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1.
Article in Chinese | WPRIM | ID: wpr-921649

ABSTRACT

The present study observed the effect of Guanxin Zhitong Capsules(GXZT) on the lipotoxicity of vascular endothelial cells and investigated the mechanism of GXZT in atherosclerosis treatment. The lipotoxicity model in human umbilical vein endothelial cells(HUVECs) was induced by palmitic acid(PA) stimulation. These cells were divided into a normal control group(NC, 15% normal serum), a model group(PA, 0.6 mmol·L~(-1) PA+15% normal serum), a high-dose GXZT group(GXZT-H, 0.6 mmol·L~(-1) PA+15% GXZT-medicated serum), a medium-dose GXZT group(GXZT-M, 0.6 mmol·L~(-1) PA+10% GXZT-medicated serum+5% normal serum) and a low-dose GXZT group(GXZT-L, 0.6 mmol·L~(-1) PA+5% GXZT-medicated serum+10% normal serum). HUVECs were detected for cell viability by cell counting kit-8(CCK-8) assay, apoptosis by flow cytometry, mitochondrial membrane potential(MMP) by JC-1 labeled laser scanning confocal microscopy, and total and phosphorylated proteins of p38, ERK1/2, and JNK1/2 in the mitogen-activated protein kinases(MAPK) signaling pathway by Western blot. The phosphorylated level was calcula-ted. Compared with the NC group, the PA group showed decreased cell viability and MMP(P<0.01, P<0.01), elevated apoptosis(P<0.01), and up-regulated phosphorylated levels of p38, ERK1/2, and JNK1/2(P<0.01, P<0.01, P<0.01). Compared with the PA group, the GXZT-H, GXZT-M, and GXZT-L groups showed increased cell viability and MMP(P<0.01, P<0.01, P<0.01), reduced apoptosis(P<0.01), and down-regulated protein expression and phosphorylated levels of p38, ERK1/2 and JNK1/2 in the MAPK signaling pathway(P<0.01, P<0.01, P<0.01). In conclusion, the results suggest that GXZT functions via blocking MAPK signaling pathway to relieve the damage of HUVECs induced by PA.


Subject(s)
Apoptosis , Capsules , Human Umbilical Vein Endothelial Cells/metabolism , Humans , MAP Kinase Signaling System , Palmitic Acid/toxicity , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism
2.
Clinics ; 75: e1865, 2020. graf
Article in English | LILACS | ID: biblio-1133469

ABSTRACT

OBJECTIVES: Hypoxia leads to endothelial cell inflammation, apoptosis, and damage, which plays an important role in the complications associated with ischemic cardiovascular disease. As an oxidoreductase, p66Shc plays an important role in the regulation of reactive oxygen species (ROS) production and apoptosis. Ketamine is widely used in clinics. This study was designed to assess the potential protective effect of ketamine against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs). Moreover, we explored the potential mechanism by which ketamine protected against hypoxia-induced endothelial injury. METHODS: The protective effects of ketamine against hypoxia-induced injury was assessed using cell viability and adhesion assays, quantitative polymerase chain reaction, and western blotting. RESULTS: Our data showed that hypoxia reduced HUVEC viability, increased the adhesion between HUVECs and monocytes, and upregulated the expression of endothelial adhesion molecules at the protein and mRNA levels. Moreover, hypoxia increased ROS accumulation and upregulated p66Shc expression. Furthermore, hypoxia downregulated sirt1 expression in HUVECs. Alternatively, ketamine was shown to reverse the hypoxia-mediated reduction of cell viability and increase in the adhesion between HUVECs and monocytes, ameliorate hypoxia-induced ROS accumulation, and suppress p66Shc expression. Moreover, EX527, a sirt1 inhibitor, reversed the protective effects of ketamine against the hypoxia-mediated reduction of cell viability and increase in adhesion between HUVECs and monocytes. CONCLUSION: Ketamine reduces hypoxia-induced p66Shc expression and attenuates ROS accumulation via upregulating sirt1 in HUVECs, thus attenuating hypoxia-induced endothelial cell inflammation and apoptosis.


