Naringin protects human umbilical vein endothelial cells against injury induced by high glucose through PI3K/AKT/eNOS pathway

AIM: To investigate the protective effect of naringin (Nar) on the injury of human umbilical vein endothelial cells (HUVECs) induced by 33 mmol/L high glucose (HG) and to explore its possible mechanisms.METHODS: The injury model was established by treating HUVECs with HG medium for the indicated time (6, 12, 24, 48 and 72 h), and then the levels of NO, eNOS and p-eNOS were detected, respectively. The effects of Nar on high glucose-induced endothelial cell injury were observed. HUVECs were treated with Nar at concentrations of 5, 10, 25, 50 and 100 mg/L for 6 h, 12 h, 24 h, 36 h and 48 h. The levels of NO in the supernatants were measured. The effects of Nar on HG-injured HUVECs were explored by treating the cells with 10 μmol/L of LY294002, a PI3K inhibitor, or 0.5 μmol/L of AKT inhibitor Ⅳ, an AKT inhibitor, and then the levels of NO, PI3K, AKT, eNOS and their phosphorylated proteins were determined by Western blot.RESULTS: Nar at concentration of 50 mg/L significantly attenuated the injury of endothelial cells induced by high glucose (P<0.01), and the protective effects of Nar were abolished by pretreating with the inhibitor of PI3K or AKT (P<0.01).CONCLUSION: Nar protects endothelial cells against the injury induced by high glucose through PI3K/AKT/eNOS pathway.     

Effects of simvastatin on cigarette smoke extract-induced sEPCR and EPCR expression in human umbilical vein endothelial cells

AIM: To investigate the effects of simvastatin on cigarette smoke extract (CSE)-induced expression levels of soluble endothelial cell protein C receptor (sEPCR) and membrane-associated endothelial cell protein C receptor (mEPCR ) in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs at passage 4 to 6 were randomly divided into control group, 5% CSE group, simvastatin groups and simvastatin+CSE groups. In simvastatin groups, HUVECs were incubated with simvastatin at the concentrations of 50, 100 and 200 μmol/L for 24 h. In simvastatin+CSE groups, the cells were treated with simvastatin at the concentrations of 50, 100 and 200 μmol/L for 2 h, and then exposed to CSE for 24 h. The protein level of sEPCR in the culture supernatants was measured by ELISA. The cells were collected for determining the mRNA expression of mEPCR by real-time PCR. RESULTS: Compared with control group, the protein level of sEPCR was significantly increased, and the mRNA expression of mEPCR was significantly decreased in 5% CSE group (both P<0.05). The protein levels of sEPCR were significantly increased, and the mRNA expression of mEPCR was significantly decreased in 100 μmol/L and 200 μmol/L simvastatin groups. However, the protein levels of sEPCR were lower, and the mRNA expression of mEPCR was significantly higher in 100 μmol/L and 200 μmol/L simvastatin groups than those in 5% CSE group. Compared with 5% CSE group, the protein levels of sEPCR in simvastatin+CSE groups were significantly decreased, but higher than those in control group and simvastatin group with corresponding concentration. On the contrary, the mRNA expression of mEPCR in simvastatin+CSE groups was significantly increased, but lower than that in control group and simvastatin group with corresponding concentration (all P<0.05). CONCLUSION: Simvastatin obviously increases the mRNA expression of mEPCR, decreases the protein level of sEPCR, and attenuates the CSE-induced endothelial injury in vitro .     

