MMP-9 Research Articles

Effects of dl-3n-butylphthalide on study and memory abilities and hippocampus expression of MMP-9 in rat model of radiation injuries of brain

Objective To study the effects of dl-3n-butylphthalide on study and memory abilities and hippocampus expression of MMP-9 in rat model of radiation injuries of brain. Methods Wistar rats were divided into radiation group radiated by linear accelerator and control group . Morris water test were taken to study the learning and memory ability of rats in each group before irradiation and 120 d after irradiation. Results Morris water maze test results showed that: in the academic test, compared with model group, high-dose butylphthalide group and low dose butylphthalide group had significantly shorter escape latency((79.61±7.17)s, (43.64±4.72)s, (42.15±4.19)s) and decreased number of errors(36.43±6.59, 14.58±4.22, 13.66±3.91)(P<0.01). In the memory test, compared with model group, high-dose butylphthalide group and low dose butylphthalide group had significantly longer the former platform quadrant time respectively (30.09±3.68)s, (31.25±3.17)s vs (17.57±4.29)s and faster swimming speed respectively(P<0.01). Conclusion Butylphthalide can significantly reduce hippocampal neurons' expression of MMP-9, which indicates that butylphthalide has curative effect on radiation brain injuries by regulating the expression of MMD-9. Key words: Butylphthalide; Radiation brain injuries; Learning and memory; Rat

Research progress of matrix metalloproteinases in endometrial carcinoma metastasis

Tumor cell metastasis is a process of extracellular matrix hydrolyzed by protease, where tumor cells traverse the defect in the extracellular matrix into the lymphatic system and the capillaries to form new metastasis hematogenously. Matrix metalloproteinases (MMPs) are a kind of enzymes closely related to the metastasis. MMP, especially the relationship between MMP-9 and endometrial cancer metastasis in this paper will be summarized to get a better understanding about it. Key words: Endometrial cancer; Matrix metalloproteinases; Metastasis

Mechanisms of astrocyte elevated gene-1 promoting cancer metastasis through regulation of the micro-RNA-885-5p/ matrix metalloproteinase 9 signaling pathway in liver cancer

Objective To investigate the relationship between astrocyte elevated gene-1 (AEG-1) and microRNA-885-5p, observe the influence of microRNA-885-5p on MHCC-97H migration and invasion, identify the target gene of microRNA-885-5p and uncover the mechanisms of AEG-1 promoting cancer metastasis. Methods The experimental study was adopted. The AEG-1 over-expressed plasmid constructed and the siRNA of silent AEG-1 synthesized were transiently transfected into MHCC-97H cells, respectively. For plasmid trans-fection, MHCC-97H cells tranfected with empty Vector NC were divided into group A as the control, and MHCC-97H cells tranfected with AEG-1 over-expressed plasmid were divided into group B. For AEG-1 siRNA transfection, MHCC-97H cells tranfected with negative siRNA were divided into group C as the control, and MHCC-97H cells tranfected with AEG-1 siRNA were divided into group D. The relative expressions of microRNA-885-5p in MHCC-97H cells of group A, B, C, D were measured by fluorescent quantitative polymerase chain reaction(PCR). For microRNA-885-5p simulator transfection, MHCC-97H cells tranfected with negative microRNA simulator were divided into group E as the control, and tranfected with microRNA-885-5p simulator were divided into group F. Transwell assay was used to evaluate MHCC-97H migration and invasion. Targetscan software was used to predict the target gene of microRNA-885-5p. MHCC-97H cells co-transfected with reporter plasmid of target gene and negative control siRNA were divided into group G as the control, and MHCC-97H cells co-transfected with reporter plasmid of target gene and microRNA-885-5p simulator were divided into group H. MHCC-97H cells co-transfected with reporter plasmid of target gene of binding region mutation and negative control siRNA were divided into group I as the control, MHCC-97H cells co-transfected with reporter plasmid of target gene of binding region mutation and microRNA-885-5p simulator were divided into group J. The luciferase activities of MHCC-97H cells in group G, H, I, J were tested. MHCC-97H cells transfected with negative control siRNA were divided into group K, and tranfected with microRNA-885-5p simulator were divided into group L. Western blot test was used to detect the relative expression of target gene of microRNA-885-5p. MHCC-97H cells transfected with siRNA of target gene of microRNA-885-5p were divided into group M, and transfected with negative control siRNA were divided into group N. Transwell assay was used to evaluate MHCC-97H migration and invasion in group M and group N. Measurement data with normal distribution were presented as ±s and comparison between groups was done using the t test. Results The relative expressions of microRNA-885-5p in MHCC-97H cells of group A and group B were (10.68±1.32)×10-4 and (5.02± 0.20)×10-4, respectively, showing significant difference between the 2 groups (t=7.357, P 0.05) . The relative expressions of MMP9 in MHCC-97H cells of group K and group L were 0.75±0.03 and 0.25±0.03, respectively, showing significant difference between the 2 groups (t=19.086, P<0.05). The results of MHCC-97H migration and invasion showed that the numbers of MHCC-97H cells in lower chambers of transwell plate were 1 210±163 and 1 192±170 in group M, 537±16 and 374±55 in group N, showing significant differences between the 2 groups (t=7.111, 7.916, P<0.05). Conclusions AEG-1 downregulates the expression of microRNA-885-5p, the target gene of which is MMP9. MicroRNA-885-5p inhabits the migration and invasion of hepatoma cells by negative regulation of MMP9. Key words: Liver neoplasms; Metastases; Astrocyte elevated gene-1; MicroRNA-885-5p; Matrix metalloproteinase 9

