroenterol Hepatol ; 34(1): 284-292, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30144357

RESUMO

BACKGROUND AND AIM: ß-Arrestins (ß-arrs) are regulators and mediators of G protein-coupled receptor signaling that are functionally involved in inflammation. Nuclear factor-κB p65 (NF-κBp65) activation has been observed early in the onset of pancreatitis. However, the effect of ß-arrs in acute pancreatitis (AP) is unclear. The aim of this study is to investigate whether ß-arrs are involved in AP through activation of NF-κBp65. METHODS: Acute pancreatitis was induced by either caerulein injection or choline-deficient supplemented with ethionine diet (CDE). ß-arr1 wild-type and ß-arr1 knockout mice were used in the experiment. The survival rate was calculated in the CDE model mice. Histological and western blot analyses were performed in the caerulein model. Inflammatory mediators were detected by real-time polymerase chain reaction in the caerulein-induced AP mice. Furthermore, AR42J and PANC-1 cell lines were used to further study the effects of ß-arr1 in caerulein-induced pancreatic cells. RESULTS: ß-Arr1 but not ß-arr2 is significantly downregulated in caerulein-induced AP in mice. Targeted deletion of ß-arr1 notably upregulated expression of the pancreatic inflammatory mediators including tumor necrosis factor α and interleukin 1ß as well as interleukin 6 and aggravated AP in caerulein-induced mice. ß-Arr1 deficiency increased mortality in mice with CDE-induced AP. Further, ß-arr1 deficiency enhanced caerulein-induced phosphorylation of NF-κBp65 both in vivo and in vitro. CONCLUSION: ß-Arr1 alleviates AP via repression of NF-κBp65 activation, and it is a potentially therapeutic target for AP.


Assuntos
Pancreatite/genética , Pancreatite/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo , Doença Aguda , Animais , Linhagem Celular Tumoral , Ceruletídeo , Deficiência de Colina/complicações , Modelos Animais de Doenças , Regulação para Baixo , Etionina , Feminino , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos Knockout , Pancreatite/induzido quimicamente , Pancreatite/patologia , Fosforilação , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/metabolismo
13.
Biochem Biophys Res Commun ; 506(1): 12-19, 2018 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-30333090

RESUMO

Although the regeneration of the adult liver depends on hepatic progenitor cells (HPCs), many uncertainties regarding hepatic regeneration in the injured liver remain. Trefoil factor family 1 (TFF1), a secretory protein predominantly expressed in the gastrointestinal tract, is responsible for mucosal restitution. Here, we investigated the role of TFF1 in liver regeneration using a mouse model of hepatic injury (choline-deficient ethionine-supplemented diet and carbon tetrachloride administration) and genetically engineered mice (TFF1 knockout (TFF1-/-)). Immunohistochemistry analysis of human liver samples revealed TFF1 expression in the hepatocytes close to ductular reaction and the regenerating biliary epithelium in injured liver. The number of cytokeratin 19 (CK19)-positive bile ducts was significantly decreased in the TFF1-/- mice after liver injury. Notch pathway in the TFF1-/- mice was also downregulated. HPCs in the control mice differentiated into biliary cells (CK19+/SRY HMG box 9 (SOX9)+) more frequently. In contrast, HPCs in the TFF1-/- mice more frequently differentiated into a hepatic lineage (alpha fetoprotein+/SOX9+) after acute liver damage. Hepatocyte proliferation was upregulated, and the liver weight was increased in TFF1-/- mice in response to chronic liver damage. Thus, TFF1 is responsible for liver regeneration after liver injury by promoting HPC differentiation into a biliary lineage and inhibiting HPC differentiation into a hepatic lineage.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/genética , Hepatócitos/metabolismo , Regeneração Hepática/genética , Células-Tronco/metabolismo , Fator Trefoil-1/genética , Animais , Ductos Biliares/citologia , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Tetracloreto de Carbono/administração & dosagem , Carcinógenos/administração & dosagem , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundário , Diferenciação Celular , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Deficiência de Colina/genética , Deficiência de Colina/metabolismo , Deficiência de Colina/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dieta/efeitos adversos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Etionina/administração & dosagem , Regulação da Expressão Gênica , Hepatite Crônica/genética , Hepatite Crônica/metabolismo , Hepatite Crônica/patologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Queratina-19/genética , Queratina-19/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Regeneração Hepática/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fator Trefoil-1/deficiência
14.
Gastroenterology ; 155(4): 1218-1232.e24, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29964040

RESUMO

BACKGROUND & AIMS: Liver fibrosis, hepatocellular necrosis, inflammation, and proliferation of liver progenitor cells are features of chronic liver injury. Mouse models have been used to study the end-stage pathophysiology of chronic liver injury. However, little is known about differences in the mechanisms of liver injury among different mouse models because of our inability to visualize the progression of liver injury in vivo in mice. We developed a method to visualize bile transport and blood-bile barrier (BBlB) integrity in live mice. METHODS: C57BL/6 mice were fed a choline-deficient, ethionine-supplemented (CDE) diet or a diet containing 0.1% 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) for up to 4 weeks to induce chronic liver injury. We used quantitative liver intravital microscopy (qLIM) for real-time assessment of bile transport and BBlB integrity in the intact livers of the live mice fed the CDE, DDC, or chow (control) diets. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and immunoblots. RESULTS: Mice with liver injury induced by a CDE or a DDC diet had breaches in the BBlB and impaired bile secretion, observed by qLIM compared with control mice. Impaired bile secretion was associated with reduced expression of several tight-junction proteins (claudins 3, 5, and 7) and bile transporters (NTCP, OATP1, BSEP, ABCG5, and ABCG8). A prolonged (2-week) CDE, but not DDC, diet led to re-expression of tight junction proteins and bile transporters, concomitant with the reestablishment of BBlB integrity and bile secretion. CONCLUSIONS: We used qLIM to study chronic liver injury, induced by a choline-deficient or DDC diet, in mice. Progression of chronic liver injury was accompanied by loss of bile transporters and tight junction proteins.


Assuntos
Bile/metabolismo , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Junções Íntimas/metabolismo , Animais , Transporte Biológico , Doença Hepática Crônica Induzida por Substâncias e Drogas/sangue , Doença Hepática Crônica Induzida por Substâncias e Drogas/etiologia , Doença Hepática Crônica Induzida por Substâncias e Drogas/patologia , Deficiência de Colina/complicações , Claudinas/metabolismo , Modelos Animais de Doenças , Etionina , Hepatócitos/patologia , Cinética , Fígado/patologia , Camundongos Endogâmicos C57BL , Permeabilidade , Piridinas , Junções Íntimas/patologia
15.
Gastroenterology ; 154(3): 689-703, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29074451

RESUMO

BACKGROUND & AIMS: Little is known about the signaling pathways that initiate and promote acute pancreatitis (AP). The pathogenesis of AP has been associated with abnormal increases in cytosolic Ca2+, mitochondrial dysfunction, impaired autophagy, and endoplasmic reticulum (ER) stress. We analyzed the mechanisms of these dysfunctions and their relationships, and how these contribute to development of AP in mice and rats. METHODS: Pancreatitis was induced in C57BL/6J mice (control) and mice deficient in peptidylprolyl isomerase D (cyclophilin D, encoded by Ppid) by administration of L-arginine (also in rats), caerulein, bile acid, or an AP-inducing diet. Parameters of pancreatitis, mitochondrial function, autophagy, ER stress, and lipid metabolism were measured in pancreatic tissue, acinar cells, and isolated mitochondria. Some mice with AP were given trehalose to enhance autophagic efficiency. Human pancreatitis tissues were analyzed by immunofluorescence. RESULTS: Mitochondrial dysfunction in pancreas of mice with AP was induced by either mitochondrial Ca2+ overload or through a Ca2+ overload-independent pathway that involved reduced activity of ATP synthase (80% inhibition in pancreatic mitochondria isolated from rats or mice given L-arginine). Both pathways were mediated by cyclophilin D and led to mitochondrial depolarization and fragmentation. Mitochondrial dysfunction caused pancreatic ER stress, impaired autophagy, and deregulation of lipid metabolism. These pathologic responses were abrogated in cyclophilin D-knockout mice. Administration of trehalose largely prevented trypsinogen activation, necrosis, and other parameters of pancreatic injury in mice with L-arginine AP. Tissues from patients with pancreatitis had markers of mitochondrial damage and impaired autophagy, compared with normal pancreas. CONCLUSIONS: In different animal models, we find a central role for mitochondrial dysfunction, and for impaired autophagy as its principal downstream effector, in development of AP. In particular, the pathway involving enhanced interaction of cyclophilin D with ATP synthase mediates L-arginine-induced pancreatitis, a model of severe AP the pathogenesis of which has remained unknown. Strategies to restore mitochondrial and/or autophagic function might be developed for treatment of AP.


