Abstract:Aim To explore possible action mechanism of mitogen-activated protein kinase (MAPK) signal transduction pathway on chemotaxis of cardiac fibroblasts (CF) induced by transforming growth factor-β1 (TGF-β1). Methods The CF of neonatal SD rats were cultured. Cell were randomly divided into blank control group, TGF-β1 group, c-Jun N-terminal kinase (JNK) inhibitor (SP600125 10 μmol/L) group, P38MAPK inhibitor (SB203580 10 μmol/L) group and extracellular signal-regulated protein kinase (ERK) inhibitor (U0126 10 μmol/L) group. Except blank control group, the other groups were given 10 μg/L TGF-β1 and corresponding inhibitors. Methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to detect cell viability of CF. Transwell chamber was applied to detect motor ability of CF. The collagen content was detected by hydroxyproline kit. The enzyme-linked immunosorbent assay (ELISA) was applied to detect levels of monocyte chemotactic protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) in CF. Western blot was applied to detect expression levels of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col-1) and matrix metalloproteinase-9 (MMP-9) in CF. Results TGF-β1 could significantly promote cell viability and chemotaxis of CF and collagen content. MAPK inhibitors could inhibit the proliferation and chemotaxis, and decrease collagen content (P＜0.05). ELISA results showed that MAPK signaling pathway inhibitors could significantly decrease increased MCP-1 and PAI-1 induced by TGF-β1 treatment (P＜0.05). Western blot results showed that MAPK signaling pathway inhibitors could significantly decrease increased expression levels of α-SMA, Col-1 and MMP-9 protein induced by TGF-β1 treatment (P＜0.05). Conclusion TGF-β1 may promote chemotaxis of CF by activating MAPK signaling pathway. The application of MAPK inhibitors can inhibit myocardial fibrosis to certain extent.

Abstract:Aim To investigate pathway mechanism about transforming growth factor-β1 (TGF-β1) activated cardiomyocyte hypertrophy. Methods Fresh isolated neonatal rat cardiomyocytes induced by TGF-β1 were used to examine formation of cell pathway. p-Smad2, Smad2, Smad2/3, p15 and c-Myc proteins were measured by Western blot. Cardiomyocyte hypertrophy was measured by PI statining and RNA expressions by real-time PCR. Smad signal pathway expression and its related protein expression were induced by TGF-β1 and siRNA interference. Results TGF-β1 could induce the expression of hypertrophic gene in cultured cardiomyocytes in vitro, and the expression of β-MHC was significantly higher than that of the control group (P＜0.01). The content of RNA in TGF-β1 group was significantly increased by PI staining (P ＜0.01). Compared with the control group, TGF-β1 significantly increased the expression of p15 and c-Myc in p-Smad2, Smad2, Smad2/3 and Smad pathway (P＜0.01). The expression of p15 of Smad2 siRNA group was less than that of TGF-β1 group (P＜0.01). Conclusion TGF-β1 may induce cardiomyocyte hypertrophy through the Smad protein pathway, and in this process the expression of p15 is increased.

Abstract:Aim To investigate the effect of peroxisome proliferators activated receptorγ (PPARγ) on the calcification of rat aortic vascular smooth muscle cells (VSMC) induced by transforming growth factor-β1 (TGF-β1). Methods Rat aortic vascular smooth muscle cells were cultured in vitro and divided into normal control group and different concentrations of TGF-β1 group (1 μg/L, 2 μg/L, 4 μg/L, 8 μg/L). Then they were divided into normal control group, calcification group (TGF-β1 4 μg/L), rosiglitazone group (RSG, 20 μmol/L), and calcification+rosiglitazone (RSG, 20 μmol/L) group. The effects of agonist RSG on calcification of VSMC, calcium content and alkaline phosphatase (ALP) activity were detected in the cells, alizarin red S staining was used to detect the formation of calcified nodules, and Western blot was used to detect vascular smooth muscle cells marker α-smooth muscle actin (α-SMA), PPARγ, protein expression of osteoblast-like marker Runt-related transcription factor (Runx2). Results Compared with the normal control group, calcium deposition and ALP activity of VSMC treated with TGF-β1 were significantly increased (P<0.05), and the effect was most obvious when TGF-β1 concentration was 4 μg/L. And Runx2 expression of osteoblast-like cell was significantly increased (P<0.05), while α-SMA expression of smooth muscle cell markers was decreased (P<0.05). Whereas calcium sulfate deposition and ALP activity of VSMC were significantly decreased (P<0.05), α-SMA and PPARγ expression was significantly increased after RSG was added(P<0.05), on the contrary, Runx2 expression was significantly inhibited (P<0.05). Conclusion TGF-β1 can induce differentiation and calcification of VSMC into osteoblast-like cells, and TGF-β1-induced calcification of VSMC can be inhibited by PPARγ agonist RSG.

