OBJECTIVETo study the changes of metalloproteinases 1, 2, 13 in granulation wound after the treatment of vacuum-assisted closure (VAC).

METHODSThe chronic wounds in 5 patients were treated with VAC. The expressions of MMP-1, 2, 13 in the granulation tissues of the chronic wounds were determined and quantified using RT-PCR technique before and at 1, 4, 7 days after the treatment.

RESULTSThe MMP-1, 13 mRNA showed obvious decrease, with the steepest variation of MMP-13. The MMP-2 mRNA also showed a decreased tendency, though in an undulatory fashion.

CONCLUSIONVAC can promote healing of chronic wounds through depressing the expressions of MMP-1, 2, 13 mRNA and protein synthesis, depressing the degradations of collagen and gelatin.

Collagen ; metabolism ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; Matrix Metalloproteinase 13 ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Negative-Pressure Wound Therapy ; Polymerase Chain Reaction ; Time Factors ; Wound Healing

Osteoarthritis (OA) is a complex chronic progressive disease attacked by biological and mechanical factors and a result from the anabolic and catabolic imbalance in chondrocyte, subchondral bone and extracellular matrix(ECM). Etiology and pathological of OA are not yet entirely clear. The degradation and destruction of collagen II caused by matrix metalloproteinase -13 (MMP-13) is considered the core factor in the occurrence and development of OA. The research of MMP-13 inhibitor provide ideas and methods for the treatment of OA. In this article,the role and determination of MMP-13 in OA and the development prospect of MMP-13 inhibitor in the treatment of OA research progress were reviewed.
Animals ; Collagen ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; analysis ; physiology ; Matrix Metalloproteinase Inhibitors ; therapeutic use ; Osteoarthritis ; drug therapy ; etiology

AIMTo explore the possible impact of endogenous hydrogen sulfide (H2S) on the content and metabolism of collagen in rats with high pulmonary blood flow.

METHODSThirty-two male SD rats, weighing 120-140 g, were randomly divided into 4 groups (n = 8), shunt group, shunt + PPG (propargylglycine, an antagonist of endogenous H2S producing enzyme) group, sham group and sham + PPG group. Rats in shunt group and shunt + PPG group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. In the sham group and sham + PPG group, rats experienced the same experimental processes except the shunting procedure. After 4 weeks of experiment, lung tissue H2S content of rat was determined by a modified sulfide electrode method. Pulmonary artery collagen I, collagen III, MMP-13 and TIMP-1 protein expressions of rat were investigated by immunohistochemistry.

RESULTSAfter 4 weeks of experiment, lung tissue H2S content increased significantly in rats of shunt group as compared with that of sham group (P < 0.05). Pulmonary artery collagen I and collagen III protein expression increased obviously in rats of shunt group as compared with that of sham group (P < 0.01). After administration of PPG for 4 weeks, lung tissue H2S content decreased significantly in rats of shunt + PPG group as compared with that of shunt group (P < 0.05). In contrast to rats in shunt group, collagen I and collagen III protein expression in pulmonary arteries of shunt + PPG group increased significantly, respectively (P < 0.05). Compared with rats of shunt group, pulmonary artery MMP-13, TIMP-1 and the ratio of MMP-13/TIMP-1 in shunt + PPG group down-regulated significantly (P < 0.05).

CONCLUSIONEndogenous H2S might play a protective regulatory role in the development of pulmonary hypertension and pulmonary vascular structural remodelling in rats by decreasing the content of pulmonary artery collagen resulting from catabolism of collagen.