Effects of angiopoietin 4 on lipopolysaccharide-induced injury of human umbilical vein endothelial cells

AIM:To observe the effects of angiopoietin 4 (Ang-4) on lipopolysaccharide (LPS)-induced injury of human umbilical vein endothelial cells (HUVECs). METHODS:The EnVision immunohistochemical method was used to identify the HUVECs. After pre-treated with different doses of Ang-4 for 0.5 h, HUVECs was exposed to LPS at concentration of 10 mg/L for 24 h. The cell viability was evaluated by MTT assay. The content of tumor necrosis factor-alpha (TNF-α) in the supernatant and the concentrations of intracellular and supernatant von Willebrand factor (vWF) were detected by ELISA. The mRNA levels of Toll-like receptor 4 (TLR4), NF-κB p65 and TNF-α were determined by real-time PCR. RESULTS:Factor Ⅷ in the cytoplasm was positive in the HUVECs.Compared with normal group, LPS reduced the cell viability (P<0.01), and significantly increased the secretion of TNF-α and vWF (P<0.01). The mRNA expression of TLR4, NF-κB p65 and TNF-α also increased (P<0.01). Ang-4 at concentration of 100 μg/L enhanced the cell viability (P<0.01), reduced the content of vWF and TNF-α, and inhibited the LPS-induced increases in the mRNA levels of TLR4, NF-κB p65 and TNF-α (P<0.01). CONCLUSION: Ang-4 antagonizes LPS-induced damage in HUVECs by inhibiting TLR4-NF-κB p65-TNF-α signaling pathways.     

Limb ischemic preconditioning protects human umbilical vein endothelial cells from oxidative stress induced by H2O2

AIM: To investigate the effects of the sera from the rats after limb ischemic preconditioning (LIPC) on human umbilical vein endothelial cells (HUVECs) injured by hydrogen peroxide (H₂O₂). METHODS: The HUVECs were divided into 5 groups: the cells in control group were cultured without any intervention; the cells in model group (M) were damaged by 1 mmol/L H₂O₂ for 2 h; the cells in early preconditioning serum (EPS) group, delayed preconditioning serum (DPS) group or sham limb ischemic preconditioning serum (SPS) group were treated with the corresponding serum at 5% for 12 h, respectively, and then treaed with H₂O₂ for 2 h. The viability of the HUVECs was analyzed by flow cytometry. The lactate dehydrogenase (LDH) in the culture media was detected. The cell adhesion molecules in the HUVECs were detected by real-time PCR. The mRNA and protein expression of heme oxygenase-1 (HO-1) was also determined. RESULTS: The viability of HUVECs incubated with 1 mmol/L H₂O₂ for 2 h significantly decreased compared with the control cells, which was accompanied with the augmentations of LDH in the medium and the cell adhesion molecules in cells, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Preincubation with EPS and DPS derived from the rats subjected LIPC attenuated these injuries. Furthermore, pretreatment with EPS and DPS increased the expression of HO-1 at mRNA and protein levels. CONCLUSION: LIPC protects the HUVECs from H₂O₂-induced injury.     

Rb1 protects human umbilical vein endothelial cells from hydrogen peroxide-induced senescence:involvement of caveolin-1

AIM: Endothelial cell senescence has been proposed to be involved in endothelial dysfunction and atherogenesis. This study aims to investigate the effects of ginsenoside Rb1, a major constituent of ginseng, on hydrogen peroxide (H₂O₂)-induced endothelial cell senescence, and to explore the expression and production of caveolin-1 in H₂O₂-induced premature senescence.METHODS: The senescence of primary human umbilical vein endothelial cells (HUVECs) was induced by H₂O₂ as judged by morphology inspection, senescence-associated β-galactosidase (SA-β-Gal) staining and cell cycle detection. The protein expression of caveolin-1 was determined by Western blot and confocal laser-scanning microscopy.RESULTS: Treatment of the HUVECs with H₂O₂ at 60 μmol/L induced premature senescence, as judged by enlarged, flattened cell morphology, increased SA-β-Gal activity and sustained growth arrest. H₂O₂ effectively increased caveolin-1 level. Pretreatment of the HUVECs with Rb1 was found to reverse endothelial cell senescence, as witnessed by a significant decrease in senescent cell numbers and a decreased percentage of G₀/G₁ phase cells. Furthermore, Rb1 administration reversed the H₂O₂-increased protein level of caveolin-1.CONCLUSION: Ginsenoside Rb1 antagonizes H₂O₂-induced endothelial cell senescence through caveolin-1 modulation.     