Assuntos
Autofagia , Estresse do Retículo Endoplasmático , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Pâncreas/metabolismo , Pancreatite/metabolismo , Doença Aguda , Animais , Arginina , Autofagia/efeitos dos fármacos , Ácidos e Sais Biliares , Sinalização do Cálcio , Ceruletídeo , Deficiência de Colina/complicações , Ciclofilina D , Ciclofilinas/deficiência , Ciclofilinas/genética , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Etionina , Predisposição Genética para Doença , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Pancreatite/patologia , Fenótipo , Ratos , Fatores de Tempo , Trealose/farmacologia
16.
J Vis Exp ; (128)2017 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-29155718

RESUMO

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.


Assuntos
Deficiência de Colina/etiologia , Modelos Animais de Doenças , Etionina/administração & dosagem , Lesão Pulmonar/etiologia , Animais , Proliferação de Células/fisiologia , Deficiência de Colina/metabolismo , Dieta , Suplementos Nutricionais , Fígado/patologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
17.
Pancreas ; 46(8): 1046-1055, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28797013

RESUMO

OBJECTIVES: We set out to examine whether berberine (BBR) might affect the severity of pancreatitis and pancreatitis-associated lung injury in choline-deficient ethionine-supplemented (CDE) diet-induced severe acute pancreatitis. METHODS: Severe acute pancreatitis was induced by feeding a CDE diet for 3 days. Berberine was administered intraperitoneally during CDE diet. Mice were killed on days 1, 2, and 3 after the onset of CDE diet. The severity of pancreatitis was assessed by evaluating changes to the pancreas and lung and survival rate. Blood, pancreas, and lung were harvested for further examination. Furthermore, the regulating mechanisms of BBR were evaluated on the pancreas. RESULTS: Administration of BBR significantly inhibited histological damage to the pancreas and lung and decreased serum level of amylase and lipase, myeloperoxidase activity, cytokine production, and the mortality rate. Furthermore, administration of BBR inhibited activation of nuclear factor kappa B, c-Jun N-terminal kinases, and p38 in the pancreas during CDE diet. CONCLUSIONS: These findings suggest that BBR attenuates the severity of pancreatitis by inhibiting activation of nuclear factor kappa B, c-Jun N-terminal kinase, and p38 and that BBR could be used as a beneficial agent to regulate AP.


Assuntos
Berberina/farmacologia , Lesão Pulmonar/prevenção & controle , Pulmão/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Pancreatite Necrosante Aguda/prevenção & controle , Amilases/sangue , Animais , Colina/isolamento & purificação , Dieta/efeitos adversos , Etionina/administração & dosagem , Feminino , Lipase/sangue , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/mortalidade , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite Necrosante Aguda/etiologia , Pancreatite Necrosante Aguda/mortalidade , Fitoterapia/métodos , Taxa de Sobrevida
18.
Biochem Biophys Res Commun ; 482(4): 1346-1352, 2017 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-27939882

