Effect of homocysteine on expression of interleukin-8 mRNA and protein in THP-1 macrophages

AIM: To investigate the effect of homocysteine (Hcy) on expression of interleukin-8 (IL-8) mRNA and protein in THP-1-derived macrophages (THP-1 macrophages). METHODS: Cultured THP-1 monocytes were induced to macrophages by 0.1 μmol/L PMA treatment for 72 hours, then the differentiated THP-1 macrophages were incubated with homocysteine (0.01 mmol/L-0.20 mmol/L) for 24 hours, or with 0.10 mmol/L Hcy for various time up to 48 hours. IL-8 protein in THP-1 supernatants was measured by ELISA, and IL-8 mRNA expression was detected by semiquantitive RT-PCR. RESULTS: Compared with control, Hcy significantly increased the expression of IL-8 protein in a concentration-dependent manner. 0.05 mmol/L, 0.10 mmol/L and 0.20 mmol/L Hcy increased IL-8 production by 1.28 fold, 1.32 fold and 1.55 fold, respectively (P＜0.01). IL-8 production were elevated significantly 3 h after treatment with 0.10 mmol/L Hcy. In addition, Hcy also increased IL-8 mRNA expression in a concentration-and time-dependent manner. CONCLUSION: Hcy may contribute to atherogenesis by inducing IL-8 expression and secretion in THP-1 macrophages.     

Role of homocysteine in tissue factor expression in human vascular smooth muscle cells

AIM: To investigate the expression of tissue factor (TF) induced by homocysteine (Hcy) and the effect of Hcy on activation of nuclear factor-κB (NF-κB) in human vascular smooth muscle cells (VSMCs).METHODS: Human umbilical artery VSMCs were cultured by tissue explanting method,and were incubated with different dosage of Hcy/ PDTC (NF-κB inhibitor) in different time.RT-PCR was used to measure the expression of TF mRNA,and flow cytometry was used to detect the expression of TF on the surface of VSMCs.Western blotting was performed to measure the NF-κB protein level in the nuclear,flow cytometry was used to examine the expression of iNOS and the expression of TF on the surface of VSMCs.RESULTS: Hcy induced VSMCs TF mRNA expression significantly after the VSMCs were incubated with Hcy at concentrations of 10,100,500 μmol/L,respectively.There was low expression level of TF protein on the surface of the control VSMCs and Hcy also induced VSMCs TF protein expressionn on the cell surface at different concentrations.Additionally,Hcy rapidly induced the activation of NF-κB and inhibited this effect significantly by PDTC.Hcy alone did not induce the expression of iNOS in VSMCs.CONCLUSION: Hcy induces human VSMCs expression of TF in mRNA and protein.These effects were partly mediated by NF-κB.These results suggest that Hcy may play an important role in atherosclerosis and thrombosis.     

Homocysteine promotes rat VSMCs proliferation and phenotypic transformation

AIM: To provide evidence for the molecular mechanism of homocysteine (Hcy) as an independent risk factor in atherosclerosis (AS) by investigating the effect of Hcy on phenotype transformation and proliferation of vascular smooth muscle cells(VSMCs) in rats. METHODS: After treated with different concentrations of Hcy for 24 h, the cultured VSMCs were assayed for cell proliferation rate by MTT method, cell cycle by flow cytometry, the expression of SM22-α mRNA by semi-quantitative RT-PCR and the observation of morphological characteristics and the phenotype transformation by transmission electron microscopy.RESULTS: Hcy increased the cell proliferation rate and gradually reduced the proportion of the cells in G0/G1 phase. Hcy down-regulated the expression of SM22α mRNA and the most significant effect was observed at concentration of 1 000 μmol/L. The observations of transmission electron microscopy revealed an abundant endoplasmic reticulum, Golgis complex, loose nucleus and puffy chromatin in VSMCs treated with high concentration of Hcy. CONCLUSION: Hcy promotes the proliferation and phenotype transformation of VSMCs simultaneously.     

