mRNA Expression Of Tissue Inhibitor Research Articles

Prunus mume Seed Exhibits Inhibitory Effect on Skin Senescence via SIRT1 and MMP-1 Regulation.

The Prunus mume seed is a by-product of the food industry, and we studied its potential as a food biomaterial, particularly for nutraceutical and inner beauty products. Alternative animal tests showed that an extract of P. mume ripened seed (PmRS) was not toxic on the skin. PmRS exhibited protective effects against ultraviolet- (UV-) induced skin aging in mice, confirmed by phenotypic indications, including increased collagen levels and decreased skin thickness. Compared with the UV-saline group, the UV-PmRS group showed increased levels of silent mating type information regulation 2 homolog 1 (SIRT1) and collagen and decreased matrix metalloproteinase- (MMP-) 1 expression. The protective effect of PmRS treatment against UVB-mediated cell viability was observed in vitro without any cytotoxicity, and PmRS prevented UVB-induced reactive oxygen species generation in HaCaT cells. PmRS downregulated MMP-1 and MMP-13 compared with the UVB-irradiated group. However, mRNA expressions of tissue inhibitor of metalloproteinase-1 and SIRT1 were upregulated by PmRS treatment. MMP-1 and SIRT1 treated with PmRS were decreased and increased, respectively, at the protein level. Moreover, PmRS treatment reduced c-Jun N-terminal kinase and p38 phosphorylation compared with the UVB-treated group. We postulate that P. mume seed could be a useful ingredient in nutraceuticals and inner beauty-purpose foods.

Interstitial changes after reperfused myocardial infarction in swine: morphometric and genetic analysis

BackgroundFollowing myocardial infarction (MI), we aimed to characterize morphometric and genetic changes in extracellular matrix (ECM) components from ischemia onset until late phases after coronary reperfusion in necrotic and salvaged myocardium.ResultsSwine were divided into one control (n = 5) and three MI groups: 90-min of ischemia without reperfusion, or followed by 1-week or 1-month reperfusion (n = 5 per group). In samples from the necrotic and salvaged areas, ECM components were morphometrically quantified and mRNA levels of factors involved in ECM remodeling were evaluated. After 90-min of ischemia, fibronectin, laminin, and elastic fibers content as well as upregulated mRNA expression of tissue inhibitors of metalloproteinases (TIMP)1, TIMP2, TIMP3 and connective tissue growth factor increased in the necrotic and salvaged myocardium. In both reperfused MI groups, collagen-I, collagen-III, elastic fibers, glycosaminoglycans, laminin, and fibronectin levels heightened in the necrotic but not the salvaged myocardium. Moreover, mRNA expression of TIMP1, TIMP2 and TIMP3, as well as metalloproteinase-2 and metalloproteinase-9 heightened in the necrotic but not in the salvaged myocardium.ConclusionsMatrix remodeling starts after ischemia onset in both necrotic and salvaged myocardium. Even if ECM composition from the salvaged myocardium was altered after severe ischemia, ECM makes a full recovery to normal composition after reperfusion. Therefore, rapid coronary reperfusion is essential not only to save cardiomyocytes but also to preserve matrix, thus avoiding impaired left ventricular remodeling.

Infiltrating CCR2+ monocytes and their progenies, fibrocytes, contribute to colon fibrosis by inhibiting collagen degradation through the production of TIMP-1

Intestinal fibrosis is a serious complication in inflammatory bowel disease (IBD). Despite the remarkable success of recent anti-inflammatory therapies for IBD, incidence of intestinal fibrosis and need for bowel resection have not significantly changed. To clarify the contribution of haematopoietic-derived cells in intestinal fibrosis, we prepared bone marrow (BM) chimeric mice (chimeras), which were reconstituted with BM cells derived from enhanced green fluorescent protein (EGFP)-transgenic mice or CC chemokine receptor 2 (CCR2)-deficient mice. After 2 months of transplantation, BM chimeras were treated with azoxymethane/dextran sodium sulphate. During chronic inflammation, CCR2+ BM-derived monocyte and fibrocyte infiltration into the colon and CC chemokine ligand 2 production increased, leading to colon fibrosis in EGFP BM chimeras. In CCR2-deficient BM chimeras, monocyte and fibrocyte numbers in the colonic lamina propria significantly decreased, and colon fibrosis was attenuated. In colon tissue, mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 but not of collagen I, transforming growth factor-β1 or matrix metalloproteinases was significantly different between the two chimeras. CCR2+ monocytes and fibrocytes showed high Timp1 mRNA expression. Our results suggest that infiltrating CCR2+ monocytes and their progenies, fibrocytes, promote colon fibrosis by inhibiting collagen degradation through TIMP-1 production.

The Protein Extract of Chlorella minutissima Inhibits The Expression of MMP-1, MMP-2 and MMP-9 in Cancer Cells through Upregulation of TIMP-3 and Down Regulation of c-Jun.

Objective Considering the bioactivities exhibited by microalgae, the effect of protein extract of Chlorella minutissimma (CP extract) was investigated on the expression of human matrix metalloproteinases-1 (MMP-1) in the breast cancer cell line MDA-MB231, and that of MMP-2 and -9 in hepatocellular cancer cell line HepG2 at different expression levels. The study aimed identification and analysis of inhibitory activity of microalgal components extracted from Chlorella minutissima against human MMPs. Materials and Methods In this experimental study, we analysed the effect of Chlorella extracts on MMP-1, -2, and -9 expression at various levels. Gelatin zymography was performed to study the inhibitory effect of Chlorella exracts on human gelatinases at the activity level, followed by western blotting to analyse the expression of all three MMPs at the protein level. The similar effect at the mRNA level along with the probable mechanism underlying inhibition of MMPs was assessed using real-time polymerase chain reaction (PCR). Results The results reveal that the treatment with CP extract decreased the mRNA expression of MMP-1, MMP-2, and MMP-9 by 0.26-, 0.29-, and 0.40-fold, respectively, at 20 μg/ml concentration as well as inhibited the activity of MMP-2 and MMP-9 by 37.56 and 42.64%, respectively, at 15 μg/ml concentration. Additionally, upregulated mRNA expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) by 1.68-fold was seen in HepG2 cells at 20 μg/ml concentration treatment group. However, CP extract did not induce any change in the mRNA expression of the TIMP-1, -2 and -4 in HepG2 and TIMP-1, -2, -3 and -4 in MDA-MB231 cells. Activator protein-1 (AP-1)-dependent c-Jun-mediated transcriptional regulation of MMP-1, -2, and -9 was also studied to elucidate the appropriate mechanism involved in the inhibition of MMPs. ConclusionThe CP extract successfully inhibited MMP-1, -2, and -9 at different expression levels through TIMP-3 upregulation and c-Jun downregulation.

Effect of medicated serum of Curcumae Radix extract on mRNA expression of TIMP-1, MMPs-13 and aI-collagen of HSC-T6 cell

To study the effect of medicated serum of Curcumae Radix (Yujin) on the mRNA expression of Tissue inhibitor of metalloproteinase-1 (TIMP-1), Matrix metalloproteinases-13 (MMPs-13) and aI-collagen of Hepatic stellate cell-T6 (HSC-T6) cell. Twenty SD rats were randomly divided into 4 groups: high dose of Yujin group (16.2gkg−1), medium dose of Yujin group (8.1gkg−1), low dose of Yujin group (4.05gkg−1) and blank control group (with the same volume of 0.9% saline). Yujin extract or 0.9% saline were administered daily by gavage to rats for 4d, after once administration of full-day dose for 1h on the fourth day, ether anaesthesia was given, and blood was taken from abdominal aortic in asepsis condition for preparation of medicated serum. HSC-T6 cells were divided into 5 groups: high dose medicated serum of Yujin group, medium dose medicated serum of Yujin group, low dose medicated serum of Yujin group, medicated serum of blank control group and negative control group (added with the same volume of PBS instead of rat serum), after 48h of simultaneous acting on HSC-T6 cells in all groups by the medicated serum with a concentration of 10%, the mRNA expression level of TIMP-1, MMPs-13 and aI-collagen was analyzed with RT-PCR. Compared with the negative control group, the mRNA expression level of TIMP-1, MMPs-13 and aI-collagen in all experimental groups increased significantly. Compared with the control group, the mRNA expression of aI-collagen and TIMP-1 was obviously inhibited in all medicated serum of Yujin groups (P<0.01), meanwhile, the mRNA expression level of MMPs-13 was effectively improved (P<0.05). The medicated serum of Yujin had an effect on the production and degradation of Extracellular matrix (ECM) of HSC-T6 cell.

Correlation between the mRNA expression of tissue inhibitor of metalloproteinase-1 and apparent diffusion coefficient on diffusion-weighted imaging in rats' liver fibrosis

To explore the correlation between the apparent diffusion coefficient (ADC) and mRNA expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in different stages of liver fibrosis in rats. Methods: A model of liver fibrosis in rats was established by intraperitoneal injection of high-fat diet combined with porcine serum. After drug administration for 4 weeks, 48 rats served as a model group and 12 rats served as a control group, then they underwent diffusion weighted imaging (DWI) scanning. The value of ADC was calculated at b value=800 s/mm2. The rats were sacrificed and carried out pathologic examination after DWI scanning immediately. The mRNA expression of TIMP-1 was detected by real time-polymerase chain reaction (RT-PCR). The rats of hepatic fibrosis were also divided into a S0 group (n=4), a S1 group (n=11), a S2 group (n=12), a S3 group (n=10), and a S4 group (n=9) according to their pathological stage. The value of ADC and the expression of TIMP-1 mRNA among the different stage groups of liver fibrosis were compared, and the correlation between ADC and the TIMP-1 mRNA were analyzed. Results: The ADC value and the TIMP-1 mRNA expression were significantly different between the control group and the liver fibrosis group (F=46.54 and 53.87, P<0.05). There were significant differences in the value of ADC between every two groups (all P<0.05), except the control group vs the S1 group, the S1 group vs the S2 group, and the S2 group vs the S3 group (all P>0.05). For the comparison of TIMP-1 mRNA, there was no significant difference between the S1 group and the S2 group, the S3 group and the S4 group (both P>0.05). There were significant differences among the rest of the groups (all P<0.05). Rank correlation analysis showed that there was a negative correlation between the ADC value and the TIMP-1 mRNA expression (r=-0.76, P<0.01). Conclusion: When the value of ADC decreases in the progress of rats' liver fibrosis, the mRNA expression of TIMP-1 increases gradually, and there is a negative correlation between them.

Methionine deprivation suppresses triple-negative breast cancer metastasis in vitro and in vivo.

Nutrient deprivation strategies have been proposed as an adjuvant therapy for cancer cells due to their increased metabolic demand. We examined the specific inhibitory effects of amino acid deprivation on the metastatic phenotypes of the human triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and Hs 578T, as well as the orthotopic 4T1 mouse TNBC tumor model. Among the 10 essential amino acids tested, methionine deprivation elicited the strongest inhibitory effects on the migration and invasion of these cancer cells. Methionine deprivation reduced the phosphorylation of focal adhesion kinase, as well as the activity and mRNA expression of matrix metalloproteinases MMP-2 and MMP-9, two major markers of metastasis, while increasing the mRNA expression of tissue inhibitor of metalloproteinase 1 in MDA-MB-231 cells. Furthermore, methionine restriction downregulated the metastasis-related factor urokinase plasminogen activatior and upregulated plasminogen activator inhibitor 1 mRNA expression. Animals on the methionine-deprived diet showed lower lung metastasis rates compared to mice on the control diet. Taken together, these results suggest that methionine restriction could provide a potential nutritional strategy for more effective cancer therapy.

Acetylsalicylic acid combined with diclofenac inhibits cartilage degradation in rabbit models of osteoarthritis.

The present study aimed to investigate the effect of different concentrations of acetylsalicylic acid combined with diclofenac on the articular cartilage of a rabbit model of osteoarthritis (OA). A total of 40 New Zealand white rabbits were divided into 5 groups. Group A was a sham-operated control group, which was treated with normal saline. Groups B-E were OA models and were treated with normal saline and acetylsalicylic acid combined with diclofenac at concentrations of 5, 10 and 20 mg/kg, respectively. A cartilage macroscopic examination and a pathological observation were performed to analyze the structure of the articular cartilage in all of the treated groups. The nitric oxide (NO) content and interleukin 1β (IL-1β) levels were detected by an enzyme-linked immunosorbent assay. In addition, the protein expression of matrix metalloproteinase 3 (MMP)-3 and MMP-13 were detected by western blot analysis. The mRNA expression of tissue inhibitor of metalloproteinases 1 (TIMP1) was detected by polymerase chain reaction (PCR). The results revealed that different concentrations of the drugs significantly reduced the scores of cartilago articularis, the NO and IL-1β levels and the protein expression of MMP-3 and MMP-13. Furthermore, PCR revealed that the mRNA expression of TIMP1 was significantly upregulated, and the effects increased with increasing drug concentration. Thus, the administration of different concentrations of acetylsalicylic acid combined with diclofenac demonstrates preventive or therapeutic effects against OA progression.

Nimesulide, a cyclooxygenase-2 selective inhibitor, suppresses obesity-related non-alcoholic fatty liverdisease and hepatic insulin resistance through the regulation ofperoxisome proliferator-activated receptor γ.

Cyclooxygenase (COX)-2 selective inhibitors suppress non-alcoholic fatty liver disease (NAFLD); however, the precise mechanism of action remains unknown. The aim of this study was to examine how the COX-2 selective inhibitor nimesulide suppresses NAFLD in a murine model of high-fat diet (HFD)-induced obesity. Mice were fed either a normal chow diet (NC), an HFD, or HFD plus nimesulide (HFD-nime) for 12 weeks. Body weight, hepatic COX-2 mRNA expression and triglyceride accumulation were significantly increased in the HFD group. Triglyceride accumulation was suppressed in the HFD-nime group. The mRNA expression of hepatic peroxisome proliferator-activated receptor γ (PPARγ) and the natural PPARγ agonist 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) were significantly increased in the HFD group and significantly suppressed in the HFD-nime group. Glucose metabolism was impaired in the HFD group compared with the NC group, and it was significantly improved in the HFD-nime group. In addition, the plasma insulin levels in the HFD group were increased compared with those in the NC group, and were decreased in the HFD-nime group. These results indicate that HFD-induced NAFLD is mediated by the increased hepatic expression of COX-2. We suggest that the production of 15d-PGJ2, which is mediated by COX-2, induces NAFLD and hepatic insulin resistance by activating PPARγ. Furthermore, the mRNA expression of tissue inhibitor of metalloproteinases-1 (TIMP-1), procollagen-1 and monocyte chemoattractant protein-1 (MCP-1), as well as the number of F4/80-positive hepatic (Kupffer) cells, were significantly increased in the HFD group compared with the NC group, and they were reduced by nimesulide. In conclusion, COX-2 may emerge as a molecular target for preventing the development of NAFLD and insulin resistance in diet-related obesity.

Adipose tissue-derived stem cell therapy for prevention and treatment of erectile dysfunction in a rat model of Peyronie's disease

Peyronie's disease (PD) is a localized connective tissue disorder that involves the tunica albuginea (TA) of the penis. While surgical correction remains the gold standard, the search for an effective and less invasive therapy continues. The objective of this study was to evaluate the effects of intratunical injection of adipose tissue-derived stem cells (ADSCs) for the prevention and treatment of erectile dysfunction in a rat model of PD. Twenty-four male Sprague-Dawley rats (300-350 g) were randomly divided into four groups: sham, PD, PD + ADSC (prevention) and PD + ADSC (treatment). All rats underwent penile injections into the TA with 50 μL vehicle (sham) or 0.5 μg transforming growth factor (TGF)-β1 (remaining groups). The ADSC groups received intratunical injections with 0.5 million rat-labelled ADSCs on day 0 (prevention) or day 30 (treatment). Forty-five days following TGF-β1 injection, rats underwent cavernous nerve stimulation (CNS) with total intracavernous-to-mean arterial pressure ratio (ICP/MAP) and total ICP recorded to measure response to therapy. Tissues were evaluated histologically and for mRNA expression of tissue inhibitors of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs) and zymographic activity of MMPs. Statistical analysis was performed by analysis of variance followed by the Tukey test for post hoc comparisons. In both prevention and treatment groups, intratunical injection of ADSCs resulted in significantly higher ICP/MAP and total ICP in response to CNS compared with the PD group. Local injection of ADSCs prevented and/or reduced Peyronie's-like changes by decreasing the expression of TIMPs, and stimulating expression and activity of MMPs. This study documents the preventive and therapeutic benefits of ADSC on penile fibrosis and erectile function in an animal model of PD.

Therapeutic Potential of Wenshen Xiaozheng Tang, a Traditional Chinese Medicine Prescription, for Treating Endometriosis

The objective of this study is to investigate the effect of Wenshen Xiaozheng Tang (WXT) on the development of endometriosis in a rat model. Sprague-Dawley rats in which endometriotic implants were induced were divided randomly into 3 groups. The rats in the low-dose and high-dose WXT groups were administered WXT 8.57 and 17.14 g/kg/d, respectively. The rats in the control groups received an equal volume of dissolvent, as did the sham-operated rats. After treatment for 4 weeks, WXT significantly decreased the mean lesion size as well as the peritoneal fluid and serum levels of tumor necrosis factor α and interleukin 1β. Cyclooxygenase-2, matrix metalloproteinase 9, plasminogen activator inhibitor 1, and intercellular adhesion molecule 1 messenger RNA (mRNA) levels were downregulated, and the mRNA expression of tissue inhibitor of metalloproteinase 1 was upregulated in the endometriotic lesions of WXT versus control group. Our data suggested that WXT may suppress the development of endometriosis by inhibiting the production of proinflammatory cytokines and regulating the expression of invasion-related genes in the endometriotic lesions.

Starfish polysaccharides downregulate metastatic activity through the MAPK signaling pathway in MCF-7 human breast cancer cells

We investigated the effects of starfish (Asterina pectinifera) polysaccharides on metastatic activity in MCF-7 estrogen receptor (ER)-positive human breast cancer cells. In wound healing assay, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration was dose-dependently decreased by the starfish polysaccharides (PS). Transcription of aromatase, which catalyzes estrogen synthesis from androgen, was reduced by PS. Also, transcription of TPA-induced cyclooxygenase-2 (COX-2), which enhances breast cancer progression and metastasis via the increase of prostaglandin E2 biosynthesis, was downregulated by the PS in a dose-dependent manner. PS decreased the expression and activity of matrix metalloproteinase (MMP)-9, an important factor in the degradation of basement membrane and extracellular matrix in the metastasis process. In contrast, mRNA expression of tissue inhibitor of matrix metalloproteinase (TIMP)-1, a MMP inhibitor, was increased by 10-120 μg/ml of PS but not that of TIMP-2. We also found that PS reversed the phosphorylations of p38, ERK and JNK but not IκBα and NF-κB. These results demonstrate that PS successfully inhibits PKC-mediated cell migration and metastatic activities in MCF-7 ER-positive human breast cancer cells via downregulation of MMP-9 activity mediated by TIMP-1 upregulation and inhibition of aromatase and COX-2 expression. Also, COX-2 and MMP-9 expressions are attenuated through the inhibition of AP-1 transcription activity via the downregulation of c-Jun expression regulated by p38, ERK and JNK signaling. In conclusion, the present investigation shows that PS may prevent COX-2- and MMP-9-mediated metastatic activities in MCF-7 ER-positive breast cancer cells through the downregulation of MAPK signaling pathways.

AB0141 Rebamipide attenuates pain severity and cartilage degeneration in a rat model of osteoarthritis by downregulating oxidative damage and catabolic activity in chondrocytes

BackgroundOsteoarthritis (OA) is a degenerative joint disease characterized by the progressive loss of cartilage and pain. The development and progression of OA are believed to involve inflammation. Many studies have...

Rebamipide attenuates pain severity and cartilage degeneration in a rat model of osteoarthritis by downregulating oxidative damage and catabolic activity in chondrocytes

The objectives were to investigate the invivo effects of treatment with rebamipide on pain severity and cartilage degeneration in an experimental model of rat osteoarthritis (OA) and to explore its mode of action. OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA). Oral administration of rebamipide was initiated on the day of MIA injection, 3 or 7 days after. Limb nociception was assessed by measuring the paw withdrawal latency and threshold. We analyzed the samples macroscopically and histomorphologically, and used immunohistochemistry to investigate the expression of matrix metalloproteinase-13 (MMP-13), interleukin-1β (IL-1β), hypoxia-inducible factor-2α (HIF-2α), inducible nitric oxide synthase (iNOS), and nitrotyrosine in knee joints. Real-time quantitative reverse transcription-polymerase chain reaction was used to quantify the mRNA for catabolic and anticatabolic factors in human OA chondrocytes. Rebamipide showed an antinociceptive property and attenuated cartilage degeneration. Rebamipide reduced the expression of MMP-13, IL-1β, HIF-2α, iNOS, and nitrotyrosine in OA cartilage in a dose-dependent manner. Nitrotyrosine expression in the subchondral bone region was decreased in the rebamipide-treated joints. mRNA expression of MMP-1, -3, and -13, and ADAMTS5 was attenuated in IL-1β-stimulated human OA chondrocytes. By contrast, rebamipide induced the mRNA expression of tissue inhibitor of metalloproteinase-1 and -3. The results show the inhibitory effects of rebamipide on pain production and cartilage degeneration in experimentally induced OA. The suppression of oxidative damage and the restoration of extracellular matrix homeostasis of articular chondrocyte suggest that rebamipide is a potential therapeutic strategy for OA.

Chronic administration of hexarelin attenuates cardiac fibrosis in the spontaneously hypertensive rat

Cardiac fibrosis is a hallmark of heart disease and plays a vital role in cardiac remodeling during heart diseases, including hypertensive heart disease. Hexarelin is one of a series of synthetic growth hormone secretagogues (GHSs) possessing a variety of cardiovascular effects via action on GHS receptors (GHS-Rs). However, the role of hexarelin in cardiac fibrosis in vivo has not yet been investigated. In the present study, spontaneously hypertensive rats (SHRs) were treated with hexarelin alone or in combination with a GHS-R antagonist for 5 wk from an age of 16 wk. Hexarelin treatment significantly reduced cardiac fibrosis in SHRs by decreasing interstitial and perivascular myocardial collagen deposition and myocardial hydroxyproline content and reducing mRNA and protein expression of collagen I and III in SHR hearts. Hexarelin treatment also increased matrix metalloproteinase (MMP)-2 and MMP-9 activities and decreased myocardial mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 in SHRs. In addition, hexarelin treatment significantly attenuated left ventricular (LV) hypertrophy, LV diastolic dysfunction, and high blood pressure in SHRs. The effect of hexarelin on cardiac fibrosis, blood pressure, and cardiac function was mediated by its receptor, GHS-R, since a selective GHS-R antagonist abolished these effects and expression of GHS-Rs was upregulated by hexarelin treatment. In summary, our data demonstrate that hexarelin reduces cardiac fibrosis in SHRs, perhaps by decreasing collagen synthesis and accelerating collagen degradation via regulation of MMPs/TIMP. Hexarelin-reduced systolic blood pressure may also contribute to this reduced cardiac fibrosis in SHRs. The present findings provided novel insights and underscore the therapeutic potential of hexarelin as an antifibrotic agent for the treatment of cardiac fibrosis.

Therapeutic potential of BaekJeol-tang in arthritis

Osteoarthritis (OA) is the most common form of arthritis, and is a public health problem throughout the world. It remains the major cause of disability in the elderly, affecting about 60% of men and 70% of women above the age of 65. Even though there are many medicines for treatment of OA throughout the world, therapeutic potential of traditional Korean medicine (TKM) is not well known. Here we discussed the therapeutic potential of BaekJeol-tang (BJT, a prescription of TKM). Celecoxib is approved in one or more countries worldwide for the relief of the signs and symptoms of OA, rheumatoid arthritis, and juvenile rheumatoid arthritis. Although celecoxib inhibited mRNA expression of matrix metalloproteinase (MMP)-3 and increased mRNA expression of tissue inhibitors of metalloproteinase (TIMP)-1, celecoxib did not significantly inhibit IL-1α-induced release of glycosaminoglycan (GAG) and only slightly reduced type II collagen. On the other hand, BJT significantly inhibited the release of GAG and type II collagen as well as regulated the mRNA expression of MMP-3 and TIMP-1. Through this article, we have tried to provide some therapeutic potential of BJT in arthritis.

Protective Effect of Sinomenine on Cartilage Degradation and Chondrocytes Apoptosis

Sinomenine (SIN), an alkaloid extracted from the stem of the Chinese medicinal plant sinomenium acutum, has been used for treating rheumatoid arthritis. But little is known whether SIN has a protective effect on osteoarthritis (OA). In this study, we investigated the protective effect of SIN on IL-1beta-induced proteoglycan degradation and apoptosis in rabbit articular cartilage and chondrocytes. Treatment with 10 ng/ml IL-1beta increased the level of glycosaminoglycan (GAG) released into the culture media, and up-regulated the activity and mRNA expression of matrix metalloproteinase 13 (MMP-13) and down-regulated the activity and mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in cartilage explants, as confirmed by the methods of GAG quantitation, MMP-13/TIMP-1 enzyme-linked immunosorbent assay (ELISA) and real-time quantitative RT-PCR. Treatment with 10 ng/ml IL-1beta resulted in marked apoptosis in chondrocytes, as demonstrated by decreased cell viability, occurrence of DNA laddering and increased caspase-3 activity and annexin V binding of phosphatidylserine. However, simultaneous treatment with SIN (10, 50 or 250 microM) inhibited the GAG release and the activity and mRNA expression of MMP-13, and enhanced the activity and mRNA expression of TIMP-1 in a dose-dependent manner in cartilage explants. Furthermore, DNA fragment, caspase-3 activity and apoptosis rate were down-regulated, and cell viability was up-regulated dose-dependently in chondrocytes. Thus, SIN has the protective capacity to antagonize cartilage degradation and chondrocyte apoptosis, which suggest that SIN may act as an agent for pharmacological intervention in the progress of OA.

Preventive effects of a fractioned polysaccharide from a traditional Chinese herbal medical formula (Yu Ping Feng San) on carbon tetrachloride-induced hepatic fibrosis

The study was to investigate the prevention effects and possible mechanism of Yu Ping Feng San fractioned polysaccharide (YPF-P) on CCl(4)-induced liver fibrosis in rats. YPF-P was prepared from root of Astragalus membranaceus, rhizome of Atractylodes macrocephaia and root of Raidix saposhnikoviae, and compared with polysaccharide from root of Astragalus membranaceus (AP). Hepatic fibrosis was induced by subcutaneous injection with carbon tetrachloride twice weekly for 12 weeks in Sprague-Dawley rats. YPF-P, AP and colchicine were administered intragastrically daily to carbon tetrachloride-treated rats. Histopathological changes of the liver and hepatic stellate cells were evaluated by Masson staining and transmission electron microscopy, respectively. Markers of fibrosis were determined by radioimmunoassay, biochemistry assay and ELISA. The mRNA expressions of tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-13 (MMP-13), procollagen I and collagen III were detected by RT-PCR. YPF-P dose-dependently alleviated the degree of liver fibrosis and inhibited hepatic stellate cell transformation into myofibroblast-like cells, markedly reduced the elevated levels of hyaluronic acid, laminin, type IV collagen, type III procollagen, hydroxyproline and transforming growth factor beta-1, suppressed procollagen I, collagen III and TIMP-1 expression, and improved the TIMP-1/MMP-13 ratio. MMP-13 expression was only promoted moderately by YPF-P. Compared with AP, YPF-P showed more potency on most markers except laminin, type IV collagen and MMP-13 mRNA. YPF-P prevented the progress of rat liver fibrosis induced by carbon tetrachloride and had a more potent preventative effect. The preventative effect may be associated with the ability of YPF-P to inhibit the synthesis of matrix collagen and balance the TIMP/MMP system.

Abstract 2341: The Subtilisin-like proprotein convertases blockade inhibits the invasiveness of human primary melanoma with altered P53, CDKN2A and N-Ras genes

Abstract Altered tumor suppressor p53 and/or CDKN2A as well as the Ras genes are frequently found in primary and metastatic melanomas. These alterations are responsible for the invasive/metastatic potential acquisition through their defective regulatory control on various MMPs and urokinase genes. Using primary human melanoma M10 cells with altered P53, CDKN2A and N-Ras genes, we found that inhibition of the proprotein convertases (PCs), enzymes involved in the proteolytic activation and/or expression of various cancer-related protein precursors reduced significantly their invasiveness. Analysis of M10 cells and their gastric and ganglion derived metastatic lesions revealed the presence of all the PCs found in the secretory pathway namely Furin, PACE4, PC5 and PC7. Expression of the general PCs inhibitor α1-PDX in these cells in a stable manner (M10/PDX) had no effect on the mRNA expression levels of these PCs. In vitro digestion assays and cell transfection experiments, revealed that M10/PDX cells present reduced PCs activity and are unable to process the PCs substrates proIGF-1R and proPDGF-A. These cells showed reduced migration, and invasion of Matrigel in vitro that paralleled decreased gelatinase MMP-2 activity and increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2. Furthermore, these cells showed decreased mRNA levels of urokinase-type plasminogen activator receptor (uPAR) and increased mRNA levels of plasminogen activator inhibitor-1 (PAI-1). Taken together, these data suggest that PCs activity inhibition has the ability to interfere with primary human melanoma cells invasiveness despite their altered P53, CDKN2A and N-Ras genes, suggesting the potential use of PCs as new therapeutic targets in melanoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2341.

Therapeutic efficacy of Traditional Chinese Medicine 319 recipe on hepatic fibrosis induced by carbon tetrachloride in rats

Background/aimsHepatic fibrosis is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. TCM 319 recipe is a Chinese Medicine formula which consists of six Chinese herbs. In this study, we investigated the anti-fibrotic efficacy and mechanisms of TCM 319 recipe. MethodsHepatic fibrosis in rats was induced by carbon tetrachloride (CCl4). 34 male adult SD rats were allocated into five groups (group 1—concomitant CCl4 and TCM 319 recipe for 8 weeks; group 2—CCl4 for 4 weeks and then CCl4 and TCM 319 recipe for 4 weeks; group 3—CCl4 alone for 8 weeks; group 4—TCM 319 recipe only for 8 weeks; group 5—untreated controls). After 8 weeks of treatment, serum ALT assay, liver tissue histological examination and immunostaining were carried out to examine the liver function and fibrosis degree. The expression levels of platelet derived growth factor (PDGF-B), PDGF-Rβ, and transforming growth factor-beta 1 (TGF-β1) were measured by quantitative RT-PCR and western blot. ResultsTCM 319 recipe reduced liver injury and attenuated hepatic fibrosis in group 1 compared with that in group 3. TCM 319 recipe suppressed the mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1). In addition, treatment with TCM 319 recipe significantly down-regulated mRNA expression of PDGF-B and PDGF-Rβ, and it also suppressed protein expression of PDGF-Rβ and TGF-β1. ConclusionsTCM 319 recipe extracts could attenuate hepatic fibrosis induced by CCl4 in rats. The anti-fibrotic effect of TCM 319 recipe is associated with the down-regulation of mRNA expression of TIMP-1, PDGF-B and PDGF-Rβ, and with the suppression of protein expression of PDGF-Rβ and TGF-β1.