Glucosyltransferases ; metabolism ; HEK293 Cells ; Humans ; Proteasome Endopeptidase Complex ; metabolism ; Protein Stability ; Proteolysis ; Smad3 Protein ; chemistry ; metabolism ; Ubiquitin ; metabolism

Transforming growth factor-β (TGF-β) is a driving force of renal fibrosis, which may lead to chronic kidney diseases and even end stage renal diseases. By activating canonical and non-canonical signaling pathways, TGF-β promotes the synthesis of extracellular matrix while preventing their degradation. In the injured kidney, TGF-β induces apoptosis, proliferation and fibrotic response of renal cells including epithelial cells, endothelial cells, podocytes, fibroblasts, pericytes and macrophages, and it also promotes transdifferentiation, activation and proliferation of myofibroblasts. Additionally, TGF-β exerts profibrotic effects by interplaying with other signaling pathways like BMP-7, Wnt/β-catenin and MAP kinase. Smad3 is the central pathological gene in renal fibrosis, and epigenetic regulation of TGF-β/Smad3 is a hot topic in kidney field. Although direct targeting TGF-β may cause side effects including tumorigenesis and immune diseases, the therapeutic strategies targeting the balance of downstream Smad3 and Smad7 may prevent or delay the progression of fibrotic kidney disease.
Epigenesis, Genetic ; Fibrosis ; Humans ; Kidney Diseases ; pathology ; Signal Transduction ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo investigate the relationship between expression of Smad3, Smad7 and ventricular remodeling in rats after myocardial infarction.

METHODSMyocardial infarction was induced by left anterior descending coronary artery ligation in rats (n = 11) and sham-operated rats were used as control (n = 10). The rats were sacrificed 8 weeks later. Heart weight/body weight (HW/BW), mean blood pressure, left ventricular end diastolic pressure (LVEDP), collagen content in the un-infarcted area were examined. The mRNA levels of transforming growth factor (TGF)beta(1), Smad 3, Smad7 were determined by RT-PCR.

RESULTCompared with controls, the level of HW/BW, LVEDP and collagen content were significant increased. The mRNA expression of TGFbeta(1) and Smad3 was significantly increased in areas of myocardial infarction, border of the infarction, interventricular septum and right ventricle. The expression of Smad7 mRNA in these areas was decreased.

CONCLUSIONThese results indicated that TGFbeta(1)-Smads signaling was correlated to the ventricular remodeling after myocardial infarction. Smad3 might promote the process while Smad7 inhibit the process.

Animals ; Male ; Myocardial Infarction ; metabolism ; physiopathology ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Smad3 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta ; metabolism ; Ventricular Remodeling

OBJECTIVETo observe the effect of single herb pilose antler (PA) on the expression of Smad2 and Smad3 in the cartilage of osteoarthritis (OA) rats.

METHODSOne hundred 3-month old female healthy SD rats, (200 +/- 20) g, were recruited and routinely fed for 1 week. They were randomly divided into 5 groups, i.e., the low dose PA group, the high dose PA group, the normal saline control group, the model group, and the normal control group, 20 in each group. The model was prepared using classic Hulth method except the normal control group. After 6-week modeling, the model was confirmed successful by pathologic observation. PA at 0.021 g/100 g and 0.084 g/1 00 g was given by gastrogavage to rats in the low dose PA group and the high dose PA group respectively. Normal saline was administered to those in the normal saline control group. No treatment was given to rats in the normal control group and the model group. Bilateral knee cartilages were harvested at week 2,4, and 6. mRNA and protein expressions of Smad2 and Smad3 were detected by immunohistochemical assay, fluorescent quantitative PCR, and Western blot.

RESULTSOA model was successfully prepared by pathological observation. Results of immunohistochemical assay showed that Smad2 and Smad3 expressed extensively in the cartilage, and located inside the chondrocyte membrane. Compared with the model group, mRNA expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 2, 4, and 6, showing statistical difference (P < 0.05). Compared with the same group at week 4 after gastrogavage, mRNA expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.05). Compared with the model group, protein expression of Smad2 and Smad3 obviously increased in the chondrocytes of the low dose PA group and the high dose PA group at week 2 and 4, showing statistical difference (P < 0.01). Compared with the same group at week 2 after gastrogavage, protein expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 4, showing statistical difference (P < 0.01). Compared with the same group at week 4 after gastrogavage, protein expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.01).

CONCLUSIONS(1) The pilose antler could repair cartilages by regulating mRNA and protein expressions of Smad2 and Smad3. (2) Up-regulating mRNA and protein expressions of Smad2 and Smad3 might be one of important mechanisms for the pathogenesis of OA.