RESUMO

Acute pancreatitis has several underlying etiologies, and results in consequences ranging from mild to complex multi-organ failure. The wide range of pathology suggests a genetic predisposition for progression. We compared the susceptibility to acute pancreatitis in BALB/c and FVB/N mice, coupled with proteomic analysis, in order to identify potential protein associations with pancreatitis progression. METHODS: Pancreatitis was induced in BALB/c and FVB/N mice by administration of cerulein or feeding a choline-deficient, ethionine-supplemented (CDE) diet. Histology and changes in serum amylase were examined. Proteome profiling in cerulein-treated mice was performed using 2-dimensional differential in gel electrophoresis (2D-DIGE) followed by mass spectrometry analysis and biochemical validation. RESULTS: Male and female FVB/N mice manifested more severe cerulein-induced pancreatitis as compared with BALB/c mice, but both strains were similarly susceptible to CDE-induced pancreatitis. Few of the 2D-DIGE alterations were validated by immunoblotting. Clusterin was markedly up-regulated after cerulein-induced pancreatitis in FVB/N but less-so in BALB/c mice. Pyrroline-5-carboxylate reductase (Pycr1), an enzyme involved in proline biosynthesis, had higher basal levels in FVB/N male and female mouse pancreata compared with BALB/c pancreata, and was relatively more resistant to degradation in FVB/N pancreata. However, serum and pancreas tissue proline levels were similar in the two strains. CONCLUSION: FVB/N is more susceptible than BALB/c mice to cerulein-induced but not CDE-induced pancreatitis. Most of the 2D-DIGE alterations in the two strains likely relate to posttranslational modifications rather than protein level differences. Clusterin levels increase dramatically in association with pancreatitis severity, while Pycr1 is higher in FVB/N versus BALB/c pancreata basally and after induction of pancreatitis. Changes in proline metabolism may represent a novel potential genetic modifier in the context of pancreatitis.


Assuntos
Clusterina/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença , Pancreatite/genética , Pirrolina Carboxilato Redutases/genética , Amilases/sangue , Animais , Ceruletídeo/química , Colina/química , Clusterina/metabolismo , Modelos Animais de Doenças , Etionina/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pancreatite/metabolismo , Prolina/química , Processamento de Proteína Pós-Traducional , Proteoma , Pirrolina Carboxilato Redutases/metabolismo , Especificidade da Espécie
19.
Int J Exp Pathol ; 97(5): 389-396, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27918111

RESUMO

Severe chronic hepatic injury can induce complex reparative processes. Ductular reaction and the appearance of small hepatocytes are standard components of this response, which is thought to have both adverse (e.g. fibrosis, carcinogenesis) and beneficial (regeneration) consequences. This complex tissue reaction is regulated by orchestrated cytokine action. We have investigated the influence of the tyrosine kinase inhibitor imatinib on a regenerative process. Ductular reaction was induced in mice by the widely used choline-deficient ethionine-supplemented diet (CDE). Test animals were treated daily with imatinib. After 6 weeks of treatment, imatinib successfully reduced the extent of ductular reaction and fibrosis in the CDE model. Furthermore, the number of small hepatocytes increased, and these cells had high proliferative activity, were positive for hepatocyte nuclear factor 4 and expressed high levels of albumin and peroxisome proliferator-activated receptor alpha. The overall functional zonality of the hepatic parenchyma (cytochrome P450 2E1 and glucose 6 phosphatase activity; endogenous biotin content) was maintained. The expression of platelet-derived growth factor receptor beta, which is the major target of imatinib, was downregulated. The anti-fibrotic activity of imatinib has already been reported in several experimental models. Additionally, in the CDE model imatinib was able to enhance regeneration and preserve the functional arrangement of hepatic lobules. These results suggest that imatinib might promote the recovery of the liver following parenchymal injury through the inhibition of platelet-derived growth factor receptor beta.


Assuntos
Mesilato de Imatinib/farmacologia , Cirrose Hepática/tratamento farmacológico , Regeneração Hepática/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Células-Tronco/efeitos dos fármacos , Animais , Deficiência de Colina/complicações , Suplementos Nutricionais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Etionina , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Mesilato de Imatinib/uso terapêutico , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Cirrose Hepática/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/uso terapêutico , Células-Tronco/fisiologia
20.
Int J Mol Sci ; 17(11)2016 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-27827847

RESUMO

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50-3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes.


Assuntos
Cádmio/toxicidade , Desenvolvimento Embrionário/efeitos dos fármacos , Exposição Ambiental/efeitos adversos , Comportamento Exploratório/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética , Adenosina/análogos & derivados , Adenosina/antagonistas & inibidores , Adenosina/metabolismo , Animais , Cátions Bivalentes , Metilação de DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Epigênese Genética/efeitos dos fármacos , Etionina/análogos & derivados , Etionina/antagonistas & inibidores , Etionina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glutationa/antagonistas & inibidores , Glutationa/metabolismo , Estresse Oxidativo , Fenótipo , Peixe-Zebra/embriologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...