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.     

Necroptosis mediates high glucose-induced injury in human umbilical vein endothelial cells

AIM: To explore whether necroptosis contributes to the high glucose (HG)-induced damage in human umbilical vein endothelial cells (HUVECs). METHODS: The protein levels of receptor-interacting protein 3 (RIP3) and cleaved caspase-3 were detected by Western blot. The intracellular levels of reactive oxygen species (ROS) were determined by DCFH-DA staining followed by photofluorography. Mitochondrial membrane potential (MMP) was measured by rhodamine 123 staining followed by photofluorography. RESULTS: Treatment of HUVECs with HG at different concentrations (10, 20 and 40 mmol/L glucose) for 24 h gradually enhanced the expression levels of RIP3. Treatment of HUVECs with HG (40 mmol/L glucose) for different time (3 h, 6 h, 9 h, 12 h and 24 h) also up-regulated the expression levels of RIP3, peaking at 9 h. Pretreatment of HUVECs with 20 μmol/L Z-VAD-FMK (an inhibitor of caspase) for 30 min before exposure to HG enhanced the expression level of RIP3. Pretreatment of HUVECs with 100 μmol/L necrostatin-1 (an inhi-bitor of necroptosis) for 1 h before exposure to HG alleviated the HG-induced injuries, such as a decrease in cell viability, an increase in ROS generation and dissipation of MMP, but up-regulated the protein level of cleaved caspase-3. CONCLUSION: Necroptosis mediates HG-induced injury in HUVECs. There is a negative interacting between necroptosis and apoptosis.     

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.     

Role of PI3K/Akt/eNOS signaling pathway in inhibitory effects of puerarin on ox-LDL-induced TF expression in vascular endothelial cells

AIM:To explore the role of phosphatidylinositiol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathways in the inhibitory effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-induced tissue factor (TF) expression in vascular endothelial cells.METHODS:The mRNA expression of TF was detected by real-time fluorescent quantitative PCR.The protein levels of TF and Akt was determined by Western blot.The content of the nitric oxide (NO) was measured by nitrate reduction method.RESULTS:Compared with control group,incubating endothelial cells with ox-LDL significantly induced TF expression at mRNA and protein levels and the dephosphorylation of Akt protein,and decreased NO production.Incubation of the endothelial cells with puerarin for 1 h and then treatment of the cells with ox-LDL decreased the TF expression at mRNA and protein levels,increased Akt protein phosphorylation and intracellular NO content.Co-incubation of the endothelial cells with PI3K inhibitor LY294002 and puerarin for 1 h and then treatment of the cells with ox-LDL augmented the TF expression at mRNA and protein levels and the Akt protein dephosphorylation,and decreased NO production.Co-incubation of the endothelial cells with eNOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) and puerarin significantly decreased the inhibitory effect of puerarin on ox-LDL-induced TF expression at mRNA and protein levels in the endothelial cells,and reduced Akt protein phosphorylation and NO production.CONCLUSION:Puerarin inhibits ox-LDL-induced TF expression at mRNA and protein levels in the human umbilical vein endothelial cells via activation of PI3K/Akt/eNOS signaling pathway.     

Role of SIRT1/eNOS/NO signaling pathway in protective effect of ginsenoside Rb1 against replicative senescence of endothelial cells

AIM: To explore the effect of ginsenoside Rb1 on replicative senescence of endothelial cells and the role of SIRT1/eNOS/NO signaling pathway in this process. METHODS: The replicative senescence model of primary human umbilical vein endothelial cells (HUVECs) was established. The morphological change of the cells, 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. The expression of eNOS and PAI-1 at mRNA and protein levels in the aging cells was determined by real-time PCR and Western blot before and after silencing of SIRT1 was performed. The NO concentration in the cell culture supernatant was measured by nitrate reductase assay. RESULTS: HUVECs with cumulative population-doubling level (CPDL) at 16 were chosen as the replicative senescence model in this research. Ginsenoside Rb1 at 80 μmol/L significantly reduced the expression of PAI-1 at mRNA and protein levels. Furthermore, ginsenoside Rb1 increased the expression of SIRT1 and eNOS at mRNA and protein levels, and increased the NO content. SIRT1 silencing inhibited the expression of eNOS at mRNA and protein levels and reduced NO generation, leading to an increase in the expression of PAI-1 at mRNA and protein levels. Upon intervention of ginsenoside Rb1, the eNOS and PAI-1 expression and the level of NO were not reversed. CONCLUSION: Ginsenoside Rb1 modulates SIRT1/eNOS/NO signaling pathway to prevent the replicative senescence of HUVECs.     