Effect of activated protein C on apoptosis in human umbilical vein endothelial cells induced by lipopolysaccharide

AIM:To study the effect of activated protein C (APC) at different concentrations on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS).METHODS:The HUVECs were induced by LPS (1.0 mg/L) as apoptotic model that was administered by different concentration of APC (10 μg/L or 50 μg/L). Meanwhile, the control group and induced apoptosis group induced by LPS (1.0 mg/L) stimulation were also set up. The changes of cellular ultrastructures were observed under electron microscope. The DNA ladder and TUNEL fluorescent staining were measured in cells. Annexin-Ⅴ/PI double staining was used to measure the cell apoptosis rate by flow cytometry. Cell survival rate was measured by MTT assay. The proliferating cell nuclear antigen (PCNA) expression levels in cells were also measured by Western blotting to reflect the proliferation of the cells.RESULTS:There were significant apoptotic changes in the cells induced by LPS, but the apoptotic changes were reduced and apoptosis rates were decreased in the cells treated with APC. Meanwhile, cell survival rate and the protein levels of PCNA were increased after APC treatment, particularly at the concentration of 50 μg/L, which showed difference when compared with those induced apoptosis group by LPS (P<0.05).CONCLUSION:APC can inhibit HUVECs apoptosis induced by LPS and promote cell proliferation, thus protect the cells from injury.     

Protective effect of anti-aging Klotho protein on human umbilical vein endothelial cells treated with high glucose

AIM: To study the protective effect of anti-aging Klotho protein on human umbilical vein endothelial cells (HUVECs) treated with high glucose (HG).METHODS: HUVECs were cultured in vitro, and divided into PBS control group, 5.5 mmol/L glucose group, 33.3 mmol/L glucose group, 0.1 μmol/L Klotho+33.3 mmol/L glucose group, 1 μmol/L Klotho+33.3 mmol/L glucose group, and 10 μmol/L Klotho+33.3 mmol/L glucose group. The viability of the HUVECs was measured by MTT assay. The content of malondialdehyde (MDA), and the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione (GSH) in cell culture supernatants were observed. The production of reactive oxygen species (ROS) in HUVECs was analyzed by flow cytometry. The levels of nitric oxide (NO), endothelin (ET-1), intercellular adhesion molecule-1 (ICAM-1) in HUVEC culture medium were detected by ELISA. The protein expression of nuclear factor-kappa B (NF-κB) in the HUVECs was determined by Western blot. RESULTS: Compared with PBS control group, 33.3 mmol/L glucose significantly decreased the HUVEC viability, increased ROS, LDH and MDA levels, reduced the activities of SOD and GSH, decreased the NO secretion, and induced the ET-1 and ICAM-1 secretion and the protein expression of NF-κB in HUVECs. When HUVECs were treated with Klotho protein at different concentrations combined with 33.3 mmol/L glucose, the cell viability was increased significantly, the ROS, LDH and MDA levels were decreased significantly, the antioxidant SOD and GSH activities were significantly increased, the secretion of NO was increased, but ET-1 and ICAM-1 releases and protein expression of NF-κB were significantly reduced.CONCLUSION: Anti-aging Klotho protein promotes the viability of HUVECs treated with HG, reduces the oxidative damage and ROS production, and restores the normal secretory function of HUVECs, thus playing a protective role in vascular endothelial cells through reducing the protein expression of NF-κB.     

Injury effect of recombinant soluble human CD40 ligand on human umbilical vein endothelial cells

AIM: To investigate the damage in human umbilical vein endothelial cells (HUVECs) induced by recombinant soluble human CD40 ligand (rshCD40L). METHODS: The cultured HUVECs were treated with rshCD40L for 12 h. The survival activity of the HUVECs was observed by MTS assay. The expression of E-selectin, intercellular adhesion molecule (ICAM)-1, tissue factor (TF) and tissue factor pathway inhibitor (TFPI) was measured by ELISA. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) were detected by the methods of thibabituric acid (TBA). RESULTS: Compared with normal group, different concentrations of rshCD40L (0.5, 1, 2, 3 mg/L) had no obvious effect on the survival activity of the HUVECs (P>0.05). rshCD40L at concentration of 0.5 mg/L promoted the secretion of E-selectin, sICAM-1, TF and TFPI in the HUVECs (P<0.01). rshCD40L at concentration of 0.5 mg/L also increased MDA content and reduced the activity of SOD in the HUVECs (P<0.05). CONCLUSION: 0.5~3mg/L rshCD40L has no obvious effect on endothelial cell survival, but already causes endothelial dysfunction by increasing endothelial inflammation and exogenous coagulation reaction, inducing lipid peroxides injury and reducing antioxidant capacity.     

Hydrogen sulfide attenuates human umbilical vein endothelial cell senescence via modulation of Sirt1/eNOS pathway

AIM: To explore the role of Sirt1/eNOS signalling pathway in the protective effect of hydrogen sulphide (H₂S) against endothelial cell senescence induced by high glucose.METHODS: High glucose (33 mmol/L) was applied to induce senescence in primary human umbilical vein endothelial cells (HUVECs). The cell viability, the proportion of senescence-associated β-galactosidase (SA-β-Gal) positive cells and the plasminogen activator inhibitor 1 (PAI-1) expression were detected to assess the senescence model. Mean while, Sirt1 siRNA was used to examine the effect of Sirt1 on eNOS expression and the senescence-related parameters.RESULTS: Treatment of HUVECs with high glucose decreased the cell viability slowly with a larger proportion of the cells stained with SA-β-Gal, and the protein expression of PAI-1 was dramatically increased. The increased cell viability, reduced SA-β-Gal positive cells and decreased protein expression of PAI-1 were detected after sodium hydrosulfide (NaHS, 100 μmol/L) treatment. Furthermore, NaHS treatment upregulated the protein expression of Sirt1 and eNOS, and eventually increased the production of nitric oxide (NO).CONCLUSION: Exogenous H₂S modulates Sirt1/eNOS/NO pathway to prevent HUVECs against high glucose-induced senescence.     

Effects of glycated serum albumin on expression of MCP-1 in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of glycated serum albumin (GSA) on the expression of monocyte chemoattratant protein-1 (MCP-1) in endothelial cells.METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured with GSA at different concentrations in the presence or absence of glycosylation-product inhibitor aminoguanidine (AG) and anti-oxidant N-acetylcysteine (NAC). The expression of MCP-1 was evaluated by the methods of immunocytochemistry and sandwich ELISA.Malondialdehyde(MDA) content and superoxide dismutase(SOD) activity were determined by the technique of thiobarbituric acid(TBA) and xanthine oxidase(XOD),respectively. RESULTS: GSA stimulated HUVECs to produce and release MCP-1. After HUVECs were treated with 50 mg/L GSA, the expression of MCP-1 at 4 h, 8 h and 12 h was 1.3, 1.9 and 2.8 folds higher than that in control group (P<0.01), respectiuely.The significant difference among the experiment groups (P<0.01) was observed, indicating that GSA took effect in a concentration-dependent manner. The release of MCP-1 in cultured supernatants in the experiment groups with 3 different concentrations of GSA was 1.6, 2.4 and 3.0 folds as much as that in control group (P<0.01), and the significant difference among the experiment groups (P<0.01) was also observed. GSA decreased the activity of SOD (P<0.05) and increased the content of MDA (P<0.01). AG and NAC obviously inhibited the upregulation of MCP-1 expression in HUVECs by GSA (P<0.01). NAC also inhibited the effect of GSA on SOD activity and MDA content in HUVECs (P<0.05). CONCLUSION: GSA stimulates the expression of MCP-1 by inducing oxidative stress in endothelial cells.     

