Role of IGF-Ⅱ in proliferation and migration of hepatocellular carcinoma Huh7 cells

AIM: To observe the effects of insulin-like growth factorⅡ (IGF-Ⅱ) at different expression levels on hepatocellular carcinoma Huh7 cell proliferation and migration, and to explore the role of IGF-Ⅱ in the development of HCC. METHODS: The Huh7 cells were transfected with the over-expression plasmid pcDNA3.1(+)- IGF-Ⅱ or RNA interference plasmid pLVX-shRNA-IGF-Ⅱ by Lipofectamine 2000. The quantitative real-time PCR and Western blotting were used to verify the expression of IGF-II. The biological behaviors of the Huh7 cells were analyzed by CCK-8 assay, plate clone formation assay, cell scratch test and Transwell chamber experiment.RESULTS: Over-expression of IGF-II promoted the growth and migration of hepatocellular carcinoma cells (P < 0.05), and the cell proliferation was significantly inhibited in the Huh7 cells with low IGF-II expression (P < 0.05).CONCLUSION: IGF-II is involved in the regulation of biological behavior of hepatocellular carcinoma Huh7 cells in vitro, which may play a promoting role in the development of hepatocellular carcinoma.     

miR-339 suppresses proliferation of pulmonary arterial smooth muscle cells via regulating IGF2BP1

AIM: The present study aimed to investigate the role of microRNA-339 miR-339 in the proliferation of pulmonary artery smooth muscle cells (PASMCs). METHODS: After treating with angiotensin Ⅱ of different concentrations for 48 h, miR-339 mimic and miR-339 inhibitor were transfected into PASMCs, respectively. CCK-8 assay and viable cell counting were performed to determine cell proliferation. The expression levels of miR-339 and PCNA mRNA were measured by RT-qPCR. The protein levels were detected by Western blot. The interaction between miR-339 and insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1) was confirmed by luciferase reporter assay. RESULTS: Angiotensin Ⅱ concentration-dependently increased cell proliferation and mRNA expression of PCNA, and decreased miR-339 expression in the PASMCs. Over-expression of miR-339 inhibited cell proliferation and mRNA expression of PCNA in the PASMCs, while mutation of miR-339 promoted cell proliferation and mRNA expression of PCNA in the PASMCs. In addition, miR-339 inhibited cell proliferation in angiotensin Ⅱ-treated PASMCs. Bioinformatics analysis showed that miR-339 targeted the IGF2BP1 3'-UTR, which was confirmed by luciferase reporter assay, and miR-339 negatively regulated the expression of IGF2BP1 in the PASMCs. More importantly, over-expression of IGF2BP1 attenuated the inhibitory effects of miR-339 on cell proliferation and mRNA expression of PCNA in the PASMCs. miR-399 over-expression suppressed phosphorylated p38 protein level but not p38 protein level. CONCLUSION: miR-339 suppresses anti-proliferative effects in PASMCs partly via regulating IGF2BP1 and p38 MAPK signaling pathway.     

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.     

Akt and ERK1/2 are involved in brain derived neurotrophic factor-induced angiogenesis of endothelial cells

AIM: To investigate the role of PI3K/Akt and MEK1/ERK pathway in the brain derived neurotrophic factor(BDNF)-induced angiogenesis in vitro and to explore the further molecular mechanisms. METHODS: The phosphorylations of Akt and ERK1/2 were detected in human umbilical vein endothelial cells(HUVECs) by Western blotting. The angiogenic activity in vitro was evaluated by transwell migration assay and tubule formation assay. Cell proliferation was determined by crystal violet staining. Cell apoptosis was analysed by FITC-Annexin-V/PI double staining and flow cytometry. RESULTS: BDNF activated the PI3K/Akt and MEK1/ERK pathway in a time-dependent manner. Ly294002 and PD98059 blocked the activation of Akt and ERK1/2 in response to BDNF. BDNF at concentration of 100 μg/L significantly increased HUVECs tube formation, migration and proliferation in vitro to a degree similar to those induced by 25 μg/L VEGF. Furthermore, tube formation and migration of HUVECs toward BDNF were significantly inhibited by treatment with 20 μmol/L Ly294002 and 20 μmol/L PD98059. BDNF-induced survival was only blocked by Ly294002 and BDNF-induced proliferation was only inhibited by PD98059. CONCLUSION: BDNF promotes angiogenesis of HUVECs in vitro. The ERK and Akt are two crucial events in BDNF-mediated signal transduction leading to HUVECs angiogenesis by different mechanisms. Moreover, the latter is more important.     

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.     

Prevention and treatment of AD by over-expression of neuroglobin via activating PI3K/Akt signaling pathway

AIM: To study the neuroprotective roles of neuroglobin (NGB) over-expression in the SH-SY5Y cells transfected with pAPPswe.METHODS: The plasmid pEGFP-NGB was successfully constructed and transfected into the SH-SY5Y cells, which were pretreated with pAPPswe. MTT assay was applied to detect the effect of NGB over-expression on the cell survival rates. JC-1 staining was used to detect the level of mitochondrial transmembrane potential. The cell apoptosis was analyzed by flow cytometry. The effects of NGB over-expression on the protein level of p-Akt, Akt and caspase-3/9 were determined by Western blotting. The generation of Aβ42 in the cells was measured by ELISA.RESULTS: The cell survival rate was remarkably increased after transfection with NGB compared with control group and empty plasmid group (P<0.05). The over-expression of NGB significantly inhibited the decrease in mitochondrial membrane potential induced by pAPPswe. The over-expression of NGB inhibited the apoptosis of the cells. Furthermore, over-expression of NGB not only inhibited the expression of caspase-3 and caspase-9, but also induced the production of p-Akt, which was prevented by LY294002, an inhibitor of PI3K/Akt. The generation of Aβ42 was inhibited in the cells with the over-expression of NGB. CONCLUSION: Over-expression of NGB significantly inhibits the SH-SY5Y cell injuries induced by pAPPswe and inhibits the expression of caspase-3/9, which is tightly related with cell apoptosis. Furthermore, the neuroprotective roles of NGB may be via activating PI3K/Akt signaling pathway.     

Retrovirus-mediated cellular repressor of E1A-stimulated genes inhibits neointima formation in the rat carotid artery after balloon injury

AIM: To evaluate the effects of over-expression of cellular repressor of E1A-stimulated genes (CREG) mediated by retrovirus on neointima formation in injured rat carotid. METHODS: The pluronic F127 containing pLNCX/CREG or pLNCX/GFP retroviral vectors was placed around the injured rat carotid.The neointima,media areas and the intima to media ratio were calculated.Expressions of CREG,SM α-actin and Ki-67 were detected. RESULTS: The GFP expression was observed at day 2 in pLNCX/GFP groups.The expression of exogenous CREG was also significantly increased in arteries at day 2 after pLNCX-CREG infection.Over-expression of CREG significantly suppressed neointima formation,attenuated the expression of Ki-67 and up-regulated SM α-actin expression. CONCLUSION: Over-expression of CREG inhibits VSMCs proliferation and promotes VSMCs differentiation after vascular injury.It suggests that modulation of CREG expression or activity may be a viable approach to treat neointimal restenosis after percutaneous coronary intervention.     

Effect of gonadotropin-releasing hormone analogue on human breast cancer cell line in vitro

AIM: To investigate the effects of gonadotropin-releasing hormone (GnRH) analogue on the growth of breast cancer cell lines MCF-7 and MDA-MB-231 in vitro and to explore the related mechanisms with PI3K/Akt or ERK/MAPK pathways. METHODS: The proliferation of human breast cancer cell lines MCF-7 and MDA-MB-231 treatment with triptorelin was detected by MTT assay and the distribution of the cell cycle was determined by flow cytometry. The phosphorylation of the ERK1/2 and Akt was evaluated by Western blotting. RESULTS: Triptorelin inhibited the proliferation of MCF-7 cells at concentration of 10^-5 mol/L after treated for 192 h or at concentration of 10^-4 mol/L after treated for 168 h and 192 h. Triptorelin inhibited the proliferation of MDA-MB-231 cells at concentration of 10^-4 mol/L after treated for 192 h (P<0.05).Treatment with triptorelin for 192 h at concentration of 10^-4 mol/L had no statistical significance effect on phosphorylation of ERK1/2 and Akt(P>0.05).CONCLUSION: Inhibitory effect of GnRH analogue triptorelin on human breast cancer cells is not just the connection with the down-regulation of pituitary hormone, but also a direct inhibitory effect. The role may not be involved in the activation of ERK/MAPK and PI3K/Akt signaling pathways.     