Abstract:Aim To investigate the effects of OPN-002-siRNA transfection on intimal hyperplasia and osteopontin （OPN）, transforming growth factor-β1 （TGF-β1）, proliferating cell nuclear antigen （PCNA） expression after carotid balloon injury in rat.Methods Through preliminary experiment, OPN mRNA in cultured vascular smooth muscle cells was tested by real-time reverse transcription polymerase chain reaction （real-time RT-PCR）, and OPN-002-siRNA was determined as the most sensitive sequence and used as transfected siRNA in the subsequent animal experiments. Seventy-two rats were randomly divided into four groups: sham group, balloon injury group, OPN-scramble-siRNA （OPN-SCR-siRNA） group and OPN-002-siRNA group. Changes of intimal hyperplasia and OPN, TGF-β1, PCNA expressions were detected by immunofluorescence, hematoxylin-eosin （HE） staining, real-time RT-PCR and Western blot, and also the effect of OPN-002-siRNA was studied on them.Results （1）There was no apparent neointima on the 3rd day after balloon injury. Intima began to thicken on the 7th day after injury, and intimal thickening was significant on the 14th day. （2）The expression of OPN, TGF-β1 mRNA and protein started to increase on the 3rd day and persisted until the 14th day. The expression of PCNA mRNA and protein started to increase on the 3rd day, peaked on the 7th day, decreased on the 14th day. （3）Compared with balloon injury group and OPN-SCR-siRNA group, the neointima thickness decreased significantly on each time point in OPN-002-siRNA group （P<0.001）, and both mRNA and protein expression of OPN, TGF-β1 and PCNA reduced significantly on each time point in OPN-002-siRNA group （P<0.001）.Conclusion OPN-002-siRNA can inhibit intima hyperplasia after artery injury by decreasing the expression of OPN, TGF-β1 and PCNA.

Abstract:Aim To investigate the expression of vascular endothelial growth factor （VEGF） and transforming growth factor-β1 （TGF-β1） on diabetic atherosclerosis in rats and the mechanism and protective effects of simvastatin.Methods SD rats were randomized into normal control group （n＝8）, normal intervention group （n＝8）, model group （n＝18） and model intervention group （n＝16）. Intervention groups were perfused with simvastatin at 20 mg/（kg·d）, and the control groups were given distilled water ［20 mL/（kg·d）］ instead. The diabetic atherosclerosis model was established by streptozotocin （STZ）＋vitamin D3 （VitD3）＋high-fat and high-cholesterol diet. The contents of fasting plasma glucose （FPG）, total cholesterol （TC）, triglyceride （TG）, low density lipoprotein （LDL）, high density lipoprotein （HDL）, fasting insulin （FINS） were detected, and insulin resistance homeostasis （HOMA-IR） was calculated. Plasma VEGF and plasma TGF-β1 were measured by enzyme-linked immunosorbent assay （ELISA）. The expression of VEGF on aortic was evaluated by immunohistochemical method. Results Compared with normal control group, TGF-β1 was significantly increased in intervention group （P<0.01）, and FPG, TC, TG, LDL, HDL, FINS, VEGF and TGF-β1 were significantly increased in model group （P<0.01, P<0.05）. Compared with model group, FPG, TC, TG, LDL, HDL and VEGF were significantly decreased （P<0.01, P<0.05）, and TGF-β1 was significantly higher in intervention group （P<0.01）. In correlation analysis, VEGF showed a negative correlation with body weight and FINS, and had a positive correlation with TC, TG, LDL, FPG and HOMA-IR. TGF-β1 showed a negative correlation with TC, LDL and FPG. In a stepwise multiple regression analysis, FPG and TC were independent risk factors for plasma VEGF level. Conclusions VEGF and TGF-β1 may participate in the occurrence of diabetic atherosclerosis. Simvastatin can decrease the level of VEGF and increase the expression of TGF-β1, and has a significant protective effect on diabetic atherosclerosis.

Abstract:Aim To explore the association of the functional －509C/T polymorphism in the promoter region of the transforming growth factor-β1 （TGF-β1） gene with coronary heart disease （CHD） and its conventional risk factors including body mass index （BMI）, lipid levels of plasma, and blood glucose in Chongqing Han population. Methods The －509C/T polymorphism of TGF-β1 was genotyped by polymerase chain reaction-restriction fragment length polymorphism methods and DNA sequence technology in 457 CHD patients and 413 controls. The sample was characterized for relevant clinical and biochemical parameters. Results The －509C/T polymorphism of TGF-β1 was significantly associated with the risk of CHD in females for the additive and dominant genetic models （adjusted OR＝1.515, 95%CI＝1.045～2.196, Padditive＝0.028 adjusted OR＝1.937, 95%CI＝1.008～3.719, Pdominant＝0.047）, but not in male for all genetic models （P>0.05）. Female individuals with TT genotype had a 2.36-fold increased risk of developing CHD （adjusted 95%CI＝1.11～5.03, P＝0.026）. In female CHD patients, both CT and TT genotype carriers were related to the decreased high density lipoprotein cholesterol （HDLC） levels and increased levels of total cholesterol （TC）, low density lipoprotein cholesterol （LDLC） and body mass index （BMI） compared with CC genotype carriers, especially in TT genotype carriers reached statistical significance （all P＜0.05）. Conclusions The functional －509C/T polymorphism in the TGF-β1 promoter region was associated with the susceptibility to CHD in Chongqing Han women. Our data show that TGF-β1 polymorphism－509C/T is associated with lipid levels, BMI and CHD in Chongqing Han women. Female individuals with TT genotype have significantly increased the risk of developing CHD.

Abstract:AimTo investigate the molecular mechanism of inflammatory protein expression via transforming growth factor-β1 (TGF-β1)/Smad pathway in ox-LDL-stimulated human umbilical vein endothelial cells (HUVEC).MethodsHUVEC were treated with 0.1 g/L of ox-LDL.TGF-β1, Smad3, VCAM-1 and ICAM-1 protein expression, and Smad3 phosphorylation were detemined by Western blot.Resultsox-LDL obviously increased the expression of VCAM-1 and ICAM-1 in HUVEC.TGF-β1 expression and Smad3 phosphorylation were enhanced in ox-LDL-stimulated HUVEC.Nuclear extract analysis showed that the phosphorylated Smad3 expression was increased in ox-LDL-treated cells.SIS3 (a specific inhibitor of Smad3) inhibited Smad3 phosphorylation in a dose-dependent manner, however, VCAM-1 and ICAM-1 expression were increased in ox-LDL-treated cells.ConclusionsTGF-β1/Smad signaling involved in the regulation of inflammatory protein expression in HUVEC induced by ox-LDL, the inhibition of Smad3 phosphorylation increased the expression of inflammatory protein, suggesting that TGF-β1/Smad3 signaling repressed the inflammatory protein expression in ox-LDL-treated endothelial cells.

Abstract:N-acetyl-seryl-aspartly-lysyl-proline （AcSDKP）is a kind of anti- fibrosis effects of short peptides, which is normally present in human plasma and circulating mononuclear cells. Recent studies found that AcSDKP was involved in some organ’s fibrosis, such as heart, lung, kidney and liver. AcSDKP could inhibit the target cells such as cardiac fibroblasts, lung fibroblasts, glomerular mesangial cells and hepatic stellate cells proliferation and collagen synthesis and expression. Through a variety of mechanisms including extracellular signal transduction AcSDKP can inhibit a target organ within the collagen deposition and reduce the causative factors leading to organ fibrosis.

Abstract:Aim To investigate the effects of Smad3 on the growth of C2C12 cells induced by transforming growth factor-β1 (TGF-β1). Methods C2C12 cells were transfected with siRNA-Smad3 in vitro, and the cell proliferations were examined with MTT methods. Results After the C2C12 cells interfered with different concentrations of TGF-β1 (0, 1, 5, 10 μg/L) for 24 h, the MTT (OD) values were 0.096±0.015, 0.177±0.014, 0.240±0.028, 0.312±0.012 respectively (P<0.01). While the C2C12 cells were transfected with 200 pmol/L siRNA-Smad3 for 24 h and then treated with 5 μg/L of TGF-β1, the MTT(OD) values in experimental group (0.063±0.011) were decreased compared with internal control group (0.137±0.016, P<0.01). Conclusion The TGF-β1 could promote C2C12 cells proliferation by the Smad3 pathway and showed dose dependent manner, siRNA-Smad3 could block the signal transduction and decreased C2C12 cell proliferation effectively.

Abstract:Aim To observe dynamically the serial concentrations of soluble intercellular adhesion molecule-1(sCIAM-1) and transforming growth factor-β1(TGF-β1) in patients with acute cerebral infarction(ACI) and its significance. Methods The serial concentrations of sICAM-1 and TGF-β1 in 50 patients with ACI and 30 healthy control subjects were measured by ELISA method. Results The serial concentrations of sICAM-1 in patients with ACI significantly exceeded those detected in the healthy control subjects at day 1st,day 3rd and day 7th,the serial concentrations of TGF-β1 in patients with ACI significantly decreased compared with those of the healthy control subjects at day 1st and day 3rd. Conclusion The dynamic changes of the serial alteration of sICAM-1 and TGF-β1 concentration may reflect the immunological and inflammatory status of the patients with ACI,meanwhile,sICAM-1 is an inflammatory damaging factor and TGF-β1 can protect brain tissue.