Decellularized porcine corneal posterior lamellae as carrier matrix for cultivating human umbilical vein endothelial cells in vitro

AIM：To investigate the feasibility of corneal posterior lamellar reconstruction with human umbilical vein endothelial cells (HUVECs) and porcine cornea acellular matrix in vitro, and to observe the physiological function of the transplantation in vivo. METHODS：HUVECs were isolated, cultured, and labeled with fluorescent dye CM-DiI. Porcine corneas were treated with 100% glycerinum, cut to a thinner structure step by step, and dried on the super-clean bench. Transmission electron microscope were used to observe the histological changes of the porcine cornea acellular matrix. Labeled HUVECs were seeded onto the porcine cornea acellular matrix, and examined by scanning electron microscopy. When the HUVECs and Descemets membrane fusion formed a monolayer, the corneal transplantation in rabbits was performed. Twenty-four New Zealand white rabbits were randomly divided into experimental group and control group (n=12 each), and their left eyes served as recipients. RESULTS：Cultured HUVECs exhibited polygonal shape. More than 90% HUVECs were labeled with CM-DiI and the cell membrane was positive with red fluorescence, which was detectable at least up to 3 generations. The histological examination indicated that porcine cornea cells were clearly extracted, and the collagen fibers were well arranged. A continuous monolayer of HUVECs on the porcine cornea acellular matrix was observed under scanning electron microscopy. The reconstructed corneal posterior lamellae were similar to the normal cornea. The observation of transplantation showed that the cornea in experimental group was substantially transparent. However, that in control group was oedematous and adiaphanous. CONCLUSION：Corneal posterior lamellae can be reconstructed in vitro by cultivating HUVECs on porcine cornea acellular matrix. After xenogeneic transplantation, the graft survives in vivo and expresses normal corneal endothelial cell biological functions. Deep lamellar corneal endothelial transplantation is an effective keratoplasty.     

Role of endoplasmic reticulum stress in trimethylamine N-oxide-induced oxidative stress in human umbilical vein endothelial cells

AIM: To investigate whether endoplasmic reticulum stress is involved in trimethylamine N-oxide (TMAO)-mediated oxidative stress in human umbilical vein endothelial cells (HUVECs). METHODS: The cell viability was examined by CCK-8 assay. The cells were stained by DCFH-DA, and the intracellular level of reactive oxygen species (ROS) was observed by phase-contrast microscopy and detected by flow cytometric analysis. The protein levels of phospho-IRE-1α, IRE-1α and GRP78/BiP were detected by Western blot. RESULTS: TMAO exerted no significant effect on the viability of HUVECs. For a long period (>24 h), even a low concentration (10 μmol/L) of TMAO increased the oxidative stress level in the HUVECs (P<0.05). TMAO increased the phosphorylation level of IRE-1α and significantly up-regulated the protein level of GRP78/BiP in HUVECs (P<0.01). Pretreatment with STF-083010, an inhibitor of IRE1α, for 1 h reduced TMAO-induced oxidative stress in HUVECs (P<0.05). CONCLUSION: Endoplasmic reticulum stress is involved in TMAO-induced oxidative stress in HUVECs.     

Effect of 27nt-miRNA on apoptosis of human umbilical vein endothelial cells induced by oxidized low-density lipoprotein