Metallothionein inhibits homocysteine-induced proliferation of rat vascular smooth muscle cells

AIM:To investigate the effect of metallothionein(MT) on proliferation of rat vascular smooth muscle cells (VSMCs) stimulated by homocysteine and its mechanism. METHODS:VSMCs proliferation was measured by [³-H]-TdR incorporation, mitogen-activated protein kinase(MAPK)activity were determined by immunoprecipitation method, the intracellular contents of MT and malondialdehyde (MDA)were assayed by -hemoglobin saturation method and TBA reaction, respectively, and lactate dehydrogenase (LDH) leakage was measured by NADH oxidation. RESULTS:Hcy(10^-6-10^-4 mmol/L) stimulated [³-H]-TdR incorporation by the VSMCs in a concentration-dependent manner. Compared with control, [³-H]-TdR incorporation in VSMCs treated with 0.1 mmol/L Hcy was increased by 4.2 fold (P＜0.01). Meanwhile, Hcy enhanced MAPK activity, MDA formation and LDH release (P＜0.01)in a concentration-dependent manner. Treatment of VSMCs with MT alone did not change above parameters, compared with control. However, MT (10^-6-10^-4 mol/L)attenuated significantly Hcy-stimulated proliferation of VSMCs (P＜0.01)in a concentration-dependent manner. And MT inhibited obviously Hcy-induced activation of MAPK activity, MDA formation and LDH release. Preincubation of VSMCs with 0.5 mmol/L ZnCl₂ for 6 h induced an increase cellular MT content by 5.7-fold (P＜0.01). The MT-overexpressed VSMCs resisted Hcy-stimulating action on MAPK activity, MDA formation and LDH leakage (P＜0.01). CONCLUSION:These results show that MT has an inhibitory effect on Hcy-induced VSMCs proliferation, and that MT could inhibit Hcy-stimulated MAPK activity and lipid peroxidation.     

Effect of peroxisome proliferator activated receptor δ on mRNA expression of MCP-1 induced by homocysteine in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of peroxisome proliferator activated receptor δ (PPARδ) on the mRNA expression of monocyte chemoattractant protein 1 (MCP-1) induced by homocysteine (Hcy) in human umbilical vein endothelial cells (HUVECs). METHODS: Collagenase was used to isolate endothelial cells from human umbilical vein, and the cells were cultured in vitro . The HUVECs were divided into blank control group, Hcy group, GW0742 (a specific agonist of PPARδ) group and diphenyleneiodonium (DPI,a specific inhibitor of NADPH oxidase) group. RT-PCR was used to examine the mRNA expression of MCP-1 and PPARδ. The protein level of PPARδ was detected by Western blotting.2',7'-Dichlorofluorescin diacetate(DCFH-DA) was added to monitor intracellular production of reactive oxygen species (ROS). RESULTS: Compared with control group, Hcy promoted the mRNA expression of MCP-1 in a concentration-dependent manner, and decreased the mRNA expression of PPARδ in HUVECs. The mRNA expression of MCP-1 was significantly elevated by Hcy at the concentration of 10^-5 mol/L, and the mRNA expression of PPARδ was decreased remarkably (P<0.01). GW0742 decreased the mRNA expression of MCP-1 compared with Hcy group (P<0.01). Hcy remarkably increased the production of ROS compared with control group. Hcy-induced production of ROS was also significantly attenuated by GW0742. CONCLUSION: The activation of PPARδ decreases the Hcy-induced mRNA expression of MCP-1 by suppressing Hcy-stimulated production of ROS.     

