Bisphosphonates have been reported to have chondroprotective activities in addition to its original functions. However, mechanisms for these just began to be elucidated. Under the hypothesis that bisphosphonates may regulate expression and activities of matrix enzymes during degradation of cartilage for bone formation, we administrated an alendronate (1 mg/kg) to newborn rats subcutaneously once a day for 4, 7, and 10 days. To identify the effects of alendronate on cartilage, thickness of cartilage layer was measured by histomorphometry on the proximal epiphysis of tibia. Immunofluorescence staining and RT-PCR were performed to investigate the expressions of matrix enzymes in both in vitro and in vivo. MTS assay revealed that at 10(-3) M in concentration, alendronate significantly reduced viability of chondrocytes. The mRNA expressions of MMP-1, MMP-9, EMMPRIN, and TIMP-3 in primary chondrocytes were decreased by the alendronate treatment. Interestingly, TIMP-1 mRNA expression was significantly increased, whereas a constitutive form, TIMP-2 was relatively unchanged by the treatment. The thickness of proliferating layer at postnatal day 7 was not significantly different, whereas thickness of hypertrophied layer was significantly thicker in the alendronate group than in the control (p<0.01). Immunofluorescence demonstrated that the expressions of MMP-9, TIMP-2 and -3 were reduced, whereas TIMP-1 expression was increased by the alendronate administration. These results suggest that the alendronate have chondroprotective properties by down-regulation of MMPs and up-regulation of TIMPs during endochondral ossification.
Alendronate ; Animals ; Antigens, CD147 ; Cartilage ; Chondrocytes ; Diphosphonates ; Down-Regulation ; Epiphyses ; Fluorescent Antibody Technique ; Humans ; Infant, Newborn ; Matrix Metalloproteinases ; Osteogenesis ; Rats ; RNA, Messenger ; Tibia ; Tissue Inhibitor of Metalloproteinase-1 ; Tissue Inhibitor of Metalloproteinase-2 ; Tissue Inhibitor of Metalloproteinase-3 ; Up-Regulation

OBJECTIVETo explore the curative mechanism of acupuncture treatment on osteoarthritis (OA).

METHODSForty cases of female SD rats were randomly divided into a normal group, a model group, an acupuncture group and a medication group, 10 cases in each group. OA animal model was established by using the method of heel tendon resection for unilateral hind limb. The acupuncture group was treated with electroacupuncture at "Xiqian"(ST 35) and "Housanli"(ST 36), and the medication group with inunction of Diclofenac cream, and the normal group and the model group without any treatment. The expression of matrix metalloproteinase-1, 3 (MMP-1, MMP-3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the cartilage were observed by immunohistochemistry.

RESULTSThere were significant differences among four groups. The expressions of MMP-1, MMP-3 and TIMP-1 in the model, acupuncture and medication groups were all significantly stronger than those in the normal group (all P < 0.01). The expressions of MMP-1 and MMP-3 in the acupuncture and medication groups were down regulated and TIMP-1 expression up-regulated with significant differences as compared with the model group (all P < 0.01), and the expressions of MMP-1 and MMP-3 in acupuncture group were significantly lower, while TIMP-1 expression significantly higher than that in the medication group (all P < 0.01).

CONCLUSIONAcupuncture can down-regulate the expression of MMP-1 and MMP-3 and up-regulate the expression of TIMP1, which is superior to that of Diclofenac cream, showing that acupuncture has a certain protective effect on cartilage from OA.

Acupuncture Therapy ; Animals ; Cartilage ; chemistry ; Female ; Immunohistochemistry ; Matrix Metalloproteinase 1 ; analysis ; Matrix Metalloproteinase 3 ; analysis ; Osteoarthritis, Knee ; metabolism ; therapy ; Random Allocation ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; analysis

OBJECTIVETo evaluate the effects of treadmill running exercise of different intensity on early repair of full-thickness defects on the patellofemoral articular surface and the changes in the expressions of matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in SD rats.

METHODSTwenty-four male SD rats with full-thickness defects on the patellofemoral articular surface were randomly assigned into sedentary control (SED) group and low-, moderate- and high-intensity running groups (LIR, MIR, and HIR groups, respectively). The running groups were trained on treadmill for 6 consecutive weeks. Blood samples were collected to detect serum MMP-3 and TIMP-1 levels using ELISA before and after the experiment, and the femoral trochlea were collected to assess tissue repair by gross appearance scoring and O Driscoll histological scoring with Safranine O-Fast Green staining and Toluidine blue staining.

RESULTSIn rats in SED group, the defect was filled with hyaline articular cartilage-like tissues, as compared to fibrous tissues in LIR and MIR groups and subchondral bone damage in HIR group. The SED group scored the highest and HIR group the lowest among the 4 groups in gross appearance scoring and O Driscoll histological scoring. No significant differences were found in MMP-3 or TIMP-1 levels among the groups before training (P>0.05), but after 6 weeks of training, serum MMP-3 and TIMP-1 levels differed significantly among the 4 groups (P<0.05), and all the 3 running groups had a significantly higher MMP-3 level than the control group (P<0.05). After the 6-week training, TIMP-1/MMP-3 ratio was significantly higher in SED group than in the 3 running groups, and was the lowest in HIR group.

CONCLUSIONBoth low- and moderate-intensity exercise failed to promote resurfacing of full-thickness cartilage defects on the patellofemoral articular surface in rats, and high-intensity exercise even induces subchondral bone damage. The expression of MMP-3 and TIMP-1 is related to exercise, and the TIMP-1/MMP-3 ratio reflects the extent of tissue repair.

Animals ; Cartilage, Articular ; metabolism ; pathology ; Male ; Matrix Metalloproteinase 1 ; metabolism ; Matrix Metalloproteinase 3 ; metabolism ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Wound Healing

PURPOSE: The purpose of this study was to analyze the expression of inducible nitric oxide synthases (iNOS), tissue inhibitors of metalloproteinase (TIMP)-3, and TIMP-4 in the gingival tissues of periodontal patients with or without type 2 diabetes mellitus (DM). METHODS: Depending on the patient's systemic condition and clinical criteria of the gingiva, each gingival sample was classified into one of three groups. Sixteen clinically, systemically healthy patients (group 1), 16 periodontal patients (group 2), and 16 periodontal patients with DM (group 3) were included. Tissue samples in each group were collected, prepared, and analyzed by western blotting. Quantification of the relative amount of TIMP-3, TIMP-4, and iNOS was performed. RESULTS: The expression levels of iNOS and TIMP-3 both increased in group 1, group 2, and group 3 in increasing order, and were significantly higher in both group 2 and group 3 as compared to group 1 (P<0.05). The expression levels of TIMP-4 increased in the same order, but significantly increased in group 2 as compared to group 1, in group 3 as compared to group 1, and group 3 as compared to group 2 (P<0.05). CONCLUSIONS: This study demonstrated that iNOS, TIMP-3, and TIMP-4 might be involved in the progression of periodontal inflammation associated with type 2 DM. It is thought that further study of these factors can be applied practically for the diagnosis and control of periodontitis in diabetics.
Blotting, Western ; Chronic Periodontitis ; Diabetes Mellitus ; Diabetes Mellitus, Type 2 ; Gingiva ; Humans ; Inflammation ; Nitric Oxide ; Nitric Oxide Synthase ; Periodontitis ; Tissue Inhibitor of Metalloproteinase-3 ; Tissue Inhibitor of Metalloproteinases

OBJECTIVETo explore the influence of tumor-associated macrophages(TAMs) on the ability of invasion and metastasis of gastric cancer cells, and its associated mechanism.

METHODSImmunohistochemistry was used to examine the expression of TAM in 10 samples of normal gastric mucosa and 15 samples of gastric cancer tissues from sample bank of Department of Pathology, Union Hospital. Phorbol 12-myristate 13-acetate(PMA) and macrophage colony stimulating factor (M-CSF) were used to make THP-1 monocytes differentiate into TAMs. AGS gastric cancer cells were divided into two groups: experiment group was cultured with RPMI/1640 condition medium containing 50% TAM and control group was cultured with RPMI/1640 complete medium. The ability of invasion and metastasis of gastric cancer cells was measured by Transwell assays. Real-time PCR and Western blot were applied to detect the expression of MMPs and its inhibitor TIMPs before and after stimulation of TAMs.

RESULTSImmunohistochemistry results showed that CD68(+) cell number in normal gastric mucosa tissue was significantly less than that in gastric cancer tissue [(11.3±0.8)/HP vs. (31.6±1.4)/HP, P<0.000 1]. When treated with PMA and M-CSF, THP-1 cells were differentiated into type M2 TAMs with high expression of specific markers CD68, CD163, CD204 and CD206. Transwell test revealed that the number of piercing cells in the experimental group was significantly more than that in control group [(36.8±1.1)/HP vs. (12.8±0.9)/HP, t=17.5, P=0.000). Compared to control group, the expression of MMP-2, MMP-9 mRNA in experimental group respectively increased by 1.61 and 1.87 folds(P=0.017 and P=0.009). Protein level of MMP-2, MMP-9 was up-regulated accordingly. The expression of TIMP-1 and TIMP-3 mRNA was not significantly different between two groups(P=0.120 and P=0.096).

CONCLUSIONSTAMs may promote the invasion and metastasis of gastric cancer cells through increasing expression of MMP-9 and MMP-2, which may be one of the mechanisms of gastric cancer development.

Cell Line, Tumor ; Humans ; Immunohistochemistry ; Macrophage Colony-Stimulating Factor ; Macrophages ; Matrix Metalloproteinase 9 ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Real-Time Polymerase Chain Reaction ; Stomach Neoplasms ; Tissue Inhibitor of Metalloproteinase-1 ; Tissue Inhibitor of Metalloproteinase-3

OBJECTIVES: The effects of superovulation on the expression of mRNA and protein of TIMP-3 and MMP-9 in murine endometrium were assessed. METHODS: Using murine pregnant uteri of gestation day (g.d.) 4, 5 and 6 after injection of PMSG 5 and 10 IU, the effects of superovulation were assessed and compared with those of natural pregnancy and pseudopregnancy groups using quantitative competitive RT-PCR and immunohistochemical staining. RESULTS: Expression of TIMP-3 mRNA and protein showed an increase in PMSG groups and pseudopregnancy group, while there was no difference in MMP-9 expression between natural pregnancy and PMSG, pseudopregnancy groups on g.d. 4 through g.d. 6. CONCLUSIONS: This study suggests that ovarian hyperstimulation by gonadotropin, which produces many oocytes and embryos, may have a detrimental effect on embryonic implantation and its relevant endometrial remodeling process by increase in expression of TIMP-3 in murine endometrium.
Animals ; Embryonic Structures ; Endometrium* ; Female ; Gonadotropins ; Mice ; Oocytes ; Pregnancy ; Pseudopregnancy ; RNA, Messenger ; Superovulation* ; Tissue Inhibitor of Metalloproteinase-3 ; Uterus

OBJECTIVETo observe the expression patterns of matrix metalloproteinase-3 and its inhibitor, tissue inhibitor metalloproteinases (TIMP)-1, in remodeling phase of mandibular distraction osteogenesis.

METHODSBilateral mandibular osteotomies were performed in 8 mature rabbits. The mandibles were lengthened 6 mm using a custom-made distractor with a rate of 1 mm/d. All animals were killed in 4 weeks after completion of distraction. The distracted calluses were harvested and processed for histology and immunohistochemistry of MMP-3 and TIMP-1.

RESULTSElevated expression of both MMP-3 and TIMP-1 was found in the distracted calluses resulting from mandibular lengthening. Positive staining for MMP-3 was seen in the osteoblasts and osteocytes, and TIMP-1 was mainly localized in osteoblasts.

CONCLUSIONMMP-3 and TIMP-1 appear to play important roles in the remodeling of new bone created by mandibular distraction osteogenesis.

Animals ; Bone Regeneration ; Bone Remodeling ; Female ; Male ; Mandible ; cytology ; metabolism ; surgery ; Matrix Metalloproteinase 3 ; biosynthesis ; Osteoblasts ; metabolism ; Osteogenesis, Distraction ; Rabbits ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis

The aim of this study was to determine the raising anticancer effects of resveratrol (Res) on paclitaxel (PA) in non-small cell lung cancer (NSCLC) cell line A549. The 10 µg/ml of Res had no effect on human fetal lung fibroblast MRC-5 cells or on A549 cancer cells and the 5 or 10 µg/ml of PA also had no effect on MRC-5 normal cells. PA-L (5 µg/ml) and PA-H (10 µg/ml) had the growth inhibitory effects in NSCLC cell line A549, and Res increased these growth inhibitory effects. By flow cytometry experiment, after Res (5 µg/ml)+PA-H (10 µg/ml) treatment, the A549 cells showed the most apoptosic cells compared to other group treatments, and after additional treatment with Res, the apoptosic cells of both two PA concentrations were raised. Res+PA could reduce the mRNA and protein expressions of COX-2, and Res+PA could reduce the COX-2 related genes of VEGF, MMP-1, MMP-2, MMP-9, NF-κB, Bcl-2, Bcl-xL, procollagen I, collagen I, collagen III and CTGF, TNF-α, IL-1β, iNOS and raise the TIMP-1, TIMP-2, TIMP-3, IκB-α, p53, p21, caspase-3, caspase-8, caspase-9, Bax genes compared to the control cells and the PA treated cells. From these results, it can be suggested that Res could raise the anticancer effects of PA in A549 cells, thus Res might be used as a good sensitizing agent for PA.
Carcinoma, Non-Small-Cell Lung ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cell Line* ; Collagen ; Fibroblasts ; Flow Cytometry ; Humans ; In Vitro Techniques* ; Lung ; Paclitaxel* ; Procollagen ; RNA, Messenger ; Tissue Inhibitor of Metalloproteinase-1 ; Tissue Inhibitor of Metalloproteinase-2 ; Tissue Inhibitor of Metalloproteinase-3 ; Vascular Endothelial Growth Factor A

OBJECTIVES: To determine the level of mRNA expression of various members of the matrix metalloproteinase and tissue inhibitors in uterine leiomyoma compared with unaffected myometrium. Materials & Method: 30 cases of portions of leiomyoma and myometrium were collected immediately followimg hysterectomy. Thirteen cases were from proliferative phase and seventeen were from secretory phase of menstrual cycle. The mean age was 43.7years old. The level of expression of mRNAs of interstitial collagenase, gelatinase, stromelysin, TIMP-1,-2,-3 was determined by reverse transcriptase-polymerase chain reaction(RT-PCR) and normalized to GAPDH(glyceraldehyde-3-phosphate dehydrogenase) mRNA. RESULTS: Myometrium and leiomyoma expressed all the members of above mentioned matrix metalloproteinase family and tissue inhibitors. Leiomyoma expressed a significantly higher level of stromelysin-3 during secretory phase, an extremely lower level of 92kDa gelatinase and a significantly lower level of TIMP-3. The immunohistochemical localization of TIMP-3 was smooth muscle cell and arteriole wall of myometrium and leiomyoma. CONCLUSIONS: The increased expression of stromelysin-3 in uterine leiomyoma compared with myometrium suggests that this MMP may be involved in the formation of a more fibrous extracellular matrix in leiomyoma. The extremely lower expression of 92kDa gelatinase of leiomyoma means that leiomyoma do not invade myometrium and forms a separated mass. Decreased expression of TIMP-3 of leiomyoma suggests that TIMP-3 is required for differentiation and homeostasis of extracellular matrix of normal myometrium and function as a suppressive role of tumor development
Animals ; Arterioles ; Extracellular Matrix ; Female ; Gelatinases ; Homeostasis ; Humans ; Hysterectomy ; Leiomyoma* ; Matrix Metalloproteinase 1 ; Matrix Metalloproteinase 3 ; Menstrual Cycle ; Mice ; Myocytes, Smooth Muscle ; Myometrium* ; RNA, Messenger ; Tissue Inhibitor of Metalloproteinase-3

OBJECTIVETo investigate the effect of the active components from Red Paeonia and Rhizoma chuanxiong (in Xiongshao Capsule, XSC) on matrix metalloproteinases (MMPs) in rabbit model of atherosclerosis.

METHODSFifty New Zealand rabbits were randomly divided into the normal control group (A), the model control group (B), the Simvastatin treated group (C), the low-dose XSC treated group (D) and the high-dose XSC treated group (E), 10 in each group. Rabbits in the normal control group were fed with normal diet, while those in the other four groups were fed with high fat diet and duplicated after two weeks feeding into model of abdominal aortic atherosclerosis by balloon angioplasty. In the 6 successive weeks feeding of high fat diet, Simvastatin 2.5 mg/kg, XSC 0.24 g/kg and 0.48 g/kg per day was given respectively to the rabbits in the three treated groups. Blood sample was collected for determining the level of blood lipids; serum MMP-3 and MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) with enzyme-linked immunoassay; and the protein expression of MMP-3 and cluster of differentiation antigen 40 ligand (CD40L) in plaque were detected with immunohistochemical method.

RESULTSCompared with Group B, the serum levels of MMP-3 and MMP-9; the expression of MMP-3 and CD40L in plaque; and the blood content of total cholesterol in the three treated groups were significantly lower (P < 0.05 or P < 0.01). Besides, the content of triglyceride and low-density lipoprotein cholesterol were significantly reduced in Group C, while the TIMP-1 showed no statistical difference among different groups (P > 0.05).

CONCLUSIONThe active components of Red Paeonia and Rhizoma chuanxiong play a definite role in stabilizing the atherosclerotic plaque in rabbits, one of their possible mechanisms may be by way of inhibiting the expressions of MMP-3, MMP-9 in vascular walls and blood serum.

Animals ; Atherosclerosis ; drug therapy ; enzymology ; Drugs, Chinese Herbal ; pharmacology ; Male ; Matrix Metalloproteinase 3 ; blood ; Matrix Metalloproteinase 9 ; blood ; Paeonia ; chemistry ; Phytotherapy ; Rabbits ; Random Allocation ; Tissue Inhibitor of Metalloproteinase-1 ; blood