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AIM: To understand whether reactive oxygen species promote the rupture of atherosclerotic plaques by regulating the balance of matrix metalloproteinase-1, 3 (MMP-1, 3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in smooth muscle cells. METHODS: Aortic smooth muscle cells from 4-6months-healthy abortive fetuses were incubated for 24 hours with xanthine (100 μmol/L) and xanthine oxidase (5 U/L) in vitro . MMP-1, 3 and TIMP-1 in the concentrated culture media were measured by Western blotting ( n =3 independent experiments). RESULTS: Incubation with xanthine/xanthine oxdiase decreased the amount of MMP-1 in the aortic smooth muscle cells (21.2%±5.5% of the control group), and pro-MMP-1 was activated completely. Reactive oxygen species (ROS) also activated pro-MMP-3, and increased the production of MMP-3 in the aortic smooth muscle cells. On the other hand, ROS inhibited the production of TIMP-1 in the aortic smooth muscle cells. CONCLUSION: It is complicated that ROS regulates the balance of MMPs and TIMPs. ROS may contribute to matrix degradation and the rupture in the atherosclerotic plaques.  相似文献   

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AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.  相似文献   

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AIM： To find out the influences of different antioxidants on the protein expression of matrix metalloproteinase 9 (MMP-9) in the membrane tissue of premature rupture cultured in vitro. METHODS：Eight pregnant women of premature rupture of membranes (PROM) who had delivered by cesarean section before labor were enrolled in the study and divided into 2 groups: term premature rupture of membranes (TPROM) and preterm premature rupture of membranes (PPROM). The fetal membrane tissues from each case was collected and divided into 3 sub-groups, which were cultured for 24 h with phosphate buffered saline (PBS, control group), 0.5 mmol/L alpha-lipoic acid (α-LA), or 28.8 g/L vitamin C+1.8 g/L vitamin E in vitro. The protein expression of MMP-9 was determined by immunohistochemistry and Western blotting. RESULTS：MMP-9 was located in the chorionic cells and neutrophils in TPROM tissues and there was no significant difference among samples of different treatments. In PPROM tissues, MMP-9 was found not only in the chorionic cells and neutrophils, but also in the amniotic epithelial cells. The integral absorbance values in antioxidant-treated samples were lower than that in PBS-treated samples, and the significant difference between α-LA-treated samples and vitamin C+vitamin E-treated samples was observed. The expression of pro-MMP-9 in TPROM tissues showed no significant difference after treated with different antioxidants, but the active MMP-9 expression was decreased after treated with antioxidants, and the influence of vitamin C+vitamin E was stronger than α-LA. The expression of pro-MMP-9/active MMP-9 in PPROM tissues was decreased after treated with antioxidants, and the influence of vitamin C+vitamin E was stronger than α-LA. CONCLUSION：Antioxidants result in the down-regulation of MMP-9 protein expression, and the influence of vitamin C+vitamin E is stronger than α-LA, indicating that these antioxidants can be used clinically to prevent premature rupture of membranes.  相似文献   

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AIM: To investigate the effect of isopsoralen(ISR) on the expression of estrogen receptor (ER) in human lens epithelial cells (HLECs). METHODS: HLECs were cultured and sub-cultured in vitro. The cultured HLECs pretreated with E2 or ISR were exposed to H2O2 at the concentration of 300 μmol/L. The expression of ERα and ERβ in HLECs was analyzed by flow cytometry. RESULTS: The expression of ERα and ERβ in H2O2 group was obviously decreased as compared to control group (P<0.01). The expression of ERα and ERβ in the cells treated with E2 and with ISR at the concentration of 10-5 mol/L, 10-6 mol/L or 10-7 mol/L plus H2O2 was obviously increased as compared to the cells treated with H2O2 only (P<0.01). A concentration-dependent effect of ISR was observed. CONCLUSION: H2O2 decreases the expression of ERα and ERβ in HLECs.E2 and ISR increase the expression of ERα and ERβ in HLECs treated with H2O2 in a concentration-dependent manner, which may account for their antioxidative effect.  相似文献   

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AIM:To study the effects of genistein on JAR/MTX cell proliferation, apoptosis and invasion and it's mechanism in vitro. METHODS:MTT assay, Annexin-Ⅴ and propidium iodide label analysis and invasion assay were used to determine the effects of genistein on proliferation, apoptosis and invasiveness in JAR/MTX methotrexate- resistant human choriocarcinoma cells. RT-PCR was used to estimate the relative mRNA amounts of estrogen receptor(ER), MTA3 and snail in the cells. Western blotting and gelatin zymography assay were used to estimate the relative protein amounts of MMP-2, MMP-9 and E-cadherin in the cells. RESULTS:After treatment of genistein, the proliferation and invasiveness of JAR/MTX cells were decreased significantly in a dose-dependent manner. 10 μmol/L genistein induced apoptosis, whereas 25, 50, 100 μmol/L genistein induced apoptosis and necrosis significantly. Genistein led to an increase in ERβ, MTA3 mRNA and E-cadherin protein expression, and decreases in the amounts for snail mRNA and MMP-2 and MMP-9 protein expression of JAR/MTX cells. CONCLUSIONS:Genistein inhibits the cell proliferation by inducing cell apoptosis and necrosis. Genistein also may inhibit JAR/MTX cell invasion in part through the upregulation of E-cadherin and downregulation of MMP-2 and MMP-9. The signal transduction pathway of invasion suppression induced by genistein in JAR/MTX cells may be as follows: MTA3→snail→ E-cadherin.  相似文献   

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AIM: To study effects of urokinase-type plasminogen activator (uPA) signal transduction on expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in giant cell tumor of bone (GCT). METHODS: Expression of uPAR, MMP-2 and TIMP-3 in GCT tissue was detected by immunohistochemistry. Phosphorylation level of mitogen-activated protein kinase (p44) in uPA/uPAR signal pathway in cultured GCT cells was detected by immunoprecipitation. The expression of MMP-2 and TIMP-3 in cultured cells after treatment with uPA-ATF or anti-uPAR antibody was also detected by Western blotting. RESULTS: 1) Urokinase-type plasminogen activator receptor (uPAR) was positive on the cell membrane and in cytoplasm of some mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs); 2) MMP-2 was positive in the cytoplasm and on the cell membrane of almost all of MSCs and some of MGCs. The polar distribution of MMP-2 in the cytoplasm of MGCs was especially obvious; 3) The expression of TIMP-3 of some MSCs and MGCs in GCT was much lower than MMP-2. The positive signal also showed a prominent polarity; 4) After treatment with uPA-ATF, the phosphorylation level of p44 in GCT cultured cells was much higher than the control. Addition of anti-uPAR antibody in the cells remarkably down-regulated the phosphorylation level of p44 as compared with the control group, suggesting that uPA-ATF participates cell signal transduction and this reaction can be inhibited by anti-uPAR antibody; 5) uPA-ATF cell signal pathway up-regulated expression of MMP-2 and TIMP-3, while anti-uPAR antibody down-regulated the expression of MMP-2 and TIMP-3. CONCLUSION: These results demonstrate for the first time that uPA-ATF directly regulates the expression of MMP-2 and TIMP-3 by signal transduction pathway, and the over-expression of MMP-2 and TIMP-3 may play an important role in local osteolysis of GCT.  相似文献   

