RESUMO
BACKGROUND: Lycium barbarum polysaccharides (LBPs) are antioxidant and neuroprotective derivative from Wolfberry. However, whether LBP has a protective effect in non-alcoholic steatohepatitis (NASH)-induced hepatic injury is still unknown. OBJECTIVE: We aimed to study the possible hepatoprotective effects and mechanisms of LBP on a diet-induced NASH rat model. METHODS AND DESIGN: In this study, female rats were fed a high-fat diet to induce NASH with or without an oral 1 mg kg(-1) LBP feeding daily for 8 weeks. After 8 weeks, blood serum and liver samples from each rat were subjected to histological analysis, biochemical and molecular measurements. RESULTS: Compared with control rats, NASH rats showed typical NASH features including an increase in liver injury, lipid content, fibrosis, oxidative stress, inflammation and apoptosis. In contrast, NASH+LBP-co-treated rats showed (1) improved histology and free fatty acid levels; (2) re-balance of lipid metabolism; (3) reduction in profibrogenic factors through the TGF-ß/SMAD pathway; (4) improved oxidative stress through cytochrome P450 2E1-dependent pathway; (5) reduction in hepatic pro-inflammatory mediators and chemokines production; and (6) amelioration of hepatic apoptosis through the p53-dependent intrinsic and extrinsic pathways. The preventive effects of LBP were partly modulated through the PI3K/Akt/FoxO1, LKB1/AMPK, JNK/c-Jun and MEK/ERK pathways and the downregulation of transcription factors in the liver, such as nuclear factor-κB and activator protein-1. CONCLUSION: LBP is a novel hepatoprotective agent against NASH caused by abnormal liver metabolic functions.
RESUMO
The carotid body (CB) plays important roles in cardiorespiratory changes in chronic and intermittent hypoxia. Pituitary adenylate cyclase activating polypeptide (PACAP) is involved in the regulation of respiratory chemoresponse. We hypothesized an upregulation of the expressions of PACAP and its receptor (PAC1) in the rat CB in chronic and intermittent hypoxia. The CB expressions of PACAP and PAC1 were examined in rats breathing 10% O(2) (in isobaric chamber for chronic hypoxia, 24 h/day) or in intermittent hypoxia (cyclic between air and 5% O(2) per minute, 8 h/day) for 7 days. Immunohistochemical studies showed that the PACAP and PAC1 proteins were localized in CB glomic clusters containing tyrosine hydroxylase. The proportional amount of cells with positive staining of PACAP and PAC1 was significantly increased in both hypoxic groups when compared with the normoxic control. In addition, the mRNA level of PAC1 expression was markedly elevated in the hypoxic groups, despite no changes in the PACAP expression. These results suggest an upregulation of PACAP and its receptor expression in the rat CB under chronic and intermittent hypoxic conditions. The PACAP binding to its receptor could activate the PKA signaling pathway leading to an increased CB excitability under hypoxic conditions.
Assuntos
Corpo Carotídeo/metabolismo , Hipóxia/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Animais , Doença Crônica , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/análise , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/análise , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Regulação para CimaRESUMO
Animal models used to study the pathogenesis of non-alcoholic fatty liver disease (NAFLD) are, in general, either genetically altered, or fed with a diet that is extremely high in fat or carbohydrates. Recent findings support the role of oxidative stress, lipid peroxidation and inflammation as probable causative factors. We hypothesize that not only the amount of dietary fat, but the quality of fat is also important in inducing NAFLD. Based on previous observations that female rats fed a diet comprising unsaturated fatty acids are susceptible to liver injury, we proposed that female rats fed with a diet containing fish oil and dextrose would develop pathological and biochemical features of NAFLD. We fed a highly unsaturated fat diet (30% fish oil) to female Sprague-Dawley rats (180-200g), consumed ad libitum for 8 weeks (NAFLD; n=6-8 ). Control animals (CF; n=6-8) were fed with an isocaloric regular rat chow. At killing, blood and liver samples were collected for serum alanine aminotransferase (ALT), histology and molecular analysis. Each histological sample was evaluated for fatty liver (graded from 0 to 4+ according to the amount of fatty change), necrosis (number of necrotic foci (no./mm2) and inflammation (cells per mm2). The amount of collagen formation was estimated based on the amount of Sirius Red staining. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out for tumor necrosis factor alpha (TNF-alpha), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), adiponectin, glutathione peroxidase (GPx), superoxide dismutase (Cu/Zn SOD) and catalase (CAT). Western Blot analysis was done for cyclooxygenases-2 (COX-2), inducible nitric oxide synthase (iNOS) and nitrotyrosine. Electrophoretic mobility shift assay was performed for nuclear factor-kappa B (NF-kB) activity. NAFLD rats had a significantly higher serum ALT level, amount of collagen formation, fatty liver, necrosis and inflammation when compared with the chow-fed control rats. mRNA and protein levels of NF-kB regulated genes, which included TNF-alpha, COX-2 and iNOS were also significantly (p<0.01; p<0.01; p<0.05 respectively) upregulated in the NAFLD group when compared with the chow-fed control rats. mRNA levels of antioxidants CAT and GPX were reduced by 35% and 50% respectively in the NAFLD group. However, Cu/Zn SOD mRNA was similar in both groups. The mRNA level of adiponectin was also reduced in NAFLD group. NF-kB activity was markedly increased in the NAFLD rats (p<0.01). The level of oxidative stress, represented by the formation of nitrotyrosine, was significantly elevated in the NAFLD rats (p<0.01). We conclude that NAFLD rats demonstrated several features of NAFLD, which included fatty liver, inflammation, necrosis, increased oxidative stress, an imbalance between pro and antioxidant enzymes mRNAs, reduced adiponectin levels and upregulation of pro-inflammatory mediators. We propose that female rats fed with a diet containing highly unsaturated fatty acids are an extremely useful model for the study of NAFLD.
Assuntos
Gorduras na Dieta/metabolismo , Modelos Animais de Doenças , Fígado Gorduroso/patologia , Hepatopatias/patologia , Adiponectina/metabolismo , Administração Oral , Alanina Transaminase/sangue , Animais , Estudos de Casos e Controles , Catalase/metabolismo , Colágeno/biossíntese , Ciclo-Oxigenase 2/metabolismo , Gorduras na Dieta/administração & dosagem , Ácidos Graxos Insaturados/administração & dosagem , Feminino , Glutationa Peroxidase/metabolismo , Inflamação/induzido quimicamente , Inflamação/etiologia , Inflamação/patologia , Hepatopatias/etiologia , Necrose/induzido quimicamente , Necrose/etiologia , Necrose/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: The purpose of this study was to investigate the ability of high-density lipoproteins (HDLs) to upregulate genes with the potential to protect against inflammation in endothelial cells. METHODS AND RESULTS: Human coronary artery endothelial cells (HCAECs) were exposed to reconstituted HDLs (rHDLs) for 16 hours before being activated with tumor necrosis factor-alpha (TNF-alpha) for 5 hours. rHDLs decreased vascular cell adhesion molecule-1 (VCAM-1) promoter activity by 75% (P<0.05), via the nuclear factor-kappa B (NF-kappaB) binding site. rHDLs suppressed the canonical NF-kappaB pathway and decreased many NF-kappaB target genes. Suppression of NF-kappaB and VCAM-1 expression by rHDLs or native HDLs was dependent on an increase in 3beta-hydroxysteroid-Delta 24 reductase (DHCR24) levels (P<0.05). The effect of HDLs on DHCR24 is dependent on SR-BI but not ABCAI or ABCGI. Silencing DHCR24 expression increased NF-kappaB (1.2-fold, P<0.05), VCAM-1 (30-fold, P<0.05), and NF-kappaB p50 (4-fold, P<0.05) and p65 subunits (150-fold, P<0.05). TNF-alpha activation of siDHCR24-treated cells increased expression of VCAM-1 (550-fold, P<0.001) and NF-kappaB (9-fold, P<0.001) that could no longer be suppressed by rHDLs. CONCLUSIONS: Results suggest that antiinflammatory effects of rHDLs are mediated partly through an upregulation of DHCR24. These findings raise the possibility of considering DHCR24 as a target for therapeutic modulation.
Assuntos
Apolipoproteína A-I/metabolismo , Arterite/prevenção & controle , Aterosclerose/prevenção & controle , Células Endoteliais/enzimologia , Lipoproteínas HDL/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Animais , Apolipoproteína A-I/administração & dosagem , Arterite/enzimologia , Arterite/etiologia , Arterite/genética , Aterosclerose/enzimologia , Aterosclerose/etiologia , Aterosclerose/genética , Células Cultivadas , Colesterol na Dieta , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Infusões Intravenosas , Lipoproteínas HDL/administração & dosagem , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Coelhos , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Homologs of the Escherichia coli surE gene are present in many eubacteria and archaea. Despite the evolutionary conservation, little information is available on the structure and function of their gene products. We have determined the crystal structure of the SurE protein from Thermotoga maritima. The structure reveals the dimeric arrangement of the subunits and an active site around a bound metal ion. We also demonstrate that the SurE protein exhibits a divalent metal ion-dependent phosphatase activity that is inhibited by vanadate or tungstate. In the vanadate- and tungstate-complexed structures, the inhibitors bind adjacent to the divalent metal ion. Our structural and functional analyses identify the SurE proteins as a novel family of metal ion-dependent phosphatases.
Assuntos
Fosfatase Ácida , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Sítios de Ligação , Cátions Bivalentes/metabolismo , Cristalografia por Raios X , Metais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Thermotoga maritima/genética , Compostos de Tungstênio/metabolismo , Compostos de Tungstênio/farmacologia , Vanadatos/metabolismo , Vanadatos/farmacologiaRESUMO
The aim of the present study was to determine the relationships between bone morphogenetic proteins (BMPs), BMP receptor type IA and carcinogenesis of oral epithelium. A retrospective study was performed on material obtained from oral mucosa, including nine cases of normal mucosa (NB), eight cases of nonspecific chronic inflammation (NCI), seven cases of hyperkeratosis (HK), five cases of squamous cell papilloma (SCP), 29 cases of squamous cell carcinoma (SCC) with various grades of differentiation and 10 cases of epithelium adjacent to carcinoma (EAC). Six cases of NB from hard palate (NHP) were chosen as a control group. The benign groups consisted of NCI, HK and SCP. The antibodies against BMP-2/4, -5, receptor BMPR-IA and purified bovine BMP (bBMP-McAb) were utilised using an immunocytochemical method. The results demonstrated that the immunostaining of BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb was weak and not consistent in normal and benign groups. The immunoreactivity level was independent of the clinical and pathological grading of SCC. All cases of SCC showed positive staining for BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb except for three cases and one case of SCC which negatively stained for BMP-2/4 and BMP-5, respectively. The staining intensity and proportion of the positively stained cells were markedly increased in SCC when compared with that of the normal and benign groups except for EAC. The metastatic carcinoma cells in lymph nodes were strongly and positively stained for BMP-2/4 and BMP-5 when compared with the primary lesions. Our results indicate that there was an overexpression of BMP-2/4, BMP-5, bBMP-McAb and BMPR-IA in the high-risk premalignant and malignant lesions of oral epithelium. Our findings suggest that BMP-2/4 and BMP-5 but not BMPR-IA might be involved in the metastasis of oral carcinoma cells.
Assuntos
Proteínas Morfogenéticas Ósseas/análise , Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/metabolismo , Proteínas de Neoplasias/análise , Lesões Pré-Cancerosas/metabolismo , Proteínas Serina-Treonina Quinases/análise , Receptores de Fatores de Crescimento/análise , Fator de Crescimento Transformador beta , Análise de Variância , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 5 , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Estudos de Casos e Controles , Membrana Celular/química , Citoplasma/química , Epitélio/química , Humanos , Imuno-Histoquímica , Leucoplasia Oral/genética , Metástase Linfática , Mucosa Bucal/química , Palato , Papiloma/genética , Estudos Retrospectivos , Estatísticas não ParamétricasRESUMO
The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.
Assuntos
Aminoácidos/química , Heme/química , Mioglobina/química , Substituição de Aminoácidos , Animais , Cristalografia por Raios X , Modelos Moleculares , Mioglobina/metabolismo , Oxigênio/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , BaleiasRESUMO
The present study investigated the relationships amongst apoptosis, terminal differentiation and telomerase activity in human colon carcinoma cells. We found that hexamethylene bisacetamide (HMBA) induced apoptosis in human colon carcinoma LoVo cells harbouring wild-type p53 but not in SW1116 cells harbouring mutant p53. HMBA reduced telomerase activity in both colon carcinoma cells but it did not induce differentiation in the colon carcinoma cells. Taken together, our results suggest that HMBA can induce apoptosis via a p53-dependent pathway, but apoptosis and terminal differentiation may be separately regulated in LoVo cells. Inhibition of telomerase activity may activate apoptosis through a p53-dependent mechanism.
Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/patologia , Telomerase/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Fosfatase Alcalina/metabolismo , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular , Neoplasias do Colo/enzimologia , Humanos , Células Tumorais CultivadasRESUMO
Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MalG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.