Animals ; Antlers ; chemistry ; Cartilage ; cytology ; metabolism ; Chondrocytes ; drug effects ; metabolism ; Female ; Medicine, Chinese Traditional ; Osteoarthritis ; drug therapy ; metabolism ; Rats ; Rats, Sprague-Dawley ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism

OBJECTIVE: To explore whether acupuncture combined with moxibustion could inhibit epithelialmesenchymal transition in Crohn's disease by affecting the transforming growth factor β 1 (TGF- β 1)/Smad3/Snail pathway. METHODS: Sixty-three patients with Crohn's disease were randomly divided into an observation group (31 cases) receiving moxibustion at 43 °C combined with acupuncture, and a control group (32 cases) receiving moxibustion at 37 °C combined with sham acupuncture using a random number table. Patients were treated for 12 weeks. Crohn's Disease Activity Index (CDAI) was used to evaluate disease activity. Hematoxylin-eosin staining and transmission electron microscopy were utilized to observe the morphological and ultrastructural changes. Immunohistochemistry was used to detect the expression of transforming growth factor β 1 (TGF-β 1), T β R1, T β R2, Smad3, Snail, E-cadherin and fibronectin in intestinal mucosal tissues. RESULTS: The decrease of the CDAI score, morphological and ultrastructural changes were more significant in observation group. The expression levels of TGF- β 1, Tβ R2, Smad3, and Snail in the observation group were significantly lower than those before the treatment (P<0.05 or P<0.01). After treatment, the expression levels of TGF-β 1, TβR2, and Snail in the observation group were significantly lower than those in the control group (all P<0.05); compared with the control group, the expression of fibronectin in the observation group was significantly decreased, and the expression of E-cadherin was significantly increased (all P<0.05). CONCLUSIONS Moxibustion at 43 °C combined with acupuncture may suppress TGF-β 1/Smad3/Snail pathway-mediated epithelial-mesenchymal transition of intestinal epithelial cells in Crohn's disease patients by inhibiting the expression levels of TGF-β 1, Tβ R2, Smad3, and Snail. (Registration No. ChiCTR-IIR-16007751).
Acupuncture Therapy ; Cadherins/metabolism* ; Crohn Disease/therapy* ; Epithelial-Mesenchymal Transition ; Fibronectins/metabolism* ; Humans ; Moxibustion ; Smad3 Protein/metabolism* ; Snail Family Transcription Factors/metabolism* ; Transforming Growth Factor beta1/metabolism*

OBJECTIVETo investigate the expression of TGF-betaR I, Smad2, Smad3 and Smad7 in keloids, normal scars and normal skins. Discuss the significance of these proteins in the course of keloid.

METHODSImmunohistochemistry method was used to detect the expression intensity and distribution of these proteins in above 3 kinds of different tissues in 44 cases. Statistics was used to analyze the data.

RESULTSThe expression of TGF-betaR I were much stronger in keloid than in the other two tissues. The expression of Smad7 were lower in keloids. The increase expression of Smad2,3 were not obvious, but they were found to accumulate in the nucleus.

CONCLUSIONSThe results indicate that over-expression of TGF-betaR I, low-expression of Smad7 and accumulation of Smad2,3 may be one of the etiological factors of keloids. This research may provide a new idea to prevent and treat keloids or other fibrosis diseases in the future.

Adolescent ; Adult ; Female ; Humans ; Keloid ; metabolism ; Male ; Middle Aged ; Protein-Serine-Threonine Kinases ; metabolism ; Receptors, Transforming Growth Factor beta ; metabolism ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Young Adult

OBJECTIVETo observe Smads protein expression in lung tissue of quartz exposed mice and to explore its association with pulmonary fibrosis in silicosis.

METHODSThe experimental mice were divided into control and quartz groups. 0.2 g/kg weight of quartz was injected intratracheally in quartz group. Samples were collected at the 1st, 3rd, 5th, 7th, 14th and 28th day after injection. Immunohistochemical methods with quantitative image analysis were used to assay the protein expression of transforming growth factor beta(1) (TGF-beta(1)), Smad 2/3, Smad 4, and Smad 7 protein levels. Protein expression level is presented by positive unit (PU).

RESULTSSmad 2/3 protein expression increased from day 3, reaching its peak level in day 14 [(42.2 +/- 2.4) PU], and decreased gradually. The elevation of Smad 4 protein level began from day 5, and the highest degree came into day 14 [(40.0 +/- 1.8) PU], decreased thereafter. The expression of Smad 7 presented a decreasing tendency at the beginning and reaching the lowest level in day 14 [(33.5 +/- 3.3) PU]. It seemed to elevate in day 28, but was still lower than the controls. There were positive correlation between Smad 2/3, Smad 4 and TGF-beta(1) (r = 0.91, r = 0.71, respectively, P < 0.05) and also between Smad 2/3 and hydroxyproline contents of lung tissue (r = 0.85, P < 0.05) except Smad 7.

CONCLUSIONSmad protein may have certain association with pulmonary fibrosis in silicosis.

