RESUMO
BACKGROUND: Senna alata L. Roxb or candle bush is a traditional medicinal plant with a wide range of biological activities including anti-inflammatory, antimicrobial and antifungal. Leaf extract of S. alata showed the anti-tumor activity in various cancer cell lines. In this study, we focused on the inhibitory mechanism of S. alata extract (SAE) on cancer metastasis including cell migration, cell invasion and signaling pathways in chondrosarcoma SW1353 cells. PURPOSE: This study aimed to evaluate the anti-metastatic mechanisms of Senna alata extract on chondrosarcoma SW1353 cells. METHODS: Screening for phytochemicals in biologically active fraction of SAE was analysed by 1H NMR spectroscopy. Cell viability and cytoxicity were determined by using MTT assay. Cell migration was observed by scratch wound healing and transwell migration assay. Cell invasion and cell adhesion assay were examined by Matrigel coated transwell chambers or plates. The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), MAPKs and PI3K/Akt signaling pathways and NF-κB were detected by Western blot analysis. RESULTS: The SAE treatment at the sub-cytoxic and non-cytotoxic concentrations significantly inhibited cell migration, cell invasion and cell adhesion of SW1353 cells in a dose-dependent manner. The results from Western blot analysis showed decreased MMP-2 and MMP-9 expression, while increased TIMP-1 and TIMP-2 expression in SAE treated cells. Moreover, SAE suppressed phosphorylation of ERK1/2, p38 and Akt but decreased NF-κB transcription factor expression in SW1353 cells. CONCLUSION: These results revealed that SAE could reduce MMP-2 and MMP-9 expression by downregulation of NF-κB which is downstream of MAPKs and PI3K/Akt signaling pathway in SW1353 cells resulting in reduced cancer cell migration and invasion. Therefore, SAE may have the potential use as an alternative treatment of chondrosarcoma metastasis.
Assuntos
Condrossarcoma/tratamento farmacológico , Metástase Neoplásica/tratamento farmacológico , Extrato de Senna/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrossarcoma/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Extrato de Senna/química , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
Chronic hepatic schistosomiasis causes portal hypertension, fibrosis and lethal hepatosplenic complications. Previous studies focused mainly on schistosomicidal drugs and neglected the therapeutic approaches against the vascular complications after portal hypertension. Investigating a novel anti-angiogenic therapy is an urgent. The current study is to evaluate the performance of Paeoniflorin (PAE) as an anti-angiogenic therapy, being a powerful anti-fibrotic, compared to artemether (ART) and praziqantel (PZQ) in schistosomiasis mansoni BALB/c mice. Thirty two laboratory bred male BALB/c Swiss albino mice. The mice were classified into four groups (8 mice each), control infected (CI), PZQ (300â¯mg/kg/12â¯h), ART (0.1â¯ml/mg/d) and PAE (50â¯mg/kg/d) treated groups for one month. All mice groups were sacrificed 15 weeks post infection for assessment of the drugs' efficacy by parasitological, histopathological and immunohistochemical studies. Our results in PAE group showed marked reduction in the mean egg count/gram stool, worm burden, egg count/gram liver tissue, granuloma diameter and pro-angiogenic factors as vascular endothelial growth factor (VEGF), Proliferating cell nuclear antigen (PCNA), alpha-smooth muscle actin (α-SMA) and CD34; conversely, there was an augmentation of the tissue inhibitor metalloproteinases-2 (TIMP-2) as an anti-angiogenic expression that was exceeded ART and PZQ treated groups compared to CI group (pË0.001). Conclusively, PAE has an anti-angiogenic impact with no vascular proliferative activity or recanalization, no micro-vessel density (MVD) changes, granuloma resolution and fibrosis regression. PAE is predicted to be a potential therapy for chronic hepatic diseases associated with fibrosis and angiogenesis, hopeful in protecting from advanced serious complications; cancer and metastasis.
