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Objective To investigate the effect and possible mechanism of tissue inhibitor of Metalloproteinases-l(TIMP-1) siRNA on human umbilical vein endothelial cells injury induced by serum of septic patient.Methods Serum samples were separately collected from septic patients and healthy controls.Human umbilical vein endothelial cells (HUVECs) were randomly divided into blank group (normal culture cells),control group (culture medium with 10％ control serum),septic group (culture medium with 10％ septic serum),negative control group (negative siRNA + 10％ septic serum),and TIMP-l siRNA group (TIMP-1 siRNA + 10％ septic serum).The survival rate of endothelial cells was detected by MTT assay.The levels of matrix Metalloproteinase-9 (MMP-9) and TIMP-1 in supernatant of culture medium were measured by enzyme-linked immunosorbent assay (ELISA).The levels of MMP-9,TIMP-1 and Thrombomoduline (TM) in endothelial cells were examined by Western blot.Results Compared with control group,the cell survival rate of septic group decreased 12 hours after the addition of serum (P＜0.05) and reached minimum 48 hours later.The levels of MMP-9 and TIMP-1 in supernatant of culture medium of septic group significantly increased (P＜0.01).The levels of MMP-9 and TIMP-1 increased in the septic group (P＜0.01),while the level of TM reduced at the same time in septic group (P＜0.01).Compared with septic group,the cell survival rate ofTIMP-1 siRNA group decreased (P＜0.05).The level of MMP-9 in supematant of culture medium of TIMP-1 siRNA group increased (P＜0.05),while the level of TIMP-1 decreased (P＜0.05).The level of MMP-9 increased in TIMP-1 siRNA group (P＜0.01),whereas the levels of TIMP-1 and TM reduced in TIMP-1 siRNA group (P＜0.01).Conclusions TIMP-1 plays a protective role in endothelial cells injury induced by septic serum.
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Objective To evaluate the effects of TIMP-1 and Ang-1 gene-modified BMSCs transplantation on the left ventricular function of rats with myocardial infarction.Methods The rat BMSCs were.transfected with eukaryotic expression plasmid encoding TIMP-1 or/and Ang-1 gene by liposome.Acute myocardial infarction was made in male rats by ligation of the left anterior descending (LAD) coronary artery.BMSCs carrying TIMP-1 or/and Ang-1 gene were injected into the ischemic myocardium after LAD ligatior.Four weeks after the administration,cardiac function was assessed by echocardiography and the hearts were harvested and sectioned for immunohistochemistry to examine the apoptosis,the collagen content and angiogenesis density.Results TIMP-1 and Ang-1 genemodified BMSCs transplantation significantly improved the cardiac function,myocardial apoptosis was alleviated,collagen content decreased and the angiogenesis density in border-zone was increased significantly (P＜0.05).Conclusions The results suggest that the combination of TIMP-1 and Ang-1-gene modified BMSCs transplantation can improve the cardiac function of rats with myocardial infarction.The increase of the blood supply,the alleviation of myocardial apoptosis and ventricle remolding after myocardial infarction possibly play important roles in the mechanism.
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Obej ctive To investigate the role of metastasis associated protein 1(MTA1)in estrogen reg-ulated expression of matrix metalloproteinase -9(MMP-9)and tissue inhibitor of metalprotease -1(TIMP-1) in estrogen receptor( ER ) positive breast cancer cells .Methods MTA1 knockdown cell model was generated based on MCF-7breast cancer cell line by transfected with MTA 1-shRNA.The mRNA and protein level of MMP-9 and TIMP-1 in wild type MCF-7(MCF-7WT)and MCF-7MTA1-shRNA before and after 17β-estradiol ( E2) treatment were examined by Real -time PCR and Western blot respectively .Results The MTA1-shRNA showed maximally 84.9%suppression of MTA1 expression in MCF-7,suggesting a satisfied MTA 1 knockdown cell model was established for subsequent experiments .After treated with E2 for 48 h,MCF-7WT showed an incre-ment of 46%(P<0.05)and 37%(P<0.05)of the mRNA and protein level of MMP -9 and a decrement of 32.3%( P<0.05)and 18.2%(P<0.05)of TIMP-1;MCF-7MTA1-shRNA showed a decrement of 32.3%(P<0.05)and 18.2%(P<0.05)of mRNA and protein expression of MMP -9 respectively but no significant differ-ence in TIMP-1 comparing with MCF-7WT before treated with Estradiol.After E2 treatment,MCF-7MTA1-shRNA didn′t show significant change of MMP -9 except decrements of 32.3%(P<0.05)and 18.2%(P<0.05)in the mRNA and protein levels of TIMP -1.Conclusion MTA1 may be involved in the pathway by which estrogen regulated the expression of MMP -9 but not TIMP-1 in ER positive breast cancer cells .
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Objective To investigate the expression and the effects of tissue inhibitor of metalloproteinases-1 (TIMP-1) on lungs of rats with sepsis.Methods Forty Sprague-Dawley (SD) rats were randomly divided into two groups,namely sham group (n =8) and sepsis model group (n =32).The rats of model group were modeled by cecal ligation and puncture (CLP),and were further divided into four subgroups as per the time after modeling,namely 6 h (n =8),12 h (n =8),24 h (n =8),48 h (n =8)subgroups.Blood and lung samples were taken 6 h,12 h,24 h and 48 h after modeling.The histological changes in lungs of the rats were observed under light microscope.Expressions of TIMP-1 mRNA,Bax mRNA and Bcl-2 mRNA in lungs were measured by RT-PCR.The immunohistochemistry was used to label the CD18 in lungs during different phases of sepsis.The data were processed by t test.Results Compared with sham group,the lung tissues of rats in model group were injured to a certain extent after CLP.The expression of TIMP-1 mRNA and the number of CD18 positive cells increased at the same time (P ＜ 0.01),and peaked 24 hours later (P ＜ 0.01).While the expression of Bax mRNA in model group decreased markedly 12-48 hours after modeling (P ＜ 0.01-0.05),and reached minimum 48 hours later (P ＜ 0.01).The expression of Bcl-2 mRNA in model group changed unnoticeable.The positive correlation between variations in number of CD18 positive cells and expression of TIMP-1 mRNA was found in model group (r =0.426,P ＜ 0.01).Conclusions The increase in expression of TIMP-1 mRNA in lungs is closely associated with the lung injury of sepsis.The mechanism of lung injury is likely attributed to the preservation of inflammatory cells from apoptosis,and the persistent inflammation response causes tissue damage,leading to organ dysfunction.
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Objective To examine whether tissue inhibitor of metalloproteinases-1 (TIMP-1) is involved in arterial calcification of chronic renal failure (CRF) rats. Methods CRF model was induced in male Wistar rats by garage daily with 2% adenine 250 mg/kg. The calcification of aorta, femoral artery, renal artery and coronary artery was evaluated histomorphometrically by van Kossa-stained sections at 2-, 4-, 6- and 8-week respectively. RT-PCR and Western blot were used to observe the expressive levels of TIMP-1 mRNA and protein. Expressions of TIMP-1, osteopentin (OPN) and core binding factor α1 (Cbfα-1) protein were analyzed by immunhistochemistry. Results Serum urea nitrogen, creatinine, inorganic phosphate, calcium-phosphorus product and intact parathyroid hormone (iPTH) increased significantly in the model animals compared with control group after 2 weeks (P<0.01). Medial calcification was found in above four arteries of model groups after 6 weeks. RT-PCR and Western blot showed that TIMP-1 expression of model group was significantly higher than that of control group (P< 0.05), and obviously elevated in a time-dependent manner. The expression of TIMP-1 and OPN in calcified aortic smooth muscle cells increased obviously (P<0.05), and positive immunostaining of Cbfα-1 was found. The expression of TIMP-1 was positively correlated with OPN and Cbfα-1 (r=0.317, P=0.000; r=0.485, P=0.000). Conclusions The pathology of arterial calcification in CRF rats induced by adenine is similar to CRF patients, which may serve as a useful model of CRF with arterial calcification. The up-regulation of TIMP-1 seems to participate in the formation and development of vascular calcification in CRF.
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Objective To study the levels of matrix metalloproteinase-3(MMP-3),matrix metalloproteinase-9(MMP-9) and inhibitor of metalloproteinases-1(TIMP-1) in patients with acute myocardial infarction(AMI) and mechanism of plaque rupture of coronary atherosclerosis.Methods Sixty-seven patients with AMI were selected,average invasion time was 3 h～5 d.40 cases of non-coronary heart disease were included in control group.The expressions of serum MMP-3,MMP-9,TIMP-1 and the levels of serum cTnI,CK-MB were analyzed by enzyme-linked immunosorbent assay.Results The serum MMP-3,MMP-9 and TIMP-1 levels in AMI group were higher than those in control group,there was significant difference between two groups(P
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Objective To observe temporal expression of matrix metalloproteinases(MMP-13) and tissue inhibitor of metalloproteina-ses-1 (TIMP-1) in lung of newborn rats with hyperoxia induced chronic lung disease (CLD),and to explore the relationship of CLD with MMPs.Methods The neonatal rats within 24 hours after birth were randomly divided into hyperoxia-exposed group(n=40) and control group(n=40).On postnatal 1,3,7,14 and 21 days,lung tissue of rats in 2 groups were collected.Lung histological changes were evaluated by hematoxylin and eosin and Masson stain;Collagen Ⅰ was detected by enzyme linked immunoadsorbent assay;MMP-13 and TIMP-1 were identifide by immunohistochemistry.Results Exposured to hyperoxia enviroment for 21 days,the number of alveolar decreased,terminal air space enlarged,inter-alveolar septa thickened,and deposition of interstitial collagen fibers.On 14 and 21 days,collagen Ⅰ in the lung of hyperoxia-exposed group increased significantly compared with that of control group(P0.05),obviously decreased on 21 day(P
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Objective To investigate the relationship between serum or urine tissue inhibitor of metalloproteinases-1 (TIMP-1)levels and the stages of chronic kidney disease (CKD).Methods Serum and urine level of TIMP-1 were measured by enzyme-linked immunoadsorbent assay (ELISA) in 122 CKD patients and 40 healthy controls.Serum cystatin-C (CystC),parathyroid hormone (PTH),low-density lipoprotein (LDL)and other markers were determined simultaneously.Correlationanalysis between TIMP-1 level and degree of renal function were performed.Results Serum and urine TIMP-1 of the patients in CKD group was significantly higher than that in control group (P
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AIM: To study the effects of Yangyu Tuji (YYTJ) on delayed healing wound of diabetic rats caused by streptozotocin (STZ). METHODS: SD male rats were randomly divided into control group (control), model group (model); and 3 different dose groups of YYTJ. 55 mg/kg STZ were given by intraperitoneal injection except for control group. After 30 days, a round skin of 1.6 cm diametre was excised on all dorsal back of rats. The healing time and healing rate were observed according to re-epithelization. The content of collagenⅠ and Ⅲ was observed by Picric acid-Sirius red staining , Matrix metalloproteinase-1, 13 (MMP-1, -13), tissue inhibitor of metalloproteinases-1 (TIMP-1) by immuno-histochemistry assay. All data were analyzed by IPP software. RESULTS: The healing time in each group treated with YYTJ was shorter than that in model group (P
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Objective To observe the inhibition of asON phosphorothioate to the TIMP-1 gene and protein expression in the liver tissue of immune- induced hepatic fibrosis rats. Methods According to the analysis of modulator, structure protein, encoding sequence of TIMP-1 genome, we designed four different groups of asONs. These asONs were injected into the hepatic fibrosis rat models through coccygeal vein. The results were observed by RT-PCR, immunohistochemistry and in situ hybridization with collagen Ⅰ、Ⅲ, special staining of collagen fiber, electron microscope. Results The asON phosphorothioate of TIMP-1 could be expressed in vivo, and could block the TIMP-1 gene and protein expression in the liver of immune- induced hepatic fibrosis rats on the level of mRNA, which could promote the degradation of collagen Ⅰ、Ⅲ(P
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Objective:To study the effects of FuYuan capsule on rat liver fibrosis induced by pig serum.Methods:The rats were randomly divided into six groups,including the normal group,the model group,the low,middle and high dose FuYuan capsule group,and the positive control group(compound Biejiaruangan-chip solution).The rat models of immune liver fibrosis were induced with pig serum.Alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum were detected with autoumatic biochemical analyzer.Hyaluronan(HA),laminin(LN).IV collagen(IV-C)and the typeⅢcollagen(PCⅢ)in serum were quantified by radiative-immune technology.The expression of TIMP-1(tissue inhibitor of metalloproteinases-1)and TGF-?1(transforming growth factor?-1)in the liver were observed by immunohistochemical assay.Results:ALT,AST,HA,LN,IV-C and PCⅢin the model group were significantly higher than those in the other group(sP