Subject(s)
Humans , Reactive Oxygen Species/metabolism , Apoptosis/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Ketamine/pharmacology , Hypoxia , Umbilical Veins , Cell Survival , Oxidative Stress , Human Umbilical Vein Endothelial Cells/metabolism , Src Homology 2 Domain-Containing, Transforming Protein 1
3.
Braz. j. med. biol. res ; 53(6): e9346, 2020. graf
Article in English | LILACS, ColecionaSUS | ID: biblio-1132516

ABSTRACT

Atherosclerosis (AS) is a common vascular disease, which can cause apoptosis of vascular endothelial cells. Notoginsenoside R1 (NGR1) is considered an anti-AS drug. MicroRNAs (miRNAs) are believed to play a vital role in cell apoptosis and angiogenesis. This study aimed to explore the mechanism of NGR1 for treating AS through miRNAs. Flow cytometry was used to detect the apoptosis rate. The levels of inflammatory cytokines interleukin (IL)-6 and IL-1β were detected using ELISA. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were measured using corresponding assay kits. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to detect miR-221-3p expression. Dual-luciferase reporter and RNA immunoprecipitation assays were carried out to examine the relationship between miR-221-3p and toll-like receptors 4 (TLR4). Also, western blot analysis was performed to determine the levels of TLR4 and nuclear factor kappa B (NF-κB) signaling pathway-related proteins. Oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) apoptosis, inflammation, and oxidative stress. NGR1 alleviated the negative effect of ox-LDL through promoting the expression of miR-221-3p in HUVECs. TLR4 was a target of miR-221-3p, and its overexpression could reverse the inhibition effects of miR-221-3p on apoptosis, inflammation, and oxidative stress. NGR1 improved miR-221-3p expression to inhibit the activation of the TLR4/NF-κB pathway in ox-LDL-treated HUVECs. NGR1 decreased ox-LDL-induced HUVECs apoptosis, inflammation, and oxidative stress through increasing miR-221-3p expression, thereby inhibiting the activation of the TLR4/NF-κB pathway. This study of the mechanism of NGR1 provided a more theoretical basis for the treatment of AS.


Subject(s)
Humans , Apoptosis/drug effects , Oxidative Stress/drug effects , Ginsenosides/pharmacology , MicroRNAs/adverse effects , Human Umbilical Vein Endothelial Cells/drug effects , Inflammation/metabolism , Lipoproteins, LDL/metabolism , Enzyme-Linked Immunosorbent Assay , Signal Transduction , Transcriptional Activation , Up-Regulation , Blotting, Western , NF-kappa B/antagonists & inhibitors , Reactive Oxygen Species , MicroRNAs/metabolism , Immunoprecipitation , Toll-Like Receptor 4/antagonists & inhibitors , Human Umbilical Vein Endothelial Cells/metabolism , Real-Time Polymerase Chain Reaction
4.
Braz. j. med. biol. res ; 50(11): e6389, 2017. tab, graf
Article in English | LILACS | ID: biblio-888946

ABSTRACT

The objective of this study was to observe the infection of human cytomegalovirus (HCMV) to human umbilical vein endothelial cells, and its effect on the expression of single-stranded DNA-binding protein (SSBP1) and on lipid metabolism in endothelial cells. We screened the differential expression of mRNAs after HCMV infection by suppression subtractive hybridization and the expression levels of SSBP1 mRNA and protein after HCMV infection by real-time PCR and western blot. After verification of successful infection by indirect immunofluorescent staining and RT-PCR, we found a differential expression of lipid metabolism-related genes including LDLR, SCARB, CETP, HMGCR, ApoB and LPL induced by HCMV infection. The expression levels of SSBP1 mRNA and protein after HCMV infection were significantly down-regulated. Furthermore, we found that upregulation of SSBP1 inhibited the expression of atherosclerosis-associated LDLR, SCARB, HMGCR, CETP as well as the accumulation of lipids in the cells. The results showed that the inhibition of SSBP1 by HCMV infection promotes lipid accumulation in the cells.


Subject(s)
Humans , Cytomegalovirus Infections/metabolism , DNA-Binding Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/virology , Lipid Metabolism/physiology , Mitochondrial Proteins/metabolism , Atherosclerosis/metabolism , Atherosclerosis/virology , Cholesterol Ester Transfer Proteins/metabolism , Cholesterol/analysis , DNA-Binding Proteins/genetics , Down-Regulation , Hydroxymethylglutaryl CoA Reductases/metabolism , Lipid Metabolism/genetics , Mitochondrial Proteins/genetics , Receptors, LDL/metabolism , Scavenger Receptors, Class B/metabolism , Time Factors
5.
Braz. j. med. biol. res ; 50(12): e6145, 2017. tab, graf
Article in English | LILACS | ID: biblio-888968

ABSTRACT

Chronic systemic inflammation and repetitive damage of vascular endothelia by incompatible dialysis system are probable causes of cardiovascular disease in patients on dialysis. The present study aimed to assess in vitro biocompatibility and anti-inflammatory effect of hemodialysis fluid supplemented with rosmarinic acid (RA) using human umbilical vein endothelial cells (HUVEC). HUVECs (5×106 cells/mL) were pre-exposed to 1 μg/mL of lipopolysaccharides (LPS) and incubated with RA-supplemented hemodialysis fluid (HDF). Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by MTT assay. Expressions of proinflammatory mediators were assessed using quantitative real-time PCR and production of NO was quantified. Phosphorylation of AKT and nuclear localization of nuclear factor kappa B (NF-κB) were examined using western blotting. Exposure of HUVECs to RA-supplemented HDF had no influence on morphology and viability. Inhibition of proinflammatory mediator production in HUVECs by RA supplementation to HDF was significant in a dose-dependent manner. Exposure to RA-supplemented HDF resulted in a decrease in nitric oxide synthase expression and reduction of NO production in LPS-stimulated HUVECs. RA supplementation of HDF suppressed Akt activation in LPS-stimulated HUVECs. In addition, the level of cellular IκB was increased in parallel to a reduced nuclear translocation of NF-κB in LPS-induced endothelial cells. Our results suggest that RA-supplemented HDF is biocompatible and significantly suppressed inflammation induced in endothelial cells. In this respect, the use of HDF supplemented with RA could alleviate inflammation and improve long-term treatment of patients with renal failure on dialysis. Further clinical studies are required to confirm the effects.


Subject(s)
Humans , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Biocompatible Materials/pharmacology , Cinnamates/pharmacology , Depsides/pharmacology , Hemodialysis Solutions/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Inflammation/drug therapy , Analysis of Variance , Cell Survival/drug effects , Cells, Cultured , Cytokines/analysis , Cytokines/drug effects , Formazans , Hemodialysis Solutions/chemistry , Human Umbilical Vein Endothelial Cells/metabolism , Immunoblotting , Inflammation/metabolism , Lipopolysaccharides , NF-kappa B/analysis , Nitric Oxide/analysis , Phosphorylation , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Tetrazolium Salts
6.
Clinics ; 70(8): 569-576, 08/2015. tab, graf
Article in English | LILACS | ID: lil-753961

ABSTRACT

OBJECTIVE: The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis, mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical vein endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells were grown and subjected to glutamine and insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and the production of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicity assays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. RESULTS: Glutamine maintains the integrity of the mitochondria by reducing the cell permeability and cytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level was significantly (p<0.005) reduced when the cells were treated with glutamine. An apoptosis assay revealed significantly reduced apoptosis (p<0.005) in the glutamine-treated cells. Moreover, glutamine alone or in combination with insulin modulated inflammatory cytokine levels. Interleukin-10, interleukin-6, and vascular endothelial growth factor were up-regulated while tumor necrosis factor-α was down-regulated after treatment with glutamine. CONCLUSION: Glutamine, either alone or in combination with insulin, can positively modulate the mitochondrial stress and cell permeability in umbilical vein endothelial cells. Glutamine regulates the expression of inflammatory cytokines and maintains the balance of the mitochondria in a cytoprotective manner. .


Subject(s)
Humans , Apoptosis/drug effects , Glutamine/pharmacology , Hyperglycemia/drug therapy , Mitochondria/drug effects , Oxidative Stress/drug effects , Cells, Cultured , Cell Membrane Permeability/drug effects , Cytochromes c/analysis , Cytokines/analysis , Cytokines/drug effects , Drug Combinations , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Hyperglycemia/metabolism , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Mitochondria/metabolism
8.
Mem. Inst. Oswaldo Cruz ; 109(7): 940-943, 11/2014. graf
Article in English | LILACS | ID: lil-728811

ABSTRACT

Endothelial dysfunction is a major component of the pathophysiology of septicaemic group B Streptococcus (GBS) infections. Although cytokines have been shown to activate human umbilical vein endothelial cells (HUVECs), the capacity of interferon (IFN)-γ to enhance the microbicidal activity of HUVECs against GBS has not been studied. We report that the viability of intracellular bacteria was reduced in HUVECs activated by IFN-γ. Enhanced fusion of lysosomes with bacteria-containing vacuoles was observed by acid phosphatase and the colocalisation of Rab-5, Rab-7 and lysosomal-associated membrane protein-1 with GBS in IFN-γ-activated HUVECs. IFN-γ resulted in an enhancement of the phagosome maturation process in HUVECs, improving the capacity to control the intracellular survival of GBS.


Subject(s)
Humans , Anti-Infective Agents/pharmacology , Human Umbilical Vein Endothelial Cells/microbiology , Interferon-gamma/pharmacology , Microbial Viability/drug effects , Streptococcal Infections/drug therapy , Streptococcus agalactiae/drug effects , Acid Phosphatase/metabolism , Bacterial Adhesion/drug effects , Endocytosis , Human Umbilical Vein Endothelial Cells/metabolism , Lysosomes/drug effects , Primary Cell Culture , Phagosomes/drug effects , Survival Analysis , Streptococcal Infections/prevention & control
9.
Biol. Res ; 47: 1-15, 2014. ilus, graf, tab
Article in English | LILACS | ID: biblio-950766

ABSTRACT

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Simultaneous silencing of VEGF and KSP, an attractive and viable approach in cancer, leads on restricting cancer progression. The purpose of this study is to examine the therapeutic potential of dual gene targeted siRNA cocktail on human hepatocellular carcinoma Hep3B cells. RESULTS: The predesigned siRNAs could inhibit VEGF and KSP at mRNA level. siRNA cocktail showed a further downregulation on KSP mRNA and protein levels compared to KSP-siRNA or VEGF-siRNA, but not on VEGF expression. It also exhibited greater suppression on cell proliferation as well as cell migration or invasion capabilities and induction of apoptosis in Hep3B cells than single siRNA simultaneously. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin. However, no sigificant difference in the mRNA and protein levels of ANG2, involving inhibition of angiogenesis was found in HUVECs cultured with supernatant of Hep3B cells treated with siRNA cocktail, compared to that of VEGF-siRNA. CONCLUSION: Silencing of VEGF and KSP plays a key role in inhibiting cell proliferation, migration, invasion and inducing apoptosis of Hep3B cells. Simultaneous silencing of VEGF and KSP using siRNA cocktail yields promising results for eradicating hepatocellular carcinoma cells, a new direction for liver cancer treatment.


Subject(s)
Humans , Kinesins/genetics , Apoptosis/genetics , Gene Silencing , RNA, Small Interfering/genetics , Vascular Endothelial Growth Factor A/genetics , Cell Proliferation/genetics , Tetrazolium Salts , Transfection , Cysteine Proteinase Inhibitors/metabolism , Down-Regulation , Cell Movement , Blotting, Western , Kinesins/metabolism , Annexin A5 , Genes, bcl-2 , Cyclin D1/metabolism , Vesicular Transport Proteins/metabolism , Cell Line, Tumor , Vascular Endothelial Growth Factor A/metabolism , Inhibitor of Apoptosis Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Real-Time Polymerase Chain Reaction , Flow Cytometry , Survivin , Mitosis/genetics
10.
Braz. j. med. biol. res ; 46(9): 765-770, 19/set. 2013. tab, graf
Article in English | LILACS | ID: lil-686568

ABSTRACT

Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of atherosclerosis, and it can stimulate the expression of a variety of inflammatory signals. As a new and highly sensitive inflammation index, OX40L may be a key to understanding the mechanisms that regulate interactions between cells within the vessel wall and inflammatory mediators during the development of atherosclerosis. To investigate whether Ox-LDL regulates OX40L expression through an oxidized LDL-1 receptor (LOX-1)-mediated mechanism, we investigated the effect of different concentrations of Ox-LDL (50, 100, 150 µg/mL) on endothelial cell proliferation and apoptosis. Stimulation with Ox-LDL increased OX40L protein 1.44-fold and mRNA 4.0-fold in endothelial cells, and these effects were inhibited by blocking LOX-1. These results indicate that LOX-1 plays an important role in the chronic inflammatory process in blood vessel walls. Inhibiting LOX-1 may reduce blood vessel inflammation and provide a therapeutic option to limit atherosclerosis progression.


Subject(s)
Humans , Apoptosis/drug effects , Cell Proliferation/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Lipoproteins, LDL/pharmacology , /metabolism , Scavenger Receptors, Class E/metabolism , Atherosclerosis/etiology , Atherosclerosis/prevention & control , Cell Cycle , Cells, Cultured , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Immunoblotting , Lipoproteins, LDL/metabolism , Lipoproteins, LDL/physiology , /genetics , Real-Time Polymerase Chain Reaction , Signal Transduction , Vasculitis/physiopathology , Vasculitis/prevention & control
11.
Indian J Biochem Biophys ; 2012 Feb; 49(1): 18-24
Article in English | IMSEAR | ID: sea-140214

ABSTRACT

Diabetes is associated with increased formation of advanced glycation end products (AGEs), which have been implicated in micro and macrovascular complications of diabetes. Our earlier reports showed proangiogenic effect of AGE-bovine serum albumin (BSA). In order to understand the mechanism of AGE-mediated angiogenesis, the possibility of involvement of peroxisome prolifeator activated receptor (PPAR) , a ligand activated transcription factor was examined. The angiogenic effect was studied in chick chorio allantoic membrane (CAM) and by analyzing angiogenic markers in human umbilical vein endothelial cells (HUVECs) in culture. The involvement of PPAR was investigated using synthetic PPAR agonist GW 1929 and antagonist GW 9662 and by RT-PCR. In CAM assay, PPAR antagonist GW 9662 reversed the AGE-induced effect on vascularity. In HUVECs in culture, GW 9662 reversed the effect of AGE-BSA and decreased the expression of CD 31, E-Selectin and VEGF. RT-PCR analysis showed that treatment with AGE-BSA caused upregulation of PPAR mRNA levels. The reversal of the effect of AGE on angiogenesis by treatment with PPAR antagonists and up-regulation of PPAR gene in HUVECs treated with AGE-BSA suggested the possible involvement of PPAR -dependent downstream pathway in mediating the angiogenic effect of AGE.


Subject(s)
Angiogenesis Inducing Agents/metabolism , Anilides/pharmacology , Animals , Platelet Endothelial Cell Adhesion Molecule-1/drug effects , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Benzophenones/pharmacology , Cells, Cultured , Chick Embryo , Chorioallantoic Membrane/drug effects , Chorioallantoic Membrane/metabolism , Diabetes Mellitus/metabolism , E-Selectin/metabolism , Glycation End Products, Advanced/pharmacology , Human Umbilical Vein Endothelial Cells/metabolism , PPAR gamma/antagonists & inhibitors , PPAR gamma/antagonists & inhibitors , PPAR gamma/drug effects , PPAR gamma/metabolism , RNA/drug effects , RNA/metabolism , Tyrosine/analogs & derivatives , Tyrosine/pharmacology , Vascular Endothelial Growth Factor A/drug effects , Vascular Endothelial Growth Factor A/metabolism
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