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.     

Antagonistic effect of angiotensin II type 1 receptor blocker candesartan on angiotensin II-induced proliferation of primary acute myeloid leukemia cells

AIM: To explore the antagonistic effect and mechanism of candesartan on angiotensin II (Ang II)-induced proliferation of primary acute myeloid leukemia (AML)cells. METHODS: MTT assay was used to observe the proliferation effect of Ang II on primary AML cells and normal bone marrow mononuclear cells, and the antagonistic effects of candesartan and PD123319 (an antagonist of AT2R) were also observed. Akt phosphorylation was detected by Western blotting when the cells were treated with candesartan and a PI3K inhibitor LY294002.RESULTS: Compared with the control cells, Ang II significantly increased the proliferation of AML cells in a dose-and time-dependent manner (P<0.05). Ang II did not stimulate the proliferation of normal bone marrow mononuclear cells. The proliferative effect of Ang II was effectively blocked by the AT1R blocker candesartan (P<0.05). PI3K inhibitor strongly repressed the Ang II-induced cell proliferation (P<0.05). Candesartan significantly reduced Akt phosphorylation promoted by Ang II on primary AML cells (P<0.05).CONCLUSION: Candesartan effectively inhibits Ang II-induced proliferation of primary AML cells by down-regulating PI3K/Akt signaling pathway, indicating a new possible treatment mechanism in some AML cells.     

Role of nephrin in preventing angiotensinⅡ-induced cytoskeletal rearrangement in glomerular podocytes

AIM: To investigate the role of nephrin, a slit diaphragm-associated protein, in angiotensinⅡ (AngⅡ)-induced cytoskeleton rearrangement in podocytes. METHODS: Immortalized mouse podocytes were exposed to AngⅡ (10^-8 mol/L) with or without AngⅡ receptor antagonist lorsatan and Akt inhibitor LY294002. FITC-conjugated phalloidin was used to stain F-actin, and semi-quantitative system with cortical F-actin score (CFS) was introduced to analyze the degree of actin cytoskeleton arrangement. The expression of nephrin was assessed by quantitative real-time RT-PCR,RT-PCR and Western blotting. Undifferentiated podocytes were transfected with pcDNA3.1-mNPHS1 plasmid containing the full length of nephrin. The stably transfected cell line was generated by G418 selection. Phosphorylation level of Akt was assessed by Western blotting, and F-actin distribution was further evaluated in transfected cells exposed to AngⅡ or not. RESULTS: Cytoskeletal rearrangements including cortical F-actin ring formation and stress fiber attenuation were observed in Ang II-and LY294002-stimulated podocytes. Pretreatment with losartan significantly prevented Ang II-induced actin cytoskeleton reorganization. The mRNA and protein levels of nephrin and phosphorylation of Akt were obviously decreased in the podocytes exposed to Ang II, which were dramatically reversed by pcDNA3.1-mNPHS1 transfection. Transfection of pcDNA3.1- mNPHS1 induced the formation of short filopodia and partially prevented AngⅡ-induced F-actin remodeling. CONCLUSION: PI3K/Akt signaling is a common downstream pathway of nephrin and Ang Ⅱ. Nephrin is able to stabilize AngⅡ-induced cytoskeletal rearrangement via PI3K/Akt signaling pathway.     

CREG gene silencing induces spontaneous apoptosis of ESCs-derived Ebs

AIM: To investigate the effect of cellular repressor of E1A-stimulated genes (CREG) on the spontaneous apoptosis of murine embryonic stem cells(ESCs)-derived embryoid bodies (EBs).METHODS: The murine ESCs R1 were infected with pDS-shRNA-CREG and pDS-shRNA-GFP retrovirus, respectively. R1, R1-GFP and R1-shCREG were cultured on STO feeder cells in DMEM supplied with leukemia inhibitory factor(LIF). Alkaline phosphatase(AKP) staining and teratoma formation assay inoculated into mouse myocardium were used to detect stemness of transfected ESCs. R1/EB, R1-GFP/EB and R1-shCREG/EB were produced by liquid suspension method. The expression of CREG and cleaved caspase-3 were analyzed by Western blotting and quantitative RT-PCR on day 7. The apoptotic rates of the 3 kinds of EBs were analyzed by flow cytometry(FCM) analysis with Annexin V/PI dual staining. RESULTS: The stably-transfected ES cells (R1-shCREG and R1-GFP) were obtained by screening the G418-resistant clones. R1-GFP and R1-shCREG had the stem cell properties similar to those of R1 detected by AKP staining. However, it was found that R1-shCREG didn't show the same almighty differentiation function as of R1 and R1-GFP by tumor experiments in mouse myocardium that it couldn't form teratomas analogue and had the lower ability of cell differentiation. Observation under phase-contrast microscope showed that the cell differentiation was inhibited while the number of cell death was increased in R1-shCREG/EB group. Compared with R1/EB and R1-GFP/EB, Western blotting analysis demonstrated that the protein expression of CREG was decreased to (78.0±1.3)% and (84.0±2.4)% on day 7, respectively. The mRNA expression of CREG was also decreased, but the expression of cleaved caspase-3 at the mRNA and protein levels was increased obviously. Annexin V/PI FCM assay indicated that the apoptotic rate of R1-shCREG/EB was significantly higher those that in other 2 groups on day 7. CONCLUSION: The down-regulation of CREG inhibits ESCs differentiation and promotes cell apoptosis.     

CREG prevents human umbilical vein endothelial cells from apoptosis by inhibiting p38 MAPK activation

AIM: To study the effect of cellular repressor of E1A-stimulated genes(CREG) and its mechanism on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by etoposide (VP-16).METHODS: Primary HUVECs were cultured. RetroviraI eukaryotic expression vectors pLNCX-CREG and pLXSN-shRNA-CREG were transfected into HUVECs. The stable cell clone was selected and obtained by screening with G418 (800 mg/L) and the puromycin (2.5 mg/L), respectively. CREG expression was detected by Western blotting. The cells with overexpression of CREG (H-C) and those with CREG down-regulation (H-S) were pretreated with apoptotic inducer VP-16 at 100 μmol/L for 6 h. The apoptotic rates of the 3 kinds of cells were analyzed by TUNEL and flow cytometry with annexin V/PI dualstaining. Furthermore, the protein levels of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in the 3 kinds of cells were analyzed by Western blotting. The p38-specific inhibitor SB203580(20 μmol/L)was used to investigate the effects of p-p38 expression on apoptosis. RESULTS: Western blotting showed that CREG expression was obviously increased up to 160% in H-C compared to HUVECs. However, CREG expression in H-S cells was identified to be down-regulated to 70% compared with HUVECs. TUNEL assay and annexin V/PI dual-color FACS showed that the apoptotic rate was dramatically increased in H-S cells,but decreased in H-C cells. Subsequently, Western blotting exhibited that p-p38 expression was increased in H-S cells compared to HUVECs and H-C cells. When the H-S was pretreated with SB203580, the apoptotic rate was decreased. CONCLUSION: CREG overexpression might prevent HUVECs from apoptosis by inhibiting p38 MAPK activition.     

miRNA-7 inhibits proliferation of human nasopharyngeal 5-8F carcinoma cells via EGFR/PI3K/Akt pathway

AIM:To investigate the relationship of microRNA-7 (miRNA-7) over-expression and epidermal growth factor receptor (EGFR)/phosphatidylinositol kinase-3 (PI3K)/protein kinase B (PKB, also called Akt) pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were transfected with miRNA-7 mimics (carrying by Lipofectamine 2000). The expression of miRNA-7 was detected by real-time PCR. The cell proliferation was analyzed by CCK-8 assay. The cell colony-forming capability was determined by cell colony formation test. The expression of EGFR/PI3K/Akt at mRNA and protein levels was examined by real-time PCR and Western blotting. RESULTS:The expression level of miRNA-7 was significantly increased in 5-8F cells compared with negative control (NC) group and control group (P<0.01). The proliferation of NPC 5-8F cells was decreased extremely after tansfected with the miRNA-7 mimics (P<0.01), so did the result of the cell colony-formation test. The expression of EGFR/PI3K/Akt at mRNA and protein levels was significantly down-regulated compared with NC group and control group (P<0.01). CONCLUSION: Over-expression of miRNA-7 significantly inhibits the proliferation and colony-forming ability of nasopharyngeal carcinoma 5-8F cells by down-regulation of EGFR/PI3K/Akt pathway.     

Effects of marrow stromal cell-mediated microenvironment on human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the effects of marrow stromal HS-5 cells on hepatocellular carcinoma SMMC-7721 cells in the tumor microenvironment. METHODS: The effects of HS-5 cell-conditioned medium (HS-5-CM) on the proliferation, migration and invasion abilities of SMMC-7721 cells were detected by MTT, wound-healing and Transwell assays. After co-culture of SMMC-7721 cells with HS-5 cells in the Transwell chamber, the expression of chemokine CCL5 and its receptor CCR5 at mRNA and protein levels in SMMC-7721 cells was examined by quantitative real-time PCR (qRT-PCR), ELISA or Western blotting. Akt and p-Akt473 protein levels in SMMC-7721 cells treated with PI3K inhibitor LY294002 were observed by Western blotting. RESULTS: HS-5-CM promoted the proliferation, migration and invasion abilities of SMMC-7721 cells. The expression of CCL5 and CCR5 at mRNA and protein levels in SMMC-7721 cells was increased after co-cultured with HS-5 cells. PI3K inhibitor LY294002 inhibited the activation of PI3K-Akt signaling pathway and the secretion of CCL5 in SMMC-7721 cells after co-cultured with HS-5 cells. CONCLUSION: HS-5 cells significantly promote the proliferation, migration and invasion abilities of SMMC-7721 cells. Co-culture of SMMC-7721 cells with HS-5 cells activates PI3K-Akt signaling pathway to increase the secretion of CCL5 in SMMC-7721 cells.     

Effects of adenovirus-mediated shRNA targeting PTEN on proliferation and apoptosis of activated hepatic stellate cells in vitro

AIM:To investigate the down-regulation of phosphatase and tensin homolog deleted on chromosome 10(PTEN) gene by adenovirus-mediated short hairpin RNA(shRNA) on proliferation and apoptosis of activated hepatic stellate cells(HSCs) in vitro and the related signaling transduction pathways. METHODS:The activated HSCs were cultured in vitro and transfected with recombinant adenovirus expressing shRNA targeting PTEN. The proliferation of HSCs was measured by MTT assay and the apoptosis was assessed by TUNEL and flow cytometry. Western blotting was used to detect the protein levels of PTEN, Bax, Bcl-2, Akt, p-Akt, ERK1/2 and p-ERK1/2 in HSCs, and real-time fluorescent quantitative PCR was applied to detect the mRNA expression of Akt and ERK1. RESULTS:The recombinant adenovirus expressing shRNA targeting PTEN was successfully transfected into activated HSCs in vitro, and significantly promoted the proliferation of HSCs in a time-dependent manner within a certain extent. The apoptotic rate of HSCs was significantly decreased 72 h after transfection(P<0.05). Meanwhile, reduced expression of Bax and elevated expression of Bcl-2 were induced 72 h after transfection(P<0.05). Furthermore, the expression of p-Akt and p-ERK1/2 were increased significantly(P<0.05), while no significant difference in the expression of Akt and ERK1 at mRNA and protein levels was observed(P>0.05). CONCLUSION:Down-regulation of PTEN by adenovirus-mediated shRNA dramatically promotes the proliferation of activated HSCs, and inhibits the apoptosis through Bcl-2/Bax pathway. In addition, the phosphorylation of Akt and ERK1/2 is increased, indicating that PI3K/Akt and ERK1/2 signal transduction pathways may play an important role in the regulation of proliferation and apoptosis of HSCs.     

Role of Toll-like receptor 4/MAPKs pathway on monocyte chemoattractant protein-1 secretion induced by oxidized low density lipoprotein in vascular smooth muscle cells

AIM: To investigate the role of Toll-like receptor 4/MAPKs pathway on the secretion of monocyte chemoattractant protein-1 (MCP-1) induced by oxidized low density lipoprotein (ox-LDL) in the vascular smooth muscle cells (VSMCs). METHODS: mRNA and protein expressions of MCP-1 in VSMCs stimulated with oxidized low density lipoprotein were determined by RT-PCR and ELISA, respectively. The phosphorylated forms of ERK1/2 and p38MAPK were determined by Western blotting. TLR4 neutralizing antibodies (a specific TLR4 inhibitor), PD98059 (ERK1/2 specific inhibitor), SB23015 (p38MAPK specific inhibitor) and SP600125 (JNK specific inhibitor) were used to investigate the underlying mechanisms. RESULTS: The mRNA and protein expressions of MCP-1 in VSMCs were up-regulated by ox-LDL (P<0.05), while those were inhibited by TLR4 neutralizing antibodies, PD98059 or SB23015 (P<0.05), but not by SP600125 (P>0.05). TLR4 had regulatory effect on the phosphorylation of ERK1/2 and p38MAPK. CONCLUSION: ox-LDL is an endogenous ligand of TLR4. The secretion of MCP-1 induced by ox-LDL in VSMCs is at least in part via TLR4/ERK1/2 and TLR4/p38MAPKs pathways.     

Erythropoietin inhibits the proliferation of neonatal rat cardiac fibroblasts induced by angiotensin II via PI3-K/Akt signaling pathway

AIM: To investigate the effects of erythropoietin (EPO) on the proliferation of rat cardiac fibroblasts induced by angiotensin Ⅱ(Ang Ⅱ) and to identify the roles of phosphatidylinositol-3-kinase/Akt (PI3-K/Akt) signaling pathway and nitric oxide synthase (NOS) in this process. METHODS: Neonatal rat cardiac fibroblasts (CFs) were isolated by collgenase, trypsinase and technique of differential attachment. EPO, Ang Ⅱ, LY294002 (an inhibitor of PI3-K), and L-NAME (an inhibitor of NOS) were added in related group respectively. Growth curves of CFs were established by cell counting and methyl thiazolyl tetrazolium (MTT). The levels of nitric oxide (NO), and the activities of NOS and its isoforms were measured by chemical enzymic method. The expressions of Akt, p-Akt, endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were detected by Western blotting. RESULTS: Ang Ⅱ markedly enhanced the proliferation of CFs. The NO level in CFs culture fluid was increased and the proliferation of CFs induced by Ang Ⅱ was suppressed by EPO in a dose dependent manner. After 4 d of administrations, the proliferation ratio of CFs was suppressed 24.4%, 41.5% and 50.5% by EPO at doses of 5×103 U/L, 1×104 U/L and 2×104 U/L respectively. The expressions of phosphated Akt, p-Akt, and eNOS were all up-regulated by EPO. The effect of EPO on NO was blocked by LY294002 and L-NAME, and the suppression of CFs proliferation induced by Ang Ⅱ was diminished similarly. However, LY294002 also down-regulated the expression of eNOS but the L-NAME had no effect on it. CONCLUSION: EPO suppresses the proliferation of neonatal rat CFs induced by Ang Ⅱ in dose dependent manner. The suppressive effects may be due to up-regulating the expression of eNOS and enhancing the production of NO via activating the PI3-K/Akt signaling pathway.     

Autophagy plays a protective role in advanced glycation end product-induced apoptosis of vascular endothelial cells

AIM: To investigate the effect of advanced glycation end products (AGEs) on autophagy in human umbilical endothelial cells (HUVECs) and to identify the role of autophagy in advanced glycation end product-induced cell apoptosis. METHODS: HUVECs were cultured and treated with AGEs or bovine serum albumin. The protein expression was detected by Western blotting. Autophagosomes were observed under electron microscope. The cell apoptotic rate was determined by flow cytometry. The cell viability was quantified by MTT assay. RESULTS: After treated with AGEs, the level of autophagy-associated protein LC3-Ⅱ in HUVECs was up-regulated, and the number of autophagosomes was increased. Compared with control group, the apoptotic rate of HUVECs increased and the viability of HUVECs was decreased in AGEs treatment group. Furthermore, pretreating the cells with an autophagy inhibitor 3-methyladenine aggravated these effects. The levels of phospho-protein kinase B(Akt) and phospho-mammalian target of rapamycin(mTOR) in HUVECs were also decreased by treatment with AGEs. Pretreatment with Akt activator insulin-like growth factor 1 (IGF-1) increased Akt phosphorylation and suppressed the AGE-induced LC3-Ⅱ expression. CONCLUSION: AGEs induce autophagy in HUVECs through PI3K/Akt/mTOR signal pathway. Autophagy plays a protective role in AGE-induced apoptosis in HUVECs.