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.     

Adiponectin attenuates H2O2-induced SH-SY5Y cell injury and tau hyperphosphorylation via activating PP2A

AIM: To study the effects of adiponectin on H₂O₂-induced cell injury and tau hyperphosphorylation in human neuroblastoma SH-SY5Y cells. METHODS: Cell viability was determined by MTT assay. H₂O₂-induced cell injury and morphological changes in the SH-SY5Y cells with or without adiponectin treatment were observed. The level of tau phosphorylation as well as the activities of protein phosphatase 2A(PP2A) and of glycogen synthase kinase-3β(GSK-3β) were examined by Western blotting. RESULTS: Adiponectin significantly attenuated H₂O₂-induced cell injury(P<0.01). Adiponectin upregulated the activity of PP2A and decreased phosphorylation levels of tau under the stimulation with H₂O₂ (P<0.01). Okadaic acid, a specific inhibitor of PP2A, blocked the protective effects of adiponectin(P<0.01). Adiponectin increased the phosphorylation level of GSK-3β at Ser9 site under H₂O₂ stimulation(P<0.01). CONCLUSION: Adiponectin protects SH-SY5Y cells against H₂O₂-induced cell injury and decreases tau hyperphosphorylation by activating PP2A and inactivating GSK-3β.     

Effect of senegenin on H2O2-induced damage in hippocampal neurons of SD rats

AIM: To observe the effect of senegenin (Sen) on hippocampal neuron injuries induced by H₂O₂.METHODS: Hippocampal neurons were isolated from neonatal SD rats. The primarily cultured neurons were divided into control group, H₂O₂ group, Sen group and Sen+H₂O₂ group. The cell viability, the content of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) in the neurons were detected after treated with Sen. The morphological changes of nucleus of the neurons were observed by Hoechst 33258 staining. The mRNA expression of bcl-2 and bax was quantified by real-time PCR. The protein levels of Bcl-2 and bax were measured by Western blotting. The activity of caspase-3 was also assayed.RESULTS: Compared with H₂O₂ group, the levels of antioxidative enzyme were increased in Sen+H₂O₂ group (P<0.05). In addition, mRNA expression of bcl-2 increased and that of bax decreased (P<0.05) in Sen+H₂O₂ group. Moreover, Sen increased the protein level of Bcl-2, and reduced the protein level of Bax and the activity of caspase-3 in the neurons exposed to H₂O₂ (P<0.05).CONCLUSION: The protective effect of Sen on hippocampal neurons with H₂O₂ -induced injury may be involved in the mechanisms of     

USP14 regulates H2O2 induced oxidative stress in H9c2 cells

AIM:To evaluate the effect of inhibiting ubiquitin-specific protease 14(USPl4) activity on oxidative stress induced by H₂O₂ of H9c2 cells.METHODS:The H9c2 cells were incubated with H₂O₂ at 25 μmol/L for 2 h to establish the oxidative stress injury model.The cells were divided into control group,H₂O₂ group,IU1 group (25 μmol/L or 50 μmol/L) and IU1+H₂O₂ group.The H9c2 cells activity was measured by MTS assay.The level of intracellular reactive oxygen species (ROS) and cell survival rate were analyzed by flow cytometry assay.The changes of the mitogen-activated protein kinase (MAPK) family related proteins were detected by Western blot.RESULTS:Compared with control group,the cell activity and the viability rate in H₂O₂ group were decreased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were increased (P<0.05).Compared with H₂O₂ group,the cell activity and the viability rate of the H9c2 cells in IU1+H₂O₂ group were increased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were decreased (P<0.05).CONCLUSION:Inhibition of USPl4 activity reduces the oxidative stress injury of the H9c2 cells.The mechanism may be related to inhibition of the MAPK signaling and down-regulation of apoptosis related proteins.     

Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability， promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.     

Astragalosides inhibit autophagy and apoptosis of rat cardiomyocytes induced by H2O2 through mTOR signaling pathway

AIM: To investigate the effects of astragalosides on autophagy and apoptosis of rat cardiomyocytes induced by hydrogenperoxide (H₂O₂).METHODS: The injury model of H9c2 cells induced by H₂O₂ was established, and the cells in astragalosides group and rapamycin group were treated with 20 mg/L astragalosides and 0.1 mg/L rapamycin, respectively. The apoptotic rate was detected by flow cytometry. The autophagy was observed by acridine orange staining. Western blot was used to detect the protein levels of p-mTOR, P70S6K, LC3 and caspase-3. RESULTS: Compared with H₂O₂ group and rapamycin group, the viability of H9c2 cells in astragalosides group was significantly increased (P<0.05). The shape of the H9c2 cells in astragalosides group was complete, the nuclei were stained with yellow-green fluorescence, and the chromatin was distributed evenly. The protein levels of p-mTOR and P70S6K in the H9c2 cells of astragalosides group were significantly increased (P<0.05), whereas the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H9c2 cells of astragalosides group were decreased significantly (P<0.05). CONCLUSION: Astragalosides enhance the viability, inhibit the apoptosis, increase the protein levels of p-mTOR and P70S6K, and decrease the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H₂O₂-induced rat myocardial H9c2 cells. The mechanism is related to the mTOR signaling pathway.     

Role of autophagy inhibitor 3-methyladenine in the injury of U251 cells induced by H2O2

AIM: To investigate the role of autophagy inhibitor 3-methyladenine(3-MA) in the injury of U251 glioma cells induced by H₂O₂. METHODS: The following groups in this study were set up: control group, 10 mmol/L 3-MA group, 1 mmol/L H₂O₂ group and 1 mmol/L H₂O₂ +10 mmol/L 3-MA group. The viability of U251 cells in each group was detected by MTT assay. Autophagic vacuoles in the cells were observed by staining with MDC. The cells were stained with Hoechst 33342 to determine the chromatin condensation. Cell apoptotic ratio was measured by flow cytometry analysis. RESULTS: Compared with control group, no effect of 3-MA on the viability of U251 cells was observed. In H₂O₂ group, the cell viability decreased and cell apoptotic ratio increased.The autophagic vacuoles and nuclear chromatin condensation in the cells were also detected. Compared with H₂O₂ group, addition of 3-MA inhibited the increase in autophagic vacuoles but exacerbated the apoptosis. CONCLUSION: Autophagy inhibitor 3-MA inhibits autophagy partially, but exacerbates apoptosis in U251 cells, indicating that autophagy exerts protective effect in the process of injury in U251 cells induced by H₂O₂.     

Effects of rapamycin on hydrogen peroxide-induced vascular endothelial cell senescence

AIM:To investigate the effects of rapamycin (Rapa) on hydrogen peroxide (H₂O₂)-induced vascular endothelial cell senescence and to explore the underlying mechanisms. METHODS:The human umbilical vascular endothelial cells (HUVECs) were divided into 4 groups:control group, senescence group, Rapa+H₂O₂ group and 3-methyladenine (3-MA)+H₂O₂ group. MTT assay was performed to assess the cell viability. Senescence-associated β-ga-lactosidase (SA-β-Gal) staining was performed to measure the senescent cells in each group. The subcellular structures were observed under transmission electron microscope (TEM). The protein levels of phosphorylated Rb (p-Rb), Rb, p21, LC3-Ⅱ and beclin-1 were determined by Western blot. RESULTS:Compared with control group, the cell viability in H₂O₂ group was significantly decreased accompanied with higher rate of SA-β-Gal staining positive cells (P<0.05) and markedly damaged structure. Additionally, the protein levels of p-Rb and p21 in senescence group were increased markedly compared with control group (P<0.05). However, the cells pre-treated with Rapa prior to stimulation with H₂O₂ showed increased viability, decreased number of senescent cells and decreased protein levels of p-Rb and p21 as compared with the cells stimulated with H₂O₂ alone (P<0.05). Moreover, the TEM observation showed that the structure of the cells in Rapa+H₂O₂ group was roughly normal and the autophagosome was captured, and the expression levels of beclin-1 and LC3-Ⅱ were increased (P<0.05). Conversely, pre-treatment with autophagy inhibitor 3-MA resulted in opposite results. The cell viability was decreased significantly, more senescent cells were stained blue, higher protein levels of p-Rb and p21 were detected (P<0.05), poor subcellular structures were captured, and no beclin-1 and LC3-Ⅱ was detected. CONCLUSION:Rapa may retard the senescence of HUVECs induced by H₂O₂, and promoting autophagy may be the underlying mechanism.     

