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1.
Drug Deliv ; 29(1): 427-439, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35098843

RESUMO

Cranberry extract (CBE) is a major source of the antioxidant polyphenolics but suffers from limited bioavailability. The goal of this research was to encapsulate the nutraceutical (CBE), into bile salt augmented liposomes (BSALs) as a promising oral delivery system to potentiate its hepatoprotective impact against dimethylnitrosamine (DMN) induced liver injury in rats. The inclusion of bile salt in the liposomal structure can enhance their stability within the gastrointestinal tract and promote CBE permeability. CBE loaded BSALs formulations were fabricated utilizing a (23) factorial design to explore the impact of phospholipid type (X1), phospholipid amount (X2), and sodium glycocholate (SGC) amount (X3) on BSALs properties, namely; entrapment efficiency percent, (EE%); vesicle size, (VS); polydispersity index; (PDI); zeta potential, (ZP); and release efficiency percent, (RE%). The optimum formulation (F1) exhibited spherical vesicles with EE% of 71.27 ± 0.32%, VS; 148.60 ± 6.46 nm, PDI; 0.38 ± 0.02, ZP; -18.27 ± 0.67 mV and RE%; 61.96 ± 1.07%. Compared to CBE solution, F1 had attenuated DMN-induced hepatic injury, as evidenced by the significant decrease in serum level of ALT, AST, ALP, MDA, and elevation of GSH level, as well as SOD and GPX activities. Furthermore, F1 exhibited an anti-inflammatory character by suppressing TNF-α, MCP-1, and IL-6, as well as downregulation of VEGF-C, STAT-3, and IFN-γ mRNA levels. This study verified that when CBE was integrated into BSALs, F1, its hepatoprotective effect was significantly potentiated to protect the liver against DMN-induced damage. Therefore, F1 could be deliberated as an antioxidant, antiproliferative, and antifibrotic therapy to slow down the progression of hepatic damage.


Assuntos
Ácidos e Sais Biliares/química , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Lipossomos/química , Extratos Vegetais/farmacologia , Vaccinium macrocarpon , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Química Farmacêutica , Dimetilnitrosamina/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Portadores de Fármacos , Liberação Controlada de Fármacos , Mediadores da Inflamação/metabolismo , Testes de Função Hepática , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores CCR2/efeitos dos fármacos , Fator de Transcrição STAT3/efeitos dos fármacos , Propriedades de Superfície , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos
2.
Appl Biochem Biotechnol ; 194(4): 1699-1715, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34855113

RESUMO

Dimethylnitrosamine (DMN) is an established carcinogen. It is toxic to several organs, viz., the liver, kidney, and lungs, and immune system. Several drugs have been used in the past to modulate its toxicity using experimental animal models. The present study was designed to investigate the effect of zinc oxide nanoparticles (ZnONPs) on renal toxicity caused by DMN in laboratory rat. Since oxidative mechanisms are mainly involved in its toxicity, the proposed study focuses on the amelioration of oxidative stress response by ZnONPs, if any. The present results show that administration of ZnONPs (50 mg/kg body weight/rat) to DMN (2 µl/100 g body weight/rat)-treated rats diminuted the concentration of malonaldehyde, H2O2, and NO in the kidney. However, reduced glutathione (GSH) concentration increased after ZnONP treatment. Results on glutathione S-transferase and glutathione peroxidase favored its antioxidative effects. These results are supported by the recovery of oxidative DNA damage and less pronounced histopathological changes in the kidney. It is hypothesized that ZnONPs might be toxic to renal tissue; however, its strong therapeutic/antioxidative potential helps in ameliorating DMN-induced renal toxicity in rat.


Assuntos
Nanopartículas , Óxido de Zinco , Animais , Antioxidantes/farmacologia , Peso Corporal , Dimetilnitrosamina/farmacologia , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo , Ratos , Óxido de Zinco/toxicidade
3.
In Vitro Cell Dev Biol Anim ; 57(8): 825-834, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34549357

RESUMO

Cholangiocarcinoma (CCA) is an aggressive bile duct cancer. Opisthorchis viverrini (O. viverrini) infection is a significant cause of CCA in the Greater Mekong subregion. Currently, there is no standard chemotherapeutic regimen for CCA. A unique hamster carcinogenesis model of O. viverrini-associated CCA was established. Molecular targets identified from the hamster CCA-comparative model are valuable for target identification and validation. Hamster CCA was induced by the administration of O. viverrini metacercariae and N-nitrosodimethylamine. Hamster-derived cancer cells were isolated and continuously cultured for more than 6 months. Ham-2 cell line was established and characterized in vitro and in vivo. Ham-2 exhibited chromosome hyperploidy. A comparative study with previously established cell line, Ham-1, demonstrated that Ham-2 acquired slower growth, higher adhesion, higher migration, and resistance to doxorubicin and 5-fluorouracil (5-FU). In BALB/c Rag-2/Jak3 double-deficient (BRJ) mice, Ham-2 subcutaneous transplantation formed mucin-producing cancers, which morphologically resemble human tubular cholangiocarcinoma. Intravenous-injected Ham-2 established the metastatic nodules in the lungs and livers of BRJ mice. Altogether, a new hamster cholangiocarcinoma cell line, Ham-2, which acquired more aggressive phenotypes in vitro and in vivo, was established. This cell line might be a valuable tool for comparative drug target identification and validation.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Mucinas/metabolismo , Animais , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/parasitologia , Carcinógenos/farmacologia , Linhagem Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/parasitologia , Cricetinae , Dimetilnitrosamina/farmacologia , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Opistorquíase/complicações , Opisthorchis
4.
Mutagenesis ; 35(2): 197-206, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32109288

RESUMO

Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(-/-)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/-)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(-/-) flies than in spel1(+/-) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(-/-) flies than in spel1(+/-) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.


Assuntos
Aberrações Cromossômicas/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Drosophila melanogaster/genética , Recombinação Genética/efeitos dos fármacos , Animais , Cromossomos/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Reparo do DNA/efeitos dos fármacos , Dietilnitrosamina/farmacologia , Dimetilnitrosamina/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Mutagênese/efeitos dos fármacos , Recombinação Genética/genética
5.
Mol Carcinog ; 59(2): 215-226, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31793679

RESUMO

The high incidence and mortality of esophageal squamous cell cancer (ESCC) is a major health problem worldwide. Precancerous lesions of ESCC may either progress to cancer or revert to normal epithelium with appropriate interventions; the bidirectional instability of the precancerous lesions of ESCC provides opportunities for intervention. Reports suggest that the upregulation of ornithine decarboxylase (ODC) is closely related to carcinogenesis. In this study, we investigated whether ODC may act as a target for chemoprevention in ESCC. Immunohistochemistry (IHC) assays indicate that ODC expression is higher in esophageal precancerous lesions compared with normal tissue controls. Its overexpression promotes cell proliferation and transformation of normal esophageal epithelial cells, and its activity is increased after N-nitrosomethylbenzylamine (NMBA) induction in Shantou human embryonic esophageal cell line (SHEE) and human immortalized cells (Het1A) cells. In addition, p38 α, extracellular regulated kinase (ERK1/2) in the mitogen-activated protein kinase pathway and protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) pathways are activated in response to NMBA treatment. Difluoromethylornithine (DFMO) is an ODC inhibitor, which inhibits NMBA-induced activation of p38 α, ERK1/2 and AKT/mTOR/p70S6K pathways; this has been verified by Western blotting. DFMO was also found to suppress the development of esophageal precancerous lesions in an NMBA-induced rat model; IHC demonstrated p38 α, ERK1/2, and AKT/mTOR/p70S6K pathways to be downregulated in these rats. These findings indicate the mechanisms by which ODC inhibition suppresses the development of esophageal precancerous lesions by downregulating p38 α, ERK1/2, and AKT/mTOR/p70S6k signaling pathways, ODC may be a potential target for chemoprevention in ESCC.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Inibidores da Ornitina Descarboxilase/farmacologia , Ornitina Descarboxilase/metabolismo , Lesões Pré-Cancerosas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinógenos/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Dimetilnitrosamina/análogos & derivados , Dimetilnitrosamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Humanos , Ornitina Descarboxilase/genética , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Transdução de Sinais/genética
6.
Cell Death Dis ; 10(1): 18, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622238

RESUMO

Hepatic fibrosis is marked by excessive synthesis and deposition of connective tissue proteins, especially interstitial collagens in the extracellular matrix of the liver. It is a result of an abnormal wound healing in response to chronic liver injury from various causes such as ethanol, viruses, toxins, drugs, or cholestasis. The chronic stimuli involved in the initiation of fibrosis leads to oxidative stress and generation of reactive oxygen species that serve as mediators of molecular events involved in the pathogenesis of hepatic fibrosis. These processes lead to cellular injury and initiate inflammatory responses releasing a variety of cytokines and growth factors that trigger activation and transformation of resting hepatic stellate cells into myofibroblast like cells, which in turn start excessive synthesis of connective tissue proteins, especially collagens. Uncontrolled and extensive fibrosis results in distortion of lobular architecture of the liver leading to nodular formation and cirrhosis. The perpetual injury and regeneration process could also results in genomic aberrations and mutations that lead to the development of hepatocellular carcinoma. This review covers most aspects of the molecular mechanisms involved in the pathogenesis of hepatic fibrosis with special emphasize on N-Nitrosodimethylamine (NDMA; Dimethylnitorsmaine, DMN) as the inducing agent.


Assuntos
Dimetilnitrosamina/farmacologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Actinas/metabolismo , Animais , Antioxidantes/metabolismo , Colágeno/metabolismo , Citocinas/metabolismo , Dimetilnitrosamina/química , Dimetilnitrosamina/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Cicatrização
7.
Xenobiotica ; 48(5): 498-505, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28608757

RESUMO

1. The objective of study was to determine the influence of ethanol and/or N-nitrosodimethyloamine (NDMA) on the inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by human neutrophils and determination of the role of NF-κB in this process. 2. Isolated polymorphonuclear leukocytes (PMNs) derived from 15 human volunteers were incubated in the presence of ethanol and/or NDMA. Expression of the tested proteins were evaluated using the Western blot method. Total NO metabolites was assayed in the cell cultures by Griess reaction. 3. In neutrophils exposed to ethanol or NDMA was observed an increased NF-κB-dependent NO production mediated by iNOS with the contribution of MAP kinases: p38 and JNK. An inhibiting effect of NF-κB signaling pathway on the MAP kinases was observed, which are involved in the iNOS-dependent NO production. By the simultaneous effect, ethanol and NDMA caused stronger generation of NO by neutrophils without the contribution of iNOS. Inhibition of NF-κB in cells simultaneously exposed to the xenobiotics caused a decreased expression of MAP kinases. 4. Individual and simultaneous effect of ethanol and NDMA may cause disorders in the response of immune system. However, the joint effect of the tested substances results in uncontrolled interactions, leading to cascading disorders of signal transduction.


Assuntos
Dimetilnitrosamina/farmacologia , Etanol/farmacologia , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/biossíntese , Dimetilnitrosamina/química , Dimetilnitrosamina/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neutrófilos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Biosci Biotechnol Biochem ; 81(10): 1941-1947, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28752795

RESUMO

The present study was designed to evaluate the hepatoprotective potential of α-lactalbumin (αLA) against dimethylnitrosamine (DMN)-induced toxic insults in the rat liver. The liver damage was induced in rats by the repeated administration of DMN (10 mg/kg, i.p.) on three consecutive days per week for three weeks. The rats were maintained on either a standard AIN-93 M or αLA-enriched diet starting one week before the DMN injection until the termination of the experiment. The DMN treatment produced a progressive increase in the plasma markers (aspartate aminotransferase, alanine aminotransferase, total bililbin, hyarulonic acid, and matrix metalloproteinase-2) in 28 days after the first DMN injection. Dietary treatment with αLA significantly reduced the DMN-induced damage toward normalcy. NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, significantly attenuated the hepatoprotective effect of αLA. These findings show that αLA has a marked suppressive effect on hepetic fibrosis through a nitric oxide-mediated mechanism.


Assuntos
Dimetilnitrosamina/farmacologia , Lactalbumina/química , Lactalbumina/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Leite/química , Óxido Nítrico/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Bovinos , Fibrose , Fígado/metabolismo , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Baço/patologia
10.
Cell Mol Biol (Noisy-le-grand) ; 62(1): 99-103, 2016 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-26828995

RESUMO

This study aims to study impact of Dicliptera chinensis polysaccharide (DCP) on hepatic fibrosis (HF) and activation of hepatic stellate cells (HSCs). Liver fibrosis model was induced by intraperitoneal injection of dimethyl nitrosamines (DMN) in rat. Rats in treatment group were administrated with different concentrations of DCP (0, 100, 300 mg/kg) by intraperitoneal injection. Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to assess histo-pathological change. α-SMA, TGF-ß1 and pSmad 2/3 were assayed by immuno-histochemistry. HSC-T6 cells were stimulated by recombined rat TGF-ß1 (1 ng/mL) to simulate an activating model in vitro and then interfered with DCP (concentration of 0, 25, 50, 100, 200, 400 µg/ml). MTT assay was used to determine cell proliferation and western blotting was used to detect α-SMA and pSmad 2/3 expression. Results demonstrated that DCP alleviated DMN-induced liver fibrosis in rat and significantly down-regulated TGF-ß1 expression, pSmad2/3 and α-SMA in liver tissue in a dose-dependent way. DCP inhibited proliferation and activation of TGF-ß1-stimulated HSC-T6 in vitro and significantly down-regulated α-SMA and pSmad2/3 expression. In conclusion, this study revealed that DCP attenuates progression of liver fibrosis through suppressing TGF-ß/Smad pathway. DCP is a potential botanical polysaccharide to management liver fibrosis.


Assuntos
Acanthaceae/química , Células Estreladas do Fígado/efeitos da radiação , Cirrose Hepática/tratamento farmacológico , Polissacarídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dimetilnitrosamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Ratos , Ratos Wistar
11.
J Immunotoxicol ; 11(3): 231-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-23971717

RESUMO

Neutrophils (PMN) play diverse regulatory and effector functions in the immune system through the release of reactive nitrogen species, including nitric oxide (NO). The enzyme responsible for NO synthesis in PMN is inducible nitric oxide synthase (iNOS) that is regulated by various signaling pathways, e.g. PI3K-Akt/PKB, and transcription factors. N-Nitrosodimethylamine (NDMA), a xenobiotic widespread in the human environment, affects immune cells. The study objective here was to examine the role of the PI3K-Akt/PKB pathway in induction of NO synthesis (with involvement of iNOS) in human PMN, as well as in autologous mononuclear cells (PBMC), exposed to NDMA. Isolated cells were incubated for 2 h with a sub-lethal dose of NDMA and then the expression of several select proteins in the cell cytoplasmic and nuclear fractions were determined by Western blot analyses. The results indicated that NDMA enhanced expression of iNOS, phospho-PI3K, and phospho-IκBα in the cytoplasmic fraction of the PMN and PBMC. The nuclear fraction of these cells also had a higher NF-κB expression. Moreover, in PMN, NDMA caused an increased expression of phospho-Akt (T308), phospho-Akt (S473), and phospho-IKKαß in the cytoplasm, and c-Jun and FosB in the nuclear fraction. Blocking of PI3K caused a decrease in expression of all these proteins in NDMA-exposed PMN. However, inhibition of PI3K led to a drop in expression of iNOS, phospho-PI3K, and phospho-IκBα in the cytoplasm, and in NF-κB in the nuclear fraction, of PBMC. The results of these studies indicated to us that NDMA activates the PI3K-Akt/PKB pathway in human PMN and that this, in turn, contributes to the activation of transcription factors NF-κB, c-Jun, and FosB involved in NO production (through modulation of iNOS expression).


Assuntos
Dimetilnitrosamina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Xenobióticos/farmacologia , Androstadienos/farmacologia , Células Cultivadas , Humanos , Leucócitos Mononucleares/imunologia , NF-kappa B/metabolismo , Neutrófilos/imunologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Wortmanina
12.
Adv Med Sci ; 58(2): 265-73, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23981673

RESUMO

PURPOSE: The role of MAP kinases in the activation of AP-1 (c-Jun, c-Fos) and NF-κB p65 engaged in the regulation of iNOS expression in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was analyzed in the study. MATERIAL AND METHODS: The study included a group of 20 healthy individuals. Isolated human PMN were incubated in the presence of NDMA. Selective MAP kinases inhibitors were used. The expression of proteins in the cytoplasmic and nuclear fractions was assessed using Western blot method. RESULTS: The results show that NDMA intensifies iNOS, c-Jun, NF-κB p65 and IκB-α expression in the analyzed PMNs. The blocking of the p38 pathway led to lower iNOS expression, and higher expression of c-Jun and c-Fos in the cytoplasmic fraction, and also lower c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. A decrease in iNOS expression in the cytoplasmic fraction, and also c-Jun in both fractions of the examined cells, was observed as a result of JNK pathway inhibition. The blocking of the ERK5 pathway led to higher iNOS, c-Jun and c-Fos expression in the cytoplasmic fraction, and higher c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. The study also demonstrated that blocking of the p38 and JNK pathways resulted in higher expression of NF-κB p65 and IκB-α in the cytoplasmic fraction and their lower expression in the nuclear fraction of these cells. CONCLUSION: Our data indicate the role of MAP kinases p38 and JNK in the activation of c-Jun and NF-κB p65 transcription factors engaged in the regulation of iNOS expression in human neutrophils exposed to NDMA. However ERK5 kinase is not involved in the regulation of iNOS and NO production by those cells.


Assuntos
Dimetilnitrosamina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Neutrófilos/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Adulto , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pessoa de Meia-Idade , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Neutrófilos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA/metabolismo , Xenobióticos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Phytomedicine ; 20(13): 1172-9, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23856441

RESUMO

Recently, the TGF-ß1/Smad signaling pathway has been investigated in the pathogenesis of hepatofibrosis, and pharmacological treatment of liver fibrosis targeted this pathway to determine its contribution to the inhibition of fibrotic development. Importantly, ethnopharmacology-derived Pueraria lobata has been reported to effectively reverse the fibrotic process in the liver. In the present study, we performed dimethylnitrosamine (DMN)-induced liver fibrosis in rats to assess the benefits of puerarin (PR), which was isolated from Pueraria lobata (Willd.), on ECM-derived hepatocytes associated with the TGF-ß1/Smad pathway. Our results showed that the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN), type III precollagen (PCIII) and type IV collagen (CIV) were significantly reduced by PR treatment, while hepatic homogenates showed decreased levels of hydroxyproline (Hyp) and collagen I (Col I). Masson's trichrome staining indicated that the DMN-induced liver fibrosis was alleviated. In addition, the protein expression levels of transforming growth factor-ß l (TGF-ß l), smad2, smad3, α-SMA and TIMP-1 were downregulated specifically by PR treatment, whereas the protein expression levels of smad7 and MMP-1 were upregulated. Furthermore, we evaluated the PR-mediated inhibitory effect on TGF-ß1-treated proliferation and activation in a rat liver stellate cell line (HSC-T6). These data resulted in inhibition of the cell growth of HSC-T6 in a dose-dependent manner and a reduction in TßRI, smad2 and smad3 expressed proteins in the presence of PR on TGF-ß1-treated HSC-T6 cells, while smad7 levels were downregulated. Taken together, these findings identify a unique effect for PR-regulation of the TGF-ß1/Smad pathway in blocking fibrotic development and provide a promising strategy for hepatofibrosis treatment.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Isoflavonas/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Pueraria/química , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Animais , Linhagem Celular , Dimetilnitrosamina/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Isoflavonas/isolamento & purificação , Isoflavonas/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Proteínas Smad/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
14.
Pathol Oncol Res ; 19(4): 785-94, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23645518

RESUMO

The results of a previous study demonstrated that prednisolone enhanced cholangiocarcinogenesis. Therefore, to clarify molecular changes during immunosuppressive cholangiocarcinogenesis, Syrian hamsters were divided into 8 groups: uninfected controls; immunosuppressed Syrian hamsters using prednisolone (P); normal Syrian hamsters administered N-nitrosodimethylamine (ND); immunosuppressed Syrian hamsters administered N-nitrosodimethylamine (NDis); normal Syrian hamsters infected with Opisthorchis viverrini (OV); immunosuppressed Syrian hamsters infected with O. viverrini (OVis); normal Syrian hamsters infected with O. viverrini and administered N-nitrosodimethylamine (CCA); and immunosuppressed Syrian hamsters infected with O. viverrini and administered N-nitrosodimethylamine (CCAis). Syrian hamster livers were used for analysis of tumor-related gene expression and immunohistochemistry through cytokeratin 19 (CK19) and proliferating cell nuclear antigen (PCNA) staining. The tumor-related gene expression results show that CCAis groups at all time points exhibited upregulation of COX-2, IL-6, SOD1, CAT and iNOS and downregulation of p53, which correlated with the predominant expression of CK19 and PCNA in liver tissue. These results suggest that prednisolone enhances cholangiocarcinoma development, which was confirmed by molecular changes.


Assuntos
Anti-Inflamatórios/farmacologia , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/genética , Análise de Variância , Animais , Neoplasias dos Ductos Biliares/imunologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Catalase/genética , Catalase/metabolismo , Colangiocarcinoma/imunologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Cricetinae , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dimetilnitrosamina/farmacologia , Regulação Neoplásica da Expressão Gênica , Interleucina-6/genética , Interleucina-6/metabolismo , Queratina-19/química , Queratina-19/metabolismo , Mesocricetus , Opistorquíase , Prednisolona/farmacologia , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1
15.
Indian J Exp Biol ; 51(1): 73-80, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23441482

RESUMO

Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of kinase regulated by extracellular signals (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells.


Assuntos
Regulação Enzimológica da Expressão Gênica , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neutrófilos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Adolescente , Adulto , Ânions , Dimetilnitrosamina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Biológicos , Nitritos/química , Oxigênio/química , Superóxidos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
J Immunotoxicol ; 10(1): 32-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22734893

RESUMO

The aim of the study was to assess the activity of AP-1 family proteins, e.g. Fra-1, Fra-2, JunB, JunD, and FosB, engaged in the regulation of inducible nitric oxide synthase (iNOS) expression and the production of NO by neutrophils (PMN) exposed to N-nitrosodimethylamine (NDMA) xenobiotic. Isolated human PMN were incubated in the presence of NDMA. iNOS mRNA expression was then analyzed using Northern blot and the expression of other proteins in the cytoplasmic and nuclear fractions were assessed using Western blot. The obtained results indicate that NDMA increased iNOS mRNA and protein expression in human PMN. Furthermore, it increased the expression of Fra-1, Fra-2, JunB, and JunD in the cytoplasmic fraction, and FosB expression in the fractions of analyzed cells. As a consequence of inhibiting p38 pathway and JNK, reduced iNOS expression and NO production was noted in PMN exposed to NDMA. Inhibition of the p38 pathway resulted in reduced expression of all analyzed proteins in the cytoplasmic fraction of PMN exposed to NDMA. Furthermore, increased Fra-2 expression and reduced FosB expression were found in the nuclear fraction of those cells. Inhibiting ERK5 pathway resulted in increased JunB expression in both fractions of the analyzed cells. Therefore, no changes in the expression of analyzed proteins in the presence of NDMA were observed in PMN pre-incubated with JNK pathway inhibitor. In conclusion, the results here indicate a role of Fra-1, Fra-2, JunB, JunD, and FosB transcription factors in the regulation of iNOS expression and NO production by human neutrophils exposed to NDMA.


Assuntos
Citoplasma/metabolismo , Regulação Enzimológica da Expressão Gênica , Neutrófilos/imunologia , Óxido Nítrico Sintase Tipo II/genética , Fator de Transcrição AP-1/metabolismo , Células Cultivadas , Dimetilnitrosamina/farmacologia , Antígeno 2 Relacionado a Fos/genética , Antígeno 2 Relacionado a Fos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Transporte Proteico , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
17.
Bioanalysis ; 5(1): 41-51, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23256471

RESUMO

BACKGROUND: A metabolomic study of biomarkers associated with dimethylnitrosamine (DMN)-induced hepatic fibrosis in Sprague-Dawley rats was performed using GC-MS. The clinical chemistry of the collected blood and the histopathology of excised liver samples were examined, and urine samples were prepared by solvent extraction. RESULTS: Through pattern analysis, the DMN-treated group was divided into two subgroups based on the aspartate aminotransferase (AST) levels compared with the control, a moderately higher group (DMN subgroup A) and a significantly higher group (DMN subgroup B). Uric acid, orotic acid, N-phenylacetylglycine and glutaric acid were biomarkers for DMN subgroup A, aminomalonic acid was a biomarker for DMN subgroup B, and arabitol level distinguished control versus DMN treatment regardless of AST level. CONCLUSION: This study suggests that the identification and profiling of AST level-related metabolites may be useful as a diagnostic tool and for the study of the mechanism of liver fibrosis induced by DMN.


