Effect of caveolin-1 and ERK1/2 on 17β-estradiol-induced inhibition of VSMC proliferation

AIM: To investigate the effect of caveolin-1 and phosphorylation of ERK1/2 on 17β-estradiol (E2) induced inhibition of vascular smooth muscle cells (VSMCs). METHODS: The proliferation in cultured VSMCs was determined by using ［3H］-thymidine incorporation. The expressions of caveolin-1, MKP-1 and ERK1/2 phosphorylation were measured by Western blotting. The expression of caveolin-1 mRNA was measured by RT-PCR. RESULTS: Exposed to fetal calf serum (FCS) for 24 h, the increase in proliferation of VSMCs was detected by ［3H］-thymidine incorporation. Pretreatment with various concentrations of E2 for 24 h inhibited VSMC proliferation induced by FCS. The results of Western blotting and RT-PCR showed that pretreated with 17β-estradiol for 24 h reserved the decrease in caveolin-1 induced by FCS. Western blotting results further proved that the expression of MKP-1 was significantly increased and the expression of ERK1/2 phosphorylation was decreased after incubated with 17β-estradiol. CONCLUSION: 17β-estradiol increases caveolin-1 and MKP-1 expressions, and decreases ERK1/2 phosphorylation, leading to the inhibition of VSMC proliferation.     

CREG inhibits proliferation of VSMCs by modulating internalization of IGF2R/IGFII

AIM: To study the molecular mechanisms of cellular repressor of E1A-stimulated genes (CREG) on the proliferation of human vascular smooth muscle cells (VSMCs) in vitro.METHODS: The pLNCX₂-CREG plasmid and the pSM₂-siCREG plasmid were transfected into VSMCs to produce the cell clones of over-expression and down-expression of CREG, respectively.BrdU assay and FACS cell cycle analysis were performed to detect the proliferation of the cells.The expression and localization of insulin-like growth factor Ⅱ receptor(IGF2R) in the hVSMCs were detected by Western blotting and immunocytochemistry.The expression and secretion of insulin-like growth factor Ⅱ(IGFII) were measured by RT-PCR and ELISA.Alexa 488-labeled rhIGFII was used to investigate the endocysis of the cells.The blockade of IGFII internalization was conducted by treating the cells with both neutralized antibody of IGF2R and recombinant IGF2R peptide to detect the effect of IGFII on HVSMCs growth.Furthermore, Western blotting and signal pathway inhibitor were used to analysis the activation of PI3K/Akt and ERK on VSMCs proliferation.RESULTS: Compared with the control cells, Western blotting identified that the expression of CREG was increased in VSMCs-CREG cells and was decreased in VSMCs-siCREG cells.Meanwhile, the over-expression of CREG in the cells inhibited the proliferation of VSMCs and enhanced the distribution of IGF2R in cellular membrane.Furthermore, over-expression of CREG also accelerated the endocytosis of IGFII in VSMCs-CREG cells, and attenuated the secretion of IGFII into cell medium.Blockade experiments showed that enhancement of IGFII secretion promoted the proliferation of HVSMCs.PI3K/Akt and ERK signal pathways mediated the effect of IGFII on the VSMCs.CONCLUSION: CREG inhibits the proliferation of VSMCs through interfering with the internalization of IGF2R-IGFII.     

Effects of extract of ginkgo biloba on human tubular epithelial-mesenchymal transition induced by transforming growth factor-β1

AIM: to investigate the effects of extract of ginkgo biloba (EGB) on human tubular epithelial-mesenchymal transition induced by transforming growth factor-β₁.METHODS: HK2 cells were induced to epithelial-mesenchymal transition by transforming growth factor-β₁ (TGF-β₁, 10 μg/L). EGB was added into the medium of HK2 cells 2 h before TGF-β₁ was added. The expressions of E-cadherin, α-smooth muscle actin (α-SMA), NADPH oxidase p67phox and superoxide dismutase (SOD) were determined by Western blotting. Malondialdehyde (MDA) in the mediums of HK2 cells was detected. RESULTS: EGB significantly attenuated the downregulation of E-cadherin, the upregulation of α-SMA and p67phox, the downregulation of SOD and the upregulation of MDA in HK2 cells induced by TGF-β₁.CONCLUSION: EGB significantly attenuates human tubular epithelial-mesenchymal transition induced by TGF-β₁, and its underlying mechanism is that EGB attenuates the upregulation of p67phox and the downregulation of SOD induced by TGF-β₁.     

Estradiol stimulated proliferation and differentiation of prostatic stromal cells through regulation of BPH-1 paracrine

AIM: To characterize the effect of estradiol on proliferation, differentiation and extracellular matrix (ECM) accumulation in stromal cells through regulation of BPH-1 paracrine. METHODS: BPH-1 cells were stimulated with different concentrations of estradiol. Conditioned media (CM) were harvested and their effects on stromal cell cultures were tested. Cell proliferation was determined by MTT assay. mRNA of smoothelin, fibronectin, collagen Ⅳ and transforming growth factor β₁(TGF-β₁) were analyzed by real-time RT-PCR. Western blotting was used to determine smooth muscle myosin heavy chain (SMMHC). ELISA and radioimmunoassay were respectively used to measure fibronectin, TGF-β₁ and collagen Ⅳ protein expressions.RESULTS: Estrodiol stimulated the expression and secretion of TGF-β₁ in BPH-1 cells. The proliferation of stromal cells increased when they were cultured with CM harvested from estrogen treated BPH-1 cells. The mRNA levels of collagen Ⅳ and smoothelin increased in stromal cells treated with CM from BPH-1 cells. The results of radioimmunoassay also showed that the collagen Ⅳ protein level up-regulated in the supernatants and cell extracts of CM-treated stromal cells. A neutralizing antibody to TGF-β₁ inhibited the stimulation of collagen Ⅳ and SMMHC by BPH-1 CM. The expression of fibronectin was only marginally changed in stromal cells cultured in the presence of BPH-1 CM. CONCLUSION: The BPH-1 cells increase ECM accumulation and differentiation of stromal cells through TGF-β₁. Estradiol stimulate differentiation of stromal cells by induction of TGF-β₁ expression. Estradiol stimulate proliferation by influencing the factors secreted from prostatic epithelial cells.     

Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.     

Role of danshensu on TGF-β signal transduction in rat’s hepatic stellate cells

AIM: To investigate the role of danshensu on Smad signal transduction in rat hepatic stellate cells (HSCs) stimulated with transforming growth factor (TGF-β1). METHODS: The rat HSCs was isolated with collagenase by in situ-liver recirculation perfusion and cultured in vitro. MTT colorimetric assay was used to detect proliferation of HSCs treated with different concentration of danshensu. The expressions of α-SMA and TβR I and II were observed by immunocytochemitry, indirect immunofluorescent staining and Western blotting when HSCs stimulated with TGF-β1 and with different concentrations of danshensu for 24 h. RESULTS: (1) Danshensu at the concentration from 0.0625 mmol/L to 1 mmol/L prevented the proliferation of HSCs in a dose-dependent manner (P<0.05). Danshensu also inhibited the proliferation of HSCs induced by TGF-β1 in a dose-dependent manner (P<0.05). (2) At concentration of 0.25 mmol/L, danshensu down-regulated α-SMA protein expression in HSCs with or without stimulation of TGF-β1 (P<0.05), and the activation of HSCs was inhibited also. (3) Danshensu down-regulated the protein expression of TβR I and II in HSCs stimulated with TGF-β1 (P<0.05, or P<0.01), these effects were correlated with the concentration. (4) TGF-β1 increased the mRNA level of Smad2, 3, and 7 in HSCs (P<0.01). Danshensu down-regulated the mRNA level of Smad2, 3 (P<0.05) and up-regulate the mRNA level of Smad7 in HSCs induced by TGF-β1 (P<0.05). CONCLUSION: Danshensu inhibits the activation and proliferation of HSCs through down-regulating the expression of TβR I and II located in cellular membrane of HSCs. Danshensu suppresses the activation of HSCs, and also inhibits the activation of HSCs stimulated by TGF-β1 through up-regulation of Smad7 mRNA and down-regulation of Smad2, Smad3 mRNA expression in HSCs.     

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells

AIM: To express human Arresten gene in eukaryotic cell,and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells (VSMCs).METHODS: COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome.48 hours after transfection,polymerase chain reaction (RT-PCR) was used to detect the expression of Arresten mRNA in the cells,while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants.VSMCs were then co-cultured with the concentrated supernatants;and its proliferation was detected using cell counting kit-8 (CCK-8) in vitro.Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chamber) with pores of 8 μm in diameter.RESULTS: RT-PCR revealed that the genome of Arresten-transferred cells contained a 449bp specific fragment of Arresten gene.Successful protein expression in supernatants was confirmed by Western blotting.CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group (P<0.01).Transwell's chamber showed that the number of control group,pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53 and 14.00±3.33 (P<0.01).CONCLUSION: Arresten protein expressed in eukaryotic cells inhibits the proliferation and migration of VSMCs effectively in vitro.     

RXRα agonist bexarotene inhibits atherosclerosis in ApoE-/- mice by regulating TGF-β1/Smad2 signaling pathway

AIM To observe the effect of retinoid X receptor α （RXRα） agonist bexarotene （Bex） on the proliferation of transforming growth factor β1 （TGF-β1）-induced vascular smooth muscle cells （VSMCs） and atherosclerosis in apolipoprotein E knockout （ApoE^-/-） mice， and to explore the underlying mechanism. METHODS Ten C57BL/6 mice were selected as normal control group， and 30 ApoE^-/- mice were randomly divided into 3 groups： ApoE^-/- group， ApoE^-/-+Bex5 （5 mg·kg^-1·d^-1 Bex） group and ApoE^-/-+Bex10 （10 mg·kg^-1·d^-1 Bex） group. Bex was intragastrically given once a day for 8 weeks. The levels of triglyceride （TG） and total cholesterol （TC） were determined by oxidase method， and select masking method was used to determine serum levels of low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C）. The protein levels of TGF-β1， p-Smad2 and Smad2 were determined by Western blot. HE staining was used to observe the intima of the thoracic aorta. The VSMCs were cultured with tissue patch method， and the proliferation of VSMCs was measured by BrdU incorporation method. RESULTS The serum levels of TG， TC and LDL-C， and the expression of TGF-β1 and p-Smad2 in thoracic aorta in ApoE^-/- group were significantly higher than those in C57BL/6 group （P<0.01）. Bex increased p-Smad2 protein level in thoracic aorta in a dose-dependent manner， inhibited the intimal plaque formation and vascular medial proliferation， and decreased the plaque area in ApoE^-/- mice （P<0.01）. No significant difference in serum levels of TG， TC， HDL-C and LDL-C， and TGF-β1 and Smad2 expression in thoracic aorta among ApoE^-/- group， ApoE^-/-+Bex5 group and ApoE^-/-+Bex10 group was observed. TGF-β1 （0.1~10 μg/L） promoted the proliferation of VSMCs， while Bex （10^-9~10^-7 mol/L） inhibited TGF-β1 （5 μg/L）-induced proliferation of VSMCs in a concentration-dependent manner. Bex （10^-7 mol/L） synergistically promoted the protein level of p-Smad2 in VSMCs induced by TGF-β1 （P<0.01）， but inhibited TGF-β1-induced nuclear translocation of p-Smad2. CONCLUSION RXRα agonist Bex inhibits the formation of atherosclerosis in ApoE^-/- mice， and its mechanism may be related to the regulation of TGF-β1/Smad2 pathway.     

Construction of FAK-shRNA retroviral vector and screening of stable HCC-LM3 cell line with persistent knockdown of FAK

AIM: To construct a recombinant retroviral vector of short interfering RNA targeting focal adhesion kinase (FAK) gene and to establish a cell line with stable knockdown of FAK.METHODS: The oligonucleotides that transcribed to short hairpin RNA (shRNA) targeting FAK gene were synthesized in vitro, cloned into retroviral vector pSuper.retro and transfected into Phoenix cell line. The stable clones were screened and high-titer virus was produced. The human hepatocellular carcinoma cell line HCC-LM3 was infected with the virus-rich supernatant. The stable LM3 cell line, which showed significantly to silence FAK and associated proteins, was selected by puromycin.RESULTS: The recombinant retroviral vector was successfully constructed. Persistent knockdown of FAK in the LM3 cell line infected with the supernatant containing the retrovirus was confirmed by Western blotting. Down-regulation of FAK resulted in the inhibition of p-Akt and p-MAPK1/2 expression and led to decreased migration and invasion of the cells. The cell cycle was blocked at G₀/G₁ phase, and apoptosis was increased. The proliferation rate also decreased significantly.CONCLUSION: FAK-shRNA virus generated by recombinant retroviral vector pSuper-FAK can inhibit the protein expression of FAK and phosphorylation of Akt and MAPK1/2 in HCC-LM3 cells. Down-regulation of FAK shows a significant impact on biological behaviors of tumor cells.     

Effects of rhTGF-β1 and TGF-β1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro

AIM: To investigate the effects of rhTGF-β₁ and TGF-β₁ gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro. METHODS: Cell growth induced by various concentrations of rhTGF-β₁ was determined by MTT proliferation assay. Under the induction of liposomes, recombinant pSecTag2-TGF-β₁MP vectors were transferred into the corneal endothelial cells. Morphological changes of transfected cells were observed by HE staining. The expression levels of TGF-β₁ were assessed by ELISA. Cell cycle analysis was assessed by flow cytometry. DNA fragment analysis was used to confirm the presence of apoptosis. RESULTS: rhTGF-β₁ in concentrations of 5-20 μg/L showed a significant suppressive effect on the proliferation of corneal endothelial cells, 0.5-1 μg/L had no effect, 0.05-0.1 μg/L facilitated cell growth, as compared with negative controls. The morphous of transfected corneal cells had no significant abnormality compared with normal cells. According to the result of ELISA, the concentration of TGF-β₁ in the supernatant was calculated to be (98±3) ng/L. Flow cytometry assay showed that S and G₂/M phase of transfected cells decreased significantly compared with that of control group, but the cell cycle recovered normally after adding 10 μg/L EGF into the culture medium. Agarose electrophoresis didn′t show marked ladders in transfected group. CONCLUSION: Effects of rhTGF-β₁ on the proliferation of corneal endothelial cells are different with various concentrations. TGF-β₁ gene transfection shows suppressive effect on the proliferation of cultured corneal endothelial cells, but does not induce cell apoptosis. EGF is the antagonist of this suppressive effect.     

Preventive effects of anti-IGFBPrP1 antibody on hepatic fibrosis in mice

AIM: To investigate the preventive effect and mechanism of anti-insulin-like growth factor binding protein related protein 1(IGFBPrP1) antibody on hepatic fibrosis induced by thioacetamide (TAA) in mice.METHODS: Twenty-four male C57BL/6 wild-type mice were randomly divided into 3 groups (n= 8 in each group): normal control group, TAA group (4 weeks) and TAA+anti-IGFBPrP1 antibody group (4 weeks). The morphological changes of liver tissues were observed. The expression levels of α-smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β₁), Smad3, phosphorylated Smad2/3 (p-Smad2/3), fibronectin (FN), collagen I, collagen Ⅲ and IGFBPrP1 were detected by the methods of immunohistochemistry and Western blotting.RESULTS: In TAA group (4 weeks), obvious injury of liver was observed, and the expression levels of α-SMA, TGF-β₁, Smad3, p-Smad2/3, FN, collagen Ⅰ, collagen Ⅲ and IGFBPrP1 were significantly increased as compared with normal control group (P<0.01). Compared with TAA group (4 weeks), the injury of the liver was alleviated and the expression levels of the proteins above were decreased in TAA+anti-IGFBPrP1 antibody group (4 weeks, P<0.01). IGFBPrP1 was positively correlated with TGF-β₁, Smad3, p-Smad2/3, FN and collagen I (P<0.01). CONCLUSION: Anti-IGFBPrP1 antibody prevents TAA-induced hepatic fibrosis in mice by inhibiting the activation of hepatic stellate cells, reducing the expression of p-Smad2/3 and inhibiting the TGF-β₁/ Smad3 signal transduction, thereby depressing the deposition of extracellular matrix in liver tissues.     

Recombinant human defensin α1 induces proliferation of rat glomerular mesangial cells in vitro

AIM: To study the effects and mechanism of recombinant human defensin α₁ on cell proliferation in cultured rat glomerular mesangial cells.METHODS: The influences of defensin α₁ at various concentrations on rat 1097 mesangial cell line cultured in vitro were evaluated with MTT assay.The different concentrations of U0126,signal-regulated protein kinase (MEK) inhibitor,were added into the culture mediums of mesangial cells to do blocking test.Incubated with a final concentration of 3 mg/L defensin α₁,the phosphorylation of extracellular signal regulated kinase (ERK)1/2 and type IV collagen of mesangial cells in different times were evaluated by Western blotting.RESULTS: Defensin α₁ at 3-20 mg/L enhanced proliferation of rat glomerular mesangial cells.The incubation times for the maximum effect on proliferation was 12 h (P<0.01),whereas defensin α₁ concentration >20 mg/L decreased cell proliferation.The cell proliferation induced by defensin α₁ was inhibited by U0126.Stimulation of the cells with defensin α₁ at concentration of 3 mg/L for 5 minutes induced a maximum effect on a ratio of phosphorylation of ERK1/2 to total ERK.After 12 h incubation with defensin α₁,an increase in type IV collagen was observed by Western blotting and continued to increase at 24 h and 48 h (P<0.01).CONCLUSION: Defensin α₁ enhances rat glomerular mesangial cell proliferation and induces type IV collagen production by MAPK signaling pathway.     

Effects of p204 expression on expression of p21 and proliferation of vascular smooth muscle cells in rats

AIM: To observe the effect of interferon-inducible protein 204 (p204) on the expression of p21 and proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Interferon alpha (IFN-α) and small interference RNA (siRNA) targeting p204 gene ( Ifi204 ) was used to intervene cultured VSMCs in vitro instantaneously, then the cell vitality was determined by MTT assay to reflect the cell proliferation. The cell cycle was analyzed by flow cytometry. The expression of p204 and p21 at mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blotting. RESULTS: In rat VSMCs, IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality and the G₁/S phase transition, and up-regulated the expression of p21 at mRNA and protein levels. Transfection of Ifi204 siRNA restrained the expression of p204 and p21, increased the cell vitality and promoted the G₁/S phase transition. CONCLUSION: The expression of p204 restrains the proliferation of rat VSMCs, probably by activating the expression of p21.     

The roles of ERK signaling pathway in osteogenic differentiation of rat MSCs promoted by quercetin

AIM:To study the roles of extracellular signal-regulated kinase(ERK) signal pathway in the process of osteogenic differentiation in rat mesenchymal stem cells(MSCs) promoted by quercetin(QUE). METHODS:The optimal concentration of QUE for promoting osteogenic differentiation of rat MSCs was determined by MTT and alkaline phosphatase(ALP) detection. The activity of ALP was detected by the ALP detection kit. The expression of bone Gla protein(BGP) and collagen typeⅠ(ColⅠ) was observed by ELISA analysis. MSCs were exposed to QUE at optimal concentration with or without ERK1/2 inhibitor PD98059. Non-phosphorylated and phosphorylated expression of ERK1/2 was analyzed by Western blotting. The mRNA expression of transforming growth factor β₁(TGF-β₁), bone morphogenetic protein 2(BMP-2) and core binding factor α1(Cbfα1) was measured by fluorescence quantitative PCR. RESULTS:QUE at concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L induced the expression of ALP in MSCs in a dose-dependent manner, and also promoted MSCs proliferation. The expression levels of ALP, BGP and ColⅠwere higher in QUE group, and was lower in PD89059 group than those in control group. Compared with control group, the level of phosphorylated ERK1/2, and the mRNA expression of TGF-β₁, BMP-2 and Cbfα1 increased in QUE group. The mRNA expression of TGF-β₁, BMP-2 and Cbfα1 in QUE+PD98059 group decreased as compared with QUE group. CONCLUSION:QUE promotes osteogenic differentiation of MSCs by activating ERK signaling pathway.