OBJECTIVESTo investigate the role of glycyrrhizin on TGFbeta1 stimulated signaling transduction in rat hepatic stellate cells (HSCs).

METHODSThe mice HSCs were isolated and cultured with or without glycyrrhizin (1 micromol/L-1000 micromol/L) in vitro after TGFbeta1 stimulation. The mRNA level of Smad2, 3, 7 were measured with RT-PCR; protein expression level of Smad2, 3, 7 and collagen I, III were analyzed with Western blot.

RESULTSTGFbeta1 increased the mRNA level and protein expression of Smad2, 3, 7 in HSC; it also increased protein expression of collagen I and III. 1 micromol/L-1000 micromol/L glycyrrhizin decreased the mRNA level and protein expression of Smad2, 3, 7; it also inhibited protein expression of collagen I and III gradually.

CONCLUSIONInterventing the TGFbeta signaling pathway and decreasing the synthesis of collagen, might be involved in the anti-fibrosis mechanism of glycyrrhizin.

Animals ; Glycyrrhizic Acid ; pharmacology ; Hepatocytes ; metabolism ; Male ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; pharmacology

The activation of hepatic stellate cells (HSCs) and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-β (TGFβ)/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations (0-10 μmol/L). Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFβ1, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different concentrations (1, 2, 3 μmol/L) or RPMI1640 alone (serving as control). The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC(50) of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 μmol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased (P<0.05), but the Smad7 mRNA expression had no significant change (P>0.05). There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 (P<0.05). However, the expression of Smad7 protein was substantially increased when compared with the control group (P<0.05). It was concluded that the inhibition of TGFβ/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors (TGFβ1, Smad3) and promote the expression of anti-fibrosis factor (Smad7), suggesting that MG132 may become a potential therapeutic alternative for liver fibrosis.
Animals ; Cell Line ; Leupeptins ; pharmacology ; Protease Inhibitors ; pharmacology ; Rats ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; metabolism

AIMTo investigate the molecular mechanism underlying the effect of linoleic acid on plasminogen activator inhibitor type-1 (PAI-1) expression in HepG2 cells.

METHODSHepG2 cells were exposed to different concentrations of linoleic acid and PAI-1 expression was determined by RT-PCR and colorimetric assay. Luciferase reporter gene plasmids containing four sequentially truncated fragments of the PAI-1 promoter region (-804 to +17) were constructed, and plasmids carrying constructs of Smad binding element (SBE)-site directed deletions in PAI-1 promoter were also generated using overlap extention PCR and transiently transfected into HepG2 cells, the transcriptional activity of PAI-1 was demonstrated by the luciferase activity.The effect of linoleic acid on Smad3 and Smad4 protein levels in cultured HepG2 cells was measured by Western blot analysis.

RESULTS(1) Linoleic acid remarkably increased PAI-1 mRNA expression and transcription in varying concentrations. (2) The level of PAI-1 transcription was gradually decreased induced by linoleic acid when transfected the SBE- site directed-deletions plasmids in PAI-1 promoter at -734/-731. (3) Protein levels of both Smad3 and 4 in HepG2 cells were increased by linoleic acid.

CONCLUSIONLinoleic acid regulated the expression of PAI-1 from transcriptional level in HepG2 cells and SBE involved in the regulation, and both Smads protein and Smad signaling pathway acted main role in this procession.

Gene Expression Regulation ; drug effects ; Hep G2 Cells ; Humans ; Linoleic Acid ; pharmacology ; Plasminogen Activator Inhibitor 1 ; genetics ; Promoter Regions, Genetic ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism

OBJECTIVETo elucidate the effects of interferon-gamma (IFN-gamma) on the transforming growth factor beta (TGF-beta)/Smad pathway in keloid-derived fibroblasts (KFb), and to investigate the underlying mechanism in the treatment of pathologic scar with IFN-gamma.

METHODSKeloid tissue of 3 patients were obtained, and then KFb were separated and cultured in vitro. KFb from passages 3 to 5 were used for the study. (1) KFb were divided into control group (incubated with serum-free DMEM), TGF-beta(1) group (treated with 10 ng/mL TGF-beta(1)), IFN-gamma group (treated with 100 ng/mL IFN-gamma), and TGF-beta(1)+IFN-gamma group (incubated with 10 ng/mL TGF-beta(1) combined with 100 ng/mL IFN-gamma). The expression level of mRNA and protein of connective tissue growth factor (CTGF), alpha smooth muscle actin (alpha-SMA) protein and expression of alpha-SMA positive KFb were detected by real-time fluorescent quantitation RT-PCR (FQ-RT-PCR), Western blot and immunofluorescence cytochemical staining. (2) Another sample of KFb was obtained and treated with 10 ng/mL IFN-gamma. The expression level of Smad 3 and Smad 7 protein was detected by Western blot before and 1, 2, 4, 6, 8 h post stimulation (PSH). The expression level of Smad 3 and Smad 7 mRNA was assessed by FQ-RT-PCR before stimulation and 30 mins post stimulation and at PSH, 1, 2, 4, 6, 8. (3) Another sample of KFb was obtained and divided into 1, 10 and 100 ng/mL IFN-gamma groups based on the concentration of IFN-gamma, treated for 4 hours; KFb without IFN-gamma treatment was set up as control group. The expression levels of the protein and mRNA of Smad 3 and Smad 7 were measured by FQ-RT-PCR and Western blot.

RESULTS(1) The level of mRNA and protein of CTGF in IFN-gamma group (0.017 +/- 0.009 and 1.198 +/- 0.004) was respectively lower than that in control group (0.024 +/- 0.013 and 1.229 +/- 0.011, P < 0.05). The level of mRNA and protein of CTGF in TGF-beta(1)+IFN-gamma group (0.634 +/- 0.138 and 1.204 +/- 0.010) was respectively lower than that in TGF-beta(1) group (1.331 +/- 0.298 and 1.727 +/- 0.004, P < 0.01). The fluorescence intensity of alpha-SMA positive KFb (0.922 +/- 0.059) and the expression level of alpha-SMA protein (0.3051 +/- 0.0031) in IFN-gamma group decreased significantly than those in control group (1.055 +/- 0.005 and 0.4513 +/- 0.0094, P < 0.01). The fluorescence intensity of alpha-SMA positive KFb (1.129 +/- 0.004) and the expression level of alpha-SMA protein (0.6734 +/- 0.0098) in TGF-beta(1)+IFN-gamma group decreased significantly than those in TGF-beta(1) group (1.270 +/- 0.005 and 1.3842 +/- 0.0024, P < 0.01). (2) The expression level of Smad 3 mRNA and protein at the first time point after IFN-gamma treatment increased temporarily then decreased gradually, and mRNA expression level reached the nadir at PSH 4, it rose gradually later, though it was still lower at PSH 8 than that before treatment (P < 0.01); protein expression level at PSH 8 was significantly lower than that before treatment (P < 0.01). The expression level of Smad 7 mRNA and protein increased gradually to the maximum at PSH 2 and 4 respectively, then decreased but was still higher at PSH 8 than that before treatment (P < 0.05). (3) Compared with those in control group, the expression levels of Smad 3 mRNA and protein in 1, 10 and 100 ng/mL IFN-gamma group were significantly lower, the expression levels of Smad 7 mRNA and protein were significantly higher (P < 0.05 or P < 0.01). The higher concentration of IFN-gamma, the more significant differences were observed.

CONCLUSIONSIFN-gamma can down-regulate the expression of Smad 3 while up-regulate the expression of Smad 7 in a time- and dose-dependent manner, and reduce the expression level of CTGF and alpha-SMA in the basic state or induced by TGF-beta(1), which shows a significant inhibitory effect on the TGF-beta/Smad signal pathway. This may be an important mechanism in the treatment of pathologic scar by IFN-gamma.

Cells, Cultured ; Fibroblasts ; drug effects ; metabolism ; Humans ; Interferon-gamma ; pharmacology ; Keloid ; metabolism ; RNA, Messenger ; genetics ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Transforming Growth Factor beta1 ; metabolism

The reduction of extracellular matrix (ECM) degradation in kidney is taken as the morphological features and pathological base in renal injury in chronic kidney disease (CKD). ECM degradation is controlled by the catabolic enzyme systems in glomerulus and renal interstitium, in which matrix metalloproteinases (MMPs) play a key role. The expression and activity of MMPs are regulated by the classical pathway, such as the genic transcription, the activation of zymogen, and the specific inhibitor. The previous studies showed that, Uremic Clearance granule, as a representation, and other prescriptions of Chinese herbal medicine, as well as some extracts from Chinese herbal medicine could intervene the pathway of ECM degradation through promoting the degradation of ECM components, affecting the expression of catabolic enzymes, regulating the genetic transcription of MMPs, and inhibiting the relative signaling transduction of MMPs.
Animals ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Matrix ; drug effects ; metabolism ; Humans ; Kidney ; cytology ; drug effects ; pathology ; Matrix Metalloproteinases ; metabolism ; Proteolysis ; drug effects ; Smad Proteins ; metabolism

OBJECTIVETo observe the effects of Wnt3a on proliferation and, activation of hepatic stellate cells (HSCs) and their the expression of the transforming growth factor beta (TGFb) and /Smad signaling factors of rat hepatic stellate cells line in vitro using a rat HSC line.

METHODSSynchronized HSC-T6 cells were stimulated with various concentrations of recombinant Wnt3a (50, 100, 200, 250 and 300 ng/mL). Unstimulated cells served as controls. Edu Effects on proliferation were determined by EdU (5-ethynyl-2'-deoxyuridine) incorporation assay and fluorescence microscopy.analysis was used to observe the proliferation of the hepatic stellate cells stimulated by different concentration of recombinant Wnt3a, and the Effects on the protein expression of TGFb/Smad signaling factors was assessed by western blot detection (gray-value analysis) of alpha-smooth muscle actin (a-SMA), a-SMA, TGFb1, Smad3, and and Smad7; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as the normalization control in the hepatic stellate cells was observed by Western blot analysis .The correlation was also observed. The significance of inter-group differences was assessed by one-way ANOVA, and correlations were determined using bivariate statistical modeling.

RESULTSIn general, HSC The proliferation of hepatic stellate cells increased after the addition of in response to Wnt3a stimulation for 24 h, reaching its peak at the maximum proliferation rate was observed with the 200 ng/mL Wnt3a concentration (63.00+/-2.30%), and it increased dramatically compared with those in which was significantly higher than the proliferation rates of the unstimulated control cells, and the cells stimulated with 50, 100 and 150 ng/mLl group (P less than 0.05), but the increase was not significantly different from that in the compared cells stimulated with 250 and 300 ng/mLl group,it had no obvious increase(P more than 0.05).; The Wnt3a stimulation also led to time-dependent increases in the protein expressions of a-SMA, TGFb1, and Smad3 increased with the addition of Wnt3a and the extension of time . For all three, The maximal amount of increased protein expression all reached to the was maximal produced by stimulation when hepatic stellate cells were treated by with 300 ng/mLl Wnt3a for 48 h hours,and the rations of(normalized gray- values:s of a-SMA, 1.0860+/-0.0101; TGFb1, 1.0346+/-0.0118; Smad3, to GAPDH were 1.0860+/-0.0101, 1.0346+/-0.0118, 1.0306+/-0.0122)respectively. However in contrast, the Wnt3a stimulation led to concentration- and time-dependent decreases in Smad7 expression varied inversely, with to them with the minimal ration of it to GAPDH the maximal decrease occurring with 300 ng/mL Wnt3a for 48 h (0.7736+/-0.0139) after being treated by 300 ng/ml Wnt3a for 48h. The comparison was remarkably discrepant, (P less than 0.05).There were positive correlations between a-SMA expression and was found to be positively correlated to TGFb1, Smad3 (r=0.968, P less than 0.05) and; Smad3 (r=0.997, P less than 0.01), but a-SMA and Smad7 had negatively correlated to Smad7 ion(r=0.960, P less than 0.05).

CONCLUSIONWnt3a can increase the stimulates proliferation as well as and activation of rat the hepatic stellate cells HSCs , and upregulate modifies the expression of TGFb/Smad signaling factors, of the hepatic stellate cells, and which may promote the hepatic fibrosis.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; metabolism ; Wnt3A Protein ; pharmacology

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.
Animals ; Disease Models, Animal ; Male ; Myocardial Infarction ; complications ; Plant Extracts ; pharmacology ; Rats, Sprague-Dawley ; Smad Proteins ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; Ventricular Remodeling ; drug effects

The purpose of this study was to investigate the effect of Geniposide on hepatic fibrosis and activation of hepatic stellate cells (HSCs) and to explore possible underlying mechanism. Human HSCs (LX-2) were treated with 5 ng/mL transforming growth factor-β1 (TGF-β1), followed by co-culture with Geniposide at various concentrations (0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100 μmol/L). Cell viability was determined by MTT assay. Then, LX-2 cells were divided into control, TGF-β1 (5 ng/mL) and TGF-β1 + Geniposide (20 μmol/L) groups, and the gene and protein expression of collagen I, fibronectin, α-smooth muscle actin (α-SMA), p-Smad2 and p-Smad3 was detected by qPCR and Western blot, respectively. BALB/c mice were treated with CCl₄ (25%, 1 mL/kg) to generate a model of hepatic fibrosis (CCl₄ group), and the control group and CCl₄ + Geniposide group were administered with olive oil and CCl₄ + 40 mg/kg Geniposide, respectively. After 4 weeks of treatment, the liver function and serum hepatic fibrosis indexes of mice were detected, histological observation was performed by HE and Masson staining, and α-SMA expression in the tissue was analyzed by immunohistochemistry. Western blot was utilized for the determination of the protein expression of α-SMA, TGF-β1, p-Smad2 and p-Smad3. The results showed that Geniposide inhibited LX-2 cell proliferation. In addition, Geniposide significantly downregulated the gene and protein expression of collagen I, fibronectin and α-SMA and the expression of TGF-β1/Smad signaling-related proteins induced by TGF-β1 in vitro. Histological observations showed that Geniposide significantly inhibited CCl₄-induced hepatic fibrosis, HSC activation and expression of TGF-β1/Smad signaling-related proteins in mice. In summary, Geniposide prevents the hepatic fibrosis and HSC activation possibly through the inhibition of the TGF-β1/Smad signaling pathway.
Animals ; Collagen Type I/metabolism* ; Fibronectins ; Hepatic Stellate Cells/pathology* ; Iridoids ; Liver Cirrhosis/pathology* ; Mice ; Signal Transduction ; Smad Proteins/pharmacology* ; Transforming Growth Factor beta1/metabolism*

OBJECTIVETo explore the effects of blocking two sites of TGF-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts.

METHODSTwo lentivirus vectors encoding soluble TGF-β receptor II (sTβRII) and mutant Smad 4-Smad 4ΔM4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50. The protein expressions of sTβRII and Smad 4ΔM4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells. The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1×10(4) cells per dish. Co-transfection group, transfected with the two previous lentivirus vectors, mixed with the ratio of 1:1 and MOI of 50, and then stimulated with 5 ng/mL TGF-β1 for 72 h; sTβRII group, transfected with lenti-sTβRII with MOI of 50, with the other treatment as above; Smad 4ΔM4 group, transfected with lenti-Smad 4ΔM4 with MOI of 50, with the other treatment as above; negative virus group, transfected with empty lentivirus vector, with the other treatment as above; positive control group, stimulated with 5 ng/mL TGF-β1 for 72 h; and blank control group, conventionally cultured without any other treatment. After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group. ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group. Data were processed with one-way analysis of variance and SNK-(q test).

RESULTS(1) HFF-1 cells transfected with lenti-sTβRII and HFF-1 cells transfected with lenti-Smad 4ΔM4 respectively expressed higher levels of sTβRII protein and Smad 4ΔM4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins. (2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (with F values respectively 53.536 and 24.365, P values below 0.001). The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60 ± 0.18 and 1.99 ± 0.40) were similar with those of negative virus group (respectively 1.60 ± 0.15 and 1.94 ± 0.28, with q values respectively 0.091 and 0.419, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 5.245 to 18.228, P values below 0.05). The protein and mRNA expressions of fibronectin in cells of co-transfection group (respectively 0.60 ± 0.05 and 0.70 ± 0.11) were significantly lower than those of sTβRII group (respectively 0.89 ± 0.13 and 1.24 ± 0.17) and Smad 4ΔM4 group (respectively 0.91 ± 0.14 and 1.28 ± 0.19, with q values from 3.964 to 4.294, P values below 0.05). (3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (with F values respectively 107.680 and 38.347, P values below 0.001). The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (with q values respectively 1.106 and 0.491, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 6.414 to 26.420, P values below 0.05). The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sTβRII group and Smad 4ΔM4 group (with q values from 3.424 to 7.143, P values below 0.05).

CONCLUSIONSIn human skin fibroblasts, blockage of two sites of TGF-β/Smad signaling can reduce the expression of scar related proteins which are up-regulated by TGF-β1 to a greater extent than that of blocking one single site.

Cicatrix ; Connective Tissue Growth Factor ; Fibroblasts ; metabolism ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; genetics ; Receptors, Transforming Growth Factor beta ; Signal Transduction ; drug effects ; Smad Proteins ; genetics ; metabolism ; Smad Proteins, Inhibitory ; genetics ; Transfection ; Transforming Growth Factor beta ; pharmacology ; Transforming Growth Factors

Liver fibrosis is a common pathological process for chronic liver injury caused by multiple etiological factors and an inevitable phase leading to liver cirrhosis. According to the previous studies, hesperidin (HDN) shows a very good protective effect on CCl4-induced chemical hepatic fibrosis in rats. In this experiment, based on the findings of the previous studies, a platelet-derived growth factor (PDGF)-induced HSC-T6 model was established to observe the inhibitory effect of HDN on HSC-T6 proliferation. The ELISA method was adopted to detect the content of collagen I in HSC-T6 supernatant. Transforming growth factor (TGF)-beta1, Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) mRNA expressions were measured by RT-PCR; TGF-beta1 and CT-GF protein expressions in HSC-T6 were determined by Western blot, in order to study HDN's effect on TGF-beta1 signaling pathway in HSC and its potential action mechanism. The results demonstrated that HDN could notably improve HSC-T6 proliferation, Collagen I growth and TGF-beta1, Smad2, Smad3 and CTGF mRNA.expressions. After being intervened with HDN, it could notably inhibit HSC-T6 proliferation and Collagen I growth, reduce TGF-beta1, Smad2, Smad3 and CTGF mRNA and TGF-beta1, CTGF protein expressions and increase Smad7 mRNA expression. HDN's antihepatic fibrosis effect may be related to the inhibition of HSC proliferation and activation by modulating TGF-beta/Smad signaling pathway.
Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; physiology ; Hesperidin ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Rats ; Signal Transduction ; drug effects ; Smad Proteins ; physiology ; Transforming Growth Factor beta1 ; physiology