AIM To investigate the effect of 27nt-miRNA （27nt-miR） on apoptosis of human umbilical vein endothelial cells （HUVECs） induced by oxidized low-density lipoprotein （Ox-LDL） and its underlying mechanism. METHODS HUVECs were cultured in vitro and grouped as below： normal control group, Ox-LDL group, 27nt-miR+Ox-LDL group, anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group. The cells in Ox-LDL group were treated with Ox-LDL at 40 mg/L for 48 h, while those in normal control group were untreated but cultured normally. The cells in 27nt-miR+Ox-LDL group, anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group were transfected with their corresponding lentiviral vectors under the same procedure, followed by treatment with Ox-LDL at 40 mg/L for 48 h to induce apoptosis. The cell viability was measured by CCK-8 assay. The migration capacity was detected by scratch assay. The caspase-3 activity was measured by caspase-3 activity assay kit. The apoptotic rate was analyzed by Hoechst 33258 and flow cytometry. The mRNA and protein expression levels of Bcl-2, Bax and caspase-3 were determined by RT-qPCR and Western blot. RESUITS： Compared with negative control+Ox-LDL group, the cell viability and migration ability were significantly decreased by over-expression of 27nt-miR in the HUVECs （P<0.05）, while the activity of caspase-3 and apoptosis induced by Ox-LDL were significantly increased （P<0.05）. Furthermore, the mRNA and protein expression levels of Bax and caspase-3 were significantly up-regulated （P<0.05）, and the mRNA and protein expression level of Bcl-2 was down-regulated in 27nt-miR+Ox-LDL group （P<0.05）. Meanwhile, all the above indexes showed an opposite tendency in anti-27nt-miR+Ox-LDL group. CONCLUSION 27nt-miR promotes Ox-LDL-induced apoptosis and inhibits the viability and migration of HUVECs in vitro, possibly through regulating the expression of apoptotic/anti-apoptotic proteins such as Bax,caspase-3 and Bcl-2.     

Effect of oxidized LDL on expression of connexin43 in cultured human umbilical vein endothelial cells

AIM: To investigate the effect of oxidized LDL (ox-LDL) on the expression of gap junction protein connexin43 in cultured human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Human umbilical vein endothelial cells cultured in normal condition were divided into blank control group, 50 mg/L,100 mg/L and 200 mg/L ox-LDL intervention groups. The mRNA expression of connexin43 in cultured HUVECs was detected with RT-PCR method; while the protein level of connexin43 was determined by the method of immunocytochemistry in the control and 100 mg/L ox-LDL intervention groups 24 h after ox-LDL was given. RESULTS: Different concentrations (50 mg/L, 100 mg/L, 200 mg/L) of ox-LDL up-regulated mRNA expression of connexin43 in cultured HUVECs after 24 h intervention (P<0.01). The protein level of connexin43 in cultured HUVECs intervened with 100 mg/L Ox-LDL for 24 h was up-regulated as compared to the control cells (P<0.01).CONCLUSION: Ox-LDL may up-regulate the expression of connexin43 at mRNA and protein levels in cultured human umbilical vein endothelial cells within short time, indicating that connexin43 plays an important role in the pro-atherosclerotic effect of Ox-LDL.     

PPARδ agonist GW501516 inhibits ox-LDL-or high glucose-induced apoptosis in human umbilical vein endothelial cells

AIM: To investigate the inhibitory effect of peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the apoptosis induced by oxidized low-density lipoprotein(ox-LDL) or high concentration of glucose in human umbilical vein endothelial cells (HUVECs). METHODS: Cell apoptosis was induced by ox-LDL or high concentration of glucose in HUVECs and was examined by flow cytometry.The HUVECs were treated with GW501516 at different concentrations. The viability of HUVECs was analyzed by MTT assay. RESULTS: The apoptosis rate of HUVECs treated with ox-LDL was 21.3%, while those of HUVECs treated with ox-LDL combined with low, medium and high concentrations of GW501516 were 17.47%, 9.72% and 3.94%, respectively. The apoptosis rate of HUVECs treated with glucose was 22.60%, while those of HUVECs treated with glucose combined with different concentrations of GW501516 were 20.23%, 17.01% and 9.38%, respectively.The results indicated that ox-LDL or glucose induced apoptosis of HUVECs and GW501516 decreased the apoptotic rates induced by ox-LDL or glucose in a dose-dependent manner. The results of MTT assay showed that glucose or ox-LDL decreased the viability of HUVECs and GW501516 attenuated the effect of glucose or ox-LDL on HUVECs. CONCLUSION: GW501516 inhibits the apoptotic effects of ox-LDL and glucose on HUVECs and increases the viability of the cells.