Assuntos
Dimetilnitrosamina/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Biomarcadores/metabolismo , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Masculino , Ratos , Ratos Sprague-Dawley
18.
Parasitol Int ; 61(1): 140-4, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21763456

RESUMO

Myristoylated alanine rich C kinase substrate (MARCKS) has been implicated in PKC-mediated membrane-cytoskeleton alterations that underlie lipopolysaccharide (LPS)-induced macrophage responses. MARCKS is postulated to be involved in inflammation-associated CCA based on its overexpression in cholangiocarcinoma (CCA) and inflammatory cells. The aims of this study were to investigate localization patterns of MARCKS in hamster and human tissue during cholangiocarcinogenesis and to examine the involvement of MARCKS in inflammation. MARCKS protein expression was found prominently in inflammatory cells of Opisthorchis viverrini-treated as well as O. viverrini plus N-nitrosodimethylamine (NDMA)-treated hamsters from week 2 to week 3 of treatment. The positive signal decreased during week 4 to week 12, then increased again at week 26 when CCA developed. At the last time point the expression of MARCKS was observed in both cancer and inflammatory cells. MARCKS protein expression was also found in inflammatory cells, including macrophages in human CCA tissues. O. viverrini excretory/secretory products or worm antigen induced MARCKS mRNA and protein expression in a dose- and time-dependent manner in the human U937 macrophage cell line. The relative mRNA expression of MARCKS in white blood cells of O. viverrini-infected patients was significantly higher than in healthy subjects (P = 0.02). Thus, MARCKS is significantly expressed in macrophages and plays a role in inflammation-related CCA induced by O. viverrini.


Assuntos
Antígenos de Helmintos/metabolismo , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Biomarcadores Tumorais/metabolismo , Colangiocarcinoma/metabolismo , Dimetilnitrosamina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Opistorquíase/metabolismo , Animais , Neoplasias dos Ductos Biliares/parasitologia , Ductos Biliares Intra-Hepáticos/parasitologia , Biomarcadores Tumorais/imunologia , Colangiocarcinoma/parasitologia , Colangiocarcinoma/patologia , Cricetinae , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Leucócitos/metabolismo , Leucócitos/parasitologia , Leucócitos/patologia , Macrófagos/parasitologia , Macrófagos/patologia , Masculino , Proteínas de Membrana/imunologia , Mesocricetus , Substrato Quinase C Rico em Alanina Miristoilada , Opistorquíase/complicações , Opistorquíase/parasitologia , Opistorquíase/patologia , Opisthorchis/imunologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Células U937/metabolismo , Células U937/parasitologia , Células U937/patologia
19.
World J Gastroenterol ; 17(36): 4090-8, 2011 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22039322

RESUMO

AIM: To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA. METHODS: Hepatic fibrosis in rats was induced throu-gh serial subcutaneous injections of dimethylnitrosamine, and expression of HMGB1 was detected by immunohistochemistry. HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000. HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis. Expression of α-smooth muscle actin (α-SMA) and collagen types I and III was evaluated by real-time PCR. Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method. Finally, collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay. RESULTS: The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen. siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner. Moreover, HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types I and III in transfected HSCs. CONCLUSION: This study suggests a significant fun-ctional role for HMGB1 in the development of liver fibrosis. It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.


Assuntos
Proteína HMGB1/metabolismo , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Dimetilnitrosamina/farmacologia , Inativação Gênica , Proteína HMGB1/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley
20.
PLoS One ; 6(11): e27710, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22110735

RESUMO

Liver fibrosis is a worldwide clinical issue. It has been well established that activated hepatic stellate cells (HSCs) are responsible for excessive extracellular matrix (ECM) deposition in chronically damaged livers. The identification of key elements that control HSCs activation will help to further our understanding of liver fibrosis and improve the outcome of clinical treatment. This study demonstrates that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of liver fibrosis as NDRG2 mRNA and protein levels were reduced during HSCs activation. In addition, enhanced NDRG2 expression reduced Smad3 transcription and phosphorylation, which inhibited HSCs activation by blocking the TGF-ß1 signal. Moreover, NDRG2 contributed to an increase in the ratio of matrix metalloproteinase 2 (MMP2) to tissue inhibitor of matrix metalloproteinase 2 (TIMP2), which may facilitate the degradation of the ECM. In dimethylnitrosamine (DMN)-induced fibrotic rat livers, adenovirus-mediated NDRG2 overexpression resulted in decreased ECM deposition and improved liver function compared with controls. In conclusion, the present findings indicate that the modulation of NDRG2 is a promising strategy for the treatment of liver fibrosis.


Assuntos
Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Metaloproteinase 2 da Matriz/metabolismo , Proteínas Smad/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adenoviridae/genética , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dimetilnitrosamina/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/enzimologia , Matriz Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Cirrose Hepática/enzimologia , Cirrose Hepática/fisiopatologia , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Supressoras de Tumor/genética
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