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 investigate the effect of Smad7 on the expressions of collagen I and alpha-smooth muscle actin (alpha-SMA) in HSC-T6 cell line activated by transforming growth factor-beta1 (TGF-beta1).

METHODSHSC-T6 cells stably expressing M2-flag protein were selected after co-infection of the cells with pTRE-Smad7-M2-flag and pTet-on. The optimal dose of doxycycline for inducing Smad7 was determined, and the effects of Smad7 over-expression on the expressions of collagen I and alpha-SMA in the cells activated by TGF-beta1 and on Smad2/3 phosphorylation were evaluated using Western blotting.

RESULTSThe optimal dose of doxycycline for inducing Smad7 expression was 2 mg/L. Smad7 over-expression induced by doxycycline decreased the expressions of collagen I and alpha-SMA in HSC-T6 cells activated by TGF-beta1, and down-regulated the level of Smad2/3 phosphorylation.

CONCLUSIONSmad7 over-expression inhibits Smad2/3 phosphorylation, and decreases the expression of collagen I and alpha-SMA in HSC-T6 cells induced by TGF-beta1 to inhibit the progression of liver fibrosis.

Actins ; genetics ; metabolism ; Cells, Cultured ; Collagen Type I ; genetics ; metabolism ; Genetic Therapy ; Hepatocytes ; cytology ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; therapy ; Smad7 Protein ; pharmacology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo construct a tetracycline-inducible eukaryotic expression vector of rat Smad7.

METHODSThe total RNA was extracted from normal rat kidney with Trizol agent. Rat Smad7 cDNA fragment was cloned by RT-PCR, and was inserted into the restriction site between Nhe I and Hind III of the inducible eukaryotic expression vector pBI-L by tetracycline. pBI-L-Smad7 was constructed by digestion and ligation, and detected by restriction endonuclease digestion and sequencing.

RESULTSThe recombinant eukaryotic expression vector pBI-L-Smad7 was constructed correctly as confirmed by restriction endonuclease digestion and sequencing. The fragment of pBI-L-Smad7 digested with restriction endonucleases and the sequence of inserted Smad7 cDNA were consistent with the results of theoretical analysis.

CONCLUSIONThe tetracycline- inducible eukaryotic expression vector of rat Smad7, pBI-L-Smad7, is constructed successfully, which may facilitate further clinical study of Smad7 gene therapy for tissue and organ fibrosis.

Animals ; Cloning, Molecular ; DNA, Complementary ; genetics ; Eukaryotic Cells ; metabolism ; Gene Expression ; drug effects ; Genetic Therapy ; Genetic Vectors ; genetics ; Rats ; Rats, Sprague-Dawley ; Smad7 Protein ; biosynthesis ; genetics ; Tetracycline ; pharmacology

OBJECTIVETo investigate the changes in transforming growth factor beta 1 (TGF-beta1)/Smads signaling pathway in rats with chemical hepatocarcinogenesis.

METHODSFresh diethylnitrosamine (DENA) solution was administered in SD rats to induce hepatocellular carcinoma (HCC). The protein expressions of TGF-beta1, phosphorylated Smad2, Smad4 and Smad7 were detected in these rats with immunohistochemistry, and the mRNA expression of Smad4 was evaluated with RT-PCR.

RESULTSCirrhotic nodules occurred in the rats 8 weeks after DENA treatment, and HCC nodules were found 16 weeks after the treatment. In the normal liver tissue, very low levels of TGF-beta1 and Smad4 expressions, low Smad7 expression and high phosphorylated Smad2 expression were detected. The development of liver cirrhosis was accompanied by increased expressions of TGF-beta1, Smad4 and Smad7 but at 8 weeks after DENA treatment, the expression of phosphorylated Smad2 was significantly decreased, followed then by gradual increment till nearly the normal level. Twenty-two weeks after DENA treatment, Smad4 expression in liver tissue decreased markedly as compared with the levels at 8 and 16 weeks. The expressions of Smad4 and phosphorylated Smad2 in the HCC tissue was significantly lower than those in normal liver tissue.

CONCLUSIONHepatocarcinogenesis involves very complex mechanisms, can can be related partially to the decreased Smad4 and phosphorylated Smad2 expression and TGFbeta1 and Smad7 overexpression in advanced stage of liver cirrhosis.

Animals ; Diethylnitrosamine ; Liver Neoplasms, Experimental ; chemically induced ; metabolism ; pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad2 Protein ; metabolism ; Smad4 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

The recombinant human Smad7 adenoviral vector was constructed by direct DNA cloning protocol and then transfected into 293 cells for virus packaging. After amplification and purification, the recombinant adenovirus was used to infect the keloid fibroblasts. The Smad7 mRNA transcription of the infected cells was detected by RT-PCR. The recombinant Adeno-Smad7 was correctly constructed and confirmed by both restriction analysis and PCR analysis. RT-PCR showed the over expression of adenovirus mediated Smad7 mRNA in keloid cells. These results demonstrated that the recombinant Smad7 adenoviral vector can be expressed in cultured cells in vitro, and it may provide a new therapeutic strategy for keloid gene therapy.
Adenoviridae ; genetics ; metabolism ; Cells, Cultured ; Fibroblasts ; metabolism ; pathology ; Genetic Vectors ; genetics ; metabolism ; Humans ; Keloid ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Smad7 Protein ; biosynthesis ; genetics ; Transfection

OBJECTIVETo observe the temporal and spatial expression of Smad7 during human tooth germ development and evaluate the effect of Smad7 on tooth germ development.

METHODSThe expression of Smad7 and its changes at different stages of human tooth germ were detected by using immunohistochemical staining.

RESULTSSmad7 was expressed at all stages of tooth germ, but the distribution patterns at various stages were different. It indicated that temporal and spatial expressing mode of Smad7 during human tooth germ development was specific, which was similar to that of TGF-beta its signal transducer Smad2/3.

CONCLUSIONSmad7 might play an important role in TGF-beta intracellular signaling for modulating the differentiation of ameloblasts and odontoblasts.

Ameloblasts ; cytology ; Cell Differentiation ; DNA-Binding Proteins ; genetics ; metabolism ; physiology ; Fetus ; Humans ; Immunohistochemistry ; Odontoblasts ; cytology ; Odontogenesis ; Signal Transduction ; Smad7 Protein ; Tooth ; growth & development ; Tooth Germ ; embryology ; Trans-Activators ; genetics ; metabolism ; physiology ; Transforming Growth Factor beta ; genetics ; metabolism ; physiology

OBJECTIVETo explore the mechanism of action of Longbixiao Capsule (LBXC, a Chinese herbal preparation) on human prostatic stromal cell cultured in vitro.

METHODSNine Japan rabbits were assigned to 3 groups. The high, low dose group was given LBXC [2.0 g/(kg x d), 1.0 g/(kg x d)] by gastro gavage respectively, while equal volume of normal saline was given by gastrog avage 60 rats in the control group, all twice a day with an infeval of 12 h, for 3 successive days. The serum collected at 3 h after the last gastro gavage was added into cell culture fluid. Rabbit's serum containing LBXC was incubated with the cultured stromal cells, and the levels of cell proliferation and apoptosis were determined using relative techniques as TUNEL, ELISA, and immunocytochemistry. Besides, Real-time RT-PCR was applied to detect the mRNA expressions of TGF-beta1, and Smad7 in the stromal cells.

RESULTSThe cell proliferation showed culture time dependence in all groups. The proliferation in the drug-serum treated groups was lower than that in the control group, and it was lower in the high dose treated group than in the low dose treated group (all P < 0.01). The unfavorable growth did not occur morphologically after being cultured for 48 h and showed insignificant difference between various groups. Cell apoptosis was not found excepting for a few appeared in the high dose treated group (with a little amount of apoptotic cells occurring). After treatment, the expressions of TGF-beta, and Smad7 were lower in the low dose treated group and high dose treated group than in the control group (P < 0.01). There was not statistical difference between the low dose treated group and high dose treated group (P > 0.05).

CONCLUSIONLBXC could reduce the expressions of TGF-beta1, and smad7 mRNA in stromal cells and inhibit the stromal cell proliferation, but its effect on promoting cell apoptosis is unobvious.

Animals ; Apoptosis ; drug effects ; Capsules ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Male ; Prostate ; cytology ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Random Allocation ; Serum ; Smad7 Protein ; genetics ; metabolism ; Stromal Cells ; cytology ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo investigate the cytotoxic effect of epigallocatechin gallate (EGCG) on human hepatocellular carcinoma cell line HepG2 cells and corresponding changes of TGF-beta1-Smad pathway.

METHODSThe cytotoxic effect of EGCG on HepG2 cells was determined by MTT assay. Cell cycle and apoptosis rate were detected by flow cytometry. RT-PCR and luciferase assay were used to verify whether TGF-beta1-Smad signaling pathway is intact in HepG2. The mRNA expression of Smad 2, Smad3, Smad4 and Smad7 was detected by real-time PCR.

RESULTSEGCG induced apoptosis in the HepG2 cells in a time- and concentration-dependent manner. The proportion of G(1) phase cells was increased gradually as the concentration increased. However, the percentage of cells in S phase was decreased gradually. Annexin V/PI assay demonstrated that early apoptosis increased as the concentration increased, and late apoptosis also increased, when treated with high-concentration EGCG. The intact TGF-beta1-Smad pathway was verified by luciferase assay and RT-PCR. There was no significant effect of EGCG on mRNA level of Smad 2, Smad 3, and Smad 4 in HepG2 cells, but downregulated mRNA level of Smad 7.

CONCLUSIONEGCG can reduce apoptosis in human hepatocellular carcinoma cell line HepG2 cells. The activation of TGF-beta1-Smad signaling pathway may be involved in its cytotoxicity mechanisms.

Anticarcinogenic Agents ; pharmacology ; Apoptosis ; drug effects ; Catechin ; analogs & derivatives ; pharmacology ; Cell Cycle ; drug effects ; Hep G2 Cells ; Humans ; RNA, Messenger ; metabolism ; Signal Transduction ; Smad Proteins ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; metabolism

The present study was designed to observe the expressions of bone morphogenetic protein-7 (BMP-7) and inhibitory Smads in kidney of rats with diabetic nephropathy (DN), and explore the possible mechanism of DN. Male Wistar rats weighing 180-220 g were single injected with streptozocin (STZ, 55 mg/kg body weight) for 2, 4, 8 and 16 weeks to induce DN. Blood glucose, kidney weight/body weight and 24-hour urine protein in the control and DN rats were examined; the expressions of BMP-7, Smad6 and Smad7 were detected by using immunohistochemical techniques, Western blot and real-time PCR. The results showed that blood glucose and 24-hour urine protein in DN rats were higher than that in the control rats and kidney weight/body weight was also elevated in DN rats, especially in 16-week STZ-induced rats. The expressions of BMP-7 and Smad6 proteins in DN rats were elevated, while BMP-7 mRNA expression was increased 2 weeks after STZ injection and decreased 16 weeks after STZ injection. The expressions of Smad7 protein and mRNA were elevated in DN rats 2 weeks after STZ injection and decreased 16 weeks after STZ injection. In addition, the expressions of transforming growth factor-beta1 (TGF-beta1) and collagen type I (COL-I) mRNA were increased in DN rats. These results suggest in the early stage of DN, increase in BMP-7 and inhibitory Smad expression may play a role in the feedback regulation and restrain the development of DN.
Animals ; Bone Morphogenetic Protein 7 ; genetics ; metabolism ; Collagen Type I ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; Diabetic Nephropathies ; metabolism ; Kidney ; metabolism ; pathology ; Male ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Smad6 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVE: To explore the mechanism of enalapril for renal interstitial fibrosis by observing the effect of enalapril on the expression of transforming growth factor-beta1(TGF-beta1), p-Smad2/3 and Smad7 in renal tissuess of unilateral urethral obstruction (UUO) rat model. METHODS: Thirty female Sprague-Dawley(SD) rats were randomly subdivided into a sham-operated group, a model group and an enalapril treated group. UUO model was induced by ligating the left ureter of rats. All rats were sacrificed 14 days after UUO. Pathological changes of the renal tissue were observed by HE and Masson staining, the protein expressions of Collagen I (ColI), TGF-beta1, p-Smad2/3 and Smad7 were detected by immunohistochemical staining,and the mRNA expressions of TGF-beta1 and Smad7 were detected by RT-PCR. RESULTS: The renal interstitial damage index, the relative Collagen area and the expression of ColI in the model group significantly increased(P<0.01). Enalapril reduced these indexes. The protein and mRNA expressions of TGF-beta1 and the protein expressions of p-Smad2/3 were low in the sham-operated group, but were strongly positive in the model group, and enalapril could decrease the expressions of TGF-beta1 and p-Smad2/3(P<0.01). The protein and mRNA expressions of Smad7 in the model group were less than that in the sham-operated group(P<0.01),and enalapril could improve the expressions of Smad7(P<0.01). CONCLUSION Enalapril could inhibit the renal interstitial fibrosis by affecting TGF-beta1, p-Smad2/3 and Smad7 of TGF-beta/smads pathway in the renal tissues of UUO rats.
Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Enalapril ; pharmacology ; Female ; Fibrosis ; prevention & control ; Kidney ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Smad2 Protein ; genetics ; metabolism ; Smad3 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Urethral Obstruction ; complications