Animals ; DNA-Binding Proteins ; immunology ; metabolism ; Lung ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Pulmonary Fibrosis ; chemically induced ; metabolism ; Quartz ; toxicity ; Smad2 Protein ; Smad3 Protein ; Smad4 Protein ; Smad7 Protein ; Trans-Activators ; immunology ; metabolism ; Transforming Growth Factor beta ; metabolism

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.
Animals ; Cells, Cultured ; Centella ; chemistry ; Collagen Type IV ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Mesangial Cells ; drug effects ; metabolism ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

AIMTo investigate the effects of transforming growth factor-beta1 (TGF-beta1) and signal protein Smad3 on rat myocardial hypertrophy.

METHODSThe total protein was analysed by flow cytometer assay to judge the hypertrophy of myocardial cell incubated with different level of TGF-beta1 in cultured myocardial cells of neonatal rats. The models of rat cardiac hypertrophy were produced with constriction of the abdominal aorta. At the different time after the operation, the rats were killed, and the left ventricular mass indexes (LVMl) were investigated. The mRNA expressions of TGF-beta1 and Smad3 of cultured cells and hypertrophic left ventricles were assessed by RT-PCR, the protein expressions of Smad3 were assessed by Western blot.

RESULTSIn cultured neonatal myocardial cells, different level TGF-beta1 could significantly increase the total protein, and TGF-beta1 (3 ng/ml) could increase the expression of mRNA and protein of Smad3 and continued for 8 h of cultured cardiomyocytes. The LVMI and the expression of TGF-beta1 mRNA and Smad3 mRNA/protein of hypertrophic left ventricle were increased at the 3rd day after the operation and continued for 4 weeks. The peak expression of them was in 2 weeks after operation.

CONCLUSIONTGF-beta1 has the effects on rat myocardial hypertrophy, signal protein Smad3 is included in the pathologic progress of rat myocardial hypertrophy.

Animals ; Cardiomegaly ; metabolism ; pathology ; Cells, Cultured ; Male ; Myocytes, Cardiac ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad3 Protein ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo observe the effects of radix astragali on expression of TGF-β₁ and Smad 3 signal pathway in hypertrophic scar of rabbits, and to analyze its therapeutic effect and mechanism on hypertrophic scar.

METHODSTwenty healthy adult Japanese big ear rabbits were inflicted with 4 full-thickness skin defects on ventral side of each ear, which formed scar later. Rabbits were divided into 5 groups: 1.00, 0.50, 0.25 g/mL radix astragali treatment groups [injected with radix astragali on post injury day (PID) 21, 25, 32, and 36 respectively], physiological saline group (PS, injected with 0.2 mL physiological saline in the same volume at the same time points as above groups), and blank control group (BC, without treatment) according to the random number table, with 32 scars in each group. Another 4 rabbits were enrolled as normal control group (NC). Structural changes of hypertrophic scar was observed with HE and Masson staining. Thickness and hardness of hypertrophic scar on PID 32 and 43 were respectively examined by chromoscope ultrasonic diagnostic equipment and hardness tester. Protein and mRNA expression of TGF-β₁ and Smad 3 in hypertrophic scar was respectively detected with RT-PCR and immunohistochemical analysis. Data were processed with t test and one-way analysis of variance.

RESULTSCompared with that in PS and BC groups, dermis of hypertrophic scar became thinner in radix astragali treatment groups on PID 32, 43, with fibroblasts and collagenous fibers arranged regularly on PID 43. Thickness and hardness of hypertrophic scar, levels of mRNA and protein of TGF-β₁ and Smad 3 decreased along with the increase in radix astragali concentration. Compared with those in PS group, levels of mRNA of TGF-β₁ and Smad 3 in 1.00 g/mL radix astragali treatment group on PID 32 decreased 26.1% and 28.2%. Protein levels of TGF-β₁ and Smad 3 in 1.00 g/mL radix astragali treatment group were 3.15 ± 0.80 and 4.72 ± 1.06, which were obviously lower than those in PS group (6.06 ± 0.85, 8.04 ± 0.63, with F value respectively 27.230 and 33.525, P < 0.05 or P < 0.01). There was significant statistical difference in all measurement indices except for mRNA of TGF-β₁ and Smad 3 among radix astragali treatment groups on PID 32 and 43 [with t values respectively 3.593-4.814 (thickness), 4.051-5.811 (hardness), 2.976-5.986 (TGF-β₁ protein), and 2.742-4.630 (Smad 3 protein), P < 0.05 or P < 0.01].

CONCLUSIONSRadix astragali injection inhibits fibroblast proliferation in hypertrophic scars through down-regulating mRNA expression and protein synthesis of TGF-β₁ and Smad 3, thus inhibits hypertrophic scars formation. Its inhibition effect is drug concentration and duration dependent. The drug may be considered as a potential agent to prevent hypertrophic scar.

Animals ; Astragalus Plant ; Cicatrix, Hypertrophic ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Rabbits ; Signal Transduction ; drug effects ; Smad3 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism