Role of hepatitis B virus in intrahepatic TGF-β1/Smads signaling pathway

AIM:To explore the effects of hepatitis B virus (HBV) on intrahepatic expression of transforming growth factor β1（TGF-β1） and Smads. METHODS:The expression of intrahepatic TGF-β1, HBsAg and HBcAg in control group and chronic hepatitis B (CHB) group was detected by immunohistochemical method.The serum HBV DNA content was determined by real-time PCR. The role of HBV in the expression of TGF-β1, Smad3 and Smad7 in human hepatic stellate cell line LX-2 in vitro was observed by cell culture and Western blotting. RESULTS:The average score of intrahepatic TGF-β1 expression in CHB group was higher than that in control group. With the increase in serum HBV DNA content, intrahepatic TGF-β1 expression was also enhanced. In the HBcAg positive hepatic tissue, there was higher TGF-β1 expression than that in the liver tissue of HBcAg negative. Compared with control group and HBV+anti-TGF-β1 group, HBV caused increased expression of TGF-β1 and Smad3 in HBV group in vitro. No difference of Smad7 protein among control group, HBV group and HBV+anti-TGF-β1 group was observed. CONCLUSION: The expression of intrahepatic TGF-β1 is related to serum HBV DNA and hepatocellular HBcAg in the patients with CHB. HBV-induced liver fibrosis mainly relies on positive regulatory mechanisms of Smad3,and the negative regulation by Smad7 almost does not function.     

Effect of tea-polyphenols on the activation of NF-κB and the expression of TGF-β1 mRNA in THP-1 cells incubated with VLDL,LDL or ox-LDL

AIM:To investigate the effect of tea-polyphenols (TP) on the activation of NF-κB and the expression of TGF-β₁ mRNA in THP-1 cells (a human acute monocytic leukemia cell line). METHODS:THP-1 cells were incubated with the different concentrations of TP, VLDL, LDL or ox-LDL. In the THP-1 cellls, the nuclear malposition rate of NF-κB was detected with immunohistochemistry technique, the positive index of the TGF-β₁ mRNA expression was detected by hybridization in situ, and accumulation of total cholesterol (TC) in cells incubated with 0.4-40 μg/L TP was determined with oxidase assay. RESULTS:The nuclear malposition rate of NF-κB, the positive index of the TGF-β₁ mRNA expression and TC in THP-1 cells incubated with 0.4-40 μg/L of TP were lower than those with 0 μg/L of TP in TP-V group, TP-L group and TP-O (P＜0.05). The differences of these markers in THP-1 cells incubated with more than 40 μg/L TP in TP-V group, TP-L group and TP-O were not statistically significant, compared with TP-C group (P＞0.05). CONCLUSION:TP inhibited the activation of NF-κB, the expression of TGF-β₁ mRNA and the foam cell formation in the mono-macrophage.     

Effect of hydrogen sulfide on myocardial fibrosis and PPARγ and NF-κB expression in rat model of diabetes

AIM: To explore the effects of hydrogen sulfide (H₂S) on the myocardial fibrosis in a rat model of diabetes and its mechanism.METHODS: Single intraperitoneal injection of streptozotocin (STZ) was utilized to establish a rat model of diabetes. Sodium hydrosulfide was used as an exogenous donor of hydrogen sulfide. Male SD rats were randomly divided into control group, STZ group, STZ+H₂S group and H₂S group. Eight weeks later, HE and VG staining methods were used to observe the collagen distribution and collagen volume fraction was measured by image analysis. The expression levels of type I collagen, PPARγ and NF-κB in the cardiac tissues were determined by Western blotting.RESULTS: Compared with control group, collagen distribution and the expression levels of type I collagen and NF-κB in the cardiac tissues were markedly increased (P<0.05), while PPARγ was significantly decreased in STZ group (P<0.05), but these indexes were reversed significantly in STZ+H₂S group (P<0.05). The expression levels of type I collagen, PPARγ and NF-κB had no significant difference between H₂S group and control group.CONCLUSION: Hydrogen sulfide attenuates cardiac fibrosis in diabetic rats, and its mechanism may be related to PPARγ-NF-κB signaling pathway.     

PPARγ mediates effects of diosgenin on proliferation and apoptosis in human glioblastoma U87MG cells

AIM:To investigate the effect of diosgenin (Dio) on the proliferation, apoptosis and expression of peroxisome proliferator-activated receptor γ (PPARγ) in human glioblastoma U87MG cells and its possible mechanism. METHODS:Human astrocytes (HA) and U87MG cells were cultured in vitro and treated with Dio (0, 10, 20, 30, 40 and 50 μmol/L) and GW9662 (5 μmol/L) for 48 h, and then the cell viability was detected by CCK-8 assay. Cell colony formation assay was used to assess the proliferation potential. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. The mRNA expression level of PPARγ was measured by RT-PCR. Western blot was used to determine the protein levels of PPARγ, cyclin D1, cyclin E1, Bcl-2 and Bax. RESULTS:Dio had no significant influence on the viabi-lity of HA (P>0.05). However, Dio remarkably reduced the viability of U87MG cells in a dose-dependent manner (P<0.05) with IC₅₀ of 24.31 μmol/L. Meanwhile, Dio remarkably diminished colony formation ability (P<0.05), induced G₀/G₁ phase arrest of the cell cycle and apoptosis (P<0.05), up-regulated the expression of PPARγ at mRNA and protein levels, increased the protein level of Bax (P<0.05), and down-regulated the protein levels of cyclin D1, cyclin E1 and Bcl-2 (P<0.05) in a dose-dependent manner. However, these effects induced by Dio were inhibited by GW9662 (P<0.05), a specific inhibitor of PPARγ. CONCLUSION:Dio may inhibit proliferation and induce apoptosis in human glioblastoma U87MG cells most likely via up-regulating the expression of PPARγ, and then down-regulating the protein levels of cyclin D1, cyclin E1 and Bcl-2, and up-regulating the protein level of Bax.     

Effect of rosiglitazone on expression of AQP1, VEGF-A and COX-2 in human peritoneal microvascular endothelial cells

AIM: To investigate the effect of rosiglitazone on the expression of aquaporin-1 (AQP1), vascular endothelial growth factor-A (VEGF-A) and cyclooxygenase-2 (COX-2) in human peritoneal microvascular endothelial cells.METHODS: Cultured peritoneal microvascular endothelial cells were divided into 4 groups. The morphological changes of the cells were observed under inverted microscope. The protein expression of AQP1, VEGF-A and COX-2 in human peritoneal microvascular endothelial cells was determined by Western blotting. The mRNA expression of AQP1, VEGF-A and COX-2 in the cells was measured by real-time PCR. RESULTS: Rosiglitazone stimulated the proliferation of the cells. Rosiglitazone up-regulated the expression of AQP1, and down-regulated the expression of VEGF-2 and COX-2 at mRNA and protein levels in the cells. The PPAR-γ antagonist GW9662 partly inhibited the up-regulation of AQP1 expression by rosiglitazone (P<0.05), but had no obvious effect on the expression of VEGF-A and COX-2 (P>0.05). CONCLUSION: Rosiglitazone up-regulates the expression of AQP1 and down-regulates the expression of VEGF-A and COX-2 in human peritoneal microvascular endothelial cells, thus promoting water transportation and attenuating peritoneal fibrosis during peritoneal dialysis.     

Phosphorothioate-modified antisense TGF-β1 oligodeoxynucleotide inhibits neointimal hyperplasia after vascular balloon injury in rats

AIM: To evaluate the effects of antisense TGF-β1 oligodeoxynucleotide (AS TGF-β1) on the expression of TGF-β1, deposition of extracellular matrix (ECM) and the neointima formation in the arteries after balloon injury. METHODS: The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed. At the same time, sense TGF-β1 oligodeoxynucleotide (S TGF-β1) with the base sequence complement to AS TGF-β1 was synthesized as a control. The oligodeoxynucleotides were introduced into in vivo and in vitro experiments, respectively. RESULTS: The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner, and S TGF-β1 did not have the same effect. Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was observed. Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis. Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs. Fibronectin (FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (001~1 μmol/L)-dependent manner. AS TGF-β1 significantly increased the mRNA expression of contractile marker SM22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla, especially at the concentration of 001 μmol/L and 01 μmol/L. After treatment with AS TGF-β1 (90 μg·kg-1·d-1) for 28 d, the neointima formation was significantly inhibited, and the area ratio of intima/media was markedly decreased by 68% compared with untreated group, but S TGF-β1 had no effect on neointimal formation. CONCLUSION：The AS TGF-β1 specifically inhibits the protein expression of TGF-β1 in the VSMCs derived from injured arteries. Moreover, it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN. Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury. The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the alteration of VSMC phenotype after balloon injury.     

Effect of lysophosphatidic acid on expression of integrin β6 in epithelial cells

AIM: To investigate the effect of lysophosphatidic acid (LPA) on the expression of integrin β₆ (ITGB6) for determining the role of transforming growth factor β (TGF-β) activation induced by LPA in this process. METHODS: Normal human bronchial epithelial (NHBE) cells were primarily cultured in 6 well plate and stimulated with LPA. The mRNA expression of ITGB6 and the level of cell surface ITGB6 protein were detected by RT-PCR and flow cytometry,respectively. The activity of active TGF-β induced by LPA was measured by the method of transformed mink lung epithelial cells (TMLC) transfected with TGF-β responsive plasminogen activator inhibitor 1(PAI-1) promoter fused with firefly luciferase reporter gene. RESULTS: After stimulated with LPA at concentration of 10 μmol/L for 2 h, the mRNA expression of ITGB6 in epithelial cells was significantly increased in a time-dependent manner. The results of flow cytometry showed that the protein level of ITGB6 on cell surface was obviously increased after treated with LPA at concentration of 10 μmol/L for 4 h. The active TGF-β induced by LPA in epithelial cells was blocked by an α_Vβ₆ blocking antibody. However, α_Vβ₆ blocking antibody failed to inhibit the mRNA expression of ITGB6 induced by LPA. CONCLUSION: LPA induces the mRNA and cell surface protein of ITGB6 in epithelial cells. The up-regulated ITGB6 expression by LPA is independent on LPA-induced TGF-β activation.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Effects of TRPC1 on TGF-β1-induced migration of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 (TGF-β1). METHODS: Silencing of TRPC1 gene expression was performed by siRNA. The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The migration and invasion abilities of the 16HBE cells were detected by wound- healing assay and Transwell assay. The protein expression of E-cadherin and vimentin was determined by Western blot. RESULTS: TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups (P < 0.01). The results of CCK-8 assay and flow cytometry indicated that there were no significant difference in proliferation and apoptosis among TRPC1 siRNA group, TGF-β1 group and control group (P > 0.05). The results of wound-healing and Transwell assays showed that migration and invasion abilities in TRPC1 siRNA + TGF-β1 group were markedly suppressed compared with TGF-β1 group (P < 0.01). The protein expression of E-cadherin and vimentin induced by TGF-β1 was inhibited by TRPC1 silencing compared with TGF-β1 group (P < 0.05). CONCLUSION: TRPC1 is involved in the migration of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vimentin.     

Relationship of microRNA-133a and TGF-β1 protein in myocardial tissues of spontaneously hypertensive rats

AIM：To observe the changes of microRNA-133a and transforming growth factor β1 (TGF-β1) protein in the myocardium of spontaneously hypertensive rats (SHR). METHODS：Male SHR (18 weeks old, n=12) and male Wistar-Kyoto rats (WKY, 18 weeks old, n=12) served as SHR group and control group, respectively. Caudal arterial blood pressure was detected by a noninvasive blood pressure measurement and analysis system. Myocardial collagen volume fraction (CVF) and perivascular collagen area ratio (PVCA) were determined by Masson staining. The level of miR-133a in the heart was detected by real-time quantitative PCR. The protein level of TGF-β1 in the heart was also analyzed by the methods of immunohistochemisty and Western blotting. RESULTS：Compared with control group, systolic and diastolic blood pressure, CVF and PVCA significantly increased, the expression of TGF-β1 protein was significantly up-regulated, and the level of miR-133a was significantly reduced in SHR group. In SHR group, the expression of miR-133a was decreased to (23.9±4.6)% in control group. A negative correlation between the levels of miR-133a and TGF-β1 protein in SHR group was observed (r=-0.791, P<0.01). CONCLUSION：The level of miR-133a is down-regulated along with the up-regulation of TGF-β1 protein expression and collagen synthesis in the myocardial tissues of SHR. miR-133a and TGF-β1 may be involved in myocardial fibrosis in SHR.     

TGF-β1 upregulates Smad2 expression in peritoneal mesothelial cells

AIM: To investigate the expression of Smad2 signal protein in peritoneal mesothelial cells and how transforming growth factor β1 (TGF-β1) affects its expression. METHODS: Rat peritoneal mesothelial cells were cultured in different levels of TGF-β1 (0,1.25,2.5,10 μg/L) for different time (0,5,15,30,60,120 min). Endogenous Smad2 expression was evaluated by RT-PCR and immunohistochemical assay. The alteration of subcellular location of Smad2 was determined by immunohistochemical assay. RESULTS: TGF-β1 induced Smad2 mRNA expression, which increased at 5 min, peaked at 30 min, and declined to baseline levels by 120 min, in a time-dependent manner. Smad2 mRNA expression induced by TGF-β1 was also in a dose -dependent manner. TGF-β1 induced Smad2 phosphorlylation and nuclear localization in both time-dependent and dose-dependent manner, which was concordant with mRNA expression. Smad2 translocated from cytoplasm to nuclear accumulation in response to TGF-β1, and peaked at 30 min. CONCLUSIONS: Smad2 is present in peritoneal mesothelial cells. TGF-β1 may activate Smad2 expression and translocation to nuclear in a time-dependent and dose-dependent manner.     

PPARγ gene and protein expression in aortic plaque of different week-old apoE-knock out mice and the relationship with the composition of aortic plaque

AIM: To investigate the relationship of PPARγ gene expression with the composition of aortic plaque in apoE-knock out mice. METHODS: PPARγ gene and protein in aortic area of 20-week-old and 40-week-old apoE-knock out mice were investigated using RT-PCR and immunoblotting. The same aged wild type mice (C57BL/6J) were served as control (n=10). The composition of aortic plaques was analyzed by Movat method and oil red O staining. The expression of antigens such as PPARγ, SM-actin and MOMA-2 in aortic plaque were compared using immunohistochemistry. The relationship of PPARγ with macrophage, smooth muscle cells (SMC), lipid, elastic fiber, collagen and proteoglycan in aortic plaque were analyzed using immunofluorescence. RESULTS: PPARγ gene and protein in aortic wall and plaque of apoE-knock out mice were more significant than that in the same aged C57BL/6J mice (P<0.05). PPARγ expression at 40-week-old apoE-knock out mice was most significant and very low in C57BL/6J mice. More PPARγ expression of gene and protein at 20-week-old C57BL/6J mice than 40-week-old C57BL/6J mice were observed. Compared with 20-week-old apoE^-/- mice, the lipid pool in aortic plaque at 40-week-old apoE^-/- mice were increased remarkably, while elastic fiber, collagen and proteoglycan in plaque were decreased and aortic remodeling was very significant. Even, upregulation of MOMA-2 and downregulation of SM-actin were also detected in latter (P<0.05). In addition to SMC of aortic tunica media, PPARγ also expressed in SMC and macrophages in the aortic plaque of apoE^-/- mice. PPARγ was very enriched in lipid pool of the plaque. CONCLUSION: PPARγ expression level decreases with aging in C57BL/6J mice, while increases with plaque progression in apoE-knock out mice. There is positive correlation between PPARγ expression and lipid composition in plaque. The observed upregulation of PPARγ gene expression in aortic plaque may be a compensatory behavior and protective mechanism in apoE-knock out mice.     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.     

Effect of erlotinib on renal injury in rats with STZ-induced diabetic nephropathy

AIM: To investigate the effect of epidermal growth factor receptor (EGFR) inhibitor erlotinib on kidney injury in diabetic nephropathy (DN) rat and the underlying mechanism. METHODS: The rat model of DN was induced by intraperitoneal injection of streptozotocin (STZ) at dose of 55 mg/kg. One week after STZ injection, the rats with blood glucose level exceeding 16.7 mmol/L were identified as diabetic. Diabetic rats were randomly divided into 2 groups:STZ group and STZ+erlotinib group. In addition, the normal rats were used as control group. The rats in STZ+erlotinib group were treated with erlotinib at 100 mg·kg^-1·d^-1 for 4 weeks(5th~8th week). The fasting blood glucose (FBG), serum creatinine (SCr) and 24 h urine protein were measured. The pathological changes of the kidney were observed by HE staining and Masson staining. The protein levels of EGFR, p-EGFR, transforming growth factor β1 (TGFβ1), Smad2/3, p-Smad2/3, collagen Ⅳ (ColⅣ) and fibronectin in the kidney tissues were determined by Western blot. The reactive oxygen species (ROS) level and malondialdehyde (MDA) content in the renal tissues were futher analyzed. RESULTS: Compared with control group, the levels of FBG, 24 h urine protein and Scr were significantly increased in STZ group (P<0.01). Compared with STZ group, the levels of FBG, 24 h urine protein and SCr in STZ+erlotinib group were markedly decreased (P<0.05). In additon, the glomerular structure was restored to normal, the proliferative degree of mesangial cells markedly attenuated, and the epithelial cells were in alignment in STZ+erlotinib group. Moreover, erlotinib significantly inhibited the protein levels of p-EGFR, TGFβ1, p-Smad2/3, ColⅣ and fibronectin in the kidney tissues of STZ rats. In addition, erlotinib also significantly inhibited the levels of ROS and MDA in the kidney tissues of STZ rats. CONCLUSION: Erlotinib ameliorates STZ-induced diabetic nephropathy possibly through inhibiting the activation of EGFR/TGFβ1-Smad2/3 signaling pathway in association with suppression of fibrosis and oxidative stress.     

Effect of insulin on the SGK1 expression and extracellular matrix synthesis in human mesangial cells cultured in high glucose

AIM:To study the effect of insulin on the serum and glucocorticoid-inducible kinase 1 (SGK1) expression and extracellular matrix synthesis in human glomerular mesangial cells (HMC) cultured in high glucose. METHODS:The HMCs were cultured in the presence of 5.5 or 25 mmol/L glucose with or without 100 nmol/L insulin (i.e. NG, HG, NI and HI groups). 4 h latter, expressions of SGK1, insulin receptor substrate-1 (IRS1) and IRS2 in corresponding groups were detected by immunofluorescence or examined by Western blotting. The phosphorylation of IRS1 and IRS2 was measured by immunoprecipitation. 24 h latter, connective tissue growth factor(CTGF) and fibronectin (FN) were also examined by RT-PCR and ELISA, respectively. RESULTS:Compared with NG, the SGK1 protein expression in HG, NI and HI groups was significantly higher (P<0.01). High glucose mainly caused IRS2 protein and its phosphorylation level increase (P<0.01). When treated with 100 nmol/L insulin, IRS1 protein and its phosphorylation in HI group apparently elevated while slight inhibition of IRS2 protein expression and its phosphorylation were observed (HI vs HG, P<0.05). High glucose enhanced the expression of CTGF and FN, and insulin strengthened this effect.CONCLUSION:Insulin and high glucose up-regulate the expression of SGK1 in mesangial cells through different target molecular pathways and ultimately enhance ECM synthesis. The effect of insulin is highly associated with IRS1 signaling cascades.     

Effects of TRPC1 on TGF-β1-induced epithelial-mesenchymal transition of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the epithelial-mesenchymal transition (EMT) of human bronchial epithelial (HBE) cells induced by transforming growth factor-β1 (TGF-β1). METHODS: EMT of 16HBE cells induced by TGF-β1 were identified by microscopy, immunofluorescence and Western blotting. Immunofluorescence, real-time PCR and Western blotting were applied to detect the mRNA and the protein expression of TRPC1 in the 16HBE cells. The influence of SKF96365 (a TRPC1 blocker) and siRNA-mediated silencing of TRPC1 on the EMT of the 16HBE cells were detected by microscopy and Western blotting. RESULTS: Treatment with TGF-β1 induced significant morphological changes of the 16HBE cells. Exposure to TGF-β1 decreased the expression of E-cadherin protein (P<0.01) and increased the expression of α-SMA protein (P<0.05) in the 16HBE cells. Immunofluorescence observation indicated that TRPC1 expression in the 16HBE cells was positive. The expression of TRPC1 at mRNA and protein levels was significantly increased in the 16HBE cells after stimulation with TGF-β1 (P<0.05). The morphological changes of the 16HBE cells induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group. The protein expression of E-cadherin and α-SMA induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group (P<0.05). CONCLUSION: TGF-β1 induces EMT with the mechanism of up-regulating TRPC1 in human bronchial epithelial cells.     

Role of TGF-β1-activated p38 MAPK in up-regulation of PAI-1 expression by TGF-β1 in human ovarian cancer cells

AIM: To investigate the relationship between up-regulation of plasminogen activator inhibitor-1(PAI-1) expression and activation of p38 mitogen-activated protein kinase(p38 MAPK) and extracellular signal-regulated kinase(ERK) pathways by TGF-β1 in human ovarian cancer cells. METHODS: PAI-1 expression in human ovarian cancer cells treated with TGF-β1(10 μg/L)was assayed by real-time PCR and Western blotting. The activation of p38 MAPK and ERK was determined by Western blotting using phosphorylated p38 MAPK and phosphorylated ERK antibodies. Specific p38 MAPK inhibitor(SB203580) or ERK inhibitor(PD98059) was used to inhibit their activation. RESULTS: TGF-β1 up-regulated the expression of PAI-1, and activated p38 MAPK and ERK pathways in the ovarian cancer cells. Inhibition of p38 MAPK activation by SB203580 resulted in significant inhibition of the mRNA expression of PAI-1 induced by TGF-β1. However, inhibition of ERK activation did not significantly alter TGF-β1-induced increase in PAI-1 mRNA level. CONCLUSION: TGF-β1-activated p38 MAPK pathway contributes to the up-regulation of PAI-1 expression by TGF-β1 in ovarian cancer cells.     

Roles of gap junction in TGF-β1-induced proliferation of vascular smooth muscle cells from spontaneous hypertensive rats

AIM: To investigate whether gap junction participates in transforming growth factor β₁(TGF-β₁)-induced proliferation of spontaneous hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). METHODS: The thoracic aorta of the rats were sampled. The primary SHR VSMCs were isolated and cultured in vitro. The cells were divided into 4 groups: control group, TGF-β₁ group,18α-glycyrrhetinic acid(18α-GA) group and TGF-β₁+18α-GA group. The proliferation of SHR VSMCs was observed by the methods of MTT and flow cytometry. The protein expression and co-localization of connexin(Cx)43 and Cx40 in SHR VSMCs were detected by immunofluorescence staining. The protein levels of Cx43 and Cx40 in the cells were also measured by Western blotting. The method of molecular dye transfer (scrape dye transfer method) was applied to detect the function of gap junction in SHR VSMCs. RESULTS: The protein expression of Cx43 and Cx40 in SHR VSMCs was positive and co-localized in the cytoplasm. Compared with control group, the percentage of S-phase detected by cell cycle and A value detected by MTT in TGF-β₁ group were obviously increased (P<0.05), indicating that the proliferation of the cells was enhanced. However, the proliferation of the cells decreased in 18α-GA group (P<0.05). Compared with TGF-β₁ group, the percentage of S-phase and A value in TGF-β₁+18α-GA group were both significantly decreased (P<0.05), indicating that the proliferation of the cells decreased. Compared with control group, the protein expression of Cx43 in TGF-β₁ group was increased (P<0.05), whereas the protein expression of Cx40 was not changed (P>0.05), and the protein expression of Cx43 and Cx40 in 18α-GA group were decreased (P<0.05). Compared with TGF-β₁ group, the expression of Cx43 in TGF-β₁+18α-GA group was significantly decreased (P<0.05),but no difference of the Cx40 protein levels between the two groups was observed. Compared with control group, the function of gap junction detected by scrape dye transfer method in TGF-β₁ group was enhanced (P<0.05), and weakened in 18α-GA group (P<0.05). Compared with the TGF-β₁ group, the function of gap junction in TGF-β₁+18α-GA group was significantly attenuated (P<0.05). CONCLUSION: TGF-β₁ enhances the function of gap junction to stimulate the proliferation of SHR VSMCs through the expression of Cx43 protein. The expression of Cx40 protein may not play a major role in this process.