Effect of homocysteine on the expression of macrophage inflammatory protein-1α in THP-1 monocytes

AIM: To investigate whether homocysteine (HCY) induce the expression of macrophage inflammatory protein-1α(MIP-1α)in cultured THP-1 monocytes. METHODS: After exposure of THP-1 monocytes to HCY at increasing concentrations (0.05,0.1 and 0.2 mmol/L) for 8 h, or at 0.1 mmol/L of HCY for different incubation times (4, 8 and 16 h), the expressions of MIP-1α mRNA and protein were determined by RT-PCR and immunocytochemistry, respectively. RESULTS: RT-PCR showed that the expression of MIP-1α mRNA increased with the concentrations of HCY compared with the control group. Meanwhile, after the treatment of 0.1 mmol/L HCY to the cells for different times, the MIP-1α mRNA expression increased at 4 h, peaked at 8 h, and then decreased at 16 h. The authenticity of RT-PCR products was confirmed by DNA sequencing. Image analysis of Immunocytochemistry assay showed the expression of MIP-1α protein in experimental groups increased in a dose- and time-dependent manner(P＜0.01). CONCLUSIONS: HCY induced monocytes to express MIP-1α mRNA and protein.     

Astragalus polysaccharides attenuate endothelial cell injury caused by homocysteine via AMPK pathway

AIM:To investigate the protective effect of Astragalus polysaccharides (APS) on human umbilical vein endothelial cells (HUVECs) injured by homocysteine (Hcy) and its mechanism. METHODS:HUVECs cultured in vitro were divided into 4 groups:control group, APS group[APS (200 mg/L) treatment for 24 h], Hcy group[Hcy (1 mmol/L) treatment for 24 h], and Hcy+APS group[Hcy (1 mmol/L) and APS (200 mg/L) co-treatment for 24 h]. The cell viability were measured by MTT assay. The activity of lactate dehydrogenase (LDH) and superoxidase dismutase (SOD), and the content of malondialdehyde (MDA) in HUVECs were detected by the commercial kits. The mRNA expression of SOD1, catalase (CAT) and NADPH oxidase 2 (NOX2) was detected by RT-qPCR. The protein levels of AMP-activated protein kinase α (AMPKα) and phosphorylated AMPKα (p-AMPKα) were determined by Western blot. RESULTS:Compared with control group, the cell viability, the activity of SOD, and the mRNA expression of SOD1 and CAT in the HUVECs were decreased, but the activity of LDH, the content of MDA, and the mRNA expression of NOX2 were increased significantly in Hcy group(P<0.05). APS inhibited the decrease in cell viability, and the increases in LDH acti-vity and MDA content induced by Hcy. APS increased SOD activity and the mRNA expression of SOD1 and CAT, but reduced the mRNA expression of NOX2. Compound C, an AMPK inhibitor, reduced the protective effect of APS on HUVECs injured by Hcy. CONCLUSION:APS protects HUVECs from Hcy-induced injury via AMPK signaling pathway to regulate intracellular oxidative stress.     

Effects of human xeroderma pigmentosum group D gene on proliferation of human vascular smooth muscle cells induced by interleukin-6

AIM: To investigate the effects of human xeroderma pigmentosum group D (XPD) gene on the proliferation of human vascular smooth muscle cells (VSMCs) induced by interleukin-6 (IL-6). METHODS: Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into VSMCs by liposome, and then these cells were incubated with IL-6 at 1×10⁵ U/L for 48 h. The cells were divided into 6 groups: blank control group; pEGFP-N2 group; pEGFP-N2/XPD group; IL-6 group; IL-6 + pEGFP-N2 group; IL-6 + pEGFP-N2/XPD group. The expression of green fluorescent protein was observed under fluorescence microscope. The cell growth was detected by MTT method. The cell cycle and apoptosis rate were examined by flow cytometre. The expression levels of XPD, Bcl-2, Bax and wild type P53 (wt-P53) were detected by RT-PCR and Western blotting.RESULTS: Green fluorescence was observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating successful transfection MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth, and reduced the positive effects of IL-6 on VSMCs growth. Flow cytometry results showed that the transfection of pEGFP-N2/XPD increased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, decreased the cell numbers in S phase, and reduced the effects that IL-6 decreased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, and increased the cell numbers in S phase. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, Bax and wt-P53, decreased the expression of Bcl-2, and reduced the effects that IL-6 decreased the expression of Bax and wt-P53, and increased the expression of Bcl-2. CONCLUSION: XPD gene inhibits VSMCs proliferation, promotes VSMCs apoptosis, and reduces the effects that IL-6 promotes VSMCs proliferation and inhibits VSMCs apoptosis. Therefore, XPD gene is likely to be potential molecular target for treatment of atherosclerosis.     