Opening of ATP-sensitive K+ channels protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting TLR4/NF-κB pathway

AIM: To investigate whether the opening of ATP-sensitive K⁺(K_ATP) channels protects H9c2 cardiac cells against high glucose(HG)-induced injury and inflammation by inhibiting the Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) pathway. METHODS: The protein levels of TLR4 and NF-κB p65 were determined by Western blot. The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by ELISA. The cell viability was measured by CCK-8 assay. Mitochondrial membrane potential(MMP) was examined by rhodamine 123(Rh 123) staining followed by photofluorography. The intracellular levels of reactive oxygen species(ROS) were detected by 2', 7'-dichlorfluorescein- diacetate(DCFH-DA) staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG(35 mmol/L glucose) for 24 h, the protein levels of TLR4 and phosphorylated NF-κB p65(p-NF-κB p65) were significantly increased. Pretreatment of the cells with 100 μmol/L diazoxide(DZ, a K_ATP channel opener) for 30 min before exposure to HG considerably blocked the up-regulation of the TLR4 and p-NF-κB protein levels induced by HG. Moreover, co-treatment of the cells with 30 μmol/L TAK-242(an inhibitor of TLR4) obviously inhibited the HG-induced up-regulation of the p-NF-κB p65 protein level. On the other hand, pretreatment of the cells with 100 μmol/L DZ had a clear myocardial protection effect, which attenuated the HG-induced cytotoxicity, inflammatory response, mitochondrial damage, oxidative stress and apoptosis, evidenced by an increase in the cell viability, and decreases in the levels of IL-1β and TNF-α, MMP loss, ROS generation and the number of apoptotic cells. Similarly, co-treatment of H9c2 cardiac cells with 30 μmol/L TAK-242 or 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries and inflammation induced by HG.CONCLUSION: The opening of K_ATP channels protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the TLR4/NF-κB pathway.     

Angiotensin-(1-7) protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis

AIM: To investigate whether angiotensin-(1-7)[Ang-(1-7)] protects H9c2 cardiac cells against high glucose (HG)-induced injury and inflammation by inhibiting the interaction between Toll-like receptor 4 (TLR4) activation and necroptosis. METHODS: The expression levels of receptor-interacting protein 3 (RIP3; an indicator of necroptosis) and TLR4 were determined by Western blot. Cell viability was measured by CCK-8 assay. The activity of lactate dehydrogenase (LDH) in the culture medium was measured with a commercial kit. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The intracellular level of reactive oxygen species (ROS) was analyzed by 2', 7'-dichlorfluorescein-diacetate (DCFH-DA) stating followed by photofluorography. Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the expression of RIP3 was obviously increased. Co-treatment of the cells with 30 μmol/L TAK-242 (an inhibitor of TLR4) attenuated the up-regulation of RIP3 induced by HG. Furthermore, the expression of TLR4 was significantly increased after the cells were exposed to HG for 24 h, and co-treatment of the cells with 100 μmol/L necrostatin-1 (Nec-1; a specific inhibitor of necroptosis) and HG for 24 h attenuated the up-regulation of TLR4 expression induced by HG. Moreover, 1 μmol/L Ang-(1-7) simultaneously blocked the up-regulation of the RIP3 and TLR4 induced by HG. On the other hand, co-treatment of the cells with 1 μmol/L Ang-(1-7), 30 μmol/L TAK-242 or 100 μmol/L Nec-1 and HG for 24 h attenuated HG-induced injuries and inflammatory response, leading to the increase in the cell viability, and the decreases in the activity of LDH, ROS generation, MMP loss as well as the releases of IL-1β and TNF-α. CONCLUSION: Ang-(1-7) protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis.     

Lycium barbarum polysaccharides attenuate oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide

AIM: To study the effect of Lycium barbarum polysaccharides (LBP) on oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide (H₂O₂). METHODS: The EA.hy926 cell model of oxidative stress injury was established by H₂O₂ treatment. The EA.hy926 cells were divided into 5 groups:control group, damage (H₂O₂ at 50 mmol/L) group, LBP (100 mg/L) group, anti-damage groups (LBP at 50 mg/L, 100 mg/L or 200 mg/L+50 mol/L H₂O₂), and LY294002 (20 μmol/L) group. The effect of LBP at different concentrations on the cell viability of EA.hy926 cells was measured by CCK-8 assay, and the optimum concentration of LBP was screened out. The apoptotic of EA.hy926 cells was analyzed by flow cytometry. Acridine orange/ethidium bromide (AO/EB) staining was used to observe the morphological characteristics of the apoptotic cells. The cell migration ability was detected by scratch method. The levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the cell culture medium were examined. The protein levels of cleaved caspase-3, Bax, Bcl-2, endothelial NO synthase (eNOS), p-eNOS and p-Akt were determined by Western blot. RESULTS: LBP at concentration of 100 mg/L significantly attenuated the injury of EA.hy926 cells induced by H₂O₂, as indicated by improved cell viability (P<0.05) and decreased apoptosis (P<0.05). Pretreatment with LBP elevated the levels of NO and VEGF (P<0.05), and promoted the migration ability of EA.hy926 cells. LBP also increased the Bcl-2/Bax ratio, down-regulated the protein level of cleaved caspase-3, and up-regulated the protein levels of eNOS and p-eNOS. The protective effect of LBP were abolished by pretreatment of the EA.hy926 cells with the inhibitor of PI3K (P<0.05). As a result, the protein level of p-Akt was down-regulated, and the level of NO was also significantly reduced. CONCLUSION: LBP has protective effect on H₂O₂ -induced EA.hy926 cells by attenuating apoptosis of the cells. The mechanism is closely related to the activation of PI3K/Akt/eNOS signaling pathway.     

Quercitrin promotes apoptosis of gastric cancer cell line SGC7901 by inhibiting PI3K/AKT signaling pathway

AIM: To investigate whether quercitrin induces apoptosis of gastric cancer cell line SGC7901 by inhibition of PI3K/AKT signaling pathway. METHODS: The human gastric cancer SGC7901 cells were selected as the research object. The cytotoxicity of quercitrin was detected by MTT assay, and IC₅₀ value of quercitrin was calculated. The SGC7901 cells were divided into control group, quercitrin group (incubated with 200 μmol/L quercitrin), insulin-like growth factor-1 (IGF-1) group (incubated with 100 μg/L IGF-1) and quercitrin+IGF-1 group (incubated with 200 μmol/L quercitrin and 100 μg/L IGF-1). After 48 h, the apoptosis of SGC7901 cells was analyzed by flow cytometry, and the protein levels of cleaved caspase-3, p-AKT (Ser473), AKT, p-PI3K (Tyr508) and PI3K were determined by Western blot. RESULTS: The viability of SGC7901 cells was significantly decreased as the concentration of quercitrin increased, starting at 100 μmol/L (P<0.05). The IC₅₀ value of quercitrin for 48 h was 275.40 μmol/L. After treatment with 200 μmol/L quercitrin for 48 h, the apoptosis rate and the protein level of cleaved caspase-3 in quercitrin group were significantly increased (P<0.05), and the phosphorylated levels of AKT and PI3K were significantly decreased compared with control group (P<0.05). Treatment with quercitrin and IGF-1 inhibited the effect of quercitrin on SGC7901 cells compared with quercitrin group. CONCLUSION: Quercitrin may induce apoptosis of gastric cancer cell line SGC7901 by inhibiting the activation of PI3K/AKT signaling pathway.     

Roles of protein phosphatase 4 in down-regulation of eNOS Ser633 phosphorylation induced by palmitic acid in human umbilical vein endotheli?al cells

AIMTo investigate the roles of protein phosphatase 4 (PP4) in down-regulation of endothelial nitric oxide synthase (eNOS) Ser633 phosphorylation induced by palmitic acid (PA). METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with PA at 25 μmol/L, 50 μmol/L, 100 μmol/L and 200μmol/L for 36 h, or treated with PA at 100 μmol/L for 12 h, 24 h, 36 h and 48 h. Protein phosphatase 2A (PP2A) family inhibitor fostriecin (FST, 20 nmol/L) or okadaic acid (OA, 5 nmol/L) was selected to pretreat the HUVECs for 30 min. Protein phosphatase 4 catalytic subunit (PP4c) siRNA or protein phosphatase 2A catalytic subunit (PP2Ac) siRNA was transfected into the HUVECs. The protein expression levels of of eNOS, PP4c and PP2Ac, as well as the level of eNOS Ser633 phosphorylation, were detected by Western blot. The intracellular nitric oxide (NO) content was measured by DAF-FM DA. RESULTS(1) Compared with control group, the levels of eNOS Ser633 phosphorylation were decreased in PA groups in which the HUVECs were treated with 25 μmol/L, 50 μmol/L, 100 μmol/L and 200 μmol/L PA for 36 h (P<0.05) and 100 μmol/L PA for 24 h, 36 h and 48 h (P<0.05). No significant difference in the level of total eNOS protein expression among all the groups was observed. (2) Compared with control group, both FST and OA pretreatment reversed the reduction of eNOS Ser633 phosphorylation (P<0.05) and the decrease in intracellular NO content (P<0.05) induced by PA. No significant difference in the level of total eNOS protein expression among all the groups was observed. (3) Compared with si-Control group, the PP4c protein expression was significantly reduced (P<0.05), while the level of eNOS Ser633 phosphorylation was significantly increased in si-PP4c group (P<0.05). Although the levels of PP2Ac protein expression declined significantly (P<0.05), the level of eNOS Ser633 phosphorylation remained unchanged in si-PP2Ac group. No significant differencein the level of total eNOS protein expression among all the groups was found. CONCLUSION PA significantly reduces the level of eNOS Ser633 phosphorylation and the content of NO in the HUVECs, which may be due to PA inducing the activation of the PP2A family member PP4 rather than PP2A.     

PPARs ligands antagonizes Ang-induced decrease in endothelial NO production by upregulating eNOS expression

AIM: We hypothesized that PPARγ ligands stimulate endothelial-derived nitric oxide (NO) release to protect the vascular wall. Thus, the purpose of this study is to investigate the effects of ciglitazone (Cig) and fenofibrate (Fen) on angiotensin Ⅱ (AngⅡ)-induced decrease in endothelial NO synthase (eNOS) expression and NO production in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were preincubated for 24 h with Cig (10-7, 10-6, 10-5, 10-4 mol/L) or Fen (10-5 and 10-4 mol/L), then incubated for 12 h with 10-7 mol/L AngⅡ. Total RNA was extracted, and the expression of mRNA and protein of eNOS was assessed by RT-PCR and Western blotting. NO production was measured by Griees method. RESULTS: In the presence of 10-7 mol/L AngⅡ for 12 h, NO production in cultured HUVECs was decreased (P<0.01). Cig and Fen pretreatments enhanced NO production (P<0.01) and antagonized Ang-induced decrease in eNOS mRNA and protein levels in HUVECs. CONCLUSION: PPARγ activator, ciglitazone, and PPARα activator, fenofibrate, antagonize Ang-induced decrease in endothelial NO production by directly upregulating eNOS expression.     

Fenofibrate increases endothelial nitric oxide synthase expression in bovine aortic endothelial cells

AIM: We hypothesize that peroxisome proliferator-activated receptor α(PPARα) agonists act directly on nitric oxide (NO) production in vascular endothelium. Thus, the purpose of this study is to investigate the effects of fenofibrate on endothelial NO synthase(eNOS) activity and its expression in cultured vascular endothelial cells. METHODS: Bovine aortic endothelial cells (BAECs) were treated with the PPARα activator fenofibrate. The eNOS activity and the expression of eNOS protein and its mRNA were determined. RESULTS: Our data show that fenofibrate increased eNOS activity in a dose-and time-dependent manner. At the concentration of 10 μmol/L or more, fenofibrate treatment caused a significant increase in eNOS activity. The maximal increase in eNOS activity(2.32±0.47 fold of the control) was observed with 50 μmol/L fenofibrate treatment for 48 h. Fenofibrate failed to increase eNOS activity at 1 and 12 h. RT-PCR analysis demonstrated that eNOS mRNA relative to β-actin mRNA significantly increased at concentrations of 5 μmol/L or more. It reached 2.08±0.33 fold of the control with 50 μmol/L fenofibrate. Significant increase in eNOS mRNA levels was observed after 6 h, and lasted for 48 h. The peak increase in eNOS mRNA levels(2.13±0.30 fold of the control,P<0.01) was observed with 50 μmol/L fenofibrate treatment for 12 h. Longer incubation of cells with 50 μmol/L fenofibrate caused no further increase. The treatment of BAECs with fenofibrate for 48 h demonstrated a concentration-dependent increase in eNOS protein levels as measured by Western blot analysis. Densitometric analysis indicated that there was a significant increase in eNOS to β-actin ratios after fenofibrate treatment at concentrations of 10,50 and 100 μmol/L(1.80±0.45, 2.70±0.42 and 2.20±0.32 fold of the control, respectively, P<0.01). The significant increase in eNOS protein levels was observed 12 h after treatment and lasted for 48 h. CONCLUSION: PPARα activator fenofibrate, enhances endothelial NO production by directly upregulating eNOS expression and activity.     

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.     

Angiotensin-(1-7)/Mas receptor axis protects cardiomyocytes against high glucose-induced injury by modulating nuclear factor-κB pathway

AIM: To study whether the angiotensin-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardiomyocytes against high glucose(HG)-induced injury by inhibiting nuclear factor-κB(NF-κB) pathway. METHODS: The cell viability was measured by CCK-8 assay. The intracellular levels of reactive oxygen species(ROS) were detected by DCFH-DA staining. The number of apoptotic cells was tested by Hoechst 33258 nuclear staining. Mitochondrial membrane potential(MMP) was examined by JC-1 staining. The levels of NF-κB p65 subunit and cleaved caspase-3 protein were determined by Western blotting. RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose(HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated(p) NF-κB p65, peaking at 60 min. Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30 μmol/L and HG for 24 h inhibited HG-induced cytotoxicity, evidenced by an increase in cell viability. On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage, indicated by decreases in the number of apoptotic cells, cleaved caspase-3 level, ROS generation and MMP loss. However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor(Mas receptor). Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG. CONCLUSION: Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway.     

Up-regulation of Snail1 expression in renal tubular epithelial cells through Akt/GSK-3β pathway under high-glucose condition

AIM: To examine the effects of high glucose (HG) on the expression of Snail1 and protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) in primary renal tubular epithelial cells (RTECs). METHODS: The primary RTECs were randomly treated with normal glucose, high glucose or D-mannitol for 30 min~72 h. RT-PCR and Western blotting were used to observe the expression of Snail1, Akt and GSK-3β at mRNA and protein levels in these cells. The primary cultured RTECs were pretreated with LY294002 (a PI3K inhibitor, 25 μmol/L) to observe the specific inhibitory effects of phosphatidylinositol 3-kinase (PI3K) on HG-induced expression of Snail1 protein. RESULTS: Treatment of RTECs with HG resulted in increased mRNA and protein levels of Snail1, Akt1, and phosphorylation of Akt and GSK-3β. LY294002 blocked the HG-induced up-regulation of p-Akt, p-GSK-3β and Snail1 expression at protein level, but no effect of LY294002 was seen on the total protein expression of Akt1 and GSK-3β. HG did not affect the expression of GSK-3β at mRNA and protein levels. CONCLUSION: HG-induced up-regulation of Snail1 may be regulated by Akt/GSK-3β pathway in RTECs.     

Interleukin-1β inhibits AKT to down-regulate eNOS Ser1177 phosphorylation in human umbilical vein endothelial cells

AIM To investigate whether interleukin-1β （IL-1β） regulates endothelial nitric oxide synthase （eNOS） phosphorylation at Ser1177 site in human umbilical vein endothelial cells （HUVECs）, and to explore its possible mechanism. METHODS The HUVECs were randomly divided into normal control group, tumor necrosis factor-α （TNF-α） group, IL-1β group, IL-6 group, SC79 ［protein kinase B （PKB/AKT） specific agonist］ group and SC79+IL-1β group. Western blot was used to determine the protein levels of eNOS, p-eNOS-Ser1177, AKT and p-AKT-Ser473 in the HUVECs. Chemical colorimetry was used to detect the nitric oxide （NO） content in the culture medium of HUVECs. RESULTS No statistically significant difference of p-eNOS-Ser1177 level in HUVECs treated with TNF-α and IL-6 was observed as compared with normal control group （P>0.05）, while the protein level of p-eNOS-Ser1177 in the HUVECs and the content of NO in the culture medium of HUVECs decreased significantly in IL-1β group （P<0.05）, and the protein level of p-AKT-Ser473 in the HUVECs was decreased as compared with normal control group （P<0.05）. The AKT agonist SC79 blocked the down-regulation effect of IL-1β on p-eNOS-Ser1177 level in the HUVECs and NO content in the culture medium of HUVECs （P<0.05）. CONCLUSION IL-1β down-regulates the protein level of p-eNOS-Ser1177 in HUVECs and affects the activity of eNOS, which may be involved in AKT/eNOS signaling pathway.     

Astragalus polysaccharides protects against free fatty acid-induced human vascular endothelial cell dysfunction via AMPK-eNOS pathway

AIM: To study the protective effect of Astragalus polysaccharides (APS) on free fatty acid-induced injury in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were divided into control group, APS group [APS (200 mg/L) treated for 24 h], free fatty acid group [free fatty acid (0.25 mmol/L) treated for 24 h], free fatty acid plus APS group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) treated for 24 h], and compound C group [free fatty acid (0.25 mmol/L) and APS (200 mg/L) and AMPK inhibitor compound C (10 μmol/L) treated for 24 h]. The cell viability was detected by MTT assay. Nitric oxide (NO) content in the medium was determined by nitrate reductase assay. The protein levels of total adenosine monophosphate-activated protein kinase (AMPK), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), endothelial nitric oxide synthase (eNOS) and phosphorylated endothelial nitric oxide synthase (p-eNOS) were measured by Western blot. RESULTS: No significant difference of all indexes between APS group and control group was observed. The cell viability in free fatty acid group decreased significantly compared with control group. The cell viability in free fatty acid plus APS group was significantly improved as compared with free fatty acid group. The cell viability in compound C group was almost the same as that in free fatty acid group. The No content and protein levels of p-AMPK and p-eNOS in free fatty acid group decreased obviously as compared with control group, while the NO content and protein levels of p-AMPK and p-eNOS in free fatty acid plus APS group increased obviously compared with free fatty acid group. No significant difference of the p-AMPK and p-eNOS protein levels between free fatty acid plus APS group and free fatty acid group was observed. No significant difference of the AMPK and eNOS protein levels in all groups was found. CONCLUSION: APS attenuates the free fatty acid-induced injury, and its mechanism is related to the AMPK-eNOS signal pathway.     

Puerarin pretreatment protects human umbilical vein endothelial cells against hypoxia/reoxygenation injury via increasing protein expression of endothelial nitric oxide synthase

AIM: To investigate the protective effects of puerarin (PUE) pretreatment on hypoxia/reoxygenation (H/R) injury in human umbilical vein endothelial cells (HUVECs), as well as its possible mechanism and the signal transduction pathways involved. METHODS: HUVECs were randomly divided into normal control group, H/R group, PUE pretreatment group and PUE+H/R group (1.0×10^-3 mol/L, PUE pretreated the cells for 24 h before H/R). The protein expression of endothelial nitric oxide synthase (eNOS) was measured by Western blot. The activity of constitutive NOS (cNOS) was determined via chemical colorimetric methods. Apoptosis of HUVECs was detected by TUNEL assay. In addition, the cells were treated with ERK inhibitor U0126 (1.0×10^-5 mol/L) or PKB/Akt inhibitor LY294002 (5.0×10^-5 mol/L) for 1 h before PUE pretreatment, and then H/R was performed.RESULTS: Compared with control group, H/R decreased the protein expression of eNOS (P<0.05), and PUE pretreatment up-regulated it (P<0.05). This effect of PUE was inhibited by U0126 or LY294002 (P<0.05). Compared with control group, the activity of cNOS decreased in H/R group (P<0.05), while it increased after PUE pretreatment (P<0.05). Compared with control group, the apoptotic index significantly increased in H/R group (P<0.01). PUE pretreatment reduced the apoptotic index (P<0.01). CONCLUSION: H/R decreases the protein expression and enzyme activity of eNOS in HUVECs, and induces apoptosis of HUVECs. PUE pretreatment up-regulates the protein expression and enzyme activity of eNOS, and reduces the apoptosis of HUVECs with H/R injury. The protective effect of PUE might be through increasing eNOS protein expression via ERK1/2 and PKB/Akt signaling pathways.