Assuntos
Inibidores da Angiogênese/farmacologia , Anti-Helmínticos/farmacologia , Glucosídeos/farmacologia , Monoterpenos/farmacologia , Paeonia/química , Esquistossomose mansoni/tratamento farmacológico , Actinas/efeitos dos fármacos , Actinas/metabolismo , Inibidores da Angiogênese/uso terapêutico , Animais , Anti-Helmínticos/uso terapêutico , Antígenos CD34/efeitos dos fármacos , Antígenos CD34/metabolismo , Artemeter/farmacologia , Artemeter/uso terapêutico , Regulação para Baixo , Fezes/parasitologia , Glucosídeos/uso terapêutico , Imuno-Histoquímica , Fígado/irrigação sanguínea , Fígado/parasitologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Monoterpenos/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/parasitologia , Contagem de Ovos de Parasitas , Praziquantel/farmacologia , Praziquantel/uso terapêutico , Antígeno Nuclear de Célula em Proliferação/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Esquistossomose mansoni/complicações , Esquistossomose mansoni/fisiopatologia , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Purpose/Aim: Intravitreal bevacizumab (Avastin) is an irreversible vascular endothelial growth factor (VEGF) inhibitor used off-label to treat severe retinopathy of prematurity in extremely low gestational age neonates. VEGF and matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) participate in lung maturation. We tested the hypothesis that intravitreal bevacizumab enters the systemic circulation and has long-lasting effects on lung MMPs. MATERIALS AND METHODS: Neonatal rats were exposed to: (1) hyperoxia (50% O2); (2) intermittent hypoxia (IH) (50% O2 with brief episodes of 12% O2); or (3) room air (RA) from birth (P0) to P14. At P14, the time of eye opening in rats, a single dose of Avastin (0.125 mg) was injected into the vitreous cavity of the left eye. A control group received equivalent volume saline. At P23 and P45, lung MMP-2 and MMP-9, and TIMP-1, and TIMP-2 were assessed in the lungs. RESULTS: At P23, Avastin increased MMP-2, MMP-9, and TIMP-1 levels in the hyperoxia group but decreased TIMP-1 levels in the IH group. The ratios of MMP-2/TIMP-1 and MMP-9/TIMP-1 were significantly elevated at P23 in the IH group treated with Avastin. At P45, the levels of MMP-2 and MMP-9 remained elevated in the hyperoxia and IH groups treated with Avastin, while a rebound increase in TIMP-1 levels was noted in the IH group. CONCLUSIONS: Avastin treatment in IH has lasting alterations in the balance between MMPs and their tissue inhibitors. These changes may lead to impaired alveologenesis and tissue damage consistent with bronchopulmonary dysplasia/chronic lung disease.
Assuntos
Bevacizumab/farmacologia , Colagenases/metabolismo , Pulmão/crescimento & desenvolvimento , Alvéolos Pulmonares/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar , Colágeno Tipo IV/metabolismo , Hiperóxia/metabolismo , Hipóxia/metabolismo , Pulmão/enzimologia , Metaloproteinases da Matriz/análise , Metaloproteinases da Matriz/efeitos dos fármacos , Ratos , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/análise , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Fator A de Crescimento do Endotélio VascularRESUMO
Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1ß (IL-1ß)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1ß induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1ß restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1ß restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.
Assuntos
Condrócitos/enzimologia , Gelatinases/efeitos dos fármacos , Interleucina-1beta/farmacologia , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Osteoclastos/fisiologia , Células 3T3 , Animais , Cartilagem Articular/citologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Condrócitos/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/fisiologia , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Monócitos/citologia , NF-kappa B/antagonistas & inibidores , Inibidores de Proteases/análise , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
BACKGROUND: Oral submucous fibrosis (OSF) is a premalignant and fibrosing disease, which is closely associated with the habit of chewing areca nut. Panax notoginseng Buck F. H. Chen is an often used antifibrotic and antitumor agent. To treat areca nut-induced OSF, we have developed a chewable tablet, in which one of the major medicines is total Panax notoginseng saponins (PNS). In this study, we have investigated the antifibrotic effect and mechanism of PNS on areca nut-induced OSF in vitro. METHODS: Through human procollagen gene promoter luciferase reporter plasmid, hydroxyproline assay, gelatin zymography, qRT-PCR, ELISA, and Western blot, the influences of PNS on areca nut extract (ANE)-induced cell growth, collagen accumulation, procollagen gene transcription, MMP-2/-9 activity, MMP-1/-13 and TIMP-1/-2 expression, cytokine secretion, and the activation of PI3K/AKT, ERK/JNK/p38 MAPK, and TGFß/Smads pathways were detected. RESULTS: Panax notoginseng saponins could inhibit the ANE-induced abnormal growth and collagen accumulation of oral mucosal fibroblasts in a concentration-dependent manner. PNS (25 µg/ml) could significantly inhibit the ANE-induced expression of Col1A1 and Col3A1, augment the ANE-induced decrease of MMP-2/-9 activity, inhibit the ANE-induced increase of TIMP-1/-2 expression, and decrease the ANE-induced transcription and release of CTGF, TGFß1, IL-6, and TNFα. PNS (25 µg/ml) also significantly inhibited the ANE-induced activation of AKT and ERK/JNK/p38 MAPK pathways in oral mucosal fibroblasts and the ANE-induced activation of TGFß/smad pathway in HaCaT cells. CONCLUSION: Panax notoginseng saponins possess excellent anti-OSF activity, and its mechanism may be related to its ability to inhibit the ANE-induced activation of PI3K/AKT, ERK/JNK/p38 MAPK, and TGFß/smad pathways.
Assuntos
Areca/efeitos adversos , Mucosa Bucal/efeitos dos fármacos , Nozes/efeitos adversos , Fibrose Oral Submucosa/patologia , Panax notoginseng , Extratos Vegetais/farmacologia , Saponinas/farmacologia , Animais , Técnicas de Cultura de Células , Linhagem Celular , Colágeno Tipo I/efeitos dos fármacos , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo III/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Hidroxiprolina/análise , Interleucina-6/análise , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Bucal/citologia , Fibrose Oral Submucosa/etiologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Extratos Vegetais/efeitos adversos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Smad/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
OBJECTIVE: Chronic isoproterenol treatment causes hypertrophy and hyperplasia of rodent salivary glands. Cell-extracellular matrix interactions play a critical role in salivary gland proliferation and matrix metalloproteinases (MMPs) are known to be involved in cell proliferation. The present study was undertaken to investigate the expression of MMP-2 and the tissue inhibitor metalloproteinase (TIMP)-2 in rat parotid gland following isoproterenol treatment. DESIGN: Female Wistar rats were daily treated with isoproterenol (25mg/kg body weight) for 0, 1, 3, and 7 days. Expression of parotid gland MMP-2 and its tissue inhibitor TIMP-2 was analysed by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Our results suggest that isoproterenol modulates expression of MMP-2 and TIMP-2 mRNAs, as well as their protein expression levels in a time dependent-manner. Interestingly, at day 1 of treatment, MMP-2 and TIMP-2 expression were higher in comparison to untreated gland. At days 3 and 7, we can observe a gradual decrease of mRNA and protein levels of MMP-2 and TIMP-2. CONCLUSIONS: Our results suggest the presence of a isoproterenol-dependent modulation of extracellular matrix components. Such a modulation seems to be associated with ß-adrenergic agonist-induced hyperplasy, occurring during the first 24h of agonist treatment, and hypertrophy of the parotid gland.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Proliferação de Células/efeitos dos fármacos , Isoproterenol/farmacologia , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Glândula Parótida/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Animais , Feminino , Hiperplasia/etiologia , Hipertrofia/etiologia , Imuno-Histoquímica , Metaloproteinase 2 da Matriz/genética , Glândula Parótida/enzimologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-2/genéticaRESUMO
Matrix metalloproteinases and tissue inhibitor of metalloproteinases play an important role in the regulation of mesangial cell proliferation and may be involved in ischemia-reperfusion injuries. Preservation solutions are thought to diminish the ischemic injury and appropriate choice of the solution should guarantee a better graft function and good prognosis for graft survival. The aim of the study was to examine the effect of preservation solutions UW and EC on the expression of matrix metalloproteinase II and tissue inhibitor of metalloproteinase II genes in rat kidney. The study was carried out on Wistar rat kidneys divided into 3 groups: kidneys perfused with 0.9% NaCl (control group), with UW, and with EC preservation solution. The results show an enhancement of MMP-2 and TIMP-2 gene expression after 12 min of cold ischemia. This increase was more expressed in kidneys preserved with UW solution in comparison with kidneys perfused with EC solution and 0.9% NaCl. After 24 h of cold ischemia the expression of MMP-2 and TIMP-2 genes in kidney perfused with UW solution decreased, while in kidneys perfused with EC it was increased. After warm ischemia the MMP-2 and TIMP-2 gene expression increased, whereas it was significantly lower in kidneys perfused with EC solution.
Assuntos
Expressão Gênica/efeitos dos fármacos , Transplante de Rim/métodos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Soluções para Preservação de Órgãos/farmacologia , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/genética , Adenosina/farmacologia , Alopurinol/farmacologia , Animais , Glutationa/farmacologia , Sobrevivência de Enxerto , Insulina/farmacologia , Rim/metabolismo , Masculino , Preservação de Órgãos/métodos , Rafinose/farmacologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/prevenção & controleRESUMO
BACKGROUND AND OBJECTIVE: Inorganic polyphosphate [poly(P)] is a biopolymer found in almost all cells and tissues, and which promotes tissue remodeling. However, there is limited information on how poly(P) affects the connective tissue in inflamed gingiva. This study examined the effects of topical application of poly(P) on gingival connective tissue and its remodeling in a rat periodontitis model. MATERIAL AND METHODS: Male Wistar rats (n = 36, 8 wk of age) were used in this 6-wk study. The rats were divided into six groups of six rats each. The control group received no treatment. In the other groups, periodontitis was ligature-induced for 4 wk. After 4 wk, the rats with periodontitis were further divided into five groups, and were left untreated (periodontitis group) or subjected to topical application of oral rinses containing 0, 0.1, 1 or 5% poly(P) for 2 wk. RESULTS: The periodontitis and 0% poly(P) groups showed a higher density of polymorphonuclear leukocytes and a lower density of collagen in gingival tissue than the control group (p < 0.05). In contrast, groups treated with more than 1% poly(P) exhibited a lower density of polymorphonuclear leukocytes (p < 0.05) and a higher density of collagen than the periodontitis and 0% poly(P) groups (p < 0.05). A higher expression of fibroblast growth factor-2 was observed in the gingiva of rats treated with 1% poly(P) than in those treated with 0% poly(P) (p < 0.05). CONCLUSION: Topical application of poly(P) may induce connective tissue remodeling, contributing to improvement of inflamed gingiva in rats.
Assuntos
Gengiva/efeitos dos fármacos , Periodontite/tratamento farmacológico , Polifosfatos/uso terapêutico , Administração Tópica , Animais , Contagem de Células , Colágeno/efeitos dos fármacos , Tecido Conjuntivo/efeitos dos fármacos , Tecido Conjuntivo/patologia , Modelos Animais de Doenças , Inserção Epitelial/efeitos dos fármacos , Inserção Epitelial/patologia , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Gengiva/enzimologia , Gengiva/patologia , Contagem de Leucócitos , Masculino , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 3 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Antissépticos Bucais/uso terapêutico , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Periodontite/enzimologia , Periodontite/patologia , Polifosfatos/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Wistar , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-3/efeitos dos fármacosRESUMO
Liver fibrosis has been reported to be inhibited in vivo by oleanolic and ursolic acids; however, the activity of other triterpenes like betulin and betulinic acid has not been examined. Butein has also been reported to prevent and partly reverse liver fibrosis in vivo, although its mechanism of action is poorly understood. Therefore, the aim of this study was to determine the antifibrotic potential of butein, betulin, and betulinic acid and examine their mechanisms of action in vitro. This study was conducted in rat stellate cells (HSCs) that were treated with acetaldehyde, which is the most reactive product of ethanol metabolism. Butein, betulin, and betulinic acid were preincubated with rat HSCs at non-toxic concentrations. Treatment effects were measured in regard to acetaldehyde-induced toxicity and cell migration, and several markers of HSC activation were evaluated, including smooth muscle α-actin (α-SMA) and procollagen I expression. In addition, changes in the release of reactive oxygen species (ROS) and cytokines such as tumor necrosis factor-α (TNF-α) and tumor growth factor-ß1 (TGF-ß1) and changes in the production of metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were determined. In vitro, HSCs were protected against acetaldehyde-induced toxicity by betulin but not by betulinic acid and butein. However, butein, betulin, and betulinic acid inhibited the production of ROS by HSCs treated with acetaldehyde and inhibited their migration. Butein also inhibited acetaldehyde-induced TGF-ß1 production. Butein, betulin, and betulinic acid down-regulated acetaldehyde-induced production of TIMP-1 and TIMP-2. Betulin decreased the acetaldehyde-induced activity of MMP-2, but butein and betulinic acid did not. The results indicated that butein, betulin, and betulinic acid inhibited the acetaldehyde-induced activation of HSCs. Each drug functioned in a different manner, whereby some were acting as either antioxidants or inhibitors of TIMPs expression and butein additionally acted as an inhibitor of TGF-ß production.
Assuntos
Chalconas/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Triterpenos/farmacologia , Acetaldeído/toxicidade , Animais , Antioxidantes/farmacologia , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Triterpenos Pentacíclicos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Ácido BetulínicoRESUMO
Statins exert anti-inflammatory effects and downregulate matrix metalloproteinases (MMPs) expression, thus contributing to restore cardiovascular homeostasis in cardiovascular diseases. We aimed at comparing the effects of different statins (simvastatin, atorvastatin, and pravastatin) on MMP-2, MMP-9, tissue inhibitors of metalloproteinases (TIMP)-1, TIMP-2, and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios released by human umbilical vein endothelial cells (HUVEC) stimulated by phorbol myristate acetate (PMA). HUVECs were incubated with statins (0.1-10 µM) for 12 h before stimulation with PMA 100 nM. Monolayers were used to perform cell viability assays and the supernatants were collected to determine MMPs and TIMPs levels by gelatin zymography and/or enzyme immunoassay. While treatment with PMA increased MMP-9 and TIMP-1 levels (by 556% and 159%, respectively; both P < 0.05), it exerted no effects on MMP-2 and TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, attenuated PMA-induced increases in MMP-9 levels (P < 0.05). Only atorvastatin decreased baseline MMP-2 levels significantly (P < 0.05). We found no effects on TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, decreased MMP-9/TIMP-1 ratio significantly (both P < 0.05), whereas atorvastatin and pravastatin, but not simvastatin, decreased MMP-2/TIMP-2 ratio significantly (both P < 0.05). Our data support the notion that statins with different physicochemical features exert variable effects on MMP/TIMP ratios (which reflect net MMP activity). Our results suggest that more lipophilic statins (simvastatin and atorvastatin), but not the hydrophilic statin pravastatin, downregulate net MMP-9 activity. However, atorvastatin and pravastatin may downregulate net MMP-2 activity. The clinical implications of the present findings deserve further investigation.
Assuntos
Ácidos Heptanoicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Pravastatina/farmacologia , Pirróis/farmacologia , Sinvastatina/farmacologia , Atorvastatina , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ácidos Heptanoicos/administração & dosagem , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Pravastatina/administração & dosagem , Pirróis/administração & dosagem , Sinvastatina/administração & dosagem , Acetato de Tetradecanoilforbol/farmacologia , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismoRESUMO
BACKGROUND: Members of the matrix metalloproteinase (MMP) family have been shown to be involved in periodontal disease. Risk factors for periodontal disease include tobacco smoking. Cigarette smoke condensate (CSC) is comprised of thousands of chemicals. Nicotine is one of the active components in tobacco. This study compares the effects of CSC and nicotine at the level in CSC on the collagen-degrading ability of human gingival fibroblasts (HGFs) and the expression of selected MMPs and tissue inhibitors of metalloproteinases (TIMPs). METHODS: HGFs were seeded in six-well collagen-coated plates, exposed to 100 µg/mL (2.4 µg/mL nicotine) of CSC or 2.4 µg/mL nicotine for 3 days, and then collagen degradation was analyzed. After 3 days exposure to CSC or nicotine, the conditioned media from HGFs was collected and the membrane proteins were extracted for gelatin zymography and Western blot analyses. The mRNA levels of MMP-2, MMP-14, and TIMP-2 were measured by reverse transcription-polymerase chain reaction. RESULTS: The CSC increased collagen degradation, and increased the levels of TIMP-2, MMP-14, and the active MMP-2 in the membrane extracts, and their mRNA levels. CSC also increased the level of active MMP-2 in the conditioned media. Nicotine at the level in CSC (2.4 µg/mL) had little influence on collagen degradation, as well as on the protein and mRNA levels of MMP-2, MMP-14, and TIMP-2. CONCLUSIONS: CSC may increase HGF-mediated collagen degradation by affecting membrane-associated MMPs and TIMPs.
Assuntos
Colágeno Tipo I/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Nicotiana , Nicotina/farmacologia , Fumaça , Western Blotting , Células Cultivadas , Colágeno Tipo I/análise , Misturas Complexas/farmacologia , Meios de Cultivo Condicionados , Meios de Cultura Livres de Soro , Ciclofilinas/análise , Ciclofilinas/efeitos dos fármacos , Precursores Enzimáticos/análise , Precursores Enzimáticos/efeitos dos fármacos , Fibroblastos/metabolismo , Gelatinases/análise , Gelatinases/efeitos dos fármacos , Gengiva/citologia , Humanos , Metaloproteinase 14 da Matriz/análise , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz Associadas à Membrana/análise , Metaloproteinases da Matriz Associadas à Membrana/efeitos dos fármacos , Inibidores de Proteases/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-2/análise , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/análise , Inibidores Teciduais de Metaloproteinases/efeitos dos fármacosRESUMO
The activation of the EGFR (epidermal growth factor receptor) signalling pathway is one of the key mechanisms underlying the development of resistance to tamoxifen in breast cancer patients. As EGCG [(-)-epigallocatechin-3-gallate], the most active catechin present in green tea, has been shown to down-regulate EGFR, we studied the effects of 10-100 µg/ml EGCG treatment on growth and invasion in a breast carcinoma cell line resistant to tamoxifen [MCF-7Tam (MCF-7 breast carcinoma cell line resistant to tamoxifen) cells] and parental MCF-7. A dose-dependent down-regulation of EGFR mRNA expression and protein level occurred after 50 µg/ml EGCG treatment of MCF-7Tam cells. EGFR molecules on the plasma membrane surface of MCF-7Tam cells significantly decreased. EGFR phosphorylation (Tyr-992, Tyr-1045 and Tyr-1068) was higher in MCF-7Tam than in MCF-7 and it was reduced by EGCG treatment. ERK (extracellular regulated kinase) and phospho-ERK p42/44 were also down-regulated by EGCG treatment and in vitro cell growth and invasion decreased. MMP-2 (matrix metalloproteinase-2) and MMP-9, which are implicated in cell invasion and metastasis, and EMMPRIN (extracellular matrix metalloproteinase inducer), a glycoprotein able to activate MMPs, were significantly reduced after 50 µg/ml EGCG treatment. In keeping with this, TIMP-1 (tissue inhibitor of metalloproteinases-1) and TIMP-2, which down-regulate MMPs, increased after EGCG treatment. Altogether, the present data demonstrated that EGCG could attenuate the tamoxifen-resistant phenotype of MCF-7Tam cells. EGCG could stop MCF-7Tam cell growth and in vitro invasion through down-regulation of EGFR and other molecules implicated in aggressive biological behaviour. The present data support the hypothesis that EGCG is an interesting molecule to be investigated in tamoxifen-resistant breast carcinoma.
Assuntos
Basigina/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Catequina/análogos & derivados , Receptores ErbB/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Basigina/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Expressão Gênica , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tamoxifeno/farmacologia , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/genéticaRESUMO
PURPOSE: Tumor necrosis factor-alpha (TNF-α) may disrupt the extracellular matrix components comprising the blood-retinal barrier (BRB) in patients with posterior uveitis, such as Behçet's disease. We investigated changes in the mRNA expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human BRB cells in the presence of TNF-α in vitro and examined the effect of infliximab addition. METHODS: Cells were cultured in the presence or absence of TNF-α, and TNF-α-exposed cells were treated with or without infliximab. We measured the expression levels of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 mRNA in human retinal microvascular endothelial ACBRI181 cells and retinal pigment epithelial ARPE-19 cells by real-time polymerase chain reaction. The cell-derived proteins degraded by MMP were observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: Expression of MMP-3 increased and TIMP-1 decreased in the presence of 10 ng/mL TNF-α in ACBRI181 cells. Expression of MMP-1 increased and TIMP-2 decreased in the presence of 10 ng/mL TNF-α in ARPE-19 cells. These altered levels of expression were reversed by the addition of infliximab. The cell-derived proteins degraded by MMP-1 and -3 were detected in each set of cells. CONCLUSIONS: The presence of TNF-α altered expression of MMPs and TIMPs in cells comprising the BRB, and infliximab counteracted this alteration.
Assuntos
Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Barreira Hematorretiniana/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Barreira Hematorretiniana/metabolismo , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Infliximab , Metaloproteinases da Matriz/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
BACKGROUND: Bone morphogenetic protein (BMP)-7 is a potent bone-inducing factor and was shown to promote periodontal regeneration in vivo and in vitro; however, to our knowledge, the specific effect of BMP-7 on cementoblasts has not been defined. We aimed to investigate the effects of BMP-7 on cementoblasts, which are cells responsible for tooth root-cementum formation. We hypothesized that BMP-7 would regulate mineralized tissue-associated genes in cementoblasts and influence the expression profile of genes associated with cementoblast extracellular matrix (ECM) and cell adhesion molecules (CAMs). METHODS: A murine immortalized cementoblast cell line (OCCM.30) was cultured with and without 50 ng/ml BMP-7. After 72 hours, total RNA was isolated, and mRNA levels for bone/cementum markers, including bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor-2 (Runx2), were investigated by real-time quantitative reverse transcription-polymerase chain reaction (Q-PCR). In vitro mineral nodule formation was assayed on day 8 using von Kossa staining. A pathway-specific gene-expression array was used to determine BMP-7-responsive ECM and CAM genes in cementoblasts. RESULTS: Mineralized tissue markers were strongly regulated by BMP-7, with an almost three-fold increase in BSP and OCN transcripts and significant increases in OPN and Runx2 mRNA expressions. BMP-7 treatment markedly stimulated cementoblast-mediated biomineralization in vitro compared to untreated cells at day 8. BMP-7 treatment altered the OCCM.30 expression profile for ECM and CAM functional gene groups. BMP-7 tended to increase the expression of collagens and matrix metalloproteinases (MMPs), mildly decreased tissue inhibitors of MMPs (TIMPs), and had mixed regulatory effects on integrins. Using Q-PCR, selected array results were confirmed, including a significant BMP-7-induced increase in MMP-3 and a decrease in TIMP-2 mRNA expression. CONCLUSION: These results support the promising applications of BMP-7 in therapies aimed at regenerating periodontal tissues lost as a consequence of disease.
Assuntos
Proteína Morfogenética Óssea 7/farmacologia , Cemento Dentário/efeitos dos fármacos , Animais , Calcificação Fisiológica/efeitos dos fármacos , Moléculas de Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colágeno/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Cemento Dentário/citologia , Proteínas da Matriz Extracelular/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Integrinas/efeitos dos fármacos , Metaloproteinase 3 da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/efeitos dos fármacos , Camundongos , Osteocalcina/efeitos dos fármacos , Osteopontina/efeitos dos fármacos , Análise Serial de Proteínas , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/efeitos dos fármacos , Raiz Dentária/citologia , Raiz Dentária/efeitos dos fármacos , Regulação para CimaRESUMO
BACKGROUND AND OBJECTIVES: In this study, we investigated the effect of basic fibroblast growth factor (bFGF) and dexamethasone (Dex) on mRNA expressions of collagen (COL) type I, III and X, matrix metalloproteinases (MMP)-1, -2, -3 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2, and also on mineralization and morphology of periodontal ligament (PDL) cells. MATERIAL AND METHODS: Periodontal ligament cells were obtained from premolar teeth extracted for orthodontic reasons. Periodontal ligament cells were cultured with Dulbecco's modified Eagle's medium containing: (1) 5% fetal bovine serum (FBS); (2) 5% FBS + ascorbic acid (AA, 50 microg/mL); (3) 5% FBS + Dex (10(-7) m) + AA; (4) 5% FBS + bFGF (10 ng/mL) + AA; or (5) 5% FBS + Dex (10(-7) m) + bFGF + AA. Cells within each group were evaluated for gene expression profile using semi-quantitative reverse transcriptase-polymerase chain reaction for COL I, III and X, MMP-1, -2, -3 and -9 and TIMP-1 and -2 on days 14 and 21 and for biomineralization by von Kossa stain in vitro on day 21. Images of PDL cells were examined using a phase contrast microscope. RESULTS: Basic fibroblast growth factor stimulated MMP-1, MMP-3 and MMP-9 mRNA expressions and inhibited TIMP-2 mRNA expression. Treatment of cells with Dex + bFGF led to downregulation of MMP-1, MMP-3 and MMP-9 transcripts. Whilst AA alone and Dex alone induced biomineralization of PDL cells, bFGF blocked the mineralization activity of the cells. In the Dex + bFGF group, more mineral nodules were noted when compared to AA alone and Dex alone groups. CONCLUSION: The addition of Dex to culture reversed bFGF-mediated inhibition of mineralization. Use of combined bFGF and Dex to regulate PDL cell function may be a good therapeutic option to obtain periodontal regeneration.
Assuntos
Dexametasona/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glucocorticoides/farmacologia , Metaloproteinases da Matriz/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo III/efeitos dos fármacos , Colágeno Tipo X/efeitos dos fármacos , DNA/efeitos dos fármacos , Regulação para Baixo , Perfilação da Expressão Gênica , Humanos , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 3 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Ligamento Periodontal/citologia , RNA Mensageiro/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacosRESUMO
It is well known that low-molecular weight hyaluronan (HA) is detected in human periodontal tissue under inflammatory conditions. HA oligosaccharide (HAoligo) has been demonstrated to induce matrix metalloproteinase (MMP)-1 expression in dendritic cells and chondrocytes, however, the bioactivities of HAoligo in periodontal ligament (PDL) cells remain unclear. In this study, we investigated the effect of HAoligo on MMP-1 expression in human PDL (HPDL) cells and the mechanisms in terms of the signal transmission. HAoligo was generated and purified from commercial human umbilical cord HA. HPDL cells were isolated from healthy ligaments, and cultured with HAoligo for 0-24h. The expression of MMP-1, tissue inhibitor of MMPs (TIMP)-1 and TIMP-2 was analyzed by real-time PCR and Western blot analyses. Effects of specific inhibitors of p38 mitogen-activated protein kinase (MAPK) activity, MAPK kinase activity and NFkB activity on HAoligo-induced MMP-1 expression in HPDL cells were also investigated by real-time PCR and Western blot analyses. HAoligo remarkably enhanced MMP-1 expression in both mRNA and protein levels, but no effect was shown on the expression of TIMP-1 and TIMP-2 mRNAs. Inhibition of p38MAPK activity decreased the MMP-1 expression. Neither inhibition of NFkB nor MAPKK activity affected the MMP-1 expression. It was suggested that HAoligo induces MMP-1 expression in HPDL cells, and p38MAPK plays a crucial role in signal transduction for MMP-1 inducted by HAoligo.
Assuntos
Ácido Hialurônico/farmacologia , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Oligossacarídeos/farmacologia , Ligamento Periodontal/enzimologia , Anti-Inflamatórios/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Metaloproteinase 1 da Matriz/análise , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , NF-kappa B/análise , NF-kappa B/antagonistas & inibidores , NF-kappa B/efeitos dos fármacos , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Piridinas/farmacologia , Sesquiterpenos/farmacologia , Sesquiterpenos de Guaiano , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/análise , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/análise , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacosRESUMO
Recently, aspirin has been shown to alleviate matrix metalloproteinase (MMP) expression, but the underlying mechanism is still unclear. In this study, the effects of aspirin on oxidative low-density lipoprotein (ox-LDL)-stimulated human monocyte-derived macrophages were examined. Following treatment of cells with aspirin, MMP-2 and MMP-9 expression and release were significantly reduced. Moreover, expression of peroxisome proliferator-activated receptors (PPARs) alpha and gamma was markedly enhanced. The effect of PPAR inhibitors on MMP levels in aspirin-treated cells was examined. RT-PCR and ELISA assays showed that inhibition of MMP-9 levels by aspirin was notably alleviated by PPAR antagonists. Interestingly, expression of nuclear factor (NF)- kappaB was also decreased by aspirin. RT-PCR study also indicated that aspirin could upregulate the expression of tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2. In all of these studies, lower dosage (50 or 100 microg/ml) exerted the best effect. These results demonstrate that aspirin could inhibit MMP-2 and MMP-9 expression through upregulation of PPARalpha/gamma expression in ox-LDL-stimulated macrophages, and could potentially inhibit MMP-2 and MMP-9 activity by induction of TIMP-1 and TIMP-2 expression. This finding may demonstrate a novel pharmacological effect of aspirin protecting against atherosclerotic plaque rupture.
Assuntos
Aspirina/farmacologia , Lipoproteínas LDL/efeitos dos fármacos , Macrófagos/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Aspirina/administração & dosagem , Linhagem Celular , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Lipoproteínas LDL/metabolismo , Macrófagos/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , PPAR alfa/efeitos dos fármacos , PPAR alfa/metabolismo , PPAR gama/efeitos dos fármacos , PPAR gama/metabolismo , Inibidores da Agregação Plaquetária/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress, we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201+/-5 vs. 163+/-7 mm Hg in diabetic rats untreated and treated with lercanidipine, respectively; P<0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P<0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P<0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P<0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2/TIMP-2 expression.
Assuntos
Antioxidantes/farmacologia , Diabetes Mellitus Experimental/complicações , Di-Hidropiridinas/farmacologia , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Aloxano , Animais , Anti-Hipertensivos/farmacologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Diabetes Mellitus Experimental/fisiopatologia , Precursores Enzimáticos/efeitos dos fármacos , Precursores Enzimáticos/metabolismo , Fluorometria , Gelatinases/efeitos dos fármacos , Gelatinases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
Remodeling of the pulmonary artery is a major feature of pulmonary artery hypertension, and CPU86017, a derivative of berberine, is known to effectively alleviate hypoxic pulmonary hypertension (HPH). CPU86017 is a racemate, possessing two chiral centers: 7N and 13aC. We have compared the effects of four CPU86017 isomers, SS [(+)-7S, 13aS-CPU86017], SR [(-)-7S, 13aR-CPU86017], RR [(-)-7R, 13aR-CPU86017] and RS [(+)-7R, 13aS-CPU86017], on HPH. Sprague-Dawley rats were exposed to hypoxic conditions (10 +/- 0.5% O2 for 8 h per day) for 4 weeks and treated with CPU86017, SS, SR, RR or RS (4 mg/kg, subcutaneously) from day 15 to 28. After 4 weeks of exposure to hypoxia, remodeling of the right ventricle and the small pulmonary arteries (<150 microm) was very pronounced, and extra-cellular matrix (ECM) had been excessively produced in association with abnormal mRNA and protein expression of matrix metalloproteinase 9 (MMP9) and mRNA of tissue inhibitor of matrix metalloproteinase 1 and 2 (TIMP1, TIMP2). Expression of endothelin receptor A was upregulated, while that connexin 40 was downregulated. The administration of CPU86017 and its four isomers attenuated the changes, with the isomer RS exhibiting the most favorable effect on HPH rats. We propose that an activated endothelin pathway associated with an unbalanced MMP-TIMP system may contribute to the over-accumulation of ECM and the remodeling of the pulmonary arterioles in HPH. CPU86017 and its four isomers attenuate ECM accumulation and vascular remodeling by normalizing both the MMP-TIMP system and the ET system. The RS isomer is superior to the racemate CPU86017 in attenuating HPH.
Assuntos
Berberina/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Receptor de Endotelina A/efeitos dos fármacos , Animais , Berberina/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Hipóxia/complicações , Masculino , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A/metabolismo , Estereoisomerismo , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Remodelação Ventricular/efeitos dos fármacosRESUMO
Clinical studies have indicated increased gelatinase activity in the airways of patients suffering from chronic obstructive pulmonary disease caused by tobacco smoke. The present study aimed to determine whether acute exposure to tobacco smoke per se causes a substantial and lasting impact on gelatinases and their inhibitors in the peripheral airways of atopic and nonatopic human subjects. Bronchoscopy with bronchoalveolar lavage (BAL) was performed on occasional smokers with and without atopy before and after smoking 10 cigarettes over a 48-h period. Samples from a group of never-smokers not exposed to tobacco smoke served as controls. Gelatinase identity and activity were measured using zymography, and gelatinase activity assay and concentrations of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of MMP (TIMP)-1 and TIMP-2 were measured using ELISA. The results revealed no pronounced changes in identity, net activity or concentration of the gelatinases or changes in concentrations of TIMP-1 and TIMP-2 in BAL fluid before and after acute exposure to tobacco smoke. In conclusion, the present experimental study indicates that acute exposure to tobacco smoke does not cause any substantial impact on gelatinases or their inhibitors in the peripheral airways, irrespective of atopy status, a finding that is compatible with the fact that it takes many years of tobacco smoking to establish chronic obstructive pulmonary disease.