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1.
Zhonghua Yi Xue Za Zhi ; 93(17): 1345-8, 2013 May 07.
Artigo em Chinês | MEDLINE | ID: mdl-24029487

RESUMO

OBJECTIVE: To evaluate the modulation of RhoA/Rho kinase (ROCK), a small Rho GTPase, on migration, invasion and proliferation of fibroblast like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. METHODS: RA FLS were collected from active RA patients. And 10% fetal bovine serum (FBS) and interleukin-1ß (IL-1ß) were used as stimuli in migration and proliferation experiments respectively. RhoA activity was measured by pull down assay while ROCK activity by Western blot. FLS migration and invasion in vitro were measured by the Transwell chamber method. And thiazolyl blue tetrazolium bromide (MTT) test was used to detect cell proliferation. RESULTS: There were increased activities of RhoA and ROCK in ex vivo FLS from RA versus OA patients and healthy control. The migrated cell number of FBS-induced, C3-treated and Y27632-treated groups was 85 ± 14, 51 ± 15 and 42 ± 11 respectively. The Matrigel invading cell number of 3 groups was 64 ± 13, 39 ± 12 and 26 ± 9 respectively. Statistical differences existed in cell number between FBS-induced, C3-treated or Y27632-treated group (P < 0.05) in above migration and invasion experiments. Inhibition of RhoA and ROCK activity also suppressed the cytoskeletal reorganization and proliferation of RA FLS. CONCLUSION: Increased RhoA/ROCK activity may contribute to abnormal migration, invasion and proliferation of RA FLS. Thus inhibition of ROCK activity may be a new therapeutic target for RA.


Assuntos
Artrite Reumatoide/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Artrite Reumatoide/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Feminino , Fibroblastos/citologia , Humanos , Masculino , Membrana Sinovial/citologia
2.
Biochem Biophys Res Commun ; 438(3): 473-8, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-23916703

RESUMO

Peritoneal membranes can be categorized as high, high average, low average, and low transporters, based on the removal or transport rate of solutes. In this study, we used proteomic analysis to determine the differences in proteins removed by different types of peritoneal membranes. Peritoneal transport characteristics in patients who received peritoneal dialysis therapy were assessed by a peritoneal equilibration test. Two-dimensional differential gel electrophoresis technology followed by quantitative analysis was performed to study the variation in protein expression from peritoneal dialysis effluents (PDE) among different groups. Proteins were identified by MALDI-TOF-MS/MS analyses. Further validation in PDE or serum was performed utilizing ELISA analysis. Proteomics analysis revealed ten protein spots with significant differences in intensity levels among different groups, including vitamin D-binding protein, complement C3, apolipoprotein-A1, complement factor C4A, haptoglobin, alpha-1 antitrypsin, immunoglobulin kappa light chain, alpha-2-microglobulin, retinol-binding protein 4 and transthyretin. The levels of vitamin D-binding protein, complement C3, and apolipoprotein-A1 in PDE derived from different groups were greatly varied (P<0.05). However, no significant difference was found in the serum levels of these proteins among different groups (P>0.05 for all groups). This study provides a novel overview of the differences in PDE proteomes of four types of peritoneal membranes. Vitamin D-binding protein, complement C3, and apolipoprotein-A1 showed enhanced expression in PDE of patients with high transporter.


Assuntos
Transporte Biológico/fisiologia , Complemento C3/metabolismo , Glomerulonefrite/fisiopatologia , Diálise Peritoneal , Peritônio/metabolismo , Proteômica , Proteína de Ligação a Vitamina D/metabolismo , Adulto , Apolipoproteína A-I/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Glomerulonefrite/terapia , Humanos , Falência Renal Crônica/fisiopatologia , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
Int J Mol Sci ; 14(5): 10369-82, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23685870

RESUMO

It has been confirmed that alternatively activated macrophages (M2) participate in tissue remodeling and fibrosis occurrence, but the effect of M2 on peritoneal fibrosis related to peritoneal dialysis (PD) hasn't been elucidated. This study was therefore conducted to assess the association between M2 and peritoneal fibrosis related to PD. In this study, peritoneal fibrosis was induced by intraperitoneal (i.p.) injection of Lactate-4.25% dialysate (100 mL/kg) to C57BL/6J mice for 28 days, and liposome-encapsulated clodronate (LC, the specific scavenger of macrophages) was used to treat the peritoneal fibrosis mice model by i.p. injection at day 18 and day 21. All animals were sacrificed at day 29. Parietal peritonea were stained with Masson's trichrome, and the expression of type I collagen (Col-I), fibronectin, mannose receptor (CD206), transforming growth factor beta (TGF-ß), chemokine receptor 7 (CCR7), chitinase 3-like 3 (Ym-1) and arginase-1 (Arg-1) was determined by Western blotting, immunofluorescence and quantitative real-time PCR. Our results revealed that peritoneal thickness, Col-I, fibronectin, CD206, TGF-ß, Ym-1 and Arg-1 were upregulated in the peritoneal fibrosis mice model, and all of these indexes were downregulated in those treated with LC. Additionally, there was no difference in the level of CCR7 between the model and treatment group. Our study indicated that peritoneal M2 played an important role in the process of peritoneal fibrosis related to PD and might be a potential target for intervention therapy of peritoneal fibrosis.


Assuntos
Ativação de Macrófagos , Macrófagos Peritoneais/metabolismo , Diálise Peritoneal , Fibrose Peritoneal/metabolismo , Animais , Arginase/genética , Arginase/metabolismo , Western Blotting , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Lectinas/genética , Lectinas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos Peritoneais/classificação , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fibrose Peritoneal/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismo
4.
Protein Expr Purif ; 89(2): 196-202, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23558012

RESUMO

PURPOSE: Despite recent progress in the proteomic analysis of peritoneal dialysate effluent (PDE), there remains unresolved problems in the development of an optimal sample preparation method. EXPERIMENTAL DESIGN: We examined five protocols for concentrating PDE proteins and the effects of immobilized pH gradient (IPG) strips with different pH ranges and sample loading techniques. In addition, we examined three kits for depleting high abundance proteins by SDS-PAGE and two-dimensional gel electrophoresis (2-DE). RESULTS: PDE proteins precipitated with 75% acetonitrile (ACN) showed the greatest number of protein spots by 2-DE, with over 800 distinct spots. Higher-resolution images were obtained using IPG strips with a pH range of 4-7. The ProteoPrep immunoaffinity albumin and IgG depletion kit removed high abundance proteins with higher efficiency and more compatibility with isoelectric focusing (IEF). Removing high abundance proteins also increased the resolution and improved the intensity of low abundance proteins. CONCLUSION AND CLINICAL RELEVANCE: High-resolution 2-DE images of PDE proteins were obtained by concentrating samples with 75% ACN, using pH 4-7 IPG strips, and depleting high abundance proteins. This optimized method will enable future studies to discover predictive biomarkers of disease in patients on dialysis.


Assuntos
Soluções para Diálise/análise , Eletroforese em Gel Bidimensional/métodos , Diálise Peritoneal , Proteínas/análise , Proteômica/métodos , Acetonitrilas/química , Adolescente , Adulto , Precipitação Química , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Concentração de Íons de Hidrogênio , Adulto Jovem
5.
Am J Emerg Med ; 31(2): 353-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23158614

RESUMO

The aim of this study was to investigate changes in Nogo receptor 1 (NgR(1)) expression in the cerebrum after cardiopulmonary resuscitation (CPR) in rats. Cardiac arrest was induced by alternating current in 50 SD rats through transcutaneous electrical epicardium stimulation, and CPR was performed with the Utstein mode 6 minutes after cardiac arrest. Rats were killed 1, 3, and 7 days after CPR. We performed immunofluorescence with antibodies against NgR(1) to map the distribution of NgR(1) in the rat cerebrum, whereas quantitative polymerase chain reaction was performed for quantitative analysis of NgR(1) messenger RNA (mRNA). There was a striking transient up-regulation of the NgR(1) protein and mRNA in both the hippocampus and cortex in response to CPR. Nogo receptor 1 proteins were strongly expressed in hippocampal neurons 1 and 3 days after CPR (P < .001 for 1 day and P < .05 for 3 days, vs the control group, respectively), which returned to the basal level 7 days after CPR. In the cortex, staining moderately increased 1 day after CPR and got the peak level after 3 days (P < .001), returning to normal expression levels on day 7. The levels of NgR(1) mRNA in the hippocampus and cerebral cortical cortex showed the same trend with staining. The changes were significantly different between day 3 and baseline in both the hippocampus and cortex (P < .05, respectively). Furthermore, there were significant differences between the hippocampus and cerebral cortical cortex at 1 day and 3 days after the CPR (P < .05, respectively). There was a transient increase in NgR(1) in the vulnerable areas of the rat brain after CPR. Blockade of NgR(1) may be important in maintaining the high regenerative capacity of neurons during the time window when NgR(1) expression increases.


Assuntos
Reanimação Cardiopulmonar , Córtex Cerebral/metabolismo , Parada Cardíaca/terapia , Hipocampo/metabolismo , Proteínas da Mielina/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Biomarcadores/metabolismo , Imunofluorescência , Proteínas Ligadas por GPI/metabolismo , Parada Cardíaca/metabolismo , Masculino , Receptor Nogo 1 , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Regulação para Cima
6.
Zhonghua Yi Xue Za Zhi ; 91(11): 742-5, 2011 Mar 22.
Artigo em Chinês | MEDLINE | ID: mdl-21600098

RESUMO

OBJECTIVE: To evaluate the modulation of RhoA/Rho kinase (ROCK) signaling pathway, a small Rho GTPase that is considered as an important modulator in inflammatory responses, on Toll-like receptor-2 mediated chemokine secretion in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. METHODS: The RhoA activity was measured by a pull-down assay. And the ROCK activity was assessed by Western blot. The secretion of chemokines was measured by ELISA (enzyme-linked immunosorbent assay). MTT test was used to detect the cellular viability. RESULTS: The stimulation of peptidoglycan (PG, 5 mg/L) increased the levels of IL-8 (interleukin-8), RANTES (regulated upon activation normal T cell expressed & secreted) and MCP-2 (monocyte chemotactic protein-2) and boosted the activities of RhoA and ROCK versus the unstimulated RA FLS. And these effects of PG were suppressed by anti-TLR-2 monoclonal antibody. Inhibition of RhoA and ROCK with a specific inhibitor inhibited the secretion of IL-8, RANTES and MCP-2 in PG-induced RA FLS. CONCLUSION: The present study provides novel evidence that the RhoA/ROCK signal pathway modulates the TLR-2-mediated secretion of chemokines in RA FLS. It suggests that the inhibition of RhoA/ROCK may be a new therapeutic approach for RA.


Assuntos
Artrite Reumatoide/metabolismo , Transdução de Sinais , Membrana Sinovial/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células Cultivadas , Quimiocina CCL5/metabolismo , Quimiocina CCL8/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Interleucina-8/metabolismo , Masculino , Líquido Sinovial/citologia , Membrana Sinovial/citologia
7.
Clin Vaccine Immunol ; 17(5): 757-63, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20200188

RESUMO

The objective of this study was to investigate the effects of glucose-based peritoneal dialysis (PD) fluids and icodextrin-based PD fluids on the expression of Toll-like receptor 2 (TLR2)/TLR4 and subsequent ligand-induced mitogen-activated protein kinase (MAPK) and NF-kappaB signaling and tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) mRNA expression in human peritoneal mesothelial cells (HPMCs). A human peritoneal mesothelial cell line (HMrSV5) was stimulated with glucose-based and icodextrin-based peritoneal dialysis fluids. Cell viability was assessed using MTT [3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]. TLR2/TLR4 expression was determined by real-time PCR, Western blotting, and an immunofluorescence assay. In addition, cells were pretreated with different PD solutions and then incubated with Pam3CSK4 or lipopolysaccharide (LPS), and the degrees of MAPK and NF-kappaB activation were reflected by detecting the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, and p65, using a Western blot method. TNF-alpha and IL-1beta mRNA expression was measured by real-time PCR. Glucose-based peritoneal dialysis fluids suppressed the expression of TLR2 and TLR4 proteins in HPMCs. Challenge of cells with either Pam3CSK4 or LPS resulted in impaired TNF-alpha and IL-1beta production. Moreover, reduced TLR2 and TLR4 levels in glucose-based peritoneal dialysis solution-treated mesothelial cells were accompanied by reduced p42/44 (ERK1/2), JNK, p38 MAPK, and NF-kappaB p65 phosphorylation upon TLR ligand engagement. No significant changes in MAPK and NF-kappaB signaling and TNF-alpha and IL-1beta mRNA expression were observed in icodextrin-based PD solution-treated mesothelial cells. Glucose-based PD solution, but not icodextrin-based PD solution, downregulates expression of TLR2/TLR4 by human peritoneal mesothelial cells and triggers hyporesponsiveness to pathogen-associated molecular patterns.


Assuntos
Soluções para Diálise/efeitos adversos , Células Epiteliais/imunologia , Glucanos/efeitos adversos , Glucose/efeitos adversos , Peritônio/citologia , Peritônio/efeitos dos fármacos , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese , Western Blotting , Linhagem Celular , Sobrevivência Celular , Compostos Cromogênicos/metabolismo , Citocinas/biossíntese , Células Epiteliais/efeitos dos fármacos , Técnica Direta de Fluorescência para Anticorpo , Humanos , Icodextrina , Diálise Peritoneal/efeitos adversos , Peritônio/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Sais de Tetrazólio/metabolismo , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia
8.
Zhonghua Yi Xue Za Zhi ; 89(27): 1881-4, 2009 Jul 21.
Artigo em Chinês | MEDLINE | ID: mdl-19953908

RESUMO

OBJECTIVE: To evaluate the inhibitory effect of blockade of Rho kinase upon mediating the secretion of proinflammatory cytokine in monocytic cells from rheumatoid arthritis (RA) patients. METHODS: Synovial fluid (SF) monocytic cells and peripheral blood monocytes (PB) from active RA patients were treated with TNFalpha or LPS respectively in the presence or absence of a specific ROK inhibitor, Y27632. ROK activity was assessed by Western blot and cytokine secretion measured by ELISA. RESULTS: Elevated ROK activity was found in synovial fluid monocytic cells from active RA patients. ROK activity was correlated with DAS, an index of disease activity of RA patients. ROK inhibitor Y27632 reduced the secretion of TNFalpha, IL-1beta and IL-6 in RA SF monocytic cells, but had no effect upon the secretion of IL-10, an anti-inflammatory cytokine. CONCLUSION: The present study provides novel evidence that ROK mediates the secretion of proinflammatory cytokines in monocytic cells from RA synovial fluids, suggesting a critical role of ROK in macrophage-mediated synovial inflammation of RA. Thus inhibition of ROK may be a new therapeutic target for RA.


Assuntos
Artrite Reumatoide/metabolismo , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Amidas/farmacologia , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Piridinas/farmacologia , Membrana Sinovial/citologia , Fator de Necrose Tumoral alfa/metabolismo
9.
Inflamm Res ; 58(8): 473-82, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19271152

RESUMO

OBJECTIVE: Activation of Toll-like receptor 4 (TLR4) in peritoneal mesothelial cells by endotoxin contributes to peritoneal inflammation and fibrosis. Here we investigated TLR4 expression induced by angiotensin II (Ang II) and functional consequences of nuclear factor-kappaB (NF-kappaB) activation and CD40 expression in rat peritoneal mesothelial cells (RPMCs). METHODS: TLR4, CD40, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were determined by reverse transcription polymerase chain reaction (RT-PCR) and TLR4, IkappaBalpha, phospho-IkappaBalpha, NF-kappaB p65, and phospho-NF-kappaB p65 were analyzed by Western blot. The intracellular distribution of NF-kappaB p65 was detected by immunofluorescence. RESULTS: Treatment of RPMCs with Ang II resulted in an increase in the expression of TLR4 mRNA and protein levels. Preincubation of RPMCs with Ang II significantly increased lipopolysaccharide (LPS)-induced phospho-IkappaBalpha and phospho-NF-kappaB p65 protein (P < 0.05 vs. LPS alone) and CD40, TNF-alpha, and IL-6 mRNA levels (P < 0.05 vs. LPS alone). A significantly increased nuclear staining of NF-kappaB p65 in cells treated with Ang II plus LPS was also observed. CONCLUSIONS: Ang II upregulates the expression of TLR4 by RPMCs, resulting in enhanced NF-kappaB signaling and induction of CD40, TNF-alpha, and IL-6 expression. Locally produced Ang II in the peritoneum may have an amplified role in LPS-induced peritoneal inflammation.


Assuntos
Angiotensina II/farmacologia , Antígenos CD40/biossíntese , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/efeitos dos fármacos , Animais , Western Blotting , Epitélio/efeitos dos fármacos , Interleucina-6/biossíntese , Masculino , Microscopia de Fluorescência , NF-kappa B/metabolismo , Peritônio/citologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/biossíntese
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