RESUMO
OBJECTIVE: To explore the risk factors for irritable bowel syndrome (IBS) among school adolescents in China. METHOD: A stratified, randomized study by cluster sampling was conducted, which recruited 51,956 students from high and primary schools in Chinese cities. All students were requested to fill in a questionnaire. RESULT: (1) Factors including class (odds ratio 1.12), excessive intake of pepper (odds ratio 1.17), fried (odds ratio 1.08) and starch-based foods (odds ratio 1.06), gastrointestinal tract infection (odds ratio 2.66), abuse of analgesic (odds ratio 1.49), inheritance (odds ratio 1.83), fatigue (odds ratio 1.32) and repression (odds ratio 1.45) were significantly associated with the presence of IBS (P < 0.05). High protein food (odds ratio 0.90) was a protective factor. CONCLUSION: Different food intake, gastrointestinal tract infection, abuse of analgesic, inheritance and psychological factors might be related to development of IBS in the students of the cities involved in this study.
Assuntos
Síndrome do Intestino Irritável/epidemiologia , Adolescente , Criança , China/epidemiologia , Humanos , Síndrome do Intestino Irritável/etiologia , Fatores de Risco , Estudos de Amostragem , Estudantes , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: To investigate the effects of small interfering RNA targeting connective tissue growth factor (CTGF) on rat transforming growth factor beta (TGF beta)/Smads signal pathway. METHODS: Chemically synthetic siRNA targeting CTGF was transfected into HSC T6 and then they were injected into rat livers through their intraportal veins. At the same time these rats also received CCl4 subcutaneously every three days for 6 consecutive weeks. Untreated HSC T6 or/and rats with random siRNA treatment served as controls. Total RNA or/and protein in HSC T6 and rat hepatic tissues were extracted. The expressions of CTGF and TGF beta 1, Smad2, 3 and 7 genes were detected by reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot. RESULTS: CTGF siRNA significantly reduced the expression of CTGF protein in HSC T6. At 48 h after CTGF siRNA treatment, the down-regulation of CTGF protein was the most significant, up to 94%+/-4% (t=46.196, P less than 0.01), but the expressions of TGF beta 1, Smad2, 3 and 7 mRNA showed no differences in HSC T6 compared with the blank controls. Six weeks after CCl4 injections, prominent up-regulations were observed in the gene expressions of CTGF and TGF beta 1 in saline control or siRNA-treated rat livers. Administering CTGF siRNA for six weeks markedly attenuated the induction of CTGF and TGF beta 1 genes; the expressions of CTGF and TGF beta 1 protein decreased by 95%+/-2% (F=21.234, P less than 0.01) and 74%+/-8% (F=13.464, P less than 0.05), respectively, whereas Smad2, 7 protein expressions were not affected. CONCLUSION: Silencing the CTGF gene can suppress the TGF beta /Smads signal pathway in rat livers.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Inativação Gênica , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Masculino , RNA Mensageiro/genética , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , TransfecçãoRESUMO
BACKGROUND: Bone marrow-derived liver stem cells (BDLSCs) are very robust cells that can differentiate into liver epithelial cells. These stem cells are promising targets for gene therapy treatment of liver diseases. Liver fibrosis results from chronic liver damage characterized by an accumulation of extracellular matrix (ECM) and levels of urokinase-type plasminogen activator (uPA) play an important role in ECM degradation. In the present study, we investigated the therapeutic effects of uPA gene-modified BDLSC transplantation on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. METHODS: BDLSCs were obtained from the bone marrow of cholestatic rats. These stem cells were selected and proliferated in medium containing 5% cholestatic serum. BDLSCs transfected with adenovirus-mediated human urokinase-plasminogen activator were transplanted into rats with CCl(4)-induced hepatic fibrosis. Liver function and the area of hepatic fibrosis were correlated with the development and prognosis of hepatic fibrosis. RESULTS: Hepatocyte-like colony-forming units were formed by bone marrow cells after 2 weeks in culture. In the uPA gene-modified BDLSC group, the areas of hepatic fibrosis were smaller and liver function was markedly ameliorated compared to controls. The expression of alpha-smooth muscle actin protein, transforming growth factor-beta1 protein and collagen types I and III mRNA were downregulated. By contrast, the levels of matrix metalloproteinases-2, -3 and -9 mRNA, hepatic growth factor mRNA and proliferating cell nuclear antigen protein increased. CONCLUSIONS: Transplantation of uPA gene-modified BDLSCs may suppress hepatic fibrosis and ameliorate liver function.