Identification of ATTM as a novel H2S donor and investigation of its protective effect on HaCaT skin cells

AIM: To investigate the ability of a metal complex ammonium tetrathiomolybdate (ATTM) to release H₂S and its cytoprotective effect on an oxidative injury model. METHODS: Released H₂S was absorbed in a reaction flask from ATTM dissolved in the cell medium. Staining with dichlorodihydrofluorescein diacetate or rhodamine 123 followed by photofluorography was conducted for the observation of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ_m) levels, respectively. Cell viability and release of lactate dehydrogenase (LDH) from the cells were measured with commercial kits. RESULTS: Similar to another H₂S donor GYY4137, ATTM had an ability to release H₂S in the cell medium in a dose-dependent manner. Treatment of human skin HaCaT cells with ATTM at concentrations of 25~400 μmol/L didn't significantly alter cell viability. Exposure of the cells to ultraviolet rays or a ROS donor H₂O₂ increased the intracellular ROS levels. Treatment with 400 μmol/L H₂O₂ significantly reduced the viability of HaCaT cells (P<0.01). However, before the treatment with H₂O₂, pretreatment with ATTM at 100 and 200 μmol/L markedly prevented the H₂O₂-induced cell injury (P<0.01). In addition, the treatment with H₂O₂ triggered ΔΨ_m loss (P<0.01) and LDH release from the cells (P<0.01). Prior to suffering from H₂O₂ injury, the preconditioning with 200 μmol/L ATTM significantly improved ΔΨ_m levels (P<0.05) and attenuated LDH release from the cells (P<0.01).CONCLUSION: ATTM is capable of releasing H₂S and protecting human skin cells against oxidative injury.     

Acetyl-L-carnitine attenuates H2O2-induced oxidative damage of PC12 cells

AIM: To investigate the effect of acetyl-L-carnitine (ALC) on H₂O₂-induced oxidative damage in PC12 cells and its possible mechanism. METHODS: A moderate oxidative damage PC12 cell model was induced by exposure of the PC12 cells to H₂O₂. ALC at different concentrations (100, 200 and 400 μmol/L) was applied to the PC12 cells cultured in vitro, and CCK8 assay was used to detect the cell viability. The cells were divided into control group, H₂O₂ group, and low-ALC, medium-ALC and high-ALC groups. The apoptosis of the cells was analyzed by flow cytometry. The protein levels of Nrf2 and cleaved caspase-3 were determined by Western blot. The nuclear translocation of Nrf2 was observed by immunofluorescence staining. RESULTS: ALC at different concentrations (100, 200 and 400 μmol/L) significantly inhibited H₂O₂-induced PC12 cell apoptosis, and the medium concentration group had the best effect. Compared with H₂O₂ group, low, medium and high concentrations of ALC significantly increased the viability of the PC12 cells induced by H₂O₂, inhibit cell apoptosis (P<0.05), significantly down-regulated the protein level of cleaved caspase-3 (P<0.05), up-regulated the protein level of Nrf2 (P<0.05), and promoted the translocation of Nrf2 from the cytoplasm to the nucleus. CONCLUSION: Acetyl-L-carnitine attenuates H₂O₂-induced oxidative damage of PC12 cells, inhibits the apoptosis and increases the viability, which is related to the activation of Nrf2 signaling pathway.     

Protective effect of HSP70 on injuried myocardial cells induced by H2O2

AIM: To investigate the role of heat-shock protein 70(HSP₇₀) in the protection of myocardial cells against ischemic injury.METHODS: Myocardial cells were cultured in vitro. HSP₇₀ was induced by hyperthermia. H₂O₂-injured myocardial cells were divided into different groups. Flow cytometry, DNA Ladder and biochemistry methods were employed to observe the myocardial cells of different groups.RESULTS: Immunohistochemistry showed hyperthermia induced the up-regulation of HSP₇₀ in myocardial cells. Apoptotic rate, activity analysis of cytochrome C and succinic dehydrogenase in H₂O₂-injuried and HSP₇₀-protected groups were obviously different. Electron micrograph shomed hyperthermia alliviated myocardial cell injury induced by H₂O₂. CONCLUSION: HSP₇₀ delays apoptosis and protects against H₂O₂-induced myocardial cell injury.     

Effects of schisandrin B on apoptosis of lens epithelial cells treated with H2O2

AIM: To investigate the effects of Schisandrin B (Sch B) on apoptosis of lens epithelial cells (LEC) treated with H₂O₂. METHODS: Eyes in SDrats were excised and lenses were separated under operating microscope in sterilized condition. Lenses were divided randomly into four groups with different treatment: control group, hydrogen peroxide group (H₂O₂), pirenoxine sodium group (PS) and schisandrin Bgroup (Sch B). Lenses were incubated in CO₂ incubator for 24 h with 300 μmol·L^-1 H₂O₂ and with or without 0.5 mmol·L^-1 Sch B. LECaoptosis and apoptosis rate were measured by TUNELmethod. Ultrastructure changes and apoptosis bodies of LECwere observed via transmitted electron microscope. RESULTS: (1) Apoptosis rate in H₂O₂ group (92.0±2.6) was significantly higher than that in control group (3.5±1.8). Apoptosis rate in Sch Bgroup (13.8±3.27) was remarkably lower than that in H₂O₂ group and PSgroup. (2) Ultrastructure observation indicated that apoptosis cells occurred in most LEC in H₂O₂ group and the changes were severe presenting different stages. While a few apoptosis cells were observed in Sch Bgroup, the changes were slight and most of them were in early and middle stages. CONCLUSION: These data indicated that Sch Bsignificantly inhibited apoptosis of LECduring experimental oxidative injury, the effects were stronger than PS.     

Hydrogen sulfide attenuates endothelial cell senescence via inhibiting over-phosphorylation of protein kinase B

AIM To explore the effect of hydrogen sulfide on the senescence of human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H₂O₂) and the possibly involved mechanism. METHODS Senescence model was established by treating HUVECs with H₂O₂ at 60 μmol/L for 1 h and then cultured for 24 h. The several parameters were detected to demonstrate the effect of hydrogen sulfide on senescence by Western blot. RESULTS In the cells treated with H₂O₂, a larger proportion of cells were stained with β-galactosidase (SA-β-Gal), and the protein expression of plasminogen activator inhibitor 1 (PAI-1) was dramatically increased (P<0.05). The less SA-β-Gal positive cells and decreased protein of PAI-1 were detected when the cells were pretreated with 100 μmol/L sodium hydrosulfide (NaHS). Furthermore, NaHS treatment inhibited the phosphorylation of protein kinase B (PKB/Akt). Meanwhile, LY294002, the specific inhibitor of Akt, was also able to reverse the cellular senescence induced by H₂O₂ in HUVECs. CONCLUSION Exogenous hydrogen sulfide prevents HUVECs against H₂O₂-induced senescence partially via the inhibition of Akt over-phosphorylation.     

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.     

Total flavonoids of onion attenuate hydrogen peroxide-induced oxidative damage in retinal pigment epithelial cells

AIM: To investigate the effects of total flavonoids of onion (FO) on hydrogen peroxide (H₂O₂)-induced oxidative damage in retinal pigment epithelial cells. METHODS: The retinal pigment epithelium ARPE-19 cells were divided into 5 groups:control group, H₂O₂ group (treated with H₂O₂), FO-L+H₂O₂ group (treated with H₂O₂ and low concentration of FO), FO-M+H₂O₂ group (treated with H₂O₂ and medium concentration of FO) and FO-H+H₂O₂ group (treated with H₂O₂ and high concentration of FO). The cell viability was measured by MTT assay. Apoptosis was analyzed by flow cytometry. DCFH-DA staining was used to detect reactive oxygen species (ROS) level in the cells. WST assay was used to detect superoxide dismutase (SOD) activity. The content of malonaldehyde (MDA) was measured by TBA method. Mitochondrial membrane potential was analyzed by JC-1 staining. The protein levels of cytochrome C (Cyt C) in the cytoplasm, and cleaved caspase-3 and cleaved caspase-9 in the cells were determined by Western blot. RESULTS: Treatment with H₂O₂ decreased ARPE-19 cell viability, increased the apoptotic rate and the level of ROS in the cells, decreased SOD activity, increased the content of MDA, decreased mitochondrial membrane potential, and increased the protein levels of Cyt C in the cytoplasm and cleaved caspase-3 and cleaved caspase-9 in the cells (P<0.05). Compared with H₂O₂ group, the cell viability in FO-L+H₂O₂ group, FO-M+H₂O₂ group and FO-H+H₂O₂ group was increased, the apoptotic rates were decreased, the levels of ROS were decreased, SOD activity was increased, the content of MDA was decreased, mitochondrial membrane potential was increased, the protein level of Cyt C was decreased in the cytoplasm, and the protein levels of cleaved caspase-3 and cleaved caspase-9 protein in the cells were decreased gradually (P<0.05). CONCLUSION: Total flavonoids of onion reduce H₂O₂-induced oxidative damage in retinal pigment epithelial cells.     

Effect of sinapine on H2O2-induced adipogenic differentiation of bone marrow mesenchymal stem cells

AIM:To investigate the effects of sinapine, an effective monomer of Chinese medicine, on hydrogen peroxide (H₂O₂)-induced adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).METHODS:The undifferentiated rat BMSCs were identified and screened by flow cytometry. The adipogenic differentiation of BMSCs was induced by H₂O₂, and the toxicity of sinapine on BMSCs was tested by CCK-8 assay. After the modeling method and the concentration range of sinapine were determined, the lipid droplets in the cells were detected by Oil Red O semi-quantitative assay, and the optimal drug concentration was selected. Finally, Oil Red O assay was observed 24 h after drug intervention, and the expression of adipogenic differentiation-related proteins, adipocyte protein 2 (aP2), peroxisome proliferator-activated receptor γ (PPARγ) and glucose transporter 4 (Glut4), at mRNA and protein levels in the BMSCs was determined by qPCR and Western blot.RESULTS:Treatment with H₂O₂ at 200 μmol/L for 1 h induced BMSCs to differentiate into adipocytes. Below the concentration of 40 μmol/L, sinapine had no toxicity to BMSCs. The best inhibitory concentration of sinapine on adipogenic differentiation was at 15 μmol/L. The number of lipid droplets in sinapine (15 μmol/L) group was significantly lower than that in model group. In sinapine group, the expression of aP2, PPARγ and Glut4 at mRNA and protein levels was lower than that in model group (P<0.01).CONCLUSION:Sinapine inhibits H₂O₂-induced adipogenic differentiation of rat BMSCs. The mechanism may be related to the PPARγ/AMPK signaling pathway.     

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.     

Involvement of 26S proteasome LMP2 subunit in development of tolerance against oxidative stress in vascular endothelial cells

AIM: To observe the expression of 26S proteasome LMP2 subunit in vascular endothelial cells (VECs) under oxidative stress, and to evaluate its role in the development of tolerance against oxidative stress in VECs. METHODS: The cell model of H₂O₂ preconditioning-induced oxidative tolerance was established in VECs. The expression of LMP2 was detected by cellular immunofluorescent labeling and Western blotting. The LMP2 anti-sense and sense oligonucleotides were transfected into VECs by Lipofectamine^TM 2000. The damages of VECs were evaluated by detecting the activity of lactate dehydrogenase (LDH) and the concentration of malondialdehyde (MDA) in the culture medium. RESULTS: H₂O₂ (500 μmol/L for 3 h) induced oxidative stress in VECss in a dose- and the activity of time-dependent manner, characterized by the increase in the concentration of MDA and LDH in the culture medium. Pretreatment with H₂O₂ (10 μmol/L for 24 h) up-regulated the expression of LMP2. Meanwhile, the capacity of cellular tolerance against oxidative stress induced by H₂O₂ was increased as the concentration of MDA and the activity of LDH in the culture medium significantly decreased. Compared with H₂O₂ group, up-regulation of LMP2 by IFN-γ pretreatment (20 μg/L for 48 h) increased the tolerance of VECs against H₂O₂ injury, and the MDA conentration and the activity of LDH in the culture medium also significantly decreased. Transfection with LMP2 antisense oligonucleotide partly inhibited the increased expression of LMP2 induced by IFN-γ in VECs and abolished the tolerance against H₂O₂. CONCLUSION: The 26S proteasome LMP2 subunit is associated with the development of the tolerance against H₂O₂-induced oxidative stress in VECs.     

An oxidative injury model of human kidney epithelial cells

AIM: To establish an injured cell model using human kidney proximal tubule epithelial cell line (HKC) to mimic the oxidative injury by hydrogen peroxide (H₂O₂). METHODS: The cell viability, the content of malondialdehyde (MDA) in the culture supernatant and the activity of intracellular superoxide dismutase (SOD) were detected to investigate the degree of cell injury. Osteopontin (OPN) expressed on the cell membrane surface were observed by laser confocal microscopy before and after cell injury. The changes of cellular morphology and the ultrastructure of membrane surface were observed under scanning electronic microscope. RESULTS: After HKC cells were treated with H₂O₂ at the concentration of 1 mmol/L for different time, the cell viability and the activity of SOD decreased and the content of MDA increased. The expression level of OPN significantly increased and reached to maximae at 1 h. The injured cells appeared shriveled and rough surface, and the shedding of most flagellae was also observed. CONCLUSION: H₂O₂ induces severer injury in HKC cells, including not only the cell viability and membrane surface ultrastructure, but also the OPN expression on the membrane, which could bind calcium oxalate crystal. Therefore, treatment with H₂O₂ at the concentration of 1 mmol/L for 1 h can be used to establish an oxidative injury model in HKC cells.     

Protective effect of ecdysterone on H9c2 cells against oxidative stress

AIM: To investigate the effect of ecdysterone (EDS) on H9c2 cardiomyocytes after oxidative stress. METHODS: H9c2 cells were cultured in vitro and divided into control group, high dose (2 μmol/L) of EDS group, middle dose (1.5 μmol/L) of EDS group, low dose (1 μmol/L) of EDS group, and H₂O₂ group. H9c2 cardiomyocytes in H₂O₂ group and high, middle and low doses of EDS groups were exposed to H₂O₂ for 6 h to establish the model of oxidative stress. The viability of the H9c2 cells was detected by CCK-8 assay. The apoptosis of H9c2 cells was analyzed by flow cytometry. The levels of lactate dehydogenase (LDH) and creatine kinase-MB (CK-MB) in the culture medium, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the H9c2 cells were measured by colorimetry. The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cytometry and confocal laser scanning microscopy. The protein levels of Bax, Bcl-2 and cleaved caspase-3 in the H9c2 cells were determined by Western blot. RESULTS: Ecdysterone at the selected concentrations had no effect on the viability of H9c2 cells. Compared with control group, the levels of LDH, CK-MB, ROS and MDA, and the apoptotic rates of the H9c2 cells were significantly increased after treated with H₂O₂, but were decreased by EDS treatment in a dose-dependent manner. The levels of SOD and mitochondrial membrane potential of the H9c2 cells in H₂O₂ group were reduced significantly compared with control group, but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mitochondrial membrane potential in H₂O₂-treated H9c2 cells. The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H₂O₂ group showed significant elevation in comparison with control group, and the protein expression of Bcl-2 declined in H₂O₂ group compared with control group, but high, middle and low doses of ecdysterone treatments down-regulated the protein levels of Bax, cleaved caspase-3 and up-regulated the expression of Bcl-2 in H₂O₂-treated H9c2 cells. CONCLUSION: Ecdysterone attenuates the effect of H₂O₂-induced oxidative stress on H9c2 cardiomyocytes. The mechanism may be involved in scavenging oxidative stress products, increasing antioxidant enzyme activity and improving mitochondrial function.