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1.
Braz. j. med. biol. res ; 54(3): e9206, 2021. graf
Article in English | LILACS | ID: biblio-1153519

ABSTRACT

Renal fibrosis is one of the most significant pathological changes after ureteral obstruction. Transforming growth factor-β (TGF-β) signaling pathway plays essential roles in kidney fibrosis regulation. The aims of the present study were to investigate effects of microRNA-302b (miR-302b) on renal fibrosis, and interaction between miR-302b and TGF-β signaling pathway in murine unilateral ureteral obstruction (UUO) model. Microarray dataset GSE42716 was downloaded by retrieving Gene Expression Omnibus database. In accordance with bioinformatics analysis results, miR-302b was significantly down-regulated in UUO mouse kidney tissue and TGF-β1-treated HK-2 cells. Masson's trichrome staining showed that miR-302b mimics decreased renal fibrosis induced by UUO. The increased mRNA expression of collagen I and α-smooth muscle actin (α-SMA) and decreased expression of E-cadherin were reversed by miR-302b mimics. In addition, miR-302b up-regulation also inhibited TGF-β1-induced epithelial mesenchymal transition (EMT) of HK-2 cells by restoring E-cadherin expression and decreasing α-SMA expression. miR-302b mimics suppressed both luciferase activity and protein expression of TGF-βR2. However, miR-302b inhibitor increased TGF-βR2 luciferase activity and protein expression. Meanwhile, miR-302b mimics inhibited TGF-βR2 mRNA expression and decreased Smad2 and Smad3 phosphorylation in vivo and in vitro. Furthermore, over-expression of TGF-βR2 restored the miR-302b-induced decrease of collagen I and α-SMA expression. In conclusion, this study demonstrated that miR-302b attenuated renal fibrosis by targeting TGF-βR2 to suppress TGF-β/Smad signaling activation. Our findings showed that elevating renal miR-302b levels may be a novel therapeutic strategy for preventing renal fibrosis.


Subject(s)
Humans , Animals , Rats , Ureteral Obstruction/pathology , Signal Transduction , Transforming Growth Factor beta/metabolism , MicroRNAs/genetics , Smad Proteins , Kidney Diseases/genetics , Fibrosis , Cell Line , Epithelial-Mesenchymal Transition , Kidney/pathology , Kidney Diseases/pathology
2.
Article in English | WPRIM | ID: wpr-827479

ABSTRACT

OBJECTIVE@#To investigate the inhibitory effect of Linggui Zhugan Decoction (LZD, ) on the ventricular remodeling (VR) after acute myocardial infarction (AMI) and related mRNA and proteins expression in transforming growth factor-beta 1 (TGF-β)/Smad signaling pathway, and explain its putative mechanism.@*METHODS@#A VR model was generated by ligation of coronary artery in mice. Two weeks after surgery, 60 mice were randomly divided into the model group, the sham-operation group (distilled water), the positive control group (2.4 mg/kg simvastatin), and the low-, medium- and high-dose LZD groups (2.1, 4.2, 8.4 g crude drug/kg, respectively) by a random number table, 10 mice in each group. Mice in each group was treated for 4 weeks. Changes of hemodynamics indices and cardiac weight index were detected by the PowerLab data acquisition and analysis recording instrument. Morphology changes of myocardial tissue were observed by hematoxylin-eosin and Masson staining. The expressions of TGF-β, Smad2, Smad3, p-Smad2 and p-Smad3 in myocardial tissue were detected by Western blotting. The mRNA expressions of TGF-β, Smad2 and Smad3 were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expressions of matrix metalloprotein 2 (MMP2), MMP9, collagen I and collagen III were observed by immunohistochemical methods.@*RESULTS@#VR mice showed significant dysfunction in hemodynamic indices and cardiac structure and function. Compared with the shamoperation group, myocardial tissue damage, interstitial fibrosis occurred in the model mice, left ventricular systolic pressure (LVSP), left ventricular pressure maximum contraction rate (+dp/dt) and left ventricular pressure maximum relaxation rate (-dp/dt) decreased significantly (all P<0.01), while left ventricular end-diastolic pressure (LVEDP), cardiac weight index and left ventricular weight index elevated significantly, meanwhile TGF-β, p-Smad2, p-Smad3, Smad2, Smad3, MMP2, MMP9, collagen I, collagen III protein expressions in myocardial tissue and TGF-β, Smad2 and Smad3 mRNA expressions increased significantly (all P<0.01). Compared with the model group, LZD could significantly improve the pathological changes of myocardial tissue, increase LVSP, +dp/dtmax and -dp/dtmax, lower LVEDP, reduce the whole heart weight index and left ventricular weight index and inhibit the over-expressions of TGF-β, p-Smad2, p-Smad3, Smad2, Smad3, MMP2, MMP9, collagen I and collagen III proteins in myocardial tissue and mRNA expressions of TGF-β, Smad2 and Smad3 (P<0.05 or P<0.01).@*CONCLUSION@#LZD can significantly suppress VR induced by AMI, and its underlying mechanism may be associated with its inhibitory effect on the TGF-β1/Smad signaling pathway.


Subject(s)
Animals , Disease Models, Animal , Male , Myocardial Infarction , Plant Extracts , Pharmacology , Rats, Sprague-Dawley , Smad Proteins , Metabolism , Transforming Growth Factor beta1 , Metabolism , Ventricular Remodeling
3.
Article in English | WPRIM | ID: wpr-719618

ABSTRACT

BACKGROUND: Transforming growth factor β (TGF-β), retinoic acid (RA), p38 mitogen-activated protein kinase (MAPK), and MEK signaling play critical roles in cell differentiation, proliferation, and apoptosis. We investigated the effect of RA and the role of these signaling molecules on the phosphorylation of Smad2/3 (p-Smad2/3) induced by TGF-β1. METHODS: A549 epithelial cells and CCD-11Lu fibroblasts were incubated and stimulated with or without all-trans RA (ATRA) and TGF-β1 and with MAPK or MEK inhibitors. The levels of p-Smad2/3 were analyzed by western blotting. For animal models, we studied three experimental mouse groups: control, bleomycin, and bleomycin+ATRA group. Changes in histopathology, lung injury score, and levels of TGF-β1 and Smad3 were evaluated at 1 and 3 weeks. RESULTS: When A549 cells were pre-stimulated with TGF-β1 prior to RA treatment, RA completely inhibited the p-Smad2/3. However, when A549 cells were pre-treated with RA prior to TGF-β1 stimulation, RA did not completely suppress the p-Smad2/3. When A549 cells were pre-treated with MAPK inhibitor, TGF-β1 failed to phosphorylate Smad2/3. In fibroblasts, p38 MAPK inhibitor suppressed TGF-β1-induced p-Smad2. In a bleomycin-induced lung injury mouse model, RA decreased the expression of TGF-β1 and Smad3 at 1 and 3 weeks. CONCLUSION: RA had inhibitory effects on the phosphorylation of Smad induced by TGF-β1 in vitro, and RA also decreased the expression of TGF-β1 at 1 and 3 weeks in vivo. Furthermore, pre-treatment with a MAPK inhibitor showed a preventative effect on TGF-β1/Smad phosphorylation in epithelial cells. As a result, a combination of RA and MAPK inhibitors may suppress the TGF-β1-induced lung injury and fibrosis.


Subject(s)
Animals , Apoptosis , Bleomycin , Blotting, Western , Cell Differentiation , Epithelial Cells , Fibroblasts , Fibrosis , In Vitro Techniques , Lung Injury , Mice , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Models, Animal , p38 Mitogen-Activated Protein Kinases , Phosphorylation , Protein Kinases , Smad Proteins , Transforming Growth Factor beta , Transforming Growth Factors , Tretinoin
4.
Article in Chinese | WPRIM | ID: wpr-777509

ABSTRACT

Hepatic fibrosis is a liver damage healing response affected by a variety of factors; its formation is associated with multiple cytokines and a variety of signaling pathways. Transforming growth factor beta1( TGF-β1) is one of the strongest fibrosis cytokines known,and involves almost all the key links in hepatic fibrosis. TGF-β1/Smads signal pathway is the most classical pathway for TGF-β1 to play its role in promoting fibrosis as well as one of the most important signaling pathways of hepatic fibrosis formation. Studies for the signal pathway have made a series of scientific research achievements in recently years. Traditional Chinese medicine has the advantages of " multiple ingredients,multiple targets and less side effects",and is widely used in the clinical treatment of hepatic fibrosis.Effective components of traditional Chinese medicine are monomer compounds,which are extracted and purified from traditional Chinese medicine. Nowadays,the molecular biology studies of effective traditional Chinese medicine have become a hotspot. Modern advanced technology and methods can be used to directly clarify the targets and the signaling pathways,reveal the mechanism of traditional Chinese medicine in treating diseases,and promote the modernization and international development of traditional Chinese medicine industry. This review summarized the structure,function and application of TGF-β1/Smads signaling pathway in the progress of anti-hepatic fibrosis,and analyzed the action mode and possible mechanism of various effective components of traditional Chinese medicine in regulating TGF-β1/Smads signaling pathway and intervening the treatment of hepatic fibrosis in the past five years,so as to put forward new ideas for innovating new targeted traditional Chinese medicine for hepatic fibrosis.


Subject(s)
Animals , Drugs, Chinese Herbal , Liver Cirrhosis , Medicine, Chinese Traditional , Signal Transduction , Smad Proteins , Transforming Growth Factor beta1
5.
Article in Chinese | WPRIM | ID: wpr-772681

ABSTRACT

OBJECTIVE@#The purpose of this study is to investigate the potential effects of sclerostin (SOST) on the biological funtions and related mechanisms of cementoblasts under mechanical stress.@*METHODS@#OCCM-30 cells were treated with varying doses of SOST (0, 25, 50, and 100 ng·mL⁻¹) and were loaded with uniaxial compressive stress (2 000 μ strain with a frequency of 0.5 Hz) for six hours. Western blot was utilized to detect the expressions of β-catenin, p-smad1/5/8, and smad1/5/8 proteins. Alkaline phosphatase (ALP) activity was determined, and reverse transcription polymerase chain reaction was used to measure the expressions of runt-related transcription factor 2 (Runx-2), osteocalcin (OCN), bone sialoproteins (BSP), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) mRNA.@*RESULTS@#The expression of p-smad
1/5/8 was significantly downregulated with increasing SOST. β-catenin and smad1/5/8 exhibited no difference. ALP activity decreased under mechanical compressive stress with increasing SOST concentrations. Runx-2 expression was reduced with increasing SOST concentrations, and a similar trend was observed for the BSP and OCN expressions. When the SOST concentration was enhanced, RANKL expression gradually increased, whereas the expression of OPG decreased.@*CONCLUSIONS@#Under mechanical comprehensive stress, SOST can adjust the bone morphogenetic protein (BMP) /smad signal pathway. Osteosclerosis inhibits the mineralization of cementoblasts under mechanical compressive stress, which may be achieved by inhibiting the expressions of osteogenesis factors (Runx2, OCN, BSP, and others) and by promoting the ratio of cementoclast-related factors (RANKL/OPG) through BMP signal pathways.


Subject(s)
Bone Morphogenetic Proteins , Metabolism , Core Binding Factor Alpha 1 Subunit , Dental Cementum , Osteocalcin , Smad Proteins , Metabolism , Stress, Mechanical
6.
Braz. j. med. biol. res ; 52(10): e8324, 2019. tab, graf
Article in English | LILACS | ID: biblio-1039243

ABSTRACT

The aim of this study was to investigate the role of kinase-insert domain-containing receptor (KDR) in intrauterine adhesions (IUA) and its mechanism. The Case group consisted of 92 patients diagnosed with IUA, and the Control group included 86 patients with uterine septum who had normal endometrium verified with an uteroscope. In addition, 50 rats were randomly assigned into Control, Sham, Model, NC-siRNA, and KDR-siRNA groups. Rats in the Model, NC-siRNA, and KDR-siRNA groups were induced by uterine curettage and lipopolysaccharide (LPS) treatment to establish the IUA model. Then, immunohistochemistry was applied for detection of VEGF and KDR expression, HE staining was used for observation of the endometrial morphology and gland counting, Masson staining for measurement of the degree of endometrial fibrosis, and qRT-PCR and western blot for the expression of KDR, VEGF, MMP-9, as well as TGF-β1/Smads pathway-related proteins. Compared with the Control group, the mRNA and protein expressions of KDR were significantly higher in IUA endometrial tissues, and the expression of KDR was positively correlated to the severity of IUA. In addition, the injection of si-KDR increased the number of endometrial glands, reduced the area of fibrosis, inhibited mRNA and protein expression of KDR and VEGF, up-regulated the expression of MMP-9 and Smad7, and decreased the expression level of TGF-β1, p-Smad2, p-Smad3, and Smad4 in rats with IUA. Highly-expressed KDR was related to patients' severity of IUA, and silencing KDR may prevent the occurrence and development of IUA via TGF-β1/Smads signaling pathway and up-regulating the expression of MMP-9.


Subject(s)
Humans , Animals , Female , Adult , Middle Aged , Rats , Young Adult , Uterine Diseases/metabolism , Signal Transduction , Tissue Adhesions/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Transforming Growth Factor beta1/metabolism , Uterine Diseases/pathology , Severity of Illness Index , Immunohistochemistry , Case-Control Studies , Tissue Adhesions/pathology , Blotting, Western , Rats, Wistar , Vascular Endothelial Growth Factor Receptor-2/genetics , Disease Models, Animal , Smad Proteins/genetics , Smad Proteins/metabolism , Transforming Growth Factor beta1/genetics , Real-Time Polymerase Chain Reaction
7.
Article in Chinese | WPRIM | ID: wpr-772124

ABSTRACT

OBJECTIVE@#To evaluate the efficacy of rapmycin for treatment of experimental autoimmune encephalomyelitis (EAE) in mice and explore the underlying mechanism.@*METHODS@#An EAE model was established in C57BL/6 mice. After immunization, the mice were divided into model group and rapamycin groups treated daily with low-dose (0.3 mg/kg) or high-dose (1 mg/kg) rapamycin. The clinical scores of the mice were observed using Knoz score, the infiltration of IL-17 cells in the central nervous system (CNS) was determined using immunohistochemistry; the differentiation of peripheral Treg cells was analyzed using flow cytometry, and the changes in the levels of cytokines were detected with ELISA; the changes in the expressions of p-Smad2 and p- smad3 were investigated using Western blotting.@*RESULTS@#High-dose rapamycin significantly improved the neurological deficits scores of EAE mice. In high-dose rapamycin group, the scores in the onset stage, peak stage and remission stage were 0.14±0.38, 0.43±1.13 and 0.14±0.37, respectively, as compared with 1.14±0.69, 2.14±1.06 and 2.2±0.75 in the model group. The infiltration of inflammatory IL-17 cells was significantly lower in high-dose rapamycin group than in the model group (43±1.83 153.5±7.02). High-dose rapamycin obviously inhibited the production of IL-12, IFN-γ, IL-17 and IL-23 and induced the anti-inflammatory cytokines IL-10 and TGF-β. The percentage of Treg in CD4+ T cells was significantly higher in high- dose rapamycin group than in the model group (10.17 ± 0.68 3.52 ± 0.32). In the experiment, combined treatments of the lymphocytes isolated from the mice with rapamycin and TGF-β induced a significant increase in the number of Treg cells (13.66±1.89) compared with the treatment with rapamycin (6.23±0.80) or TGF-β (4.87±0.85) alone. Rapamycin also obviously up-regulated the expression of p-Smad2 and p-Smad3 in the lymphocytes.@*CONCLUSIONS@#Rapamycin can promote the differentiation of Treg cells by up-regulating the expression of p-Smad2 and p-smad3 to improve neurological deficits in mice with EAE.


Subject(s)
Animals , Anti-Inflammatory Agents , Therapeutic Uses , Cell Differentiation , Encephalomyelitis, Autoimmune, Experimental , Drug Therapy , Metabolism , Interferon-gamma , Metabolism , Interleukins , Metabolism , Lymphocytes , Cell Biology , Mice , Mice, Inbred C57BL , Sirolimus , Therapeutic Uses , Smad Proteins , Metabolism , T-Lymphocytes, Regulatory , Cell Biology , Transforming Growth Factor beta , Metabolism , Up-Regulation
8.
Article in English | WPRIM | ID: wpr-740115

ABSTRACT

TGF-β signaling plays a tumor suppressive role in normal and premalignant cells but promotes tumor progression during the late stages of tumor development. The TGF-β signaling pathway is tightly regulated at various levels, including transcriptional and post-translational mechanisms. Ubiquitination of signaling components, such as receptors and Smad proteins is one of the key regulatory mechanisms of TGF-β signaling. Tripartite motif (TRIM) family of proteins is a highly conserved group of E3 ubiquitin ligase proteins that have been implicated in a variety of cellular functions, including cell growth, differentiation, immune response, and carcinogenesis. Recent emerging studies have shown that some TRIM family proteins function as important regulators in tumor initiation and progression. This review summarizes current knowledge of TRIM family proteins regulating the TGF-β signaling pathway with relevance to cancer.


Subject(s)
Carcinogenesis , Humans , Smad Proteins , Transforming Growth Factor beta , Ubiquitin , Ubiquitin-Protein Ligases , Ubiquitination
9.
Article in Chinese | WPRIM | ID: wpr-771465

ABSTRACT

OBJECTIVE@#To investigate the effect of acupuncture on TGF-β1/Smads signaling pathway in the lung tissue of mice with airway remodeling.@*METHODS@#Thirty specific pathogen-free mice were randomly divided into blank group, model group and acupuncture group (=10). Mouse models of asthma were established in the model group and the acupuncture group, and the mice in the latter group received 7 acupuncture therapies (at bilateral Fei Shu, Da Zhui and Zu Sanli, 20 min each time) every other day, starting on the 10th day after the modeling. At 24 h after the last acupuncture, the mice were subjected to inhalation of 1% OVA for 3 days, and 24 h after the last challenge, the mice were given methacholine chloride (Mch) inhalation at different concentrations for measurement of lung resistance using a noninvasive stroke volume meter. HE staining was used to observe the pathological changes in the lung tissues, and TGF-β1 levels in the the bronchoalveolar lavage fluid (BALF) and serum were detected using ELISA; Western blotting was used to detect the differential protein expressions in the airway smooth muscles between the two groups. The airway smooth muscle cells were isolated from the mice in the acupuncture group and treated with a TGF- β1 inhibitor (LY2157299), and the relative expressions of type-Ⅰ and Smads proteins were detected using Western blotting.@*RESULTS@#The mice in the model showed obvious tracheal fistula with airway pathologies including lumen narrowing, bronchial mucosa thickening, dissociation of the epithelial cells, and thickening of the alveolar septum and airway smooth muscles. These pathological changes were obviously milder in the acupuncture group. The asthmatic mice exhibited significantly increased lung resistance in positive correlation with Mch concentration. Serum TGF-β1 level was significantly elevated in asthmatic mice ( < 0.05); TGF-β1 levels in the serum and BALF were significantly lower in the acupuncture group than in the model group ( < 0.05). In the model group, the expressions of -SMA, TGF-β1 and Smads in the airway smooth muscles were significantly higher than those in the other two groups (both < 0.05). In cultured airway smooth muscle cells, the expressions of type-Ⅰ and Smads were significantly higher in cells treated with LY2157299 than in the control cells (>0.05).@*CONCLUSIONS@#Acupuncture can inhibit airway remodeling by inhibiting the expression of airway TGF-β1 and down-regulating the expression of Smads and -SMA to reduce airway inflammatory response. Airway expressions of type-Ⅰ and Smads proteins remain high after inhibiting TGF-β1. Acupuncture may control asthma progression through the TGF-β1/Smads pathway.


Subject(s)
Acupuncture Points , Acupuncture Therapy , Airway Remodeling , Airway Resistance , Animals , Asthma , Metabolism , Pathology , Therapeutics , Bronchi , Pathology , Disease Progression , Lung , Metabolism , Mice , Muscle, Smooth , Random Allocation , Smad Proteins , Metabolism , Transforming Growth Factor beta1 , Metabolism
10.
Article in Chinese | WPRIM | ID: wpr-773790

ABSTRACT

OBJECTIVES@#Stably expressed transforming growth factor -beta 1(TGF-β1)MCs were obtained and the effects of centellaasiatica (CA) granule on the expressions of Smad 2/3, Smad 7 and collagen Ⅳ and the level of Smad 2/3 phosphorylation were observed.@*METHODS@#Lipofectin method was used to transfect TGF-β1 vector into MC, and the stably expressed TGF-β1 cell lines were selected by G418. The cells were divided into three groups. Control group:normal MC + RPMI 1640 + 10% normal rat serum; TGF-β1 group:stably expressed TGF-β1 MC + RPMI 1640 + 10% normal rat serum; CA group:stably expressed TGF-β1 MC + RPMI 1640 + 10% rat serum containing high CA. The experiments were repeated for five times. The contents of TGF-β1 and collagen Ⅳ in the culture medium were detected with ELISA, the expressions of mRNA and protein of TGF-β1, Smad 2/3, Smad 7 and the level of Smad 2/3 phosphorylation were detected by using real time quantitative polymerase chain reaction and Western blot.@*RESULTS@#The contents of TGF-β1 and collagen Ⅳ in the culture medium of stably-expressed TGF-β1 MC were increased significantly, and the CA could reverse the effects of TGF-β1. The expressions of mRNA and protein of TGF-β1, Smad 2/3 and the level of Smad 2/3 phosphorylation were increased significantly in TGF-β1 transfected MC, and CA could dramatically reduce the expressions of mRNA and protein of TGF-β1, Smad 2/3 and the level of Smad 2/3 phosphorylation. The high expression of TGF-β1 decreased the expression of Smad 7 mRNA and protein, and the CA could antagonize the effect of mRNA expression.@*CONCLUSIONS@#The MCs stably-expressed TGF-β1 can activate the TGF-β1/Smad signal pathway and increase the expression of collagen Ⅳ. CA can decrease the occurrence of diabetic nephropathy(DN) by reducing the production of collagen Ⅳ through inhibiting the TGF-β1/Smad signal pathway.


Subject(s)
Animals , Cells, Cultured , Centella , Chemistry , Collagen Type IV , Metabolism , Drugs, Chinese Herbal , Pharmacology , Mesangial Cells , Metabolism , Rats , Signal Transduction , Smad Proteins , Metabolism , Smad2 Protein , Metabolism , Smad3 Protein , Metabolism , Smad7 Protein , Metabolism , Transforming Growth Factor beta1 , Metabolism
11.
Article in English | WPRIM | ID: wpr-773628

ABSTRACT

Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes mellitus. Renal fibrosis is closely related to the deterioration of renal function. The present study aimed to investigate protective effect of Taxus chinensis on high-fat diet/streptozotocin-induced DN in rats and explore the underlying mechanism of action. The rat DN model was established via feeding high fat diet for 4 weeks and subsequently injecting streptozotocin (30 mg·kg body weight) intraperitoneally. The rats with blood glucose levels higher than 16.8 mmol·L were selected for experiments. The DN rats were treated with Taxus chinensis orally (0.32, 0.64, and 1.28 g·kg) once a day for 8 weeks. Taxus chinensis significantly improved the renal damage, which was indicated by the decreases in 24-h urinary albumin excretion rate, blood serum creatinine, and blood urea nitrogen. Histopathological examination confirmed the protective effect of Taxus chinensis. The thickness of glomerular basement membrane was reduced, and proliferation of mesangial cells and podocytes cells and increase in mesangial matrix were attenuated. Further experiments showed that Taxus chinensis treatment down-regulated the expression of TGF-β1 and α-SMA, inhibited phosphorylation of Smad2 and Smad3. These results demonstrated that Taxus chinensis alleviated renal injuries in DN rats, which may be associated with suppressing TGF-β1/Smad signaling pathway.


Subject(s)
Albumins , Animals , Blood Glucose , Metabolism , Creatinine , Blood , Diabetic Nephropathies , Blood , Drug Therapy , Genetics , Urine , Drugs, Chinese Herbal , Humans , Kidney , Metabolism , Male , Phosphorylation , Rats , Rats, Sprague-Dawley , Signal Transduction , Smad Proteins , Genetics , Metabolism , Taxus , Chemistry , Transforming Growth Factor beta1 , Metabolism
12.
Article in English | WPRIM | ID: wpr-773610

ABSTRACT

The present study was designed to elucidate whether the mechanism by which osthole decreases collagenI/III contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts (CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pcDNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·mL of osthole for 24 h, the mRNA and protein expression levels of collagensIand III were reduced. The collagen I/III ratio was also reduced. The mRNA and protein expression levels of TGF-β1, TβRI, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen I and III expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.


Subject(s)
Actins , Genetics , Animals , Cells, Cultured , Collagen , Genetics , Coumarins , Pharmacology , Fibroblasts , Metabolism , Gene Expression Regulation , Mice , Myocardium , Cell Biology , Protein-Serine-Threonine Kinases , Genetics , RNA, Messenger , Genetics , Real-Time Polymerase Chain Reaction , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta , Genetics , Signal Transduction , Smad Proteins , Genetics , Transforming Growth Factor beta1 , Genetics
13.
Article in English | WPRIM | ID: wpr-812418

ABSTRACT

Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes mellitus. Renal fibrosis is closely related to the deterioration of renal function. The present study aimed to investigate protective effect of Taxus chinensis on high-fat diet/streptozotocin-induced DN in rats and explore the underlying mechanism of action. The rat DN model was established via feeding high fat diet for 4 weeks and subsequently injecting streptozotocin (30 mg·kg body weight) intraperitoneally. The rats with blood glucose levels higher than 16.8 mmol·L were selected for experiments. The DN rats were treated with Taxus chinensis orally (0.32, 0.64, and 1.28 g·kg) once a day for 8 weeks. Taxus chinensis significantly improved the renal damage, which was indicated by the decreases in 24-h urinary albumin excretion rate, blood serum creatinine, and blood urea nitrogen. Histopathological examination confirmed the protective effect of Taxus chinensis. The thickness of glomerular basement membrane was reduced, and proliferation of mesangial cells and podocytes cells and increase in mesangial matrix were attenuated. Further experiments showed that Taxus chinensis treatment down-regulated the expression of TGF-β1 and α-SMA, inhibited phosphorylation of Smad2 and Smad3. These results demonstrated that Taxus chinensis alleviated renal injuries in DN rats, which may be associated with suppressing TGF-β1/Smad signaling pathway.


Subject(s)
Albumins , Animals , Blood Glucose , Metabolism , Creatinine , Blood , Diabetic Nephropathies , Blood , Drug Therapy , Genetics , Urine , Drugs, Chinese Herbal , Humans , Kidney , Metabolism , Male , Phosphorylation , Rats , Rats, Sprague-Dawley , Signal Transduction , Smad Proteins , Genetics , Metabolism , Taxus , Chemistry , Transforming Growth Factor beta1 , Metabolism
14.
Article in English | WPRIM | ID: wpr-812399

ABSTRACT

The present study was designed to elucidate whether the mechanism by which osthole decreases collagenI/III contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts (CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pcDNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·mL of osthole for 24 h, the mRNA and protein expression levels of collagensIand III were reduced. The collagen I/III ratio was also reduced. The mRNA and protein expression levels of TGF-β1, TβRI, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen I and III expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.


Subject(s)
Actins , Genetics , Animals , Cells, Cultured , Collagen , Genetics , Coumarins , Pharmacology , Fibroblasts , Metabolism , Gene Expression Regulation , Mice , Myocardium , Cell Biology , Protein-Serine-Threonine Kinases , Genetics , RNA, Messenger , Genetics , Real-Time Polymerase Chain Reaction , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta , Genetics , Signal Transduction , Smad Proteins , Genetics , Transforming Growth Factor beta1 , Genetics
15.
Article in English | WPRIM | ID: wpr-690628

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of YOD1 overexpression on the proliferation and migration of human oral keratinocytes (HOKs), and to clarify whether the mechanisms involve transforming growth factor-β (TGF-β) signaling.</p><p><b>METHODS</b>HOKs were transfected with the plasmid pEGFP-N3-YOD1 containing YOD1. The mRNA levels of YOD1 and TGF-β were determined by qPCR. The protein expressions of YOD1, TGF-β, Smad2/3, Smad4, and phospho-Smad2/3 were determined by western blotting. Cell proliferation and migration were evaluated by Cell Counting Kit-8 assay and wound healing assay, respectively.</p><p><b>RESULTS</b>The mRNA and protein levels of YOD1 were higher in HOKs transfected with YOD1. YOD1 overexpression significantly enhanced the migration of HOKs. The mRNA and protein levels of TGF-β3 were increased by YOD1 overexpression. HOKs transfected with YOD1 exhibited increased phospho-Smad2/3 levels.</p><p><b>CONCLUSION</b>YOD1 overexpression enhances cell migration by promoting TGF-β3 signaling which may play an important role in lip and palate formation. YOD1 mutation may contribute to aberrant TGF-β3 signaling associated with decreased cell migration resulting in NSCLP.</p>


Subject(s)
Cell Movement , Physiology , Cell Proliferation , Cells, Cultured , Endopeptidases , Genetics , Metabolism , Humans , Keratinocytes , Physiology , Signal Transduction , Physiology , Smad Proteins , Genetics , Metabolism , Thiolester Hydrolases , Genetics , Metabolism , Transforming Growth Factor beta3 , Genetics , Metabolism
16.
Article in English | WPRIM | ID: wpr-691399

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the possible mechanism of San-Cao Granule (SCG, ) mediating antiliver fibrosis.</p><p><b>METHODS</b>A total of 60 male Sprague-Dawley rats were randomly divided into the normal control group, porcine serum-treated group, ursodesoxycholic acid (UDCA, 60 mg/kg), SCG (3.6 g/kg) group, SCG (1.8 g/kg) group and SCG (0.9 g/kg) group, with 10 rats in each group. Liver fibrosis was induced with porcine serum by intraperitoneal injection for 8 weeks, except for the normal control group. Then, the rats in the three SCG-treated groups and UDCA group were administered SCG and UDCA respectively for 4 weeks. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), albumin (ALB), total bilirubin (TBIL), hyaluronic acid (HA), laminin (LN), and type IV collagen (IVC) were examined using commercial kits and hepatic histopathology was examined with hematoxylin and eosin and Masson staining. Moreover, the protein expression levels of high mobility group box-1 protein (HMGB1), transforming growth factor β1 (TGF-β1), phosphorylated mothers against decapentaplegic homolog 3 (p-Smad3), Smad7, toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB) and α-smooth muscle actin (α-SMA) were determined by western blot, immunohistochemistry and real time quantitative-reverse transcription polymerase.</p><p><b>RESULTS</b>Both SCG (3.6 and 1.8 g/kg) and UDCA significantly ameliorated the liver fibrosis induced by porcine serum as indicated by retarding the serum levels increasing of ALT, AST, TBIL, HA, LN and IVC and preventing the serum level reducing of ALB compared with the model group (all P<0.01). Meanwhile, the collagen deposition was attenuated by SCG and UDCA treatment. Furthermore, SCG markedly reduced the expressions of HMGB1, TGF-β1, p-Smad3, TLR4, MyD88, NF-κB and α-SMA, and enhanced the expression of the Smad7 compared with the model group (all P<0.01).</p><p><b>CONCLUSION</b>SCG ameliorates hepatic fibrosis possibly through inhibiting HMGB1, TLR4/NF-κB and TGF-β1/Smad signaling pathway.</p>


Subject(s)
Animals , Disease Models, Animal , Drugs, Chinese Herbal , Therapeutic Uses , HMGB1 Protein , Metabolism , Liver , Metabolism , Pathology , Liver Cirrhosis , Drug Therapy , Metabolism , Pathology , Male , Rats , Rats, Sprague-Dawley , Signal Transduction , Smad Proteins , Metabolism
17.
Article in English | WPRIM | ID: wpr-20675

ABSTRACT

BACKGROUND/OBJECTIVES: Gallus gallus domesticus (GD) is a natural mutant breed of chicken in Korea with an atypical characterization of melanin in its tissue. This study investigated the effects of melanin extracts of GD on osteoblast differentiation and inhibition of osteoclast formation. MATERIALS/METHODS: The effects of the melanin extract of GD on human osteoblast MG-63 cell differentiation were examined by evaluating cell viability, osteoblast differentiation, and expression of osteoblast-specific transcription factors such as bone morphogenetic protein 2 (BMP-2), small mothers against decapentaplegic homologs 5 (SMAD5), runt-related transcription factor 2 (RUNX2), osteocalcin and type 1 collagen (COL-1) by reverse transcription-polymerase chain reaction and western blotting analysis. We investigated the inhibitory effect of melanin on the osteoclasts formation through tartrate-resistant acid phosphatase (TRAP) activity and TRAP stains in Raw 264.7 cell. RESULTS: The melanin extract of GD was not cytotoxic to MG-63 cells at concentrations of 50-250 µg/mL. Alkaline phosphatase (ALP) activity and bone mineralization of melanin extract-treated cells increased in a dose-dependent manner from 50 to 250 µg/mL and were 149% and 129% at 250 µg/mL concentration, respectively (P < 0.05). The levels of BMP-2, osteocalcin, and COL-1 gene expression were significantly upregulated by 1.72-, 4.44-, and 2.12-fold in melanin-treated cells than in the control cells (P < 0.05). The levels of RUNX2 and SMAD5 proteins were higher in melanin-treated cells than in control vehicle-treated cells. The melanin extract attenuated the formation of receptor activator of nuclear factor kappa-B ligand-induced TRAP-positive multinucleated RAW 264.7 cells by 22%, and was 77% cytotoxic to RAW 264.7 macrophages at a concentration of 500 µg/mL. CONCLUSIONS: This study provides evidence that the melanin extract promoted osteoblast differentiation by activating BMP/SMADs/RUNX2 signaling and regulating transcription of osteogenic genes such as ALP, type I collagen, and osteocalcin. These results suggest that the effective osteoblastic differentiation induced by melanin extract from GD makes it potentially useful in maintaining bone health.


Subject(s)
Acid Phosphatase , Alkaline Phosphatase , Blotting, Western , Bone Morphogenetic Protein 2 , Calcification, Physiologic , Cell Differentiation , Cell Survival , Chickens , Collagen Type I , Coloring Agents , Gene Expression , Humans , Korea , Macrophages , Melanins , Osteoblasts , Osteocalcin , Osteoclasts , Smad Proteins , Smad5 Protein , Transcription Factors
18.
Article in English | WPRIM | ID: wpr-203542

ABSTRACT

BACKGROUND: The transforming growth factor-β/SMAD (TGF-β/SMAD) pathway plays an important role in tissue repair and collagen synthesis. Low-level light therapy (LLLT) is increasingly used to alleviate pain and inflammation and promote wound healing. However, few studies have directly compared the effects of different wavelengths of light-emitting diodes (LEDs) or examined their individual effects at the molecular level. OBJECTIVE: Here we used a mouse model to investigate the effect of blue (410 nm), red (630 nm), and infrared (830 nm) LEDs on wound closure and assessed the underlying changes in a signal transduction pathway. METHODS: A full-thickness wound was created on the dorsal skin of mice using a 6-mm-diameter punch. In part I, the wounds were irradiated using blue, red, and infrared LEDs. In part II, the wounds were irradiated at different time points. Photo documentation, serial skin biopsies, wound measurements, and immunohistochemical staining using TGF-β/SMAD pathway-related molecules were performed. RESULTS: The overall wound closure percentage was highest during the first 10 days when an 830-nm LED was used. The wound closure process was accelerated when the irradiation was initiated immediately after wounding. Irradiation using 830-nm LED upregulated TGF-β and collagen-1 but downregulated SMAD7. CONCLUSION: Our findings show that LLLT using an 830-nm wavelength LED delivered immediately after wound formation may have the best effect on wound healing by upregulating the TGF-β/SMAD signaling pathway.


Subject(s)
Animals , Biopsy , Collagen , Inflammation , Low-Level Light Therapy , Mice , Signal Transduction , Skin , Smad Proteins , Wound Healing , Wounds and Injuries
19.
Braz. j. med. biol. res ; 49(10): e5526, 2016. graf
Article in English | LILACS | ID: lil-792523

ABSTRACT

Pseudomonas aeruginosa is one of the common colonizing bacteria of the human body and is an opportunistic pathogen frequently associated with respiratory infections. Inactivated P. aeruginosa (IPA) have a variety of biological effects against inflammation and allergy. Transforming growth factor-β (TGF-β) signaling plays a critical role in the regulation of cell growth, differentiation, and development in a wide range of biological systems. The present study was designed to investigate the effects of IPA on TGF-β/Smad signaling in vivo, using a hypoxia-induced pulmonary hypertension (PH) rat model. Sprague Dawley rats (n=40) were exposed to 10% oxygen for 21 days to induce PH. At the same time, IPA was administered intravenously from day 1 to day 14. Mean pulmonary artery pressure (mPAP) and the right ventricle (RV) to left ventricle plus the interventricular septum (LV+S) mass ratio were used to evaluate the development of PH. Vessel thickness and density were measured using immunohistochemistry. Primary arterial smooth muscle cells (PASMCs) were isolated and the proliferation of PASMCs was assayed by flow cytometry. The production of TGF-β1 in cultured supernatant of PASMCs was assayed by ELISA. The expression levels of α-smooth muscle actin (α-SMA), TGF-β1 and phospho-Smad 2/3 in PASMCs were assayed by western blot. Our data indicated that IPA attenuated PH, RV hypertrophy and pulmonary vascular remodeling in rats, which was probably mediated by restraining the hypoxia-induced overactive TGF-β1/Smad signaling. In conclusion, IPA is a promising protective treatment in PH due to the inhibiting effects on TGF-β1/Smad 2/3 signaling.


Subject(s)
Animals , Male , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/prevention & control , Hypoxia/metabolism , Myocytes, Smooth Muscle/physiology , Pseudomonas aeruginosa/physiology , Smad Proteins/metabolism , Transforming Growth Factor beta1/metabolism , Actins/analysis , Actins/metabolism , Blotting, Western , Cell Proliferation/physiology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Hypertension, Pulmonary/etiology , Hypoxia/complications , Immunohistochemistry , Rats, Sprague-Dawley , Reproducibility of Results , Signal Transduction/physiology , Smad Proteins/analysis , Transforming Growth Factor beta1/analysis
20.
Article in English | WPRIM | ID: wpr-301016

ABSTRACT

<p><b>OBJECTIVE</b>To explore the mechanism of Bushen Qiangji Granule (, BSQJ) in restraining the osteogenic differentiation of ankylosing spondylitis (AS) fifibroblasts.</p><p><b>METHODS</b>Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement and healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery as a control. Finite fifibroblast lines were established from these tissue samples to observe the effect of BSQJ on suppressing osteogenic differentiation of fifibroblasts. The expression of osteogenic marker gene corebinding factor a1 (Cbfa1) and Smad family proteins were examined by Western blot and real-time quantitative polymerase chain reaction (qPCR).</p><p><b>RESULTS</b>The mRNA expression level of Cbfa1 was significantly higher in AS fibroblasts than that in normal fibroblasts and the expression of pSmad1, pSmad5, Smad4 and Cbfa1 in AS fibroblasts was also higher, demonstrating the activation of the BMP/Smads signal pathway in AS fifibroblasts. BSQJ-medicated serum not only restrained the mRNA and protein expression levels of Cbfa1 and inhibited protein expression level of Smad4 but also decreased the expression quantities of pSmad1 and pSmad5.</p><p><b>CONCLUSIONS</b>BSQJ can inhibit osteogenic differentiation of AS fifibroblasts in vitro by suppressing the activation of the BMP/Smads signal pathway. This may be the important molecular mechanism of BSQJ in regulating AS ossifification.</p>


Subject(s)
Adult , Bone Morphogenetic Proteins , Metabolism , Cell Differentiation , Core Binding Factor Alpha 1 Subunit , Genetics , Metabolism , Drugs, Chinese Herbal , Pharmacology , Fibroblasts , Metabolism , Pathology , Humans , Middle Aged , Osteogenesis , Genetics , Phosphorylation , RNA, Messenger , Genetics , Metabolism , Serum , Metabolism , Signal Transduction , Smad Proteins , Metabolism , Spondylitis, Ankylosing , Genetics , Pathology , Young Adult
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