PURPOSE: The degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis. MATERIALS AND METHODS: Cirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and alpha-smooth muscle actin (alpha-SMA) protein in the liver, transforming growth factor beta1 (TGF-beta1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-beta1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction. RESULTS: Liver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-beta1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group. CONCLUSION: Thalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-beta1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats.
Actins ; Animals ; Carbon Tetrachloride/toxicity ; Collagen Type III/metabolism ; Down-Regulation ; Extracellular Matrix/metabolism ; Immunohistochemistry ; Immunosuppressive Agents/*pharmacology ; Liver Cirrhosis, Experimental/chemically induced/*metabolism/pathology/*prevention & control ; Male ; RNA, Messenger/analysis/metabolism ; Rats ; Rats, Wistar ; Thalidomide/*pharmacology ; Tissue Inhibitor of Metalloproteinase-1/biosynthesis/*drug effects ; Transcription Factor RelA/biosynthesis/drug effects ; Transforming Growth Factor beta1/biosynthesis/*drug effects ; Transforming Growth Factors/metabolism

OBJECTIVETo investigate the effect of an anti-fibrotic tetra peptide Ac-SDKP on vascular fibrosis by regulating extracellular regulated protein kinase (ERK1/2) activity through Ang II.

METHODSRat vascular adventitial fibroblasts were cultured in vitro. They were randomly divided into control group, Ang II (10(-6) mmol/L) group, Ang II and Ac-SDKP joint action group, PD98059 group. Type I, III collagen contents in adventitia fibroblasts were measured by RT-PCR and the expressions of matrix metalloproteinases (MMP-2) and transforming growth factor-beta1 (TGF-beta1) were determined by Western blot.

RESULTSAc-SDKP could reduced Ang II-induced expression of type I, III collagen secretion and TGF-beta1 at mRNA,and increase MMP-2 expression, PD98059 could inhibit the above effect.

CONCLUSIONThe results suggested that Ac-SDKP could inhibit the formation and development of vascular fibrosis through blocking ERK1/2 pathway mediated by Ang II. Ac-SDKP therefore served as an antifibrotic factor in vascular fibrosis.

Angiotensin II ; adverse effects ; Animals ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flavonoids ; pharmacology ; MAP Kinase Signaling System ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Oligopeptides ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the effect of Qindan capsule (QC) on collagen synthesis and the mechanism underlying the process in spontaneously hypertensive rats (SHRs).

METHODSTwentyfour SHRs were divided into three groups: the hypertension model group, the QC treatment group, and the losartan treatment group. Eight Wistar Kyoto (WKY) rats were used as the normal control group. The systolic blood pressure (SBP) of the rats was monitored, and the thoracic aorta adventitia of the rats was segregated. The expressions of transforming growth factor 1 (TGF-β1), Smad3, and collagens I and were measured by histological staining and reverse transcription polymerase chain reaction.

RESULTSThe SBP was significantly higher in the model group than in the normal control group (P<0.01). However, a significant SBP-lowering effect was observed in QC or losartan treatment groups (P<0.05 or P<0.01) after 3 weeks of treatment. QC-treated rats showed a decrease of approximately 40 mm Hg, and the losartan-treated rats showed a decrease of approximately 50 mm Hg at the end of treatment compared with the beginning of treatment. The protein and gene levels of TGF-β1, Smad3, and collagens I and in the model group were significantly increased compared with those in the normal control group (P<0.01). However, the levels were significantly decreased in the QC or losartan treatment group compared with the model group (P<0.05 or P<0.01). However, there was no significant difference between the QC and losartan treatment groups (P<0.05).

CONCLUSIONSQC could exert its antihypertensive effect through down-regulating TGF-β1-stimulated collagen expressions. The TGF-β1/Smad3 signaling pathway may be involved in this process.

Adventitia ; drug effects ; metabolism ; pathology ; Animals ; Blood Pressure ; drug effects ; Blood Vessels ; drug effects ; metabolism ; pathology ; Capsules ; Collagen ; biosynthesis ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Losartan ; pharmacology ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Smad3 Protein ; genetics ; metabolism ; Staining and Labeling ; Systole ; drug effects ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo investigate the effects of Bushen Huoxue Fang on the proliferation of rat cardiac fibroblasts and collagen production in the cells.

METHODSRat cardiac fibroblasts were isolated and cultured in DMEM containing 10% (group A) or 20% (group B) or no (group C) serum from rats treated with Bushen Huoxue Fang, with cells cultured in DMEM containing 10% FBS as the control (group D). After 72 h of cell culture, the proliferation of the fibroblasts was detected using CCK-8 kit, and collagen mRNA and protein expressions were examined using RT-PCR and Western blotting, respectively.

RESULTSCompared with that in groups C and D, the cell proliferation decreased significantly in groups A and B, and especially in the latter (P<0.05). RT-PCR demonstrated significant reductions of the mRNAs of type 1 and 3 collagens in groups A and B (P<0.05), and their protein levels were also significantly lowered (P<0.05).

CONCLUSIONBushen Huoxue Fang can effectively inhibit the proliferation of rat cardiac fibroblasts and reduced collagen type 1 and 3 productions in the cells in vitro.

Animals ; Animals, Newborn ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; cytology ; metabolism ; Fibrosis ; prevention & control ; Myocardium ; cytology ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of interleukin-17 (IL-17) on the proliferation, transformation and collagen synthesis of the lung fibroblasts in mice with bleomycin-induced pulmonary fibrosis.

METHODSIn a mouse model of pulmonary fibrosis established by intratracheal administration of 5 mg/kg bleomycin, the dynamic expressions of IL-17/IL-17 receptor (IL-17R) mRNAs were detected by RT-PCR. At 14 days following bleomycin administration, the pulmonary fibroblasts were isolated, cultured and identified. MTT assay was used to assess the proliferation of the pulmonary fibroblasts in response to IL-17 treatment at different concentrations, and RT-PCR and Western blotting were employed to examine the mRNA and protein expressions of α-smooth muscle actin (α-SMA) and types I and III collagen.

RESULTSIL-17/IL-17R mRNA levels were increased obviously in the pulmonary fibroblasts of rats with pulmonary fibrosis, and the highest expressions occurred at 14 days following bleomycin administration. Exogenous IL-17, at the optimal concentration of 50 ng/ml, significantly promoted the proliferation of the pulmonary fibroblasts in primary culture and obviously increased α-SMA expression and types I and III collagen synthesis in the fibroblasts.

CONCLUSIONIL-17 can promote the proliferation, transformation, and collagen synthesis of the pulmonary fibroblasts from rats with bleomycin-induced pulmonary fibrosis.

Animals ; Bleomycin ; Cell Proliferation ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Epithelial-Mesenchymal Transition ; Fibroblasts ; metabolism ; pathology ; Interleukin-17 ; genetics ; metabolism ; Lung ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis ; chemically induced ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Receptors, Interleukin-17 ; genetics ; metabolism

Acetaldehyde ; pharmacology ; Animals ; Anthracenes ; pharmacology ; Apoptosis ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Collagen ; biosynthesis ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Flow Cytometry ; Hepatic Stellate Cells ; drug effects ; metabolism ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; Liver Cirrhosis, Alcoholic ; pathology ; prevention & control ; Rats ; Signal Transduction ; drug effects

To investigate the effect of different frequencies of cyclic tensile strain on extracellular matrix (ECM) of vascular smooth muscle cells (VSMCs) and to research the relationship between tensile strain and vascular remodeling, the aortic vascular smooth muscle cells of rats grown on dishes coated with collagen I were subjected to 10% elongation and various frequencies of mechanical strain using the Flexercell 4000 Strain Unit. The expression of extracellular matrix including fibronectin, collagen I and collagen III was detected by Real-time RT-PCR, and p38 activity by western blot. The result showed that the expression of extracellular matrix was induced by mechanical strain in a nonlinear frequency-dependent manner, which was mediated by p38 pathway. These results demonstrate that the variety of frequencies of cyclic tensile strain could modulate the expression of ECM. It may have important influence on vascular remodeling.
Animals ; Aorta ; cytology ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Extracellular Matrix ; metabolism ; Fibronectins ; biosynthesis ; Male ; Mechanotransduction, Cellular ; physiology ; Muscle, Smooth, Vascular ; cytology ; physiology ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical

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

OBJECTIVETo suppress COL1A1 and COL3A1 gene expressions in human skin fibroblasts (HSFs) by means of RNA interference (RNAi).

METHODSThree small interfering RNA (siRNA) expression cassette (SEC) sequences were designed for each of the COL1A1 and COL3A1 gene sequences available in GenBank. The synthesized SECs capable of effective gene suppression were transfected into cultured HSFs, either after cloning into the expression vector or mediated by Lipofectamine 2000, and the suppression of the target genes at both mRNA and protein levels was determined by quantitative fluorescence RT-PCR and Western blotting, respectively.

RESULTSTransfection of the SECs into HSFs resulted in specific depression of COL1A1 and COL3A1 expressions (down to 5.00% and 6.48%, respectively). The expression vector-mediated RNAi established a HSF cell line with persistent gene knockdown for over 30 days (to 25.21% and 22.12%, respectively).

CONCLUSIONCOL1A1 and COL3A1 gene expressions can be specifically and efficiently inhibited in HSFs by either liposome- or vector-mediated SEC transfection.

Blotting, Western ; Cells, Cultured ; Collagen Type I ; biosynthesis ; genetics ; Collagen Type III ; biosynthesis ; genetics ; Fibroblasts ; cytology ; metabolism ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Skin ; cytology ; Transfection ; methods

OBJECTIVETo investigate the anti-fibrotic effects of Qishen Yiqi Dropping Pills (QYDP) in rats with liver fibrosis (LF).

METHODSThe LF model was induced by intraperitoneal injection with dimethylnitrosamine (DMN). Sixty Wistar rats were randomly divided into the normal group, the model group A, the QYDP intervened group , the model group B , and the QYDP treated group B. The degree of LF was evaluated according to 6-phase grading method. The expressions of collagen type I and III and tissue inhibitor of metalloproteinase-1 (TIMP-1) in liver tissues were determined by immunohistochemistry and the levels of collagen type I and III and TIMP-1 mRNA determined by semi-quantitive RT-PCR.

RESULTSCompared with the model group A and B, the degree of LF, the positive expressions of TIMP-1 mRNA and the content of collagen type I and III in liver tissue in the QYDP intervened and treated groups were significantly lower.

CONCLUSIONQYDP could reduce the pathological changes and degree of LF in rats, which may be partially through inhibiting the expressions of collagen type I and III and TIMP-1.

Animals ; Collagen Type I ; biosynthesis ; genetics ; Collagen Type III ; biosynthesis ; genetics ; Drugs, Chinese Herbal ; therapeutic use ; Immunohistochemistry ; Liver Cirrhosis, Experimental ; drug therapy ; Male ; Phytotherapy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics