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.     

Effects of high glucose on proliferation,migration, adhesion and secretion of rat late endothelial progenitor cells

AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.     

Insulin protects endothelial progenitor cells against functional damage caused by high glucose

AIM: To investigate the impact of various levels of glucose on endothelial progenitor cells (EPCs) proliferation, senescence, and nitric oxide (NO) secretion,and the effect of insulin under high glucose conditions.METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation, cultured with medium 199, and identified to be EPCs at 7th day by flk-1 and AC133 double staining. EPCss were harvested and incubated with glucose (5, 10, 20, 40 mmol/L) or insulin (0.1, 1, 10, 100 nmol/L) under high glucose conditions for 24 h or 7 days. Proliferative capacity, senescence level and NO secretion (after 24 h of incubation) were subsequently determined.RESULTS: High glucose (40 mmol/L) markedly inhibited EPCs proliferation, accelerated EPCs senescence, and decreased NO production (all P<0.05). Compared with high glucose (40 mmol/L) group, insulin intervention promoted EPCs proliferation, inhibited EPCs senescence (prominent at 1 nmol/L, P<0.05), and enhanced NO secretion (prominent at 10 nmol/L, P<0.05).CONCLUSION: High glucose harms EPCs proliferation and function, while insulin alleviates this jeopardy, indicating the protective role of insulin for the cardiovascular system.     

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.     

Protective effect of BNDF on vascular endothelial cells with H2O2-induced oxidative injury

AIM: To study the protective effect of brain-derived neurotrophic factor (BDNF) on vascular endothelial cells with H₂O₂-induced oxidative injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and the oxidation injury model of HUVECs was established by treatment with H₂O₂. The oxidatively injured HUVECs were cultured with different concentrations (1, 10 and 100 μg/L) of BDNF. At the same time, the control group (no injury), PBS treatment after H₂O₂ injury group and TrkB inhibitor group (with 100 μg/L BDNF and 1: 1 000 TrkB inhibitor) were also set up. The viability of the HUVECs was detected by MTT assay. The levels of LDH, MDA, SOD and GSH were measured. The releases of NO, ET-1 and ICAM-1 were analyzed by ELISA. The changes of ROS production and cell apoptosis were evaluated by flow cytometry. The protein levels of TrkB, p-TrkB, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with uninjured control group, in H₂O₂ oxidative injury plus PBS treatment group, the viability of the cells was decreased significantly, the LDH and MDA levels were increased significantly and the activities of SOD and GSH were decreased significantly. The NO secretion was decreased, and the ET-1 and ICAM-1 concentrations were increased significantly. The ROS content and apoptotic rate were increased significantly. The protein levels of cleaved caspase-3 and Bax were increased but Bcl-2 protein expression was decreased significantly. Compared with PBS treatment group, in H₂O₂-injured HUVECs treated with different concentrations of BDNF, the cell viability was gradually increased, the LDH and MDA levels were decreased and the activities of SOD and GSH were increased gradually. The secretion of NO was increased but ET-1 and ICAM-1 were decreased gradually. The ROS content and apoptotic rate were decreased significantly. The TrkB and p-TrkB levels were significantly increased significantly, the protein expression of cleaved-caspase 3 and Bax was decreased gradually and the Bcl-2 protein expression increased gradually. The role of BDNF was inhibited by TrkB inhibitor. CONCLUSION: BDNF protects HUVECs from oxidative injury by binding with TrkB to activate the BDNF-TrkB signaling pathways.     

Effect of water extract propolis on expression of vascular endothelial cell adhesion molecules

AIM: To explore the mechanism of propolis on the inhibition of atherosclerosis and thrombosis in injured human umbilical vascular endothelial cells (HUVECs) induced by tumor necrosis factor alpha (TNF-α)in vitro.METHODS: TNF-α at the concentration of 50 μg/L was used to induce the injury of HUVECs. The injured HUVECs were treated with water extract propolis (WEP) at the concentrations of 50, 100 and 200 mg/L for 6 h, 12 h and 24 h. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was examined by flow cytometry.RESULTS: The expression of ICAM-1 and VCAM-1 was significantly higher in injured HUVECs (P<0.01) than that in the control cells. The expression of ICAM-1 and VCAM-1 was downregulated by WEP treatment in a dose-dependent manner. Between the groups of 100 and 200 mg/L WEP, the difference was significant. In the injured HUVECs treated with 50 mg/L WEP, the inhibitory effect on the expression of ICAM-1 and VCAM-1 was presented in a time-dependent manner. Compared to the single administration, the use of WEP combined with fluvastatin showed better inhibitory effect on the expression of ICAM-1 and VCAM-1 in the injured HUVECs induced by TNF-α (P<0.01).CONCLUSION: WEP may be helpful for the protection of vascular endothelial cells by inhibiting the expression of ICAM-1 and VCAM-1 in a time-and dose-dependent manner. The protective effect of WEP on endothelial cells may be synergic with the inhibitor of HMG-CoA reductase such as fluvastatin sodium.     

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.     

Genipin inhibits oxidative stress and apoptotic damage in high glucose-induced H9c2 cardiomyocytes

AIM:To explore the effects of genipin (GEN) on high glucose (HG)-induced oxidative stress injury and apoptosis in H9c2 cardiomyocytes.METHODS:H9c2 cells were cultured in vitro and HG-induced injury model was established. H9c2 cells were divided into 4 groups:normal control (NC) group (glucose at 5.6 mmol/L), HG group (glucose at 50 mmol/L), NG+GEN group and HG+GEN group. The concentration of genipin was used at 10 μmol/L. The viability of the H9c2 cells was measured by CCK-8 assay. The intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined by enzyme labeling and WST-1 methods, respectively. The activity of lactate dehydrogenase (LDH) in the cell culture supernatant was detected by microplate method. Fluorescent probe DCF was used to detect intracellular levels of reactive oxygen species (ROS). Nucleosome fragments was measured to evaluate cell apoptosis by ELISA. The intracellular mitochondrial membrane potential was detected by JC-1 method. The protein levels of Mn-SOD, cytochrome C (Cyt C), Bax and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with HG group, the cell viability in HG+GEN group was increased significantly (P<0.05), the levels of MDA and LDH were decreased (P<0.05), SOD activity was increased (P<0.05), the levels of ROS and nucleosome fragments in HG+GEN group were decreased (P<0.05), and the mitochondrial membranes potential was notably increased (P<0.05). Compared with NG group, the activation of Mn-SOD was decreased, but the protein levels of Cyt C, Bax and cleaved caspase-3 were increased in HG group (P<0.05). Compared with HG group, the activation of Mn-SOD was increased, and the protein levels of Cyt C, Bax and cleaved caspase-3 were decreased in HG+GEN group (P<0.05).CONCLUSION:Genipin protects HG-induced H9c2 cardiomyocytes against oxidative stress injury and apoptosis.     

Crocin promotes mobilization of EPCs and re-endothelialization of damaged blood vessels in mice with carotid artery injury

AIM: To study the effect of crocin on the mobilization of endothelial progenitor cells (EPCs) in the peripheral blood of the mice with carotid arterial injury and its mechanism.METHODS: The carotid artery injury model of the C57BL/6 mice was established by the method of wire injury. The animals were divided into sham operation group, saline-treated model group, and low dose, medium dose and high dose (10, 50 and 100 μmol·kg^-1·L^-1, respectively) of crocin treatment groups. The mobilization of the EPCs in peripheral blood of the mice with carotid artery injury was detected by flow cytometry at 3 d. The changes of vascular endothelial growth factor (VEGF), stromal-derived factor-1 (SDF-1), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and matrix metalloproteinase-9 (MMP-9) in the peripheral blood of the mice with carotid artery injury were detected by enzyme-linked immunosorbent assay at 7 d. The vascular re-endothelialization and intimal hyperplasia of the mice with carotid artery injury were detected by Evans blue and hematoxylin-eosin staining. At the same time, real-time PCR was used to detect the mRNA expression of vascular repair factor-related receptors, vascular endothelial growth factor receotor-2 (VEGFR-2), CXC chemokine receptor-4 (CXCR4), basic fibroblast growth factor receptor (bFGFR) and epidermal growth factor receptor (EGFR), in the injured segments of carotid arteries.RESULTS: Compared with sham group, the EPCs mobilization and the content of vascular repair factors VEGF, SDF-1, bFGF, EGF and MMP-9 in peripheral blood were increased in model group (P<0.05). The area of vascular endothelium was decreased, while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were increased significantly (P<0.05). The expression levels of VEGFR-2, CXCR4, bFGFR and EGFR were also increased in the injured segments of carotid arteries (P<0.05). Compared with model group, the EPCs mobilization and the content of vascular repair factors VEGF, SDF-1, bFGF, EGF and MMP-9 in peripheral blood were significantly increased in different concentrations of crocin-treated mice with carotid artery injury (P<0.05). The area of vascular endothelium was gradually increased, while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were gradually decreased (P<0.05). The expression levels of VEGFR-2, CXCR4, bFGFR and EGFR were also gradually increased in the injured segments of cartid arteries (P<0.05).CONCLUSION: Crocin promotes the mobilization of EPCs and the re-endothelialization of damaged blood vessels in the mice with carotid artery injury, thus repairing the injured vasculature.     

he effects of constant magnetic field on apoptosis,adhesion molecule expression in endothelial cells and their adhesion rates with monocytes

AIM: To investigate the effects of constant magnetic field on apoptosis, secretion and expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC), and their adhesion rates with THP-1 monocytes induced by angiotensin Ⅱ (AngⅡ).METHODS: The third passage of cultured HUVEC was used.There were six groups: control group, Ang Ⅱ (10^-6 mol/L) group, Ang Ⅱ with 1, 5, 10 or 20 gausses of constant magnetic field group.Samples were collected 24 h after incubation with or without magnetic field.Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and propidinm iodide staining with flow cytometry.Secretion and expression of ICAM-1 and VCAM-1 were detected by ELISA and immunocytochemistry, respectively.Adhesion rate between HUVEC and THP-1 was measured by counting method.RESULTS: Ang Ⅱ at concentration of 10^-6mol/L induced apoptosis in HUVECs (P<0.05 vs control), whereas in 1, 5, 10 and 20 gausses group, apoptosis of HUVECs was significantly lower than that in Ang Ⅱ group (P<0.05).Ang Ⅱ at concentration of 10-6 mol/L significantly increased secretion and expression of ICAM-1 and VCAM-1 (P<0.05 vs control), whereas secretion and expression of ICAM-1 and VCAM-1 in 1, 5, 10 and 20 gausses group significantly decreased, compared with Ang Ⅱ group (P<0.05).The adhesion rates between HUVEC and THP-1 significantly increased 24 h after incubation of HUVEC with Ang Ⅱ (P<0.05 vs control), in contrast, the adhesion rates between HUVEC and THP-1 in 1, 5, 10 and 20 gausses group significantly decreased, compaed with Ang Ⅱ group (P<0.05).CONCLUSIONS: One gauss to 20 gausses of constant magnetic field antagonizes the effects of Ang Ⅱ on HUVEC, decreases apoptosis and expression of ICAM-1 and VCAM-1 in HUVEC, and also decreases the adhesion rates between HUVEC and monocytes induced by Ang Ⅱ.     

Angiotensin 1-7 attenuates angiotensin Ⅱ-induced human glomerular endothelial cell injury via Mas receptor

AIM:To investigate the effect of angiotensin 1-7 (Ang1-7) on the human glomerular endothelial cells (HGECs) injury induced by angiotensin Ⅱ (Ang Ⅱ) and its possible mechanism. METHODS:Cultured HGECs were divided into 6 groups randomly:control group, Ang Ⅱ group, Ang1-7 group, Ang Ⅱ +Ang1-7 group, Ang Ⅱ +Ang1-7+A779 (an inhibitor of Mas receptor) group and A779 group. The apoptotic rate and reactive oxygen species (ROS) of HGECs were analyzed by flow cytometry and photographed by fluorescence microscopy. The levels of lactate dehydrogenase (LDH), nitric oxide (NO), endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) in the supernatant of cell cultures were measured. RESULTS:Compared with the control group, the apoptotic rate and the average fluorescence intensity of ROS were increased in the Ang Ⅱ group, IL-6, TNF-α, TGF-β, ICAM-1 and MCP-1 in cell supernatants were also increased in the Ang Ⅱ group (P<0.05). Compared with the Ang Ⅱ group, the apoptotic rate, ROS level, and the above inflammatory factors were decreased in Ang Ⅱ +Ang1-7 group (P<0.05). Compared with the Ang Ⅱ +Ang1-7 group, adding A779 increased the cell apoptotic rate, ROS production and the releases of the above inflammatory factors in cell supernatants (P<0.05). Compared with the Ang Ⅱ group, adding Ang1-7 inhibited the LDH leakage, ET-1 secretion and promoted the release of NO in a dose-dependent manner (P<0.05). CONCLUSION:Ang1-7 attenuates the HGECs injury induced by Ang Ⅱ by inhibiting the Mas receptor.     

Transforming growth factor α promotes the proliferation,migration and adhesion of human endothelial progenitor cells

AIM: To explore the effects of transforming growth factor-α (TGF-α) in the monoclonal formation, proliferation, migration and adhesiveness of human endothelial progenitor cells (EPCs). METHODS: The isolated and cultured EPCs were treated with various concentrations of TGF-α (final concentrations of 1, 5, 10 μg/L, respectively). At the same time, the PBS control and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) group (10 μg/L TGF-α plus 1: 1 000 EGFR-TKI) were set. The effects of TGF-α on monoclonal formation, proliferation, migration and adhesiveness of EPCs were determined by clone formation experiment, thiazolyl blue tetrazolium bromide (MTT), EdU, Transwell and adhesion assays, respectively. The expression of epithelial growth receptor (EGFR) and vascular endothelial growth factor (VEGF) were measured by Western blotting. RESULTS: Different concentrations of TGF-α all significantly induced the monoclonal formation, proliferation, migration and adhesiveness of EPCs (P<0.01), which were inhibited by EGFR-TKI. The results of Western blotting showed that TGF-α also induced the expression of EGFR and VEGF with a certain concentration effect (P<0.01). CONCLUSION: By combining with EGFR induced the expression of VEGF, TGF-α significantly promotes the related cell function of monoclonal formation, proliferation, migration, adhesiveness in EPCs.     

CHIP participates in high glucose-induced vascular endothelial cell injury

AIM To investigate the effects of carboxy terminus of heat shock protein 70-interacting protein (CHIP) on high glucose (HG)-induced vascular endothelial cell injury. METHODS Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with 5.5 mmol/L glucose (normal glucose, NG) or 25.5 mmol/L glucose (HG) for 24 h. Down-regulation of CHIP expression by RNA interference was conducted. Before the experiment, mannitol was used to eliminate the interference of osmotic pressure. Subsequently, the cells was divided into 4 groups: NG+siRNA NC group, NG+siRNA CHIP group, HG+siRNA NC group, and HG+siRNA CHIP group. Additionally, MTT assay and TUNEL staining were used to detect the viability and apoptosis. The level of endothelin-1 (ET-1) was measured by ELISA, and the level of reactive oxygen species (ROS) was detected by fluorescence probe dihydroethidium. The level of nitric oxide (NO), and the activity of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in the cells were detected by their respective kits. The mRNA expression of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) was detected by RT-qPCR. The protein levels of CHIP, NADPH oxidase (NOX) 2, NOX4, p38, p65, p-p38, p-p65, Bax and Bcl-2 were determined by Western blot. RESULTS Compared with NG+siRNA NC group, the cell viability was decreased, the apoptosis rate, the mRNA expression of IL-8 and MCP-1, and the level of ROS were increased (P<0.05), the activity of SOD was decreased (P<0.05), while the levels of ET-1, NO and iNOS and the protein levels of p-p38, p-p65 and Bax were increased in HG+siRNA NC group (P<0.05). Compared with HG+siRNA NC group, the inflammatory response, the oxidative stress, the apoptosis rate, and the protein levels of p-p38, p-p65 and Bax were significantly increased in HG+siRNA CHIP group (P<0.05). CONCLUSION Down-regulation of CHIP expression aggravates HG-induced vascular endothelial cell injury.     

Effects of Xijiao Dihuang and Yinqiao San decoction on ICAM-1 and VCAM-1 expression in mouse lung tissues and rat pulmonary microvascular endothelial cells infected with influenza virus

AIM：To investigate the effects of Xijiao Dihuang and Yinqiao San decoction (XDY) on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in mouse lung tissues and rat pulmonary microvascular endothelial cells (RPMVECs) infected with influenza virus, and to explore its mechanism for treatment of viral pneumonia. METHODS：Fifty-four male BALB/c mice were randomly divided into normal group, model group and XDY group (n=18 in each group). The viral pneumonia model was established by intranasally dripping influenza A (H1N1) virus into the mice. The mice in XDY group were treated with XDY 1 h after dripping the virus. The expression of ICAM-1 and VCAM-1 in lung tissues was examined by immunohistochemical staining 2, 4 and 6 d after infection. On the other hand, RPMVECs were obtained from male Wistar rats and primarily cultured. The cells were randomly divided into control group, virus group, virus+XDY group, tumor necrosis factor α (TNF-α) group and TNF-α+XDY group. The mRNA and protein expression of ICAM-1 and VCAM-1 was evaluated by real-time PCR and flow cytometry 24 h after infection. RESULTS：Virus exposure increased ICAM-1 and VCAM-1 expression in mouse lung tissues (P<0.01), and XDY treatment attenuated this effect (P<0.01). Virus and TNF-α both led to the increases in mRNA and protein expression of ICAM-1 and VCAM-1 in RPMVECs (P<0.01), which were also reduced by treatment with XDY (P<0.01). CONCLUSION：Treatment with XDY decreases virus-induced ICAM-1 and VCAM-1 expression, suggesting an important role of XDY in treatment of viral pneumonia.     

CD137 signaling molecules promote proliferation of pulmonary artery endothelial cells by aerobic glycolysis

AIM:To investigate whether CD137 signaling molecules promote the proliferation of pulmonary artery endothelial cells (PAECs) by aerobic glycolysis. METHODS:The experiments of mouse PAECs were performed as follows. (1) Stimulating factors TNF-α (10 μg/L), ET-1 (10 mmol/L) and 5-HT (1 μmol/L) were used to stimulate the cells for 24 h. (2) After stimulation with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group (treatment with 5 mg/L CD137L recombinant protein to activate CD137-CD137L signaling), c-Myc inhibitor group (pretreatment with 10 μmol/L c-Myc inhibitor 10074-G5, dissolved in DMSO, for 30 min, followed by treatment with 5 mg/L CD137L recombinant protein) and DMSO group (pretreated with DMSO at the same volume to c-Myc inhibitor group for 30 min followed by CD137L recombinant protein treatment). (3) After stimulated with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group and 2-deoxyglucose (2-DG) group (pretreatment with 10 mmol/L glycolysis inhibitor 2-DG for 30 min followed by CD137L recombinant protein treatment).The expression of membrane protein and total protein of CD137 in the PAECs was detected by flow cytometry and Western blot, respectively. The protein levels of glycolytic enzymes such as hexokinase (HK2), 6-phosphofructo-2-kinase/fructose-2,6-diphosphatase 3 (PFKFB3) and c-Myc were measured by Western blot. The enzyme activity of HK2 and PFKFB3 was detected by HK2 kit and PFK kit, respectively. Glucose oxidase method was used to measure the glucose uptake rate, and lactate colorimetric assay was conducted for analyzing lactic acid production. CCK-8 assay and EdU staining were used to detect proliferation of the PAECs. RESULTS:Compared with control group, TNF-α, ET-1 and 5-HT significantly increased the expression of CD137 membrane protein and total protein in the PAECs (P<0.05). The protein levels and enzyme activity of HK2 and PFKFB3 protein in CD137 agonist group were significantly higher than those in control group (P<0.05). Compared with control group, the lactic acid production and glucose consumption in CD137 agonist group were significantly increased. The protein level of c-Myc was significantly higher than that in control group after stimulation with CD137L recombinant protein, while c-Myc inhibitor 10074-G5 significantly inhibited the promoting effect of CD137L recombinant protein on glycolysis (P<0.05). The results of CCK-8 assay and EdU staining showed that the cell proliferation in CD137 agonist group was significantly increased compared with control group, while glycolysis inhibitor 2-DG significantly inhibited the proliferation-enhancing effect of CD137 signaling activation on the cells (P<0.05). CONCLUSION:CD137 signaling molecules may modulate the aerobic glycolysis by up-regulating c-Myc, thus promoting the proliferation of mouse PAECs.     

Effect of mininally modified low density lipoprotein on the adhesive function of vascular endothelial cell and its mechanism

AIM: To observe the effect of MM-LDL (minimally modified LDL) on the interaction between human umbilical vein endothelial cell (HUVEC) and U937 monocyte-like cell line and the exporession of vascular cell adhesion-1 (VCAM-1), intercellular adhesion molecule-1(ICAM-1), P-selectin.METHODS:The adhesive percentage between HUVEC treated with MM-LDL and U937 was determined by counting and the expression of VCAM-1, ICAM-1, P-selectin were examined by ELISA. RESULTS: Treatment of HUVEC with MM-LDL (75 mg/L) for 4 hours significantly increased adhesion of U937 to HUVEC ( P <0.01) and did not induce the surface expression of VCAM-1, ICAM-1, P-selectin . Recombination tumor necrosis factor-alpha (rTNF_α) 5.0 μg/L, as a positive control, induced the expression of these adhesion molecules ( P <0.05). Prolonged (18 h) exposure to MM-LDL resulted in the expression of P-selectin, but not VCAM-1.CONCLUSION: the adhesion of monocytes to endothelial cells induced by MM-LDL is not mediated by VCAM-1, ICAM-1. P-selectin induction may be partly involved in the process.     

Effects of endothelial progenitor cell-conditioned medium on proliferation and differentiation of type Ⅱ alveolar epithelial cells exposed to hyperoxia

AIM: To investigate the effect of hyperoxia exposure on the paracrine function of endothelial progenitor cells (EPCs), and to explore the effects of paracrine factors of EPCs on the proliferation and differentiation of type Ⅱ alveolar epithelial cells (AECⅡ) exposed to hyperoxia. METHODS: Bone marrow-derived mononuclear cells were isolated and cultured in EGM-2MV medium for 7~10 d to obtain and identify EPCs. EPCs were cultured in room air (RA) or 60% O₂. The normoxia EPC-conditioned medium (E-CM-RA) and hyperoxia EPC-conditioned medium (E-CM-O₂) were collected. The levels of VEGF, FGF10, PDGF-BB and EGF in E-CM-RA and E-CM-O₂ were detected by ELISA. AECⅡ from adult rats were isolated, purified and cultured for 2 d, then divided into RA group, O₂ group, O₂+E-CM-RA group and O₂+E-CM-O₂ group. The proliferation of AECⅡ was detected by MTT assay and cell counting. The mRNA expression of SP-C and AQP5 was quantified by real-time PCR. RESULTS: The expression of VEGF and FGF10 in E-CM-O₂ group decreased significantly compared with E-CM-RA group (P<0.01). There were significant differences in AECⅡ viability and number among the 4 groups at 12 h, 24 h, 2 d and 3 d (P<0.01). Compared with RA group, AECⅡ viability and number in O₂ group decreased significantly at 12 h, 24 h, 2 d and 3 d (P<0.05). The AECⅡ viability and number in O₂+E-CM-RA group were significantly higher than those in O₂ group at 12 h, 24 h, 2 d and 3 d (P<0.05). However, no significant difference in AECⅡ viability and number between O₂+E-CM-O₂ group and O₂ group at 12 h, 2 d and 3 d was observed. There were significant differences in the mRNA expression of SP-C and AQP5 in the 4 groups at 24 h, 2 d and 3 d (P<0.01). Compared with RA group, the mRNA expression of SP-C in O₂ group was significantly inhibited (P<0.01), but the mRNA expression of AQP5 was promoted (P<0.01) at 24 h, 2 d and 3 d. Compared with O₂ group, the mRNA level of SP-C in O₂+E-CM-RA group and O₂+E-CM-O₂ group (P<0.05) at 24 h, 2 d and 3 d was increased, and the mRNA expression of AQP5 (P<0.01) at 2 d and 3 d was inhibited.CONCLUSION: EPCs secrete VEGF and FGF10, and hyperoxia impairs this paracrine function. Hyperoxia exposure inhibits AECⅡ proliferation and the mRNA expression of SP-C, but promotes the mRNA expression of AQP5. EPC-conditioned medium improves the proliferation of hyperoxia-exposed AECⅡ, and inhibits the transformation of AECⅡ. Hyperoxia exposure impairs the paracrine function of EPCs, and weakened the effects of E-CM-O₂ on AECⅡ.     