Pathogenesis and treatment of overwhelming inflammation induced by Pseudomonas aeruginosa keratitis in rabbit

Background Infectious keratitis is common blinding eye disease in China. Inflammatory runaway reaction often occurs in infectious keratitis, and its mechanism and treating approach are worthy of research. Objective This study was to investigate the clinical manifestation, mechanism and treatment outcome of doxycycline in overwhelming inflammation induced by Pseudomonas aeruginosa keratitis. Methods Pseudomonas aeruginosa suspension was intrastromal injected in 60 right eyes of 60 New Zealand white rabbits, and gatifloxacin eye drops was frequent instilled for consecutive 3 days to establish corneal inflammatory runaway reaction models, and 42 eyes with worse keratitis were defined as overwhelming inflammation. Then the models were randomly divided into gatifloxacin treatment group (15 rabbits), combined treatment group (gatifloxacin with doxycycline treatment, 15 rabbits) and balance salt solution (BSS) control group (12 rabbits), and corresponding eye drops was topically administered in the rabbits 8 times per day for 14 days. The symptoms of the models were observed under the slit lamp microscope on day 1, 3, 7 and 14 after treatment, and the corneal infiltration area was calculated. The corneal morphology was examined by using hematoxylin-eosin staining. The expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the corneal tissue were examined by immunohistochemistry. The contents of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in corneal homogenate were detected by ELISA. The use and care of the animals complied with the ARVO Statement. Results The inflammatory scores were lower and infiltration area of cornea was smaller 7 days after treatment than those before treatment in the combined treatment group and gatifloxacin treatment group (both at P<0.05). Hematoxylin-eosin staining result showed that the inflammation response of cornea subsided 14 days in comparison with 5 days after treatment in the combined treatment group, with the proliferation and rearrangement of fiberal tissue. The MMP-2 and MMP-9 were strongly expressed in corneal tissue with overwhelming inflammation in day 5 after treatment, however, the expression intensity weakened in 14 days after treatment in the combined treatment, showing significant differences in the absorbance between them (all at P<0.05). ELISA assay showed that the contents of TNF-α and IL-1β in corneal tissue were significantly lower in the combined treatment group than those in the BSS group (P=0.00, 0.03). In addition, contents of TNF-α and IL-1β in corneal tissue were significantly declined at day 14 in comparison with day 5 and day 7 after treatment in the combined treatment group (all at P<0.05). Conclusions Overwhelming inflammatory keratitis models can be successfully established by corneal intrastromal injection of Pseudomonas aeruginosa suspension and immediately frequent 3-day instillation of gatifloxacin eye drops in rabbits. The topical application of doxycycline plays treating effect on overwhelming inflammatory keratitis by inhibiting the release of pro-inflammatory cytokines and MMPs. Key words: [Key words] Infectious eye disease, corneal; Bacteria; Pseudomonas aeruginosa; Antibacterial agents; Overwhelming inflammation; Keratitis/drug therapy; Doxycycline; Disease models, animal; Rabbits

Hepatic matrix metalloproteinase-10 exerts a hepatoprotective role after acute liver injury

After injuries that lead to a loss of liver tissue a regenerative and reparative response is performed in order to restore an adequate hepatic mass. The remodeling of the extracellular matrix, accompanies the liver regeneration and when the reparative reaction goes awry in the setting of chronic liver injury, could be involved in the carcinogenic process (1,2). Following the damage, a provisional matrix is deposed, intended to be successively replaced, which has the function of stabilizing the lesional area and constitutes a support for guiding regenerating cells. Matrix metalloproteinases are increasingly recognized as important modulators of the matrix remodeling process. Matrix metalloproteinase-10 (MMP-10) has been implicated in the reparative process in other organs and has effects on the plasminogen system, which plays a fundamental role in liver repair (3). The hepatic expression of MMP10 in animal models of acute liver injury was tested in order to investigate the role of MMP-10 in liver repair and regeneration. The liver regeneration after two thirds partial hepatectomy (PH) and bile duct ligation (BDL) models were examined. Hepatic MMP-10 expression, analyzed by immunohistochemistry, western blot and qPCR showed a rise early after injury. In the MMP10-deficient mice a diminished and delayed resolution of necrotic lesions, enhanced fibrogenesis and a fibrinogen/fibrin and fibronectin compromised turnover were observed. These findings showed that the MMP10 expression plays a role in the hepatic wound healing response probably through its profibrinolytic activity.

PAR1 activation induces the release by Schwann cells of factors promoting cell survival and neuritogenesis

Schwann cells (SCs) regulate a wide variety of axonal functions in the peripheral nervous system, providing a supportive growth environment following nerve injury (1). Here we show that rat SCs express the protease-activated receptor-1 (PAR1) both in vivo and in vitro. PAR1 is a G-protein coupled receptor eliciting cellular responses to thrombin and other proteases (2). To investigate if PAR1 activation affects the neurotrophic properties of SCs, this receptor was activated by a specific agonist peptide (TFLLR) and the conditioned medium was transferred to PC12 pheocromocytoma cells for assessing cell survival and neurite outgrowth. Culture medium from SCs treated with 10 µM TFLLR reduced significantly the release of LDH and increased the viability of PC12 cells with respect to the medium of the untreated SCs. Furthermore, conditioned medium from TFLLR-treated SCs increased neurite outgrowth on PC12 cells respect to control medium from untreated cells. To identify putative neurotrophic candidates we performed proteomic analysis on SC secretoma and real time PCR experiments after PAR1 activation. Stimulation of SCs with TFLLR increased specifically the release of a subset of five proteins: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). At the same time there was a significant decrease in the level of three proteins: Complement C1r subcomponent (C1r), Complement component 1 Q subcomponent-binding protein (C1qbp) and Angiogenic factor with G patch and FHA domains 1 (Aggf1). These data indicate that PAR1 stimulation does induce the release by SCs of factors promoting cell survival and neuritogenesis. Among these proteins, Mif, Sdc, Dcn and Mmp2 are of particular interest.

Possible interactions between HDACs and TGF-beta/Smads pathway in Glioblastoma

Glioblastoma (GBM) is the most common and aggressive tumor of the Central Nervous System (CNS). Unfortunately, patients afflicted with this disease have a very poor prognosis, due to high level of invasiveness and resistance to standard therapies (1). Although the molecular profile of GBM has been extensively investigated, the events responsible for its pathogenesis and progression remain largely unknown. Reports have indicated that HDAC (Histone deacetylases) dependent epigenetic modifications (2) and the Tgfβ/Smad pathway (3) play roles in GBM tumorigenesis. The aim of this study was to evaluate the involvement and the possible interaction between these two molecular cascades in the pathogenesis, therapeutic responsiveness and prognosis of GBM. Immunohistochemestry (IHC) was performed on microdissected GBM samples, collected from 14 patients (n.8 men and n.6 women) ranging in age from 43 to 74 years. The patients were previously divided, on the basis of their overall survival (OS), into three groups: low, intermediate and high OS. Patients with poor prognosis showed hyperexpression of HDAC4 and HDAC6, an activation of the Tgfβ/Smad pathway, with high levels of IL-13, Smad2, PDGF and MMP3 expression, compared to the intermediate and high OS groups, whereas the expression of Smad7 was reduced. The high OS group also exhibits an increase in p21 immunostaining, which represents a common target of the two cascades. The IHC data was confirmed by Immunoblotting. Our results suggest that both HDAC4 and HDAC6 together with the Tgfβ/Smad pathway are involved in progression of GBM and could be a useful prognostic markers and may predict responsive to therapy.

In vitro comparison of new bisphosphonic acids and zoledronate effects on human gingival fibroblasts viability, inflammation and matrix turnover

Bisphosphonates (BPs) are well known clinically used drugs, commonly applied to treat osteoclast-mediated bone resorption. Some clinically used BPs were demonstrated to be able to inhibit the activity of matrix metalloproteinases (MMPs) (1), a protease family required to fully degrade all the components of the extracellular matrix during connective tissue remodelling (2). Combining the arylsulfonamide function with the bisphosphonic group, several compounds were synthesized to obtain selective inhibitors of MMPs. The aim of the present work is to compare the effects on cell adhesion, cytotoxicity, inflammatory response occurrence and matrix turnover process in an in vitro model of primary human gingival fibroblasts (HGFs) treated with newly synthesized sulfonamide BPs and with zoledronic acid (ZA), a clinically used drug. Western blot was used to measure Procollagen I, β1 integrin MMP-8 and MMP-9, phase contrast and MTT for cell viability, LDH was performed for toxicity evaluation, ELISA for Prostaglandin E2 (PGE2) secretion assessment. When compared with ZA, the treatment with the newly synthetized compounds shows increasing viability, Procollagen I expression and decreased expression of β1 integrin in HGFs. Higher levels of released LDH, PGE2 and MMP-9 expression are recorded in ZA-treated HGFs. Increased levels of MMP-8 are recorded in newly synthetized compounds-treated samples.These findings imply that new BPs could accelerate the physiological matrix turnover, they are more able to preserve the soft tissue surrounding bone as they have neither inflammatory effects nor toxicity, along with reduced effects on the cell viability, which are instead typical side effects of ZA administration. We can conclude that the newly synthesized compounds are better tolerated, leading to the hypothesis that their use leads to connective tissues side effects reduction compared to clinically used drugs, even though several studies are required to deeply investigate the signaling cascades involved in the mechanism of action of these new BPs.

Increased MG-63s invasion potential mediated by HFs

During a malignant transformation, the crosstalk between the stroma and the cancer cells is described as a growing network of physical and paracrine signals, and it seems to have a direct influence on the phenotypic, genetic and epigenetic changes that affect the cells (1). In order to invade and metastasize to distant tissues, cancer cells transform themselves via ECM, induce tumor angiogenesis as well as undergo proliferation, detachment, migration, and invasion through secretion of various tumor derived factors (2). In this study we decided to analyze morphological and molecular aspects due to the coexistence between tumor cells MG-63s and fibroblasts HFs, verifying in particular the ability of MG-63s of invasion and microenvironment modulation. Monolayers of co-cultured cells were morphologically analyzed in time-laps by HR-SEM microscopy and a trans-well migration assay was performed over 24 h, 48 h, 72 h, and 96 h. The expression of several proteins, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis (TNF alpha, IL-6, YKL-40, MMP-1, MMP-9, and VEGF) was validated by Western blotting analysis. The images in time-laps for HR - SEM showed that fibroblasts in contact with MG-63 lost their spatial orientation, while the MG-63 quickly reached the confluence advancing towards HF cells, invading their space and overlying them. The increased MG-63s invasion mediated by the coexistence with HFs was confirmed by invasion assays in transwell co-culture. The protein levels of TNF-alpha, IL-6, YKL-40 and VEGF confirmed that tumor cells can regulate the development of a “tumor-stroma” via the aberrant expression of growth factors in the stromal compartment. Our results showed how tumor-stroma interactions play a significant role in tumor development and progression.

The correlation analysis of MMP-13 and AGG and Col-II in the chondrocytes caused by nutritional deficiencies in rabbits

Objective To explore the relationship among the expression level of MMP-13, AGG and Col-II in the chondrocytes caused by nutritional deficiencies in rabbits. Methods 30 New Zealand white rabbits were randomly divided into autologous chondrocyte transplantation group (control group,n=10), nutrition block group (surgery group,n=10), and peripheral nutrition block group (sham surgery group,n=10). 4 weeks after treatment, the rabbits were sacrificed for undergoing the observations on general and histological level; real-time PCR assay was employed for testing the expression level of MMP-13, AGG and Col-II; cellular apoptosis percentage was observed through TUNEL stain. The relationship among the apoptosis level, cartilage cells histological Mankin score as well as the expression level of MMP-13, AGG and Col-II were analyzed. Results Based on the Mankin score, there was a statistic difference between surgery group and control group. On the other side, there were no statistic differences between sham surgery group and control group. 4 weeks after treatment, surgery group presented a higher apoptotic percentage compared with control group; this value between sham surgery group and control showed no significant differences. There was an increased mRNA expression level of MMP-13 and a decreased mRNA expression level of AGG and Col-II in surgery group compared with control group; no statistic differences of all these values was found between sham surgery group and control group. Histological Mankin score and apoptotic percentage presented positive correlation (r=0.922,P <0.001), the regression equation: Y=-0.548+ 0.404X,R2=0.844 (F=157.735,P <0.001); the mRNA expression level of MMP-13 and apoptotic percentage presented positive correlation (r=0.942,P <0.001), the regression equation: Y=0.951+0.116X,R2=0.883 (F=219.054,P <0.001). There was a negative correlation between the mRNA expression level of MMP-13 and the mRNA expression level of AGG as well as Col-II (r=-0.956, -0.945,P <0.001). Conclusion Damage of cartilage cells causes the up-regulation of the MMP-13 expression which could exacerbate the degeneration of cells. It could induce the down-regulation of AGG and Col-II mRNA expression, which will cause the extracellular matrix synthesis disorder. Key words: Rabbits; Chondrocytes; Apoptosis; Gene expression

Influence of acidity microenvironment of degenerative intervertebral disc on nucleus pulpous - derived mesenchymal stem cells and adipose- derived mesenchymal stem cells

Objective To compare the viability, proliferation and matrix metabolism of nucleus pulpous-derived mesenchymal stem cells (NPMSCs) compared with adipose-derived mesenchymal stem cells (ADMSCs) under intervertebral disc (IVD)like acidic conditions. Methods In our vivo experiment, ADMSCs and NPMSCs from Sprague Dawley rats were cultured under four different pH levels representing the standard condition (pH 7.4) and the normal, mildly degenerated and severely degenerated IVD (pH 7.1, 6.8 and 6.5, separately). By using annexin-V-FITC/propidium iodide staining and a CCK-8 assay, we examined cell viability and cell proliferation respectively. The expression of aggrecan, collagen-Ⅰ , collagen-Ⅱ, MMP-2, ADAMTS4 and TIMP-3 at mRNA and protein levels were measured by RT-PCR and Western blotting. Results Flow cytometry and CCK-8 assays revealed that the viability and proliferation of ADMSCs and NPMSCs decreased with increasing acidity from pH 7.4 to 6.5. Compared with ADMSCs, NPMSCs had significantly fewer apoptotic and necrotic cells under acidic conditions (P<0.05). Cell proliferation of NPMSCs was significantly greater than that of ADMSCs under IVD-like acidic conditions (P<0.05). RT-PCR and western blotting indicated that, in both cell types with increasing acidity, the expression of aggrecan, collagen-Ⅰ , collagen-Ⅱ and TIMP-3 at mRNA and protein levels decreased, while that of MMP-2 and ADAMTS4 increased. Compared with ADMSCs, NPMSCs expressed significantly higher levels of aggrecan and collagen-Ⅱ, and lower levels of collagen-Ⅰ , MMP-2 and ADAMTS4. Conclusion An acidic environment can significantly restrain the IVD regeneration by ADMSCs or NPMSCs. NPMSCs appeared less sensitive to inhibition by acidic pH, and might be promising candidates for cell-based IVD regeneration. Key words: Intervertebral disc degeneration; Mesenchymal stem cells; Cell proliferation

The role of extracellular and intracellular proteolytic systems in aneurysms of the ascending aorta.

Aneurysms of the ascending aorta are an outstanding challenge to clinicians as they may persist asymptomatic until they present with dissection or rupture. Intensive research is performed to reveal the molecular mechanisms causing aneurysm formation. Calpains are ubiquitous non-lysosomal cysteine proteases which are classically activated by calcium signaling. The two major forms of the calpain-family are calpain-I and calpain-II. Calpastatin specifically inhibits the proteolytic activity of calpain-I and -II. Recently it has been demonstrated in aneurysm tissues from ascending aortas obtained from Marfan syndrome patients that calpain-II expression is increased and calpastatin expression is decreased. Thus, we were interested in the probable role of calpains in aneurysms of ascending aorta in non-Marfan patients. Therefore, ascending aortic samples of dilated and non-dilated aortas were analyzed according to their calpain-I, -II and calpastatin content as well as the expression levels of MMPs and elastin as well as the infiltration of inflammatory cells. We have found significant differences in calpain-I and calpastatin protein expression and serum levels in patients with aneurysm of the ascending aorta. Furthermore, MMP-1 and MMP-3 expression levels correlate with calpain-I protein levels. Due to our findings we conclude that calpain-1 seems to be related to fibrotic alteration in aortic aneurysm tissue in our experimental group. The change in calpain-1 modulates the structure of aortic tissue causing alteration in elastin structure, thus enabling macrophage infiltration and elevation of MMP levels. Circulating levels of calpain-1 may be used as a prognostic marker in the future if further correlation analyses are done.

In vitro activation of peripheral blood mononuclear cells and its effects on the proliferation of and production of matrix metalloproteinases by cultured human fibroblasts

Objective To study the effects of different stimulators on the production of matrix metalloproteinases (MMPs) by peripheral blood mononuclear cells (PBMCs) , and to evaluate the effects of the culture supernatant of activated PBMCs, named conditioned media (CM) , on the proliferation of and production of MMPs by cultured human fibroblasts. Methods PBMCs were isolated from the venous blood samples of healthy volunteers and divided into three groups to be stimulated by phytohemagglutinin (PHA group) , the combination of antibodies against CD3 and CD28 (double-antibody group) , or the RPMI 1640 medium containing 10% fetal calf serum (control group). After 72-hour stimulation, CM was collected from all the three groups, diluted to several different degrees. Cultured human fibroblasts were classified into several groups to be treated with different dilutions of CM from the three groups for 48 or 24 hours, with the fibroblasts untreated with CM serving as the control group. Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity, semi-quantitative reverse transcription (RT) -PCR to detect the expressions of MMP-1, MMP-3 and MMP-9 mRNAs in cells, and enzyme-linked immunosorbent assay (ELISA) to measure the levels of interleukin (IL) -6, MMP-1, MMP-3 and MMP-9 proteins in the culture supernatant of cells. Statistical analysis was carried out mainly by using one-way analysis of variance (ANOVA) , Tukey HSD test, and Games-Howell test. Results Compared with the control group, the PHA group showed increased cellular proliferative activity, IL-6 and MMP-3 protein levels in the culture supernatant of activated PBMCs (all P 0.05) , and MMP-9 mRNA expression was undetected in the treated fibroblasts. Conclusions PBMCs can be induced to express MMP-1 mRNA and secret MMP-3 protein after activation. However, the culture supernatant of activated PBMCs has no capacity to stimulate the expressions of MMP-1, MMP-3 and MMP-9 mRNAs or proteins by fibroblasts, suggesting that inflammatory cells may function through self-production of MMPs. Key words: Leukocytes, mononuclear; Fibroblasts; Matrix metalloproteinases; Cell proliferation; Phyto-hemagglutinins

Effects of glycerin fructose combined with dexamethasone on the expressions of aquaporins-4 and matrix metalloproteinases-9 and neurological function in mice after cerebral hemorrhage

Objective To assess the effects of glycerin fructose combined with dexamethasone on neurological function and the expressions of aquaporins (AQP)-4 and matrix metalloproteinases (MMP)-9 in mice after cerebral hemorrhage. Methods Totally 96 male mice(kunming) were randomly divided into the control group (n=24), the model group (n=24), dexamethasone treatment group (n=24), glycerin fructose combined with dexamethasone treatment group (combination group, n=24). The cerebral hemorrhage model was set up in the last three groups, which were given corresponding drug treatment. Mice in each group were subgrouped into 12 h , 1 d, 3 d and 5 d groups according to the time after treatment (n=6, each group). The neurological function was determined according to the reformed mNSS. The dry and wet weight method was used to the brain water content. RT-PCR technology was used to analysis the expressions of AQP-4 and MMP-9 mRNA in mice brain tissue. Results Each treatment group as compared with model group, mNSS was the highest (F=4.56, P<0.05). the brain water content was the lowest (F=5.38, P<0.05), the mRNA expressions of AQP-4 and MMP-9 was the least in combination Group(F=4.02, F=4.68, P<0.05) Conclusions Dexamethasone may relieve cerebral edema, improve the neural function in mice after brain hemorrhage and protect the brain tissue by regulating the transcriptional expression of AQP-4 and MMP-9. If combined with the high permeability dehydrant glycerin fructose, dexamethasone will further inhibit the progress of neurological impairment and brain edema. Key words: Fructors; Dexamethasone; Cerebral hemorrhage; Aquaporin 4; Matrix metalloproteinase 9

Inhibitory effect of tetramethylpyrazine on ultraviolet A-induced senescence and matrix metalloproteinase-1 and -3 mRNA expressions in human dermal fibroblasts

Objective To explore the inhibitory effect of tetramethylpyrazine (TMP) on ultraviolet A-induced senescence as well as matrix metalloproteinase-1 (MMP-1) and -3 (MMP-3) mRNA expressions in human dermal fibroblasts (HDFs) . Methods HDFs were isolated from the prepuce by enzymatic digestion, and subjected to primary culture. Cultured HDFs were randomly divided into several groups: control group cultured in high-glucose DMEM medium and receiving no treatment, three TMP groups treated with 20, 50 and 100 mg/L TMP respectively, UVA group receiving UVA radiation alone, UVA+ TMP groups pretreated with 20, 50 and 100 mg/L TMP respectively for different durations followed by UVA radiation. UVA radiation was given once daily for 5 consecutive days. The 55th passage HDFs served as the P55 group (senescence control group). Subsequently, CCK-8 assay was performed to evaluate the proliferative activity of HDFs in vitro, optical microscopy to observe the morphologic changes of HDFs after UVA radiation, β-galactosidase staining to estimate the senescence in HDFs, and real-time fluorescence-based quantitative PCR to quantify the mRNA expressions of MMP-1 and MMP-3 in HDFs. Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) -t test or Dunnett's T3 test. Results Compared with the control group, the proliferation of HDFs was significantly but transiently inhibited in vitro after the treatment with 100 mg/L TMP for 48 hours (P< 0.05) , but showed no significant changes after the treatment with 20 or 50 mg/L TMP for 24, 48 or 72 hours or after the treatment with 100 mg/L TMP for 24 or 72 hours (all P< 0.05). The pretreatments with TMP of 20, 50 and 100 mg/L for 24, 48 and 72 hours all promoted the proliferation of HDFs to a certain degree in the UVA+ TMP groups compared with the UVA group, with significant differences in cellular proliferative activity among the UVA group, UVA+ TMP groups and control group at 24, 48 and 72 hours (F= 17.451, 15.231, 23.535, all P< 0.01). Compared with the UVA group, the proliferative activity of HDFs was significantly increased in UVA+ 100-mg/L TMP group at 24, 48, 72 hours, UVA+ 50-mg/L TMP group at 24 and 72 hours and UVA+ 20-mg/L TMP group at 72 hours. After repetitive UVA radiation, HDFs in the UVA group experienced an increase in cell volume, granule acount, and β-galactosidase expression, which was similar to the changes in the P55 group, while the pretreatments with 20, 50 and 100 mg/L TMP for 24 hours suppressed these UVA-induced changes in HDFs. The percentage of β-galactosidase-positive HDFs was 68.417% ± 1.181% in the UVA group, 58.167% ± 5.620% in the UVA+ 20-mg/L TMP group, 45.167% ± 5.502% in the UVA+ 50-mg/L TMP group, 43.000% ± 2.000% in the UVA+ 100-mg/L TMP group, 33.667% ± 5.865% in the control group, and 76.000% ± 6.557% in the P55 group, with significant differences among these groups (F= 45.918, P< 0.01). Furthermore, the UVA group significantly differed from the UVA+ TMP groups and control group in the percentage of β-galactosidase-positive HDFs and mRNA expressions of MMP-1 and MMP-3 (all P< 0.05) . Conclusion TMP can protect HDFs against senescence induced by repetitive UVA radiation, and down-regulate the mRNA expressions of MMP-1 and MMP-3 during senescence. Key words: Tetramethylpyrazine; Fibroblasts; Cell aging; Matrix metalloproteinase 1; Matrix metalloproteinase 3; Ultraviolet rays; Cell proliferation

Impact of siRNA-mediated down-regulation of CD147 on human breast cancer cells

To investigate the influence of siRNA-mediated down-regulation of CD147 on growth, proliferation and movement of human breast cancer cell line MDA-MB-231. The protein expression of CD147, MMP-2 and TIMP-2 of the MDA-MB-231 cells were analyzed by ABC. Lentiviral expression vector of CD147 gene was constructed and transfected into MDA-MB-231 cells. RT-PCR and Western blot were used to detect the mRNA and protein level changes of CD147 genes to identify the optimal time point, followed by detection of changes of mRNA and protein expression of MMP-2 and TIMP-2 genes. CCK-8 reagent method and cell scratch test were used to detect the proliferation and migration change of MDA-MB-231 cells. The nude mouse model of breast cancer by hypodermic injection with MDA-MB-231 cells was established to document the effect of CD147 siRNA on the tumor transplants. After transfection of lentiviral expression vector of CD147 gene, protein of CD147, MMP-2 and TIMP-2 were weakly or negative expressed, significantly weaker than those of control group (P < 0.01). After 72 hours of transfection, average down-regulation rate of CD147 and MMP-2 were 96.03% ± 0.84% and 96.03% ± 0.84%, respectively. Both CD147 mRNA and MMP-2 mRNA expression were down-regulated (P < 0.05), while TIMP-2 mRNA expression showed no significant deference (P > 0.05). No less than 2 days after transfection, cell growth of MDA-MB-231 cell line was found significantly inhibited (P < 0.05). After 24 hours of transection, average migration distance of MDA-MB-231 cell line and control group were (0.64 ± 0.12) mm and (4.69 ± 0.85) mm, respectively, which indicated a lower migrate speed. Down regulation of CD147 led to reduction of volume and mass of nude mouses. The growth of the carcinoma transplant was inhibited upon siRNA-mediated down-regulation of CD147 (P < 0.05), with an average tumor mass of (1.85 ± 0.98) g and both reduction of tumor size and tumor mass. CD147 may alter the MMP-2/TIMP-2 balance in MDA-MB-231 cells. CD147 gene silencing inhibits the proliferation and migration of MDA-MB-231 cells and the growth of carcinoma transplants in nude mice.

Effect of therapy time from myocardial infarction onset to percutaneous coronary intervention on matrix metalloproteinases level and left ventricular remodeling in patients with acute anterior wall myocardial infarction

Objective To investigate the effect of time from myocardial infarction (MI) onset to percutaneous coronary intervention (PCI) on plasma matrix metalloproteinases (MMPs) level and left ventricle (LV) remodeling in patients with acute ST-segment elevation myocardial infarction of anterior wall, and the relationship between MMPs and left ventricular remodeling. Methods All patients with anterior wall STEMI undergoing PCI were divided into early PCI group (PCI within 18 h after MI onset) and delayed PCI group (PCI between 2 and 3 weeks after MI onset). Plasma MMP-2 and MMP-9 activities were assayed on admission, and at 2 days, 1 week after admission. One-year follow-up was finished after PCI. Left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV) and left ventricle ejection fraction (LVEF) were measured by echocardiography at baseline and one year later to elucidate the effects of time from onset to PCI on LV remodeling and the relationship between MMP-2, -9 levels and LV remodeling. Results The MMP-9 activity at 2 days after myocardial infarction was lower in early PCI group than in delayed PCI group〔(46±26)μg/L vs. (66±40) μg/L, P=0.000〕. The changes in LVEDV and LVESV (ΔLVEDV and ΔLVESV) were lower and the change in LVEF (ΔLVEF) was higher in early PCI group than in delayed PCI group〔(10.9±6.2) ml vs. (15.0±6.0) ml, (–1.1±5.7) ml vs. (2.9±4.6) ml, (5.5±4.0) % vs. (3.8±3.4) %, P=0.000, 0.000 and 0.015〕. MMP-9 had positive correlations with ΔLVEDV and ΔLVESV, and a negative correlation with ΔLVEF at admission and after 1-year follow-up (r=0.32, 0.36 and–0.29, respectively, P=0.000, 0.000 and 0.001). Conclusions MMP-9 activity at admission is correlated with LV remodeling and LV function. Early PCI can reduce MMP-9 activity and improve LV remodeling after myocardial infarction. Key words: Myocardial infarction; Ventricle remodeling; Matrix metalloproteinases

Suppressing effects of MMPs inhibitor GM6001, MMP-2/9 inhibitorI, MMP-2/9 inhibitorII on migration of human lens epithelial cells

Background The primary pathologic mechanism of posterior capsular opacification (PCO) is proliferation, migration and epithelial-mesenchymal transformation of residuary lens epithelial cells (LECs) following cataract surgery. Matrix metalloproteinases (MMPs) play a role during the migration of LECs. Researches showed that GM6001, a broad inhibitor of MMPs, can arrest the migration of LECs, but as specific inhibitors of MMPs, the efficacy and safety of MMP-2/9 inhibitorⅠand Ⅱ on LECs migration remain unclear. Objective This study was to determine and compare the inhibitory efficacy among GM6001, MMP-2/9 inhibitorⅠand Ⅱ on human LECs and search the clinical medication to prevent PCO. Methods Human LECs were cultured and passaged in vitro, and the cells of 3-4 generation were incubated in 6-well plates. Then the cells of 70% confluent monolayer were cultured in DMEM without fetal bovine serum for 12 hours. GM6001, MMP-2/9 inhibitorⅠand Ⅱ at different concentrations (0. 25, 0. 50, 1. 00, 2. 00, 4. 00, 8. 00, 16. 00, 32. 00, 64. 00, 128. 00 μmol/L) were added into the culture medium for 24 hours separately, and regularly cultured cells served as the control group. A bare area was made by a 200 μl sterile spear on the cell layer, and the migrated distance and inhibitory rate were calculated. The second or third generation of cells were incubated in 96-well plates at a density of 5×105/ml (200 μl/well). GM6001 (128. 00 μmol/L), MMP-2/9 inhibitorⅠ(64. 00 μmol/L) and Ⅱ (32. 00 μmol/L) were added into the culture medium for 24 hours, and the cell viability was assayed by using MTT assay. Results Cultured cells grew well with irregular arrangement and presented the polygon in shape. The migrated distance was gradually reduced as the increase of concentrations of GM6001, MMP-2/9 inhibitorⅠand Ⅱ, showing significant differences among the various concentration groups (GM6001: F=248. 647, P 0. 05). Conclusions GM6001, MMP-2/9 inhibitorⅠ and Ⅱ reduce the mobility of human LECs effectively but do not affect the viability of the cells in vitro. MMP-2/9 inhibitorⅡappears to be most dominant in inhibiting migration of human LECs. Key words: Matrix metalloproteinase/inhibitor; Epithelial cells/eye; Lens/ophthalmology; Migration/drug effect; Posterior capsule opacification

Expression and pathological mechanism of MMP-9 and HIF-2α in CD133(+) lung cancer stem cells

To investigate the expression and pathological mechanism of matrix metalloproteinase (MMP)-9 and hypoxia-inducible factor (HIF)-2α in CD133⁺ lung cancer stem cells. Sixty-two cases of lung cancer paraffin embedding tissues were collected from the First Affiliated Hospital of Soochow University between January 2009 and December 2009. Immunohistochemistry (IHC) was used for detection of CD133 expression in lung cancer tissues and the clinical significance was analyzed. Real-time polymerase chain reaction (PCR) was used for the investigation of expression of tumor metastasis associated genes, including MMP-1, MMP-2, MMP-9, HIF-1α, HIF-1β, HIF-2α and tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, TIMP-4. Scrambled siRNA or CD133 siRNA were used to transfect the lung cancer cell line A549, then Control-si-A549 cells and CD133-si-A549 cells were generated respectively. PCR was used to analysis CD133, MMP-9 and HIF-2α genes expression and transwell invasion assay was used to study the invasion ability of A549 cells in two groups. 51.6% of lung cancer tissues expressed CD133 (P<0.05); the expression level of CD133 was related to tumor metastasis and patients' survival rate (P<0.05). The gene expression of HIF-2α and MMP-9 were increased in CD133⁺ lung cancer cells compared with CD133⁻ cancer cells (1.58 ± 0.39 vs 1.10 ± 0.31, 1.67 ± 0.38 vs 1.05 ± 0.21, all P<0.05), whereas no difference was found in gene expression of MMP-1, MMP-2, HIF-1α, HIF-1β and TIMP-1, TIMP-2, TIMP-3, TIMP-4 (all P>0.05). Compared with the Control-si-A549 cell, the expression of CD133, HIF-2α and MMP-9 (0.24 ± 0.10 vs 0.85 ± 0.23, 0.19 ± 0.09 vs 0.54 ± 0.18, 0.31 ± 0.17 vs 1.12 ± 0.31, all P<0.05) in CD133-si-A549 cell were remarkably decreased. The number of CD133-si-A549 cells migrated to below room was significantly smaller than that of Control-si-A549 cells (207 ± 25 vs 82 ± 10, P<0.05). The CD133⁺ lung cancer stem cell is correlated to the tumor metastasis and patients' survival. CD133⁺ tumor stem cell can promote the tumor invasion and metastasis via the up-regulation of HIF-2α and MMP-9 expression.

Effects and mechanisms of ghrelin on plaque neovascularization in rabbits with atherosclerosis

Objective The aim of our study was to investigate the effects and mechanisms of ghrelin on neovascularization in atherosclerosis plaque. Methods 30 male New Zealand rabbits were randomly divided into normal control group(CON group), atherosclerosis model group(AS group), and ghrelin treatment group(ghrelin group), and each group of 10 rabbits. The AS group and ghrelin group underwent balloon-induced arterial wall injury and then fed with high fat diet, the CON group was fed only on a regular diet. They were all fed for 3 months. Then the ghrelin group was given ghrelin 25 μg·kg-1·d-1, the other two groups received the same amount of sterile normal saline only. Four weeks later, body weight and blood lipids were detected. The thickness ratio of the intima to media was measured by HE staining. Degree of intra-plaque angiogenesis was evaluated by CD31+ cells immunohisto-chemistry. The vascular endothelial growth factor(VEGF) and vascular endothelial growth factor receptor 2(VEGFR2) were detected by quantitative realtime PCR and Western blot. The expressions of matrix metalloproteinase(MMP)-2 and MMP-9 were detected by immunohistochemistry and Western blot. Results (1)No significant differences in body weight and blood lipids were found between the AS group and the ghrelin group(P>0.05), but both items were significantly higher than those of the CON group(P<0.05). (2)The thickness ratio of the intima to media in the ghrelin treated group was distinctly less than that in the AS group(P<0.05). (3)Compared with the AS group, the ghrelin group showed significantly decreased microvascular density and the expressions of VEGF and VEGFR2(P<0.05). (4)Compared with the AS group, ghrelin dramatically inhibited the plaque contents of MMP-2 and MMP-9(P<0.05). Conclusions Ghrelin is able to inhibit the growth of neovascularizationin in the atherosclerotic plaque and the development of plaque. And these beneficial effects derive from downregulation of VEGF, VEGFR2, MMP-2, and MMP-9 at the advanced stage of atherosclerosis in rabbits. (Chin J Endocrinol Metab, 2015, 31: 717-724) Key words: Ghrelin; Neovascularization; Atherosclerosis; Plaque stability