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AIM: To investigate the hypothesis that low-molecular weight heparin (LMWH) regulates in vitro cytotrophoblast invasiveness and production of metalloproteinases-2 (MMP-2), tissue inhibitor of metalloproteinas-2 (TIMP-2). METHODS: Chorionic villi tissue of normal 6-8 weeks pregnancy was obtained. Trophoblastic cells were collected by trypsin-collagenase digestion and Percoll gradient centrifugation. The cytotrophoblastic cells were cultured for 24 h and divided into 4 groups according to the concentrations (1.0×102 IU/L, 1.0×103 IU/L or 1.0×104 IU/L) of LMWH adding into the medium. The contents of MMP-2 and TIMP-2 in cell culture supernatants were measured by the method of ELISA. Cytotrophoblast invasiveness was determined by Transwell chamber assay. RESULTS: With the increasing concentrations of LMWH, the invasion activity of cytotrophoblastic cells and MMP-2 secretion were increased. At concentration of 1.0×103IU/L, LMWH greatly enhanced cytotrophoblast invasiveness and the expression of MMP-2 (P<0.05). The levels of TIMP-2 were decreased after intervention with LMWH. At concentration of 1.0×103IU/L or 1.0×104 IU/L, LMWH induced a significant decrease in TIMP-2 expression. No significant difference between group 1×103IU/L and group 1.0×104 IU/L was observed (P>0.05). CONCLUSION: LMWH might regulate cytotrophoblast invasiveness in vitro by influencing the expression of MMP-2 and TIMP-2 in cytotrophoblastic cells.  相似文献   

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AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.  相似文献   

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AIM: To characterize the effect of prostatic epithelial cell paracrine on aromatase expression in prostatic stromal cells.METHODS: Conditioned medium (CM) of prostatic epithelial cell lines (BPH-1, LNCap, DU-145 and PC3) were collected and used to treat prostatic stromal cells. Expression of aromatase was determined by real-time RT-PCR and Western blotting. Expression of cyclooxygenase-2 mRNA in prostatic epithelial cell lines and prostaglandin (PGE2) in CMs were examined by real-time RT-PCR and ELISA, respectively. The CM of BPH-1 cells cultured with NS-398, specific inhibitor of cyclooxygenase-2, were collected, and the effect of NS-398 and PGE2 on aromatase expression was analyzed.RESULTS: CM of human benign prostate hyperplasia epithelial cell line (BPH-1) stimulated expression of aromatase mRNA and protein in stromal cells. But CM of prostate cancer epithelial cell lines (LNCap, DU145, PC3) had no effect on aromatase expression. COX-2 mRNA level in BPH-1 was much higher than that of other cell lines and PGE2 concentration in BPH-1 CM was much higher than that of other CMs. PGE2 concentration of the CM from BPH-1 cultured with NS-398 significantly decreased. CM from BPH-1 cultured with NS-398 failed to stimulate aromatase expression, while PGE2 induced aromatase expression in prostatic stromal cells.CONCLUSION: BPH-1 could induce aromatase expression in prostatic stromal cells through paracrine of PGE2.  相似文献   

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AIM To investigate the effects of histone demethylase inhibitor IOX1 (5-carboxy-8-hydroxyquinoline) on the proliferation, apoptosis and extracellular matrix (ECM)-related protein expression in transforming growth factor-β (TGF-β)-induced human hepatic stellate LX2 cells. METHODS The proliferation and apoptosis of the LX2 cells were determined by real-time cell analysis and flow cytometry, respectively. The level of histone H3 lysine 9 dimethylation (H3K9me2) and the protein expression of ECM-related molecules [α-smooth muscle actin (α-SMA), collagen type I (Col I), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1)] in the LX2 cells were detected by Western blot. RESULTS Treatment with IOX1 at 50~300 μmol/L significantly inhibited LX2 cell proliferation, and 300 μmol/L IOX1 significantly promoted the apoptosis of the LX2 cells. In addition, different concentrations of IOX1 increased the levels of H3K9me2 and MMP-1, and down-regulated the expression of α-SMA, Col I and TIMP-1 in TGF-β-induced LX2 cells (P<0.05). CONCLUSION Treatment with IOX1 inhibits the proliferation of LX2 cells induced by TGF-β, promotes the cell apoptosis, and regulates the synthesis and metabolism of ECM by elevating H3K9me2 level, thus attenuating hepatic fibrosis.  相似文献   

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AIM: To study the effect of idazoxan on the permeability of inflammatory blood-brain barrier (BBB) model in vitro and the expression of tight junction protein ZO-1.METHODS: In vitro BBB model was established by murine brain endothelial cell line bEnd.3 incubated for 7 d. The cells were treated with TNF-α (10 nmol/L) for additional 24 h to establish the inflammatory BBB model, which was pretreated with IDA at doses of 50, 100 and 200 μmol/L, respectively. The permeability was measured using fluorescein isothiocyanate-conjugated dextran (FD-40, MW 40,000), the expression of ZO-1 was detected by Western blot analysis, the distribution of ZO-1 was observed by immunofluorescence, and the mRNA expression of MMP-9/TIMP-1 was measured by RT-PCR.RESULTS: After incubated for 7 d, b.End3 cells converged to be confluent monolayer with low permeability. The inflammatory BBB model induced by TNF-α treatment displayed much higher permeability with decreased expression of tight junction protein ZO-1, destroyed distribution of ZO-1 and increased mRNA expression of MMP-9. When pretreated with IDA, the permeability was greatly decreased, the expression of ZO-1 was greatly increased, the abnormal distribution of ZO-1 was greatly ameliorated and the mRNA expression of MMP-9 was obviously reduced. The effect was most significant in IDA (200 μmol/L)-pretreated group (P<0.01).CONCLUSION: IDA directly acts on brain endothelial cells to reduce the expression of MMP-9, increase the expression of tight junction protein ZO-1 and ameliorate the destroyed distribution of ZO-1 in the inflammatory BBB, thus reversing the abnormally elevated permeability in a inflammatory BBB model in vitro induced by TNF-α.  相似文献   

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AIM: To investigate the effect of estrogen antagonists on the in vitro growth of human prolactinomas. METHODS: RT-PCR was applied to the detection of estrogen receptor (ER) mRNA expressed in a human prolactinomas CH3 cell strain. Estradiol and 4-hydroxytamoxifen (OHTam) were added respectively at different concentrations into the culture medium. Cell number and levels of ER mRNA were examined. RESULTS: The growth of CH3 cells became slower in estrogen-deprived medium than that in nomal culture and was higher in medium containing estrogen(E2) at concentration of 10-8 mol/L than at concentration of 10-6 mol/L. OHTam (10-6mol/L) inhibited the growth of CH3 cell strain treated with E2. The expression of ER mRNA in CH3 cells was observed, the levels of ER mRNA in the E2 (10-8mol/L) group, higher than those in estrogen deprived group. OHTam (10-6mol/L) obviously inhibited the expression of ER mRNA. CONCLUSION: The growth of CH3 cells depends on estrogen, estrogen antagonists inhibits the growth of CH3 cells and decline the levels of ER mRNA. ER levels in human prolactinomas cell lines can be auto-regulated.  相似文献   

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AIM:To observe the effect of intravitreal injection of pirenzepine on form-deprivation myopia in chicks. METHODS:Forty-one day old chicks were randomly divided into 4 groups:normal control group, form deprivation group, vehicle control group and pirenzepine injection group. The right eyes of all chicks were used as experimental eyes. The deprived eyes in vehicle control group and pirenzepine injection group received daily intravitreal injection of vehicle control solution(0.01 mol/L PBS) and pirenzepine(1%in PBS), respectively. Optical examinations such as refraction, axial length and equatorial diameter were made at the end of the 5th day. The mRNA and protein levels of matrix metalloproteinase 2(MMP-2) and tissue inhibitor of metalloproteinase-2(TIMP-2), and the activity of MMP-2 were detected by RT-PCR, Western blotting and zymography analysis,respectively. RESULTS:Refraction, axial length and equatorial diameter of the eyes in pirenzepine injection group were significantly lower than those in form deprivation group and vehicle control group(P<0.05), but those were higher(P<0.05) and the eyes were relatively myopic as compared with normal control group. The mRNA expression, protein le vels and activity of MMP-2 in pirenzepine group were significantly higher than those in normal control group(P<0.01), and were significantly lower than those in form deprivation group and vehicle control group(P<0.01, P<0.01). The mRNA and protein levels of TIMP-2 in pirenzepine group were significantly lower than those in normal control group(P<0.01), and was significantly higher than those in form deprivation group and vehicle control group(P<0.01, P<0.01). CONCLUSION:Intravitreal injection of pirenzepine may partly prevent form-deprivation myopia by modulating the expression of MMP-2 and TIMP-2 in the fibrous layer of sclera.  相似文献   

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AIM:To investigate the effects of Maxing-Shigan decoction on airway remodeling and expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the lung tissues of asthmatic mice, and to explore its possible mechanism in treatment of asthma. METHODS:The BALB/c mice were divided into blank control group, model group, low-dose Maxing-Shigan decoction group, middle-dose Maxing-Shigan decoction group, high-dose Maxing-Shigan decoction group and positive control group. The mice were sensitized and challenged with ovalbumin to establish asthma model. The mice in blank control group and model group were given saline by oral administration before 30 min of suscitation. The mice in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups were given Maxing-Shigan decoction at 5.0 g/kg, 10.0 g/kg and 20.0 g/kg, respectively, by oral administration before 30 min of suscitation. The mice in positive control group was given dexamethasone at 0.005 g/kg by oral administration before 30 min of suscitation. After consecutive administration for 7 d, the variations of airway responsiveness, the percentage of the goblet cells, the collagen deposition, and the eosinophil (EOS) counts in bronchoalveolar lavage fluid (BALF) of each group were observed. The protein levels of MMP-9 and TIMP-1 in the lung tissues were determined by ELISA and Western blot. The mRNA expression of MMP-9 and TIMP-1 was detected by RT-qPCR. RESULTS:Compared with blank control group, the airway responsiveness, the goblet cell percentage, the collagen deposition, the EOS counts in BALF, the protein levels of MMP-9 and TIMP-1, and the mRNA expression of MMP-9 and TIMP-1 were significantly increased in model group (P<0.01). Compared with model group, all of the indexes were reversed in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups and positive control group (P<0.05 or P<0.01). CONCLUSION:Maxing-Shigan decoction improves airway remodeling in asthma model mice by down-regulating the expression of MMP-9 and TIMP-1.  相似文献   

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AIM: To observe the effect of different concentrations of glucose and homocysteine on the expression of MMP-9 in fibroblasts, and to investigate the relationship between homocysteine and diabetic foot.METHODS: Fibroblasts were obtained from the dermis of 1-day-old normal SD rat and cultured in DMEM containing 10% FBS. After deprivation of serum (using 0.5% FBS) for 24 h, the fibroblasts in 2-4 passages were cultured for 6 h under the conditions of normal glucose, high glucose and high glucose with high concentration of homocysteine, respectively. The expression of MMP-9 at mRNA and protein levels was assessed by RT-PCR and Western blotting, and the activity of MMP-9 was determined by gelatin zymography analysis. RESULTS: The expression of MMP-9 at mRNA and protein levels and activity of MMP-9 in high glucose (22 mmol/L) group were 1.15 folds, 1.59 folds and 1.34 folds of those in normal glucose group (P<0.05), respectively. The effect of high glucose became more pronounced when co-treated with high concentration of homocysteine (100 μmol/L), which were 1.25 folds, 2.63 folds and 2.52 folds of those in normal glucose group (P<0.05), respectively.CONCLUSION: The increase in the expression of MMP-9 is concentration-dependen with glucose content in the culture. High concentration of homocysteine promotes this process, suggesting that hypercysteinemia might play an important role in the expression of MMP-9 in fibroblast.  相似文献   

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