Effects of Danggui Buxue decoction on endothelial progenitor cells,serum VEGF and SDF-1 in atherosclerotic rabbits

AIM: To investigate the effects of Danggui Buxue decoction (DBD) on serum vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1), as well as the activity of circulating endothelial progenitor cells (EPCs) in atherosclerotic rabbits. METHODS: The model of atherosclerosis was established using immune injury and fatty diet for 4 weeks in New Zealand rabbits (n=25). All modeled rabbits were randomized into 5 groups with 5 animals in each group. The rabbits in atherosclerosis group were intragastrically administered with distilled water. The rabbits in simvastatin group were treated with simvastatin at the dose of 1.7 mg/kg. The rabbits in DBD high-dose, middle-dose and low-dose treatment groups were given DBD at the doses of 6 g/kg, 3 g/kg and 1.5 g/kg, respectively. All drugs were given once a day for 2 weeks. After treatment, the levels of serum VEGF and SDF-1 were measured. The mononuclear cells isolated from the rabbit peripheral blood were cultured for 7 days in vitro, and then attached cells were cultured with both DiI-ac-LDL and FITC-UEA-1 for identification. The proliferation was detected by MTT method. The cell migration was observed using Transwell chambers. The adhesion determination and in vitro angiogenesis assay were also performed. RESULTS: Compared with atherosclerosis group, the levels of serum VEGF and SDF-1 were elevated (P<0.05), the proliferation, migration, adhesion and angiogenesis of EPCs were all improved in DBD high-dose, middle-dose treatment groups and simvastatin group (P<0.05). CONCLUSION: DBD elevates the levels of serum VEGF and SDF-1 to improve the activity of EPCs in the process of atherosclerosis.     

Sinomenine inhibits TNF-α-induced VCAM-1 expression on human umbilical vein endothelial cells

AIM: To investigate whether sinomenine(SN) can decrease TNF-α-induced VCAM-1 expression in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were isolated from freshly collected umbilical cords.Positive control samples were stimulated with TNF-α, omitting SN. Negative control samples were treated in the same way, omitting TNF-α and SN. Experiment samples were co-cultured with TNF-α and SN at different concentration (0.25, 0.5, and 1.0 mol/L),or TNF-α and dexamethasone(Dex) at concentration of 1.0×10^-6mol/L.Cells were harvested after cultivation with the drugs for 12 hours. VCAM-1 mRNA expression was detected by real-time quantitative PCR, and VCAM-1 expression was detected by flow cytometry (FCM).RESULTS: VCAM-1 mRNA and VCAM-1 were induced by TNF-α. Compared with the positives, the relative VCAM-1 mRNA expression decreased to varying degrees in the experiment groups (P<0.05), and SN at concentration of 0.5 mol/L and 1.0 mol/L inhibited expression of VCAM-1 (P<0.05). SN at concentration of 1.0 mol/L decreased VCAM-1 expression by 28.8%(P<0.05), and SN at concentration of 0.5 mol/L reduced VCAM-1 expression by 21.68%(P<0.05). But SN at concentration of 0.25 mol/L and Dex at concentration of 1.0×10^-6mol/L didnt depress expression of VCAM-1. CONCLUSION: SN may inhibit TNF-α-induced VCAM-1 expression in HUVECs in vitro.     

Low density lipoprotein from patients of diabetes mellitus induces apoptosis of murine macrophages via activating caspase-12 pathway

AIM:To explore the effect of low density lipoprotein from the patients of diabetes mellitus (DM-LDL) on the activation of caspase-12 an important molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, in the murine macrophages, and to clarify the underlying molecular mechanisms of apoptosis. METHODS:Murine macrophage RAW264.7 was exposed to DM-LDL (25, 50 and 100 mg/L), normal low density lipoprotein (n-LDL, 50 mg/L), or tunicamycin (TM, 4 mg/L) for 24 h. Additionally, RAW264.7 macrophages were precultured with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then exposed to DM-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were detected by MTT assay and flow cytometry with Annexin V-FITC/propidium iodide staining, respectively. Lactate dehydrogenase (LDH) activity in the media was measured by assay kit. The protein level of caspase-12 was determined by Western blot. RESULTS:Similar to TM (an ERS inducer), treatment with DM-LDL caused significantly decrease in the viability and increase in LDH activity in the media and apoptotic rate of the RAW264.7 macrophages (P<0.05). Additionally, DM-LDL induced activation of caspase-12 especially at the dose of 50 and 100 mg/L (P<0.01). However, the ERS inhibitor PBA protected RAW264.7 macrophages from DM-LDL-induced decrease in viability and increase in LDH activity and apoptosis (P<0.05). Furthermore, PBA attenuated DM-LDL-induced activation of caspase-12 (P<0.05). CONCLUSION:DM-LDL may induce apoptosis in RAW264.7 macrophages, and the mechanism may be related to the activation of caspase-12.