Animals ; Collagen ; metabolism ; Hydrogen Sulfide ; metabolism ; Lung ; blood supply ; metabolism ; Male ; Matrix Metalloproteinase 13 ; metabolism ; Pulmonary Artery ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

OBJECTIVE: To explore and verify that transient receptor potential vanilloid 4(TRPV4) affects chondrocyte degeneration. METHODS: Neonatal SD rats were selected, primary chondrocytes were extracted, and identified by toluidine blue staining and alcian blue staining;an in vitro chondrocyte inflammation model was constructed by IL-1β, and TRPV4 inhibitor was used to treat chondrocytes under inflammatory conditions, and the chondrocytes were treated by RT-PCR method was used to detect matrix metallopeptidase 13(MMP-13), a disintegrin and metalloproteinase with thrombospondin 5, (ADAMTS-5)、nitric oxide synthase 2(NOS2)、Collagen, type II alpha 1(Col2α1)and aggrecan (Acan) mRNA in chondrocytes; primary chondrocytes were treated with different concentrations of TRPV4 overexpression plasmid, and the optimal overexpression dose was screened. The mRNA expressions of TRPV4, MMP-13, ADAMTS-5, NOS2, Col2α1 and Acan in chondrocytes under the optimal TRPV4 overexpression dose were detected. RESULTS: Toluidine blue staining and Alcian blue staining identified the extracted cells as primary chondrocytes;RT-PCR showed that TRPV4, MMP-13, ADAMTS-5, NOS2 mRNA in chondrocytes treated with TRPV4 inhibitor under inflammatory conditions. The expression of Col2α1 mRNA was significantly decreased (P<0.05), and the expression of Col2α1 mRNA was increased (P<0.05). Although there was no significant difference in the expression of Acan mRNA, the overall trend was also increasing. The expression of Col2α1 and Acan mRNA in chondrocytes was significantly decreased (P<0.05), and the expression of NOS2 mRNA was increased(P<0.05), but there was no significant difference in MMP-13 and ADAMTS-5 (P>0.05). CONCLUSION Inhibiting the expression of TRPV4 can down-regulate the expression of genes related to chondrocyte degeneration.
Animals ; Rats ; Aggrecans/metabolism* ; Cartilage, Articular ; Cells, Cultured ; Chondrocytes ; Interleukin-1beta/metabolism* ; Matrix Metalloproteinase 13/metabolism* ; Rats, Sprague-Dawley ; RNA, Messenger/metabolism* ; TRPV Cation Channels/metabolism*

BACKGROUND: One of the characteristics of spinal stenosis is elastin degradation and fibrosis of the extracellular matrix of the ligamentum flavum. However, there have been no investigations to determine which biochemical factors cause these histologic changes. So we performed the current study to investigate the hypothesis that matrix metalloproteinases (MMPs), which possess the ability to cause extracellular matrix remodeling, may play a role as a mediator for this malady in the ligamentum flavum. METHODS: The ligamentum flavum specimens were surgically obtained from thirty patients with spinal stenosis, as well as from 30 control patients with a disc herniation. The extents of ligamentum flavum elastin degradation and fibrosis were graded (grade 0-4) with performing hematoxylin-eosin staining and Masson's trichrome staining, respectively. The localization of MMP-2 (gelatinase), MMP-3 (stromelysin) and MMP-13 (collagenase) within the ligamentum flavum tissue was determined by immunohistochemistry. The expressions of the active forms of MMP-2, MMP-3 and MMP-13 were determined by western blot analysis, and the blots were quantified using an imaging densitometer. The histologic and biochemical results were compared between the two conditions. RESULTS: Elastin degradation and fibrosis of the ligamentum flavum were significantly more severe in the spinal stenosis samples than that in the disc herniation samples (3.14 +/- 0.50 vs. 0.55 +/- 0.60, p < 0.001; 3.10 +/- 0.57 vs. 0.76 +/- 0.52, p < 0.001, respectively). The expressions of the active form of MMPs were identified in all the ligamentum flavums of the spinal stenosis and disc herniation patients. The expressions of active MMP-2 and MMP-13 were significantly higher in the spinal stenosis samples than that in the disc herniation samples (both p < 0.05). The expression of active MMP-3 was slightly higher in the spinal stenosis samples than that in the disc herniation samples, but the difference was not statistically significant (p = 0.131). MMP-2, -3, and -13 were positively stained on the ligamentum flavum fibroblasts. CONCLUSIONS: The current results suggest that the increased expression of active MMPs by the ligamentum flavum fibroblasts might be related to the elastin degradation and fibrosis of the ligamentum flavum in the patients who suffer with lumbar spinal stenosis.
Aged ; Blotting, Western ; Elastin/*metabolism ; Extracellular Matrix/metabolism/pathology ; Female ; Fibrosis ; Humans ; Immunohistochemistry ; Ligamentum Flavum/*metabolism/pathology ; *Lumbar Vertebrae ; Male ; Matrix Metalloproteinase 13/metabolism ; Matrix Metalloproteinase 2/metabolism ; Matrix Metalloproteinase 3/metabolism ; Matrix Metalloproteinases/*metabolism ; Middle Aged ; Spinal Stenosis/*metabolism/pathology

The present study aims to investigate the effect of Cbl-b, a member of E3 ubiquitin ligase family, on interleukin-1 (IL-1) pathway in synoviocytes. The protein expression levels of Cbl-b and IL-1-induced matrix metalloproteinase 13 (MMP-13) in synoviocytes were analyzed by Western blot. Collagen substrates were incubated with the conditioned medium collected from synoviocytes cultures and then subjected to SDS-PAGE for analysis of collagen degradation. The results showed that compared with wild-type cells, Cbl-b-deficient cells expressed more MMP-13 protein and had enhanced ability to degrade collagens under IL-1 stimulation. These data suggest that Cbl-b may negatively regulate IL-1-triggered degradation of collagen matrix in synoviocytes.
Collagen ; metabolism ; Epithelial Cells ; enzymology ; Humans ; Interleukin-1 ; metabolism ; Matrix Metalloproteinase 13 ; metabolism ; Proto-Oncogene Proteins c-cbl ; metabolism ; Signal Transduction

BACKGROUND: Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway. METHODS: Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting. RESULTS: IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells. CONCLUSION HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.
Cell Line, Tumor ; Cells, Cultured ; Chondrocytes/metabolism* ; Glycogen Synthase Kinase 3 beta/genetics* ; Histone Deacetylases/genetics* ; Humans ; Interleukin-1beta/pharmacology* ; Matrix Metalloproteinase 13/metabolism* ; Matrix Metalloproteinase 3 ; Repressor Proteins ; Wnt Signaling Pathway ; Wnt3A Protein/genetics* ; beta Catenin/metabolism*

OBJECTIVEThis study was to explore the cytotoxic effect and the related injury mechanism of deoxynivalenol (DON) on articular chondrocytes in human embryo.

METHODSArticular cartilage cells were isolated from knees of human embryo and cultured in DMEM/F12 medium. The cells of the 4th generation were divided into five groups and incubated with varying concentrations of DON as the followings: control group and group with DON of 0.1, 0.2, 0.4, 1.0 µg/ml. The effects of DON were observed 72 hours after incubation. Cell apoptosis was assayed by flow cytometry (FCM) with Annexin V-FITC/PI staining; MMP-13 and PGE2 were detected by ELISA kits; NO was measured by Griess assay with spectrophotometer. Inducible nitric oxide synthase (iNOS) and collagen II in cells were detected by FCM. The expression levels of iNOS, mRNA and collagen II mRNA were measured with RT-PCR.

RESULTSThe rates of cell apoptosis in DON groups were 6.78% - 19.05%, which were significantly higher than that in control (1.20%, F = 174.761, P < 0.05). The levels of NO in DON groups were 20.8 - 40.7 µmol/L, which were significantly higher than that in control (10.2 µmol/L, F = 91.966, P < 0.05). The levels of MMP-13 in DON groups were 0.25 - 0.56 µmol/L, which were significantly higher than that in control (0 µmol/L, F = 78.420, P < 0.05). The levels of PGE2 in DON groups were 3.2-20.6 µmol/L, which were significantly higher than that in control (11.6 µmol/L, F = 276.453, P < 0.05). The proportions of cells with positive iNOS in DON groups were 14.8% - 56.8% which were significantly higher than that in controls (7.1%, F = 214.614, P < 0.05). The proportions of cells with positive collagen II in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 56.7% and 52.7%, which were significantly lower than that in control (62.2%, F = 5.134, P < 0.05). The relative absorbance values of iNOS mRNA in DON groups were 1.07 - 1.33, which were significantly higher than that in control (0.62, F = 8.358, P < 0.05). The levels of collagen II mRNA in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 0.83 and 0.82, which were significantly lower than that in control (1.14, F = 7.887, P < 0.05).

CONCLUSIONDON could promote anabolism of NO in articular cartilage cells by which up-regulated the expression of PGE2 and MMP-13, which both promoted resolution of articular cartilage matrix such as collagen II. DON induced apoptosis in articular cartilage cells.

Cartilage, Articular ; cytology ; embryology ; Cells, Cultured ; Chondrocytes ; drug effects ; metabolism ; Dinoprostone ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; metabolism ; Nitric Oxide ; biosynthesis ; Trichothecenes ; toxicity

OBJECTIVESTo demonstrate the gene expression of MMP-13 in the progressive phase of ethanol-induced experimental liver fibrosis in rats.

METHODS34 SD rats were randomized into two groups. The rats in experimental group (n=24) were given ethanol (44%, 7g/kg) every day, and the rats in control group (n=10) were given equality normal saline. Liver samples were harvested from experimental rats at the 4th, 12th and 24th weeks respectively. The dynamic expression of MMP-13 mRNA was assayed by semi-quantity reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSIn normal rat liver, a faint band of MMP-13 mRNA was observed by RT-PCR (0.24+/-0.41). The gene expression of MMP-13 increased in the livers of rats treated with ethanol for 4 weeks (0.62+/-0.54), but it was not considered statistically, when compared with that in normal rats livers. And the livers from 12-week-treated rats showed a markedly MMP-13 mRNA expression (1.65+/-0.47, t=-4.363, P<0.01). Once the fibrosis became prominent (24 weeks), a faint band of MMP-13 mRNA was observed (0.39+/-0.25).

CONCLUSIONMMP-13 participates in the degradation of newly-formed matrix in the early phase of rat liver fibrosis induced by ethanol, but it expresses in a distinct time frame

Animals ; Collagenases ; genetics ; metabolism ; Disease Progression ; Ethanol ; Gene Expression ; Liver Cirrhosis ; chemically induced ; metabolism ; Matrix Metalloproteinase 13 ; Rats ; Rats, Sprague-Dawley

AIMTo investigate the effect of IH764-3 on the expression of MMP-13 and TIMP-1 by H2O2-stimulated hepatic stellate cell and the alteration of FAK during this process.

METHODSThe expression of MMP-13 and FAK mRNA was examined by RT-PCR. TIMP-1 mRNA was analyzed by in-situ hybridization. FAK and TIMP-1 were evaluated at protein level through Western blotting method.

RESULTSBeing incubated for 2 h, compared with control group, MMP-13 mRNA was upregulated by IH764-3, but TIMP-1 transcription was reduced in a dose-dependent manner, accompanied with the decrease of FAK mRNA. The expression of TIMP-1 and FAK protein in HSC also decreased after being exposed by IH764-3 for 24 h.

CONCLUSIONIH764-3 can induce the expression of MMP-13 and inhibit the expression of TIMP-1. Down-regulating the expression of FAK mRNA may be one of its mechanisms.

Animals ; Cells, Cultured ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Hydrogen Peroxide ; adverse effects ; Matrix Metalloproteinase 13 ; metabolism ; Rats ; Salvia miltiorrhiza ; chemistry ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism