The precise mechanism underlying the therapeutic efficacy of an extraction powder of Angelica gigas (AGE) for the treatment of degenerative osteoarthritis was investigated in primary cultured rabbit chondrocytes and in a monosodium-iodoacetate (MIA)-induced osteoarthritis rat model. The treatment with AGE (50 μg/mL) effectively inhibited NF-B activation. The anti-inflammatory mechanism was clarified by gelatin zymography and western blotting measurements of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities. The AGE (50 μg/mL) treatment significantly reduced MMP-9 activity. The constituents of AGE— decursinol, decursin, and decursinol angelate—were determined by LC-MS/MS after a 24 hr treatment of rabbit chondrocytes. The contents of the major products, decursin and decursinol angelate, were 3.62±0.47 and 2.14 ±0.36 μg/mg protein, respectively in AGE-treated (50 μg/mL) rabbit chondrocytes. An in vivo animal study on rats fed a diet containing 25, 50, and 100 mg/kg AGE for 3 weeks revealed a significant inhibition of the MMPs in the MIA-induced rat articular cartilage. The genetic expression of arthritic factors in the articular cartilage was examined by RT-PCR of collagen Type I, collagen Type II, aggrecan, and MMP (MMP3, MMP-9, MMP13). Specifically, AGE up-regulated the expression of collagen Type I, collagen Type II, and aggrecan and inhibited MMP levels at all tested concentrations. Collectively, AGE showed a strong specific site of action on MMP regulation and protected against the degeneration of articular cartilage via cellular regulation of MMP expression both in vitro and in vivo.
Extracellular matrix component is degraded by enzymes of thematrix metalloproteinases(MMPs). MMPs are produced by both hemopoietic and structural cells. Increased activity of MMP-3 in periodontium is strongly associated with inflammatory periodontal disease. The purpose of the present study was to determine the effect of tetracycline analogues on the activity of MMP-3. Tetracycline-HCl, doxycycline-HCl, and minocycline-HCl were applied to huamn gingival fibroblasts at various concentrations of 10, 25, 50, 100, 200${\mu}g$/ml, and 1 hour later IL-$1{\beta}$ of 25ng/ml was added. After incubation for 24 hours the cells were reacted by enzyme-linked immunosorbent assay using proMMP-3 ELISA kit. The optical density was measured by microwell plate reader at 450nm. The relative activity of MMP-3 was calculated as the percentage of the optical density of each experimental group to that of the control. The difference of the optical density and the relative activity of MMP-3 between the experimental groups and the control wasstatistically analyzed by one way ANOVA. The results were as follows: 1. Tetracycline-HCl showed the tendency to inhibit the activity of MMP-3 at the concentration lower than 25${\mu}g$/ml, but increased significantly the activity of MMP-3 at the concentration of 200${\mu}g$/ml(p<0.05). 2. Doxycycline-HCl inhibited significantly the activity of MMP-3 at the concentration lower than 100${\mu}g$/ml, but increased significantly the activity of MMP-3 at the concentration of 200${\mu}g$/ml(p<0.05). 3. Minocycline-HCl inhibited the activity of MMP-3 at the concentration in the range of 10 to 200${\mu}g$/ml. Within the limit of the present study, the above results suggested that the low concentration of tetracycline analogues could inhibit the activity of MMP-3 induced by IL-$1{\beta}$ in human gingival fibroblasts.
Tooth eruption requires remodeling of surrounding tissues. This study was aimed to investigate the effect of indomethacin on the dental follicle and paradental tissues during tooth eruption by observing the distribution and expression of MMP by the immunohistochemical method. Ten mongrel dogs of ten to twelve weeks old were divided into 5 groups; four experimental groups administered indomethacin 2 mg/Kg/day and 8 mg/Kg/day orally 2 times a day for 14 days and 7 days respectively, and the control group was administered a placebo. Permanent teeth before eruption and their surrounding tissues were selected and excised. H&E staining and immunohistochemical stainings of MMP-3 and -9 were performed and examined under the light microscope. Osteoclasts, osteoblasts, periodontal ligament cells, ameloblasts and odontoblasts of the control group all expressed MMP-3 and -9. In the experimental group, osteoclasts, osteoblasts and periodontal ligament cells showed reduced expression of MMP-3 and -9. Magnitude of MMP reduction In the experimental group showed a time and dose of indomethacin administration dependent manner. These results show that indomethacin inhibited MMP-3 and -9 expression in the dental follicle and surrounding tissues and suggest that when indomethacin is administered for long periods, tooth eruption could be delayed.
Saururus chinensis is a perennial plants, its flavonoid compound is known to exhibit anti-oxidative activity. This study was aimed to investigate the effect of Water Extract and Solvent Fractions of Saururus chinensis on antioxidant, anti-inflammatory and anti-invasive of Phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase (MMP-2) and MMP-9 activities. Plant samples were fractionated into hexane, CHCl3, ethyl acetate, butanol, and water fractions, and each of these was assayed individually. The water fraction showed the highest extraction yield at 9.25%(w/w). Anti-oxidative activity was analyzed by DPPH assay. Cell viability was detected by the MTS assay. Anti-inflammatory activity was assayed by the nitric oxide (NO) production in mouse macrophage Raw 264.7 cells. The activity and mRNA expression of MMP-2 and MMP-9 in human oral squamous carcinoma YD-10B cells were examined by zymography and RT-PCR. As results, MMP-2/-9 activation was increased in PMA induced YD-10B cells. In PMA-treated YD-10B cells, the increased mRNA expression and protein activation of MMP-2/-9 were significantly inhibited in the ethyl acetate fraction. The ethyl acetate fraction showed the highest anti-oxidative activity at 73.38%. The ethyl acetate fraction at non-cytotoxic concentrations significantly exhibited the anti-inflammatory activity of Raw 264.7 cells in dose-dependent manner. In conclusion, these findings demonstrate that the ethyl acetate fraction obtained from a chinensis water extract potentiates a promising therapeutic anti-invasive agent and, therefore, as an anti-cancer drug for cancer prevention and therapy in oral cancer.
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which degrade extracellular matrix (ECM) during embryogenesis, wound healing, and tissue remodeling. Dysregulation of MMP activity is also associated with various pathological inflammatory conditions. In this study, we examined the expression pattern of MMPs during PMA-induced differentiation of THP-1 monocytic cells into macrophages. We found that MMP1, MMP8, MMP3, MMP10, MMP12, MMP19, MMP9, and MMP7 were upregulated during differentiation whereas MMP2 remained unchanged. Expression of MMPs increased in a time-dependent manner; MMP1, MMP8, MMP3, MMP10, and MMP12 increased beginning at 60 hr post PMA treatment whereas MMP19, MMP9, and MMP7 increased beginning at 24 hr post PMA treatment. To identify signal transduction pathways involved in PMA-induced upregulation of MMPs, we treated PMA-differentiated THP-1 cells with specific inhibitors for PKC, MEK1, NF-${\kappa}B$, PI3K, p38 MAPK and PLC. We found that inhibition of the MEK1 pathway blocked PMA-induced upregulation of all MMPs to varying degrees except for MMP-2. In addition, expression of select MMPs was inhibited by PI3K, p38 MAPK and PLC inhibitors. In conclusion, we show that of the MMPs examined, most MMPs were up-regulated during differentiation of monocyte into macrophage via the MEK1 pathway. These results provide basic information for studying MMPs expression during macrophage differentiation.
Journal of the Korean Association of Oral and Maxillofacial Surgeons
/
v.27
no.4
/
pp.314-320
/
2001
Matrix metalloproteinases have long been viewed as ideal candidates for proteinases that enables tumor cells to permeated basement membrane defenses and invade surrounding tissue. There is growing evidence that the MMPs have an expanded role, as they are important for the creation and maintenance of a microenvironment that facilitates growth and angiogenesis of tumors at primary and metastatic sites. MT-MMPs are not secreted but instead remaining attached to cell surfaces. Although not all of the MT-MMPs are fully characterized, MT-MMPs have important role in localizing and activating secreted MMPs. The MMP genes are transcriptionally responsive to a wide variety of oncogene, growth factors, cytokine, and hormones. Currently, a number of MMP inhibitors are being developed and some have reached clinical trials as anti-metastatic or anti-cancer therapies. MT1-MMP is involved in the activation of proMMP-2. MT1-MMP is significant not only as a tumor marker but as a new target for chemotherapy against cancer. The purpose of this study was to evaluate the effects of protein kinase C inhibitor(genistein) on the proliferation of HT1080 and expression of MT1-MMP mRNA. Human fibrosarcoma cell line HT1080 was cultured and divided 2 groups. The experimental group was treated with $100{\mu}M$ genistein and incubated 12h, 24h for $[3^H]-thymidine$ uptake assay and northern hybridization individually. And the control group was treated with same amount of PBS for the above procedures. $[3^H]-thymidine$ incorporation was measured with ${\beta}$ ray detector. And RT-PCR and northern blotting for MT1-MMP mRNA was performed. The results were as follows 1. $[3^H]-thymidine$ uptake was reduced in experimental group with statistical significance. 2. MT1-MMP mRNA expression was significantly reduced in experimental group. These results showed that protein kinase C inhibitor (genistein) inhibited proliferation of HT1080 and almost completely blocked transcription of MT1-MMP mRNA. So, it is possible to use the protein kinase inhibitor (genistein) as anti-metastatic and anti-proliferative agent.
Lee, Choae;An, Jaewoo;Kim, Ji Hee;Kim, Eun Sun;Kim, Soo Hyun;Cho, Yeon Kyung;Cha, Dong Hyun;Han, Man Yong;Lee, Kyu Hyung;Sheen, Youn Ho
Clinical and Experimental Pediatrics
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v.58
no.11
/
pp.415-420
/
2015
Purpose: Bronchopulmonary dysplasia (BPD) is characterized by inflammation with proteolytic damage to the lung extracellular matrix. The results from previous studies are inconsistent regarding the role of proteinases and antiproteinases in the development of BPD. The aim of the present study was to investigate whether matrix metalloproteinase (MMP)-8, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2, and TIMP-1 levels in the serum of preterm infants at birth are related to the development of BPD. Methods: Serum was collected from 62 preterm infants at birth and analyzed for MMP-8, MMP-9, TIMP-2, and TIMP-1 by using enzyme-linked immunosorbent assay. MMPs and TIMPs were compared in BPD (n=24) and no BPD groups (n=38). Clinical predictors of BPD (sex, birth weight, gestational age, etc.) were assessed for both groups. The association between predictors and outcome, BPD, was assessed by using multivariate logistic regression. Results: Sex, birth weight, and mean gestational age were similar between the groups. BPD preterm infants had significantly lower TIMP-2 levels at birth compared with no BPD preterm infants ($138.1{\pm}23.0ng/mL$ vs. $171.8{\pm}44.1ng/mL$, P=0.027). No significant difference was observed in MMP-8, MMP-9, and TIMP-1 levels between the two groups. Multivariate logistic regression analysis indicated that the TIMP-2 levels were predictive of BPD after adjusting for sex, birth weight, gestational age, proteinuric preeclampsia, and intraventricular hemorrhage (${\beta}=-0.063$, P=0.041). Conclusion: Low TIMP-2 serum levels at birth may be associated with the subsequent development of BPD in preterm infants.
Intermediate-wavelength solar radiation, also known as ultraviolet B (UVB: 290-320 nm) radiation, may cause premature aging and oxidative damage-dependent skin cancer in humans. UVB-induced formation of reactive oxygen species (ROS)-often a consequence of excessive exposure to these rays-could activate matrix metalloproteinases (MMPs) such as MMP-1 and MMP-3. These enzymes break down type I collagen in human fibroblasts. In this study, we assessed the antioxidant and anti-aging effects of ethyl acetate extract of blueberry (EEB). An antioxidant test in blueberries evaluated ROS production using CCD-986sk cells and DPPH assay. In order to evaluate the anti-wrinkle efficacy of blueberries, the MMP-1 production and type 1 procollagen synthesis evaluated and the expression of MMP 1, 3 were tested through Western blot and RT- PCR. EEB exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and reduced the production of UVB-induced ROS. Also, EEB inhibited UVB-induced processes associated with photoaging and skin cancer, such as reduction in procollagen production and increase in MMP-1 production. More precisely, EEB (50 ㎍/ml) markedly suppressed mRNA and protein levels of MMP-1 and -3. The anti-aging effects are attributable to the antioxidant activity of EEB. These findings indicate that EEB has a protective effect against UVB-induced aging in human fibroblast cells by regulating the levels of type-1 procollagen, MMP-1, and MMP-3.
The pathogenesis of endometriosis is unknown, but retrograde menstruation is widely accepted as an etiology. Refluxed endometrium from endometriosis patients is more prone to implant and invade peritoneum possibly through the action of extracellular proteolysis. This proteolytic action may involve plasminogen activators and the collagenase system. Plasminogen activators (PAs) and matrix metalloproteinases (MMPs) play a critical role in the breakdown of extracellular matrix components and basement membrane in the processes of implantation and tumor invasion. PAs are inhibited by plasminogen activator inhibitor (PAI) and MMPs activity is inhibited by tissue inhibitor of metalloproteinase (TIMP). To test the hypothesis that lower expression of PAI-1 and TIMP-3 in endometrium from women with endometriosis, we investigated their PAI-1 and TIMP-3 expression by quantitative competitive RT PCR in endometrium from women with and without endometriosis. Endometrial tissues were obtained from 14 patients with severe endometriosis and 14 patients without endometriosis. Total RNA was extracted and reverse transcribed into cDNA, and quantitative competitive PCR (QC PCR) was performed to evaluate PAI-1 and TIMP-3 mRNA expression. Endometrium from patients with endometriosis showed decreased expression of PAI-1 and TIMP-3 mRNA compared to endometrium from control in luteal phase (p<0.05). Our results suggest that endometrium from women with endometriosis expresses lower levels of PAI-1 and TIMP-3 than endometrium from normal women. Endometrium from endometriosis patients may be more invasive and prone to peritoneal implantation than control because of higher PA and MMP enzymatic activity. Thus, increased proteolytic activity may be one of the reasons for the invasive properties of the endometrium resulting in the development of endometriosis.
Objectives : This study aimed at evaluation of the effects of Rhodiola rosea on brain edema and expressions of matrix metalloproteinases (MMPs) related to blood-brain barrier (BBB) disruption. Methods : Brain edema following intracerebral hemorrhage (ICH) was induced by the stereotaxic intrastriatal injection of bacterial collagenase type VII in rats (Sprague-Dawley). Then ethanol extract of Rhodiola rosea was treated once a day for 3 days. Brain edema % and water contents, and BBB leakage were examined. Immunohistochemistry was processed for MMP-9, MMP-12, and iNOS expressions in the brain sections and each immuno-labeled cells were analyzed with image analysis software. Results : 1. Ethanol extract of Rhodiola rosea reduced brain edema following ICH in rats significantly. 2. Ethanol extract of Rhodiola rosea reduced excessive brain tissue water contents following ICH in rats significantly. 3. Ethanol extract of Rhodiola rosea reduced BBB leakage in the cerebral cortex following ICH in rats. 4. Ethanol extract of Rhodiola rosea reduced cellular edema of neurons in peri-hematoma and the cerebral cortex following ICH in rats significantly. 5. Ethanol extract of Rhodiola rosea reduced MMP-9 positive cells in the cerebral cortex following ICH in rats significantly. 6. Ethanol extract of Rhodiola rosea reduced MMP-12 positive vessels in the cerebral cortex following ICH in rats significantly. 7. Ethanol extract of Rhodiola rosea reduced iNOS positive cells in the cerebral cortex and external capsule following ICH in rats significantly. Conclusions : These results suggest that Rhodiola rosea reveals protective effect against brain edema and cytotoxic edema of neurons by means of down-regulation of MMPs and iNOS expressions, and inhibition of BBB leakage.
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