No abstract available.
Blotting, Western ; Cells, Cultured ; Connective Tissue Cells/drug effects/metabolism ; Endothelin-1/*biosynthesis ; Fibroblasts/*drug effects/metabolism ; Humans ; Transforming Growth Factor beta1/*pharmacology

OBJECTIVETo investigate the role of connective tissue growth factor (CTGF) induced TGF-beta 1 in the transdifferentiation of human hypertrophic scar fibroblast (HSFb).

METHODSHuman hypertrophic scar fibroblasts were cultured in vitro, 5 cell samples were stimulated with TGF-beta 1 (0, 2.5, 5.0, 7.5, 10.0 ng/mL, respectively) for 48 hours; other cell samples were divided into: normal control (NC) group, CTGF group (with addition of 10 ng/mL rhCTGF into culture medium), TGF-beta 1 group (with addition of 10 ng/mL TGF-beta 1 into culture medium), CTGF ASODN group (with addition of 10% FBS-DMEM after transfection of CTGF ASODN), CTGF ASODN + TGF-beta 1group (with addition of 10 ng/mL TGF-beta 1 after transfection of CTGF ASODN). Expression of CTGF was determined by Western blotting with stimulation of different concentration of TGF-beta 1. Expression of alpha-smooth muscle actin (alpha-SMA) was measured by Western blotting. Positive cell rate of alpha-SMA was examined by flow cytometry.

RESULTSWith stimulation of 10.0 ng/mL TGF-beta 1, the expression of CTGF was obviously higher than that of non-stimulation (P < 0.05). Expression of alpha-SMA in the CTGF group and the TGF-beta 1 group was obviously higher than that in NC group (P < 0.01), while there was no obvious difference among NC, CTGF ASODN, CTGF ASODN + TGF-beta 1 groups (P > 0.05). The positive cell rate of alpha-SMA in NC, CTGF, TGF-beta 1, CTGF ASODN, CTGF ASODN + TGF-beta 1 groups was (10.8 +/- 2.8)%, (29.1 +/- 4.0)%, (28.7 +/- 4.8)%, (10.7 +/- 2.3)%, (14.3 +/- 2.9)%, respectively, which was similar to expression of alpha-SMA on statistic analysis.

CONCLUSIONSCTGF is one of the most important downstream efforts for TGF-beta 1 in inducing the transdifferentiation of HSFb.

Cell Differentiation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; Connective Tissue Growth Factor ; pharmacology ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVESTo observe the effects of pioglitazone on morphological changes and connective tissue growth factor (CTGF) expression of the transforming growth factor beta (TGF b)-induced rat hepatic stellate cells (HSCs) in vitro, and to investigate the anti-fibrotic mechanism of pioglitazone.

METHODSCultured rat HSCs were divided into a no-treatment control group, a TGF b-treated group, and a TGFb plus different dosage pioglitazone-treated group. The morphological changes of the cultured HSCs were observed. The expression of CTGF was assessed by immunohistochemistry and flow cytometry. The level of collagen type III in the culture supernatant was measured by ELISA.

RESULTSTGFb induced morphological changes, and increased the expressions of CTGF and collagen type III of the HSCs (P less than 0.05). Pioglitazone prevented the TGFb induced morphological changes of the HSCs. The expression of CTGF and the levels of collagen type III in the pioglitazone group were lower than the TGF b-treated group (P less than 0.05). This prevention effect was dose-dependent (P less than 0.05).

CONCLUSIONPioglitazone blocks the excretion of CTGF and collagen type III of cultured HSCs, preventing the development of liver fibrosis.

Animals ; Cells, Cultured ; Collagen Type III ; secretion ; Connective Tissue Growth Factor ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Rats ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta ; pharmacology

OBJECTIVETo investigate the effects of transforming growth factor-β1 (TGF-β1) on growth controlling and the expression of connective tissue growth factor mRNA(CTGF mRNA) in urethra epithelium cells and fibroblasts cultured in vitro.

METHODSUrethra epithelial cells and fibroblasts were cultured in vitro and identified. The fourth generation cells were divided into control group (cultured by cell medium without TGF-β1) and experimental groups(cultured by cell medium containing TGF-β1 1, 2, 4 and 8 µg/L), the vital force of cells were examined by MTT and cell counting, the expression of CTGF mRNA were examined by RT-PCR after 24 hours.

RESULTSThe optical density and cell count decreased in experimental groups of urethra epithelium cells and increased in experimental groups of fibroblasts with the concentration of TGF-β1 being heightened compared with the control group (P < 0.05). The expression of CTGF mRNA increased with the heightening concentration of TGF-β1 in all experimental groups of urethra epithelium cells and fibroblasts by RT-PCR (P < 0.05).

CONCLUSIONSTGF-β1 can inhibit the growth of urethra epithelium cells and promote the growth of fibroblasts in vitro, it can induce the expression of CTGF mRNA in two cells above-mentioned.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; metabolism ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Male ; Mucous Membrane ; cytology ; RNA, Messenger ; genetics ; Rabbits ; Transforming Growth Factor beta1 ; pharmacology ; Urethra ; cytology

Cell Differentiation ; drug effects ; Cell Line ; Connective Tissue Growth Factor ; metabolism ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; Kidney Tubules, Proximal ; cytology ; Lead ; toxicity ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo detect the effect of connective tissue growth factor (CTGF) on podocalyxin expression in mouse podocytes exposed to high glucose in vitro and explore the possible pathway involved.

METHODSThe expression vector carrying a small interfering RNA (siRNA) targeting CTGF was transfected into mouse podocytes cultured in the presence of 1 g/L glucose (normal control), 4.5 g/L glucose (high glucose group), 1 g/L glucose + 3.5 g/L mannitol (iso-osmolar control group). The changes in the protein expression levels of podocalyxin, CTGF and ERK1/2 in the cells in response to the treatments were investigated using Western blotting.

RESULTSHigh glucose exposure for 24 and 48 h resulted in significantly decreased expression of podocalyxin and increased CTGF in the podocytes (P<0.05). Phosphorylation of ERK1/2 occurred as early as 30 min after the exposure, and the activation was maintained till 24 h. Transfection of the cells with siRNA targeting CTGF significantly inhibited these changes.

CONCLUSIONCTGF is an important mediator of high glucose-induced podocyte damage and decreases the protein level of podocalyxin by the ERK1/2 pathway. CTGF-specific siRNA can alleviate high glucose-induced podocyte injury, suggesting its potential value in treatment of diabetic nephropathy.

Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Diabetic Nephropathies ; Glucose ; adverse effects ; MAP Kinase Signaling System ; drug effects ; Mice ; Podocytes ; cytology ; drug effects ; metabolism ; RNA, Small Interfering ; genetics ; Sialoglycoproteins ; metabolism

OBJECTIVETo study the effect of dracorhodin perchlorate on high glucose-induced connective tissue growth factor (CTGF) expression in human mesangial cells (HMC), and its mechanism of prevention and treatment on renal fibrosis in diabetic nephropathy (DN).

METHODThe HMC were divided into low glucose group (LG group, 5.5 mmol x L(-1) D-glucose), high glucose group (HG Group 25 mmol x L(-1) D-glucose), each group was located with dracorhodin perchlorate (7. 5 micromol x L(-1)) as comparison, and was examined at 24, 48 h. The expression of CTGF mRNA was assessed by semi quantatiue RT-PCR, and the expression of CTGF protein was assessed by Western blot.

RESULTCompared to HG group, the expression of CTGF mRNA and protein were reduced in LG group after stimulating 24, 48 hours (P < 0.01). Compared to HG group, the expression of CTGF mRNA and protein were reduced in HG group by added with dracorhodin perchlorate after stimulating 24, 48 hours (P < 0.01).

CONCLUSIONDracorhodin perchlorate can inhibit high glucose-induced connective tissue growth factor expression in human mesangial cells, and this may be its mechanism of prevention and treatment on renal fibrosis in diabetic nephropathy (DN).

Benzopyrans ; pharmacology ; Connective Tissue Growth Factor ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Gene Expression Regulation ; drug effects ; Glucose ; pharmacology ; Humans ; Mesangial Cells ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Time Factors

OBJECTIVETo explore the role of exogenous connective tissue growth factor (CTGF) in the collagen III synthesis of human renal tubular epithelial cell line HK2 in vitro.

METHODSCultured HK2 cells were randomly assigned to three groups: placebo-control, low-dose CTGF-treated (2.5 ng/mL) and high-dose CTGF-treated groups (20 ng/mL). Cell morphological changes were observed under an inverted microscope. Collagen III alpha mRNA expression was detected using RT-PCR. Immunohistochemistry staining was used to assess the levels of intracellular collagen III alpha protein.

RESULTSAfter 48 hrs of low- or high- dose CTGF treatment, the appearances of HK2 cells were changed from oval to fusiform. High-dose CTGF treatment increased collagen III alpha mRNA expression (0.4461+/-0.0274 vs 0.2999+/-0.0115; P<0.05) as well as the protein expression of collagen III alpha (0.4075+/-0.0071 vs 0.3503+/-0.0136; P<0.05) compared with the placebo-control group.

CONCLUSIONSCTGF can induce morphological changes of human renal tubular epithelial cells in vitro. High concentration of CTGF may increase the synthesis of collagen III alpha.

Cells, Cultured ; Collagen Type III ; biosynthesis ; genetics ; Connective Tissue Growth Factor ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Immediate-Early Proteins ; pharmacology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Kidney Tubules ; drug effects ; metabolism ; RNA, Messenger ; analysis

To explore the angiotensin peptide [Ang (1-7)]-mediated inhibition of Ang II in human hepatic stellate cells (HSCs) and determine the involvement of the ACE2-Ang (1-7)-Mas axis. The human HSC line, LX2, was used in all experiments, and divided into control (unstimulated) and Ang II-stimulated (10-6 mol/L) groups. The Ang II-stimulated cells were further divided among several pre-treatment (prior to Ang II) groups: ROCK-inhibited (Y27632 blocking agent, 10-6 mol/L); irbesartan-inhibited (AT-1 receptor antagonist, 10-6 mol/L); and Mas receptor-inhibited (A779 Mas receptor antagonist, 10-6 mol/L). To explore the potential inhibitory effects of various Ang family members, the Ang II-stimulated and pre-treated LX2 cells were exposed to Ang (1-7) (10-6 mol/L) for 24 h. Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and QuantiGene assay were used to assess changes in protein and mRNA expression levels of RhoA, ROCK, and connective tissue growth factor (CTGF). Compared with the control group, Ang II-stimulated cells showed significantly increased levels of RhoA protein (0.337+/-0.074 vs. 0.870+/-0.093), ROCK2 mRNA (0.747+/-0.061 vs. 0.368+/-0.023), and CTGF mRNA (0.262+/-0.007 vs. 0.578+/-0.028) (all, P less than 0.01). Pre-treatment with irbesartan or Y27632 eliminated these responses. Ang (1-7) inhibited the Ang II-stimulated up-regulation of RhoA, ROCK, and CTGF. Ang (1-7) can inhibit the Ang II-stimulated up-regulation of RhoA, ROCK and CTGF in hepatic stellate cells, indicating that the ACE2-Ang (1-7)-Mas axis, an important branch of the renin-Ang-aldosterone system is involved in the occurrence and development of liver fibrosis.
Angiotensin I ; pharmacology ; Angiotensin II ; pharmacology ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Humans ; Peptide Fragments ; pharmacology ; RNA, Messenger ; genetics ; Signal Transduction ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism

The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.
Cells, Cultured ; Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/*metabolism ; Glucose/*pharmacology ; Immediate-Early Proteins/metabolism ; Immediate-Early Proteins/*physiology ; Mesangial Cells/cytology ; Mesangial Cells/*metabolism ; Protein-Serine-Threonine Kinases/metabolism ; Protein-Serine-Threonine Kinases/*physiology ; Signal Transduction/drug effects