Assuntos
Cirrose Hepática Experimental/genética , Fígado/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Adenoviridae/genética , Animais , Ductos Biliares/lesões , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Tetracloreto de Carbono/toxicidade , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Feminino , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Ligadura , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Masculino , Distribuição Aleatória , Ratos , Ratos Endogâmicos F344 , TransfecçãoRESUMO
OBJECTIVE: To investigate the anti-fibrogenesis property of intraportal vein injection of small interfering RNA targeting connective tissue growth factor (CTGF) in a rat model of liver fibrosis and its effect on the accumulation of extracellular matrix (ECM). METHODS: Thirty male rats were randomly divided into five groups. Some rats received CCl4 subcutaneously every three days for 6 consecutive weeks, and in the meantime they also received either siRNA targeting CTGF (preventive group), saline (model group) or siRNA (siRNA control group) by intraportal vein injections. Other rats received CCl4 by subcutaneous injection for 2 weeks, followed by CCl4 and CTGF siRNA intraportal vein injection for 4 more weeks (as treatment group). The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot respectively. Hepatic histology was evaluated by HE and Sirius red stained sections. The collagen staining areas were measured quantitatively using a computer-aided manipulator with slight modifications. Serum procollagen type III and hyaluronic acid were determined by radioimmunoassay. RESULTS: Six weeks after CCl4 injection, prominent upregulation of gene expressions of CTGF, type I and III collagen, and laminin in saline or siRNA-treated rat livers were observed. The expressions of CTGF at mRNA and protein level and type I and III collagen at mRNA level were markedly reduced in rats with CTGF siRNA treated for four or six weeks. Expressions of CTGF at mRNA and protein levels decreased by 76%+/-8%, 80%+/-3% (F = 68.630) and 95%+/-2%, 93%+/-3% (F = 21.234, P < 0.01); type I and III collagen and laminin at mRNA levels decreased by 74%+/-8%, 78%+/-8%, 31%+/-7% and 57%+/-6%, 59%+/-10%, 43%+/-9% (F = 24.219, 16.315, 9.716, P < 0.01) compared with rats in the model group at 72 h. The CTGF siRNA treatment markedly reduced serum levels of procollagen type III and hyaluronic acid and the degrees of liver fibrosis. CONCLUSION: Intraportal vein siRNA injection targeting CTGF could significantly inhibit CTGF gene expression in rats, thereby attenuating liver fibrosis by reducing ECM accumulation.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Matriz Extracelular/metabolismo , Inativação Gênica , Cirrose Hepática Experimental/patologia , Animais , Tetracloreto de Carbono , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/metabolismo , Masculino , RNA Interferente Pequeno , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To explore the most common bowel frequency and the prevalence rates of functional bowel disorders among adolescents in China. METHODS: A questionnaire survey was conducted among 51,956 students from high and primary schools in 6 Chinese cities distributed in the whole China collected by stratified, randomized, cluster sampling to study the epidemiology of functional bowel disorders. RESULTS: (1) 88.05% +/- 0.28% of the students had bowel frequency between 1 - 2 times/day and 1 time/two days. Girl students were found to have a lower bowel frequency than boy students (P < 0.01). (2) The prevalence rates of irritable bowel syndrome, chronic constipation, and chronic diarrhea were 20.19% (10 490), 25.92% (13 467), and 8.77% (4557) respectively. CONCLUSION: (1) The normal bowel frequency among adolescents in China may be defined as bowel movements between 1 - 2 times per day and 1 time per two days. (2) Irritable bowel syndrome, chronic constipation and chronic diarrhea are common disorders among the adolescents in China.
Assuntos
Doenças Funcionais do Colo/epidemiologia , Inquéritos e Questionários , Adolescente , Criança , China/epidemiologia , Constipação Intestinal/epidemiologia , Diarreia/epidemiologia , Feminino , Humanos , Síndrome do Intestino Irritável/epidemiologia , Masculino , PrevalênciaRESUMO
OBJECTIVE: To explore the prevalence of irritable bowel syndrome (IBS) and its distribution characteristics among adolescents in China. METHODS: A stratified and randomized study by cluster sampling was employed, the study recruited 51 956 students from high and primary schools in different Chinese cities. All students were requested to fill in a questionnaire. RESULTS: The prevalence of IBS in China was 53.5% according to the Manning criterion and 20.2% according to the Rome II criterion. The prevalence in male and female students showed no significant difference (P>0.05), but there was higher prevalence of IBS in high school students. The prevalence of IBS was 53.3% according to the Manning criterion and 19.6% according to the Rome II criterion in south China. The prevalence of IBS was 51.2% according to the Manning criterion and 18.9% according to the Rome II criterion in North China. The prevalence of IBS was 58.0% according to the Manning criterion and 23.4% according to the Rome II criterion in west China. CONCLUSION: IBS is a common disorder among the adolescents and the prevalence of IBS is increasing with increase of age in adolescents.
Assuntos
Síndrome do Intestino Irritável/epidemiologia , Adolescente , Criança , China/epidemiologia , Feminino , Humanos , Masculino , Prevalência , Estudos de Amostragem , Estudantes , Inquéritos e QuestionáriosRESUMO
BACKGROUND/AIMS: Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the ECM turnover. We investigate the effect of modified synthetic small interfering RNA (siRNA) targeting TIMP-2 in rat model of liver fibrosis. METHODS: Rat hepatic fibrosis was induced by CCl4 for 8 weeks. After the 2-week CCl4 injection period, rats in the three siRNA groups simultaneously received a different dosage (0.05, 0.1 and 0.2 mg.kg(-1), respectively) of modified synthetic siRNA targeting TIMP-2 via the tail vein every 3 days for 6 weeks. The pathological changes in liver tissues were observed by light microscopy and transmission electron microscopy. Portal vein pressure and proliferating cell nuclear antigen were measured. Expression of TIMP-2, MMP-2, MT1-MMP, MMP-13, hepatocyte growth factor, collagen type I, collagen type III and alpha-SMA were evaluated by quantitative real-time polymerase chain reaction or Western blotting or gelatin zymography. RESULTS: Modified synthetic siRNA targeting TIMP-2 induced a dose-dependent inhibition of the TIMP-2 expression in the rat model of liver fibrosis with a similar trend in MMP-2 and MT1-MMP, but an increase in MMP-13. Rats administered siRNA targeting TIMP-2 showed promotion of ECM degradation, reduction in activated hepatic stellate cells and enhancement of hepatocyte regeneration. Furthermore, portal hypertension was also ameliorated after treatment with siRNA targeting TIMP-2. CONCLUSIONS: Knock-down of TIMP-2 expression attenuates CCl4-induced liver fibrosis and is a potential pharmacological target for gene therapy in liver fibrosis.
Assuntos
Terapia Genética/métodos , Cirrose Hepática Experimental/terapia , RNA Interferente Pequeno/uso terapêutico , Inibidor Tecidual de Metaloproteinase-2/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Matriz Extracelular/ultraestrutura , Hidroxiprolina/sangue , Fígado/química , Fígado/ultraestrutura , Cirrose Hepática Experimental/patologia , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/química , Ratos , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-2/análise , Inibidor Tecidual de Metaloproteinase-2/genéticaRESUMO
AIM: To investigate the effect of arg-gly-asp-mannose-6 phosphate (RGD-M6P) on the activation and proliferation of primary hepatic stellate cells in vitro. METHODS: Hepatic stellate cells (HSCs) were isolated from rats by in situ collagenase perfusion of liver and 18% Nycodenz gradient centrifugation and cultured on uncoated plastic plates for 24 h with DMEM containing 10% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, HSCs were cultured in 2% FBS/DMEM with transforming growth factor beta1, M6P, RGD, or RGD-M6P, respectively. Cell morphology was observed under inverted microscope, smooth muscle alpha-actin (alpha-SMA) was detected by immunocytochemistry, type III procollagen (PC III) in supernatant was determined by radioimmunoassay, and the proliferation rate of HSCs was assessed by flow cytometry. RESULTS: RGD-M6P significantly inhibited the morphological transformation and the alpha-SMA and PC III expressions of HSCs in vitro and also dramatically prevented the proliferation of HSCs in vitro. Such effects were remarkably different from those of RGD or M6P. CONCLUSION: The new compound, RGD-M6P, which has a dramatic effect on primary cultured HSCs in vitro, can inhibit the transformation of HSCs in culture caused by TGFbeta1, suppresses the expression of PC III and decreases proliferation rate of HSC. RGD-M6P can be applied as a selective drug carrier targeting at HSCs, which may be a new approach to the prevention and treatment of liver fibrosis.
Assuntos
Proliferação de Células/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Manose/análogos & derivados , Oligopeptídeos/farmacologia , Actinas/análise , Animais , Células Cultivadas , Colágeno Tipo III/análise , Citometria de Fluxo , Hepatócitos/química , Hepatócitos/fisiologia , Imuno-Histoquímica , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , Masculino , Manose/farmacologia , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologia , Fator de Crescimento Transformador beta1RESUMO
OBJECTIVE: To study COX-2 expression in H. pylori infected gastric mucosal epithelia and its significance in the carcinogenesis of the stomach. METHODS: Rapid urease test and histological examination with basic magnenta staining were used to assess the status of H. pylori infection in the stomach. COX-2 was detected immunohistochemically. RESULTS: COX-2 immunostaining was positive in 1 out of 12 cases with H. pylori-negative gastric mucosa and also in 1 out of 10 cases with H. pylori-positive gastric mucosa without macroscopic alterations, while COX-2 expression was found to be positive in 5 out of 9 cases with H. pylori related superficial gastritis with mucosal erosions. COX-2 expression was detected in 5 out of 10 cases with H. pylori-positive mild atrophic gastritis, 8 out of 10 cases with H. pylori-positive moderate-severe atrophic gastritis and intestinal metaplasia, and 6 out of 8 cases with H. pylori-positive moderate-severe dysplasia. COX-2 expression was positive in 22 out of 32 cases of gastric cancer. CONCLUSION: H. pylori may induce COX-2 expression of gastric mucosal epithelia in chronic superficial gastritis, which is related to the development of mucosal injury. According to gastric mucosal carcinogenesis pattern up-regulation of COX-2 expression is associated with gastric mucosal carcinogenesis, and involved in the early development of premalignant lesions.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Gastrite/enzimologia , Infecções por Helicobacter/enzimologia , Helicobacter pylori , Adulto , Idoso , Ciclo-Oxigenase 2/genética , Feminino , Mucosa Gástrica/enzimologia , Gastrite/microbiologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Playing a central role in hepatic fibrosis, hepatic stellate cell (HSC) has made itself the major target of research. The limited supply of HSC, however, can not meet the ever growing need of experiment. Establishment and identification of novel immortalized HSC line thus may be urgently required. METHODS: Primary HSCs were isolated from a normal adult male Sprague-Dawley rat by in situ perfusion with collagenase IV and pronase E, and then were purified by single-step density gradient centrifugation with nycodenz. Once they reached total activation in culture, a new immortalized myofibroblast-like HSC line was established through cellular cloning. Its characteristics were identified by means of immunocytochemical staining, light microscopy, transmission electron microscopy, and growth curve analysis. RESULTS: The novel HSC line, termed HSC-PQ, had a doubling time of about 75 hours in the Dulbecco's modified Eagle medium (DMEM) containing 20% fetal bovine serum. Most of the main morphological characteristics of the differentiated primary HSC could be detected in HSC-PQ cell, while functional features of activated HSC such as alpha-smooth muscle actin, desmin, collagen type I, collagen type III, fibronectin, laminin and other extracellular matrix proteins could also be found in it except for collagen type IV. In contrast, fat droplets and autofluorescence of vitamin A disappeared in the HSC-PQ line. This cell line had been maintained in culture for over 30 passages and more than 1 year with little alternation in biological characteristics. CONCLUSION: A new rat HSC line (HSC-PQ) has been successfully established. It consistently retains the characteristics of activated primary HSC, and has proved to be immortalized.
Assuntos
Linhagem Celular , Hepatócitos/citologia , Hepatócitos/fisiologia , Análise de Variância , Animais , Apoptose/fisiologia , Linhagem Celular Transformada , Proliferação de Células , Citometria de Fluxo , Imuno-Histoquímica , Cirrose Hepática/patologia , Cirrose Hepática/fisiopatologia , Masculino , Microscopia Eletrônica de Varredura , Probabilidade , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade , Coleta de Tecidos e ÓrgãosRESUMO
Tetrandrine is a bisbenzylisoquinoline alkaloid derived from the root of a Chinese herbal medicine Stephania tetrandra S. Moore, which has been used traditionally for the treatment of hepatofibrogenic disease in China for several decades. In the present study, the inhibitory effects of tetrandrine lower concentrations (0.25, 0.5, 1, 2 mg/L) on culture-activation and transforming growth factor-beta(1) (TGF-beta(1))-stimulated activation of quiescent rat hepatic stellate cells (HSCs) in vitro were assessed, and the possible relations between the underlying mechanism of these effects and TGF-beta signaling via its receptors were investigated. As shown by the examination of alpha-SMA using immunocytochemical staining or Western blot, tetrandrine inhibited both culture-activation and TGF-beta(1)-stimulated activation of HSCs. Further investigations revealed that, in this process, TGF-beta(1) mRNA expression was suppressed significantly in contrast to an up-regulation of Smad 7, while the expressions of type I and type II TGF-beta(1) receptors and Smad 3 mRNA were insignificantly changed by tetrandrine. These results suggest that tetrandrine at lower concentrations has a significant inhibiting effect on culture-activation and TGF-beta(1)-stimulated activation of rat HSCs, and that it may be due to an up-regulation of Smad 7 which in turn blocks TGF-beta(1) expression and its downstream signaling.
Assuntos
Alcaloides/farmacologia , Benzilisoquinolinas/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/farmacologia , Actinas/biossíntese , Animais , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Técnicas In Vitro , Indicadores e Reagentes , Raízes de Plantas/química , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Receptores de Fatores de Crescimento Transformadores beta/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1 , Regulação para Cima/efeitos dos fármacosRESUMO
AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-beta (TGF-beta) signaling in vitro. METHODS: HSCs were isolated from rats by in situ perfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 h with DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1, 2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscle-alpha-actin (alpha-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type III procollagen (PCIII) in supernatants were determined by radioimmunoassay. TGF-beta1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively. RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while alpha-SMA, LN and PCIII expressions were inhibited. As estimated by gray values, the expression of alpha-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2% (control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P<0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCIII to 84.6%, 81.5%, 75.7% or 80.7% respectively (P<0.05 vs control), with no significant difference among tetrandrine groups. RT-PCR showed that TGF-beta1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-beta1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r=-0.755, P<0.01), and both were significantly correlated with alpha-SMA protein expression (r=-0.938, P<0.01; r=0.938, P<0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L) was confirmed by Western blotting as well. CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-beta1 expression and signaling.
Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Benzilisoquinolinas/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína Smad7 , Transativadores/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs. METHODS: HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation. SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immunohistochemical staining. RESULTS: SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc. CONCLUSION: The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.
Assuntos
Cirrose Hepática/etiologia , Fígado/citologia , Receptores de Somatostatina/genética , Animais , Fígado/metabolismo , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To investigate changes in the expression of the somatostatin receptor (SSTR) of the pancreas and of pancreatic blood flow, and its relationship to the metabolism of eicosanoids in order to elucidate the effectiveness of octreotide, an analog of somatostatin, in acute necrotizing pancreatitis (ANP). METHODS: A model of ANP was induced in rats with injection of sodium taurocholate via the pancreaticobiliary duct. The SSTR was detected using a radioligand binding assay (RBA) with 125I-somatostatin-14, and the SSTR2 mRNA of the pancreas was analyzed using in situ hybridization. Pancreatic blood flow and the metabolites of eicosanoids were also determined. RESULTS: The SSTR of the pancreas was 109.70 +/- 58.32 fmol/mg protein in normal rats. A significant decrease in the SSTR, together with the signals of SSTR2 mRNA, was shown at 3, 6 and 12 h after onset of ANP. Pancreatic blood flow was reduced and thromboxin-2 was increased significantly in the course of ANP. In the ANP group treated with octreotide, both the decrease in pancreatic blood flow and the abnormal metabolism of eicosanoids were corrected, and the pathological damage was relieved. CONCLUSION: SSTR expression of the pancreas is significantly reduced in ANP. Correction of the abnormal metabolism of eicosanoids and improvement in pancreatic microcirculation may be the major mechanism of somatostatin analogs in the treatment of ANP and inhibition of pancreatic enzymes via their receptors plays a minor role.
Assuntos
Fármacos Gastrointestinais/uso terapêutico , Octreotida/uso terapêutico , Pancreatite Necrosante Aguda/metabolismo , Receptores de Somatostatina/metabolismo , 6-Cetoprostaglandina F1 alfa/sangue , Animais , Dinoprostona/sangue , Masculino , Pâncreas/irrigação sanguínea , Pâncreas/metabolismo , Pancreatite Necrosante Aguda/tratamento farmacológico , Pancreatite Necrosante Aguda/fisiopatologia , Ratos , Ratos Sprague-Dawley , Tromboxano B2/sangueRESUMO
AIM: To evaluate the therapeutic effectiveness of oxaliplatin on human gastric carcinoma and to explore its mechanisms. METHODS: Twenty-two cases of stage IV gastric carcinoma received 4-6 (mean 4.6) cycles of first line combined chemotherapy with oxaliplatin (oxaliplatin 85 mg/m(2), iv, gtt, 1 h, d 1; leukovorin 200 mg/m(2), iv, gtt, 1 h, d 1 and d 2; 5-FU 300 mg/m(2),iv, d 1 and d 2, 5-FU, continuous iv, gtt, 48 h; 1 cycle/2 wk). Response rate, progression-free survival (PFS), total survival time, toxic side effects were evaluated. The inhibitory effect of oxaliplatin on human gastric cell line SGC-7901 was detected and IC(50) was calculated by MTT. Transmission electron microscopy, flow cytometry and TUNEL were performed to evaluate the apoptosis of cell line induced by the drug. The expression of Caspase-3 m-RNA was detected by RT-PCR. AC-DEVD-CHO, a Caspase-3 specific inhibitor, was used to elucidate the role of activated Caspase-3 in the process of apoptosis induced by oxaliplatin. RESULTS: Total response (complete and partial) occurred in 9 (40.9%) patients. Mean PFS was 4.2 mo and mean total survival time was 7.2 mo. Cumulative neurotoxicity (all grade I-II), vomiting and diarrhea, myelosuppression appeared in 93.5%, 20%, 32.9% patients, respectively. IC(50) was calculated to be 0.71 mg/L by MTT assay. A maximal inhibitory rate reached 85.3%. Apoptosis index was elevated after incubated with 1 mmol/L oxaliplatin for 30 min, but without statistic significance (P>0.05). However it could be detected at a much higher degree both by flowcytometry and by TUNEL with a statistical significance (68.47+/-7.92% and 8.23+/-2.67%, respectively, P<0.05) after incubated with 1 mmol/L oxaliplatin for 2 d. By means of RT-PCR, we detected an enhancement of Caspase-3 m-RNA expression induced by oxaliplatin which was also in positive correlation with the apoptotic level. AC-DEVD-CHO, a Caspase-3 specific inhibitor, could significantly inhibit and delay apoptosis induced by oxaliplatin. CONCLUSION: Oxaliplatin is effective and well-tolerated in patients with advanced gastric carcinoma. Oxaliplatin could significantly inhibit the growth of human gastric cell line SGC-7901. The induction of Caspase-3 m-RNA expression, activation of Caspase-3 and promotion of apoptosis may be some of the therapeutic mechanisms of oxaliplatin on gastric carcinoma. Annexin-V-fluorescein labeling flow cytometry is much more sensitive than TUNEL in detecting early stage apoptosis.
Assuntos
Antineoplásicos/uso terapêutico , Compostos Organoplatínicos/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Adulto , Idoso , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Oligopeptídeos/farmacologia , Oxaliplatina , Neoplasias Gástricas/patologia , Neoplasias Gástricas/ultraestruturaRESUMO
AIM: To explore the effects of endothelin-1(ET-1) on hepatic stellate cells (HSCs) DNA uptake, DNA synthesis, collagen synthesis and secretion, inward whole-cell calcium concentration ([Ca2+]i) as well as the blocking effect of verapamil on ET-1-stimulated release of inward calcium (Ca2+) of HSC in vitro. METHODS: Rat hepatic stellate cells (HSCs) were isolated and cultivated. 3H-TdR and 3H-proline incorporation used for testing DNA uptake and synthesis, collagen synthesis and secretion of HSCs cultured in vitro; Fluorescent calcium indicator Fura-2/AM was used to measure [Ca2+]i inward HSCs. RESULTS: ET-1 at the concentration of 5X10(-8) mol/L, caused significant increase both in HSC DNA synthesis (2,247+/-344 cpm, P<0.05) and DNA uptake (P<0.05) when compared with the control group. ET-1 could also increase collagen synthesis (P<0.05 vs control group) and collagen secretion (P<0.05 vs control group). Besides, inward HSC [Ca2+]i reached a peak concentration (422+/-98 mol/L, P<0.001) at 2 min and then went down slowly to 165+/-51 mol/L (P<0.01) at 25 min from resting state (39+/-4 mol/L) after treated with ET-1. Verapamil (5 mol/L) blocked ET-1-activated [Ca2+]i inward HSCs compared with control group (P<0.05). Fura-2/AM loaded HSC was suspended in no Ca2+ buffer containing 1 mol/L EGTA, 5 min later, 10(-8) mol/L of ET-1 was added, [Ca2+]i inward HSCs rose from resting state to peak 399+/-123 mol/L, then began to come down by the time of 20 min. It could also raise [Ca2+]i inward HSCs even without Ca2+ in extracellular fluid, and had a remarkable dose-effect relationship(P<0.05). Meanwhile, verapamil could restrain the action of ET-1(P<0.05). CONCLUSION: Actions of ET-1 on collagen metabolism of HSCs may depend on the transportation of inward whole-cell calcium.
Assuntos
Cálcio/metabolismo , Colágeno/biossíntese , Endotelina-1/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , DNA/biossíntese , Matriz Extracelular/metabolismo , Hepatócitos/citologia , Masculino , Prolina/farmacocinética , Ratos , Ratos Wistar , Timidina/farmacocinética , Trítio , Verapamil/farmacologiaRESUMO
AIM: To investigate the pathway via which 17beta-estradiol (beta-Est) exerts suppressive effects on rat hepatic fibrosis. METHODS: In vivo study was done in CCl4-induced female hepatofibrotic rats. Fibrosis-suppressive effect of beta-Est (20 microg/kg/d) was evaluated in intact and ovariectomized rat models. Six weeks after the treatment, all the rats were sacrificed and specimens of serum or liver tissue were collected for the studies. Serum liver enzymes, fibrosis markers and estradiol levels were determined by standard enzymatic methods, ELISA and RIA, respectively. Degrees of fibrosis and areas of hepatic stellate cells (HSCs) positive for alpha-smooth muscle actin (alpha-SMA) in the liver were determined by van Gieson (VG) stain and immunohistochemistry. In vitro studies, HSCs were isolated by a combination of pronase-collagenase perfusion and density gradient centrifugation. First-passage HSCs were randomly divided into 10 groups, and different concentrations of beta-Est, 2-hydroxyestradiol (2OHE) or 2-methoxyestradiol (2MeOE) were separately added to the cell groups. After incubation for 72 h, the degree of cell proliferation, collagen production, alpha-SMA or estrogen receptor (ER) expression was determined by MTT assay, ELISA and immunohistochemistry, respectively. RESULTS: Beta-Est treatment reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT), hyaluronic acid (HA) and type IV collagen (C IV) in sera, suppressed hepatic collagen content, decreased the areas of HSCs positive for alpha-SMA significantly in both intact and ovariectomized female hepatofibrotic rats. There was a negative correlation between the percentage of fibrotic area of liver tissue and the serum estradiol level; the calculated correlation coefficient was -0.57 (P<0.01). Beta-Est and its metabolites concentration-dependently (10(-9) mol/L-10(-7) mol/L) inhibited HSC proliferation and collagen synthesis. At the concentration of 10(-7) mol/L, they could inhibit alpha-SMA expression. The order of potency was 2MeOE>2OHE>beta-Est. CONCLUSION: Beta-Est may suppress hepatic fibrosis probably via its biologically active metabolites.
Assuntos
Tetracloreto de Carbono/toxicidade , Estradiol/metabolismo , Fibrose/induzido quimicamente , Cirrose Hepática Experimental/patologia , Animais , Células Cultivadas , Feminino , Fibrose/metabolismo , Fibrose/patologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
AIM: To assess the effects of somatostatin on proliferation and apoptosis of activated rat hepatic stellate cells (HSCs). METHODS: HSCs isolated from the livers of adult Sprague-Dawley rats (weighing 400-500 g) by in situ perfusion and purified by single-step density gradient centrifugation with Nycodenz, became activated after 10 days' cultivation. Then the apoptotic rate of HSCs treated with different doses of somatostatin for 72 h, was assayed by acridine orange/ethidium bromide fluorescent staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, transmission electron microscopy and flow cytometry, while the proliferation of HSCs was measured by MTT assay. Furthermore, the mechanisms of somatostatin were investigated by cytodynamic analysis. RESULTS: Somatostatin at the concentration of 10(-6)-10(-9) mol/L could decrease the proliferative rate, and promote the apoptosis of activated rat HSCs in a dose-dependent way. Its action was most significant when the concentration reached 10(-6) mol/L or 10(-7) mol/L (P<0.05-0.01). An obvious cell-cycle arrest (G(0)/G(1) arrest) was the important way for somatostatin to exert its action. CONCLUSION: Antiproliferative and proapoptotic effects of low-dose somatostatin on activated rat HSCs can be obtained. These findings reveal its potential antifibrotic action.