CNKI引文数据库《园艺学报》高被引频次论文排序

(截至2006年6月,排名前100位)序号被引文献题名被引文献作者被引文献来源被引频次1蔬菜硝酸盐累积的研究———Ⅰ.不同蔬菜硝酸盐沈明珠,翟宝杰,东惠茹,李俊国园艺学报/1982/04266和亚硝酸盐含量评价2温室土壤次生盐渍化的形成和治理途径研究童有为,陈淡飞园艺学报/1991/021323     

Effects of tetrahydroxystilbene-2-O-β-D-glucoside on apoptosis and expression of bcl-2/bax/caspase-3 in HUVECs treated with homocysteine

AIM：To explore the effects of tetrahydroxystilbene-2-O-β-D-glucoside (TSG) from Polygonum multiflorum on the apoptosis and the mRNA expression of bcl-2, bax and caspase-3 in human umbilical vein endothelial cells (HUVECs) treated with homocysteine (Hcy). METHODS：Cultured HUVECs were treated with Hcy (3 mmol/L) to establish a Hcy-damaged model. HUVECs in TSG treated groups were pre-incubated with TSG at concentrations of 1 μmol/L and 10 μmol/L for 2 h before treated with Hcy. Cell nuclear damage was detected by Hoechst 33342 staining. Cell apoptosis was determined by flow cytometry. The mRNA expression of bcl-2, bax and caspase-3 was measured by real-time fluorescence quantitative RT-PCR. RESULTS： After treatment with Hcy at concentration of 3 mmol/L, the nuclear damage and apoptotic rate of HUVECs were higher than that in normal group. The expression of bcl-2 was lower, and the expression of Bax and caspase-3 was higher than that in normal group. On the other hand, pre-incubation with TSG at concentrations of 1 μmol/L and 10 μmol/L decreased the nuclear damage and cell apoptosis, increased the expression of bcl-2, and decreased the expression of bax and caspase-3 as compared with the cells only treated with Hcy. CONCLUSION：TSG reduces the apoptosis of HUVECs induced by Hcy, and the mechanism might be associated with regulating the expression of bcl-2, bax and caspase-3.     

Effect of homocysteine on the apoptosis of cultured human umbilical vein endothelial cells

AIM: To investigate the effect of homocysteine(Hcy) on the apoptosis of endothelial cells (EC). METHODS: First-passaged human umbilical vein endothelial cells (hUVEC) were cultured with M199 containing 3 mmol/L Hcy. hUVEC apoptosis was detected as follow: demonstration of nuclear changes by Hoechst 33258 staining, agarose gel electrophoresis of DNA fragments, detection of apoptotic cells by flow cytometry following Annexin V-PI doubled stain, Western blot for P53 and Bax protein detection and colorimetry detecting caspase-3 activity. RESULTS: Compared with control, homocysteine induced characteristic apoptotic changes in hUVEC. The chromosomal DNA of hUVEC appeared "DNA ladder" by agarose gel electrophoresis. Apoptotic cells were increased significantly (P<0.01, n=3). Hcy promoted the expression of protein Bax, P53 (P<0.01, n=3) and enhanced the activity of caspase 3 (P<0.05, n=3). CONCLUSION: Homocysteine induces apoptosis in cultured hUVEC.     

Effects of interleukin -13 on interleukin-12 production in mesangial cells induced by LPS

AIM: To investigate the effect of interleukin-13 (IL-13) on interleukin-12 (IL-12) production in mesangial cells.METHODS: The protein synthesis of IL-12 in mesangial cells was measured by ELISA.The expression of IL-12 mRNA in mesangial cells was evaluated by RT-PCR.RESULTS: The production of IL-12 in mesangial cells stimulated by lipopolysaccharide(LPS) was significantly increased (P<0.01).IL-13 (1-100 μg/L) inhibited the protein and mRNA expression of IL-12 in a dose-dependent manner (P<0.05 or P<0.01).CONCLUSION: IL-13 inhibits IL-12 expression induced by LPS in mesangial cells.IL-13 may regulate immune responses by balancing Th1/Th2 in glomerulonephritis.     

Mechanism underlying the antagonistic effect of folic acid on homocysteine-induced apoptosis in endothelial cell

AIM: To investigate the effects of homocysteine (Hcy) on apoptosis in SV40-transformed aortic rat endothelial cell line and the anti-apoptosis effects of folic acid. METHODS: Cells were treated with different concentrations of Hcy and folic acid, apoptosis was detected by TUNEL and annexin-V/ PI staining methods. Immunohistochemical assay was used to examine the expression of Bax and Bcl-2 in all groups. RESULTS: Both annexin-V/PI staining and TUNEL method showed that Hcy increased endothelial apoptosis in a dose-dependent manner, while folic acid reduced cell apoptosis. Hcy increased expression of Bax and Bcl-2 in endothelial cells, and folic acid decreased it. Bax/Bcl-2 ratio in 0.5 mmol/L Hcy and 5.0 mmol/L Hcy group were upregulated compared with control group (P<0.05 and P<0.01, respectively). Addition of 0.1 mmol/L folic acid decreased Bax/Bcl-2 ratio compared with the corresponding group without folic acid (P<0.05). Correlation analysis showed a strong relation between Bax/Bcl-2 ratio and apoptotic rate (P<0.01). CONCLUSION: Folic acid attenuates the apoptosis induced by Hcy in endothelial cells. Hcy may promote endothelial cell apoptosis via upregulation of Bax /Bcl-2 ratio, which can be partially antagonized by folic acid.     

Effects of lentivirus-mediated transfection of shRNA targeting Adra1d gene on calcium and calmodulin in rat aortic vascular smooth muscle cells

AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α_1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca²⁺) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca²⁺ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×10¹¹ TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca²⁺ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca²⁺ concentration and CaM expression in rat aortic VSMCs.     

Effect of fibrin on expression of interleukin-6 in rat brain vascular endothelial cells

AIM:To investigate the kinetics of interleukin-6 (IL-6) protein and mRNA expressions in the co-culture of rat brain vascular endothelial cells(VECs) with fibrin. METHODS:The IL-6 concentration were measured by rat IL-6 ELISA kit and the IL-6 mRNA was measured by real-time PCR after development an in vitro model of in situ fibrin polymerization on rat brain vascular endothelial cells. RESULTS:Fibrin induced IL-6 expression. The IL-6 antigen concentration increased significantly in 1.0 g/L media group compared to the low dose fibrin group or control media group (P<0.01). Fibrin-induced IL-6 expression in rat brain VECs as early as 2 h post-fibrin stimulation, increased significantly at 8 h, keeping at high level till 24 h (P<0.05). Collagen did not induce IL-6 expression at 24 h. Real-time PCR analysis showed that IL-6 antigen increase in fibrin treated rat brain VECs was due to fibrin induced elevation of steady state mRNA expression. CONCLUSION:Fibrin is a potent vascular endothelial cell activator. It induces IL-6 expression in time and dose dependent fashions.     

Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model. Oil red O staining was used to determine whether the model was established successfully. miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h. The intracellular lipid droplets were observed by Oil red O staining. The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot. The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05). The intracellular cholesterol content was increased gradually (P<0.05), and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group. Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05). No diffe-rence of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.