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.     

Functional changes of retinal ganglion cells in rd1 mice during midterm of retinal pigmentosa

AIM: To investigate how the function of retinal ganglion cells (RGCs) change in the midterm of retinal pigmentosa (RP) in rd1 mice (a transgenic animal model of RP). METHODS: The action potentials from multiple RGCs in rd1 mice at postnatal 20 d (P20) or normal C57 mice (control) were simultaneously recorded by multi-electrode array recording. The functional changes of surviving ganglion cells were evaluated by comparing spontaneous and light-evoked activities of RGCs between rd1 and control mice. The extent of photoreceptor degeneration was verified by immunohistochemical staining. RESULTS: Immunohistochemistry results showed the thickness of the retinal photoreceptor layer of rd1 mice was significantly lower than that in normal mice at P20. According to the light response properties, we classified ganglion cells into 6 subgroups: ON sustained, ON transient, ON-OFF sustained, ON-OFF transient, OFF sustained and OFF transient RGCs, with a very tiny percentage of OFF sustained RGCs (1.0%~3.1%). The percentage of RGCs remaining light responsive in rd1 mice was significantly lower than that in C57 mice. The average spontaneous spiking rate for rd1 RGCs was overall significantly increased compared to that in C57 cells, whereas different RGC types had different changes. The light-induced responses and light sensitivities of all types of RGCs in rd1 mice were both significantly lower than those in C57 mice. CONCLUSION: The photoreceptors of rd1 mice are severely degenerated in the midterm of retinal degeneration. The functions of RGCs in rd1 mice in the midterm of degeneration decay obviously, with variance in different RGCs types.     

Effects of recombinant basic fibroblast growth factor on retinal ganglion cells of rats

AIM:To investigate the pharmacological effects of recombined basic fibroblast growth factor (rbFGF) on retinal ganglion cells (RGCs) of rats.METHODS:Using calibrated cross-action forceps a moderate crush injury was inflicted on the nerve. After crush injury, rbFGF, saline and VB₁₂ were administered by retrobublar injection. Four weeks after injury, the apoptosis of RGCs was measured with flow cytometer.RESULTS:Four weeks after operation, it was shown that the rbFGF, but not saline or VB₁₂ injection could significantly improve the maintainance of RGCs of rats. After 800 U, 1600 U and 2400 U rbFGF injection, the injured RGCs were rescued by 24.5%, 27.3% and 28.5% respectively. Furthermore, it was also found that rbFGF injection could effectively prevent the axons from injury. The flow cytometer showed that the rate of apoptosis was reduced markedly on 7 days at rbFGF group. CONCLUSION:rbFGF can significantly promote the functional repair of injured optic nerve.     

Protective effect of salidroside on rats with hypoxic pulmonary hypertension by suppressing oxidative stress

AIM: To study whether salidroside plays a protective role in hypoxia-induced pulmonary hypertension by suppressing oxidative stress. METHODS: Sprague-Dawley rats were randomly divided into 4 groups:normoxia (N) group, hypoxia for 4 weeks (H₄) group, low-dose salidroside (hypoxia for 4 weeks and treatment with salidroside at 16 mg/kg, H₄S16) group and high-dose salidroside (hypoxia for 4 weeks and treatment with salidroside at 32 mg/kg, H₄S32) group. The mean pulmonary arterial pressure (mPAP), the weight ratio of right ventricle/(left ventricle+septum)[RV/(LV+S)] and vessel wall area/vessel total area (WA/TA) were evaluated. The levels of malondialdehyde (MDA) in the serum and lung tissues were detected by colorimetric method. The levels of 8-iso-prostaglandin F_2α (8-iso-PGF_2α) in the serum and lung tissues were measured by ELISA. The activity of superoxide dismutase (SOD) in the serum was analyzed by hydroxylamine method. The expression of NAPDH oxidase 4 (NOX4) and SOD1 in the lung tissues was determined by Western blot. RESULTS: Compared with N group, the levels of mPAP, RV/(LV+S) and WA/TA in H₄ group were significantly increased, which were apparently attenuated by salidroside injection in a dose-dependent manner. Meanwhile, salidroside administration apparently decreased the levels of MDA and 8-iso-PGF_2α in the serum and lung tissues, as well as the expression of NOX4 in the lung tissues. Besides, compared with N group, the activity of SOD in the serum and the expression of SOD1 in the lung tissues in H₄ group were significantly decreased, while administration of salidroside increased the activity of SOD in the serum and the expression of SOD1 in the lung tissues in a dose-dependent manner. CONCLUSION: Salidroside protects the pulmonary vessels from remodeling and attenuates hypoxia-induced pulmonary hypertension by inhibiting oxidative stress.     

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.     

Effects of retinal ganglion cell apoptosis on delayed rectifier K+ currents

AIM: To investigate the effects of rat retinal ganglion cell (RGC) apoptosis on delayed rectifier K⁺ currents (I_K). METHODS: The retinas of 2~3 d newborn Sprague-Dawley rats were dissociated into cell suspension by trypsin digestion. The RGCs were cultured and divided into control group, pressure 0.5 h group, pressure 1 h group, pressure 1.5 h group and pressure 2 h group. The cells were cultured regularly for 6 d in control group, and the cells in other groups were cultured regularly for 6 d and gave pressure of 80 mmHg for 0.5 h, 1 h, 1.5 h and 2 h, respectively. The rhodamine 123 fluorescence from labeled RGC mitochondrion was detected by continuous wavelength multifunctional microplate detection instrument. The membrane capacitance (C_m) in different groups and I_K in the pressure 1 h group were recorded from the RGCs by whole-cell patch-clamp technique. RESULTS: No difference of rhodamine 123 fluorescence in the RGC mitochondria between control group and pressure 0.5 h group was observed. Rhodamine 123 fluorescence in the other 3 groups was significantly lower than that in control group (P<0.05). No difference of the C_m between control group and pressure 0.5 h group was found, and the C_m in the other 3 groups was significantly lower than that in control group (P<0.05). The amplitudes of I_K were higher than that in control group. At the test potential from -10 mV to 60 mV, the current density in pressure 1 h group was significantly higher than that in control group (P<0.05). The maximal conduction (G_max) in pressure 1 h group was significantly higher than that in control group. The voltage for I_K channel half-activation (V_1/2) in pressure 1 h group declined comparison with control group (P<0.01), and the k value had no significant difference between the 2 groups. CONCLUSION: Retinal ganglion cell apoptosis accompanies with delayed rectifier K⁺ current enhancement.     

Dependence of adrenoceptor regulation on oxidative stress in cardiac injury induced by high sympathetic activity in rats

AIM：To investigate the dependence of the adrenoceptor regulation on oxidative stress in the rats with cardiac injury induced by high sympathetic activity. METHODS：Healthy SD rats were randomly divided into 7 groups: control, model, propranolol (Pro), prazosin (Praz), Pro+Praz, vitamin E (VE) and Pro+Praz+VE. The rats were intraperitoneally injected with norepinephrine (NE) for continuous 16 d to reproduce cardiac injury, and treated with the respective drugs. During the experimental process, the body weight was recorded. At the end of the experiments, the following parameters were measured: the ventricular remodeling indexes (cardiac index and hydroxyproline of the left ventricle), histopathologic examination, oxidative/antioxidative indexes ［MDA, SOD, catalase (CAT), GSH-Px and total antioxidant capacity (T-AOC)］, and energy metabolism (Na+-K+ ATPase and Ca2+-Mg2+ATPase). RESULTS：The increase of body weight in model group was significantly slower than that in control group after 9 d of treatment (P<0.05). The cardiac index and left ventricular hypertrophy were significantly increased. Oxidation/antioxidation and energy metabolism were disturbed. In Pro, Praz, Pro+Praz and VE groups, the body weight, cardiac index, left ventricular fibrosis and oxidative/antioxidative dysfunction were ameliorated. Pro, Praz and Pro+Praz increased the activity of Na+-K+ ATPase and Ca2+-Mg2+ATPase. Treatment with Pro+Praz showed the best result in all of the indexes (P<0.05). CONCLUSION：The dependence of adrenoceptor regulation plays an important role in the formation of oxidative stress in the process of rat cardiac injury induced by high sympathetic 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.     

Salvianolate attenuates oxidative stress injury of human endothelial EA.hy926 cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolate on oxidative damage induced by hydrogen peroxide in human endothelial EA.hy926 cells.METHODS: EA.hy926 cells were cultured in vitro and divided into the following groups:control group, damage group, and anti-damage groups (salvianolate+damage groups). The cell viability was measured by CCK-8 assay. The migration ability of the EA.hy926 cells was detected by Transwell assay. The content of nitric oxide (NO) in the culture supernatant of the EA.hy926 cells was examined. The levels of vascular endothelial growth factor (VEGF) were detected by ELISA. The apoptosis,mitochondrial membrane potential and intracellular superoxide anion content of the EA.hy926 cells were analyzed by flow cytometry. The protein levels of caspase-3, cleaved caspase-3, Bcl-2, Bax, NF-κB and p53 were determined by Western blot. RESULTS: Compared with damage group, the viability of EA.hy926 cells pretreated with salvianolate at different concentrations was significantly increased (P<0.05). The apoptotic rate was significantly decreased (P<0.05). Savianolate enhanced the migration ability of the cells. The levels of VEGF, NO and mitochondrial transmembrane potential were increased (P<0.05), and the intracellular ROS level was significantly decreased (P<0.05). The protein levels of NF-κB, p53, Bax and cleaved caspase-3 were significantly decreased, and the protein level of Bcl-2 was markedly increased(P<0.05). CONCLUSION: Savianolate reduces the damage of EA.hy926 cells by hydrogen peroxide exposure, and its mechanism may be related to the blocking of NF-κB signaling pathway.     

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

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

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.     

Effect of shikonin on high glucose-induced apoptosis and oxidative stress in vascular endothelial cells

AIM:To investigate the effect of shikonin on the apoptosis and oxidative stress induced by high concentration of glucose in vascular endothelial cells. METHODS:Rat thoracic aortic endothelial cells were randomly divided into 5 groups:normal control group (with glucose at concentration of 5.5 mmol/L in cell culture medium), high glucose group (with glucose at concentration of 33 mmol/L in cell culture medium), high glucose+low shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 0.1 μmol/L in cell culture medium), high glucose+medium shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 1 μmol/L in cell culture medium), and high glucose+high shikonin group (with glucose at concentration of 33 mmol/L and shikonin at concentration of 10 μmol/L in cell culture medium). After treatments, the cell viability was measured by CCK-8 assay and cell apoptotic rate was analyzed by flow cytometry. In addition, the status of oxidative stress was evaluated by determining the levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activation of Nrf2/HO-1 signaling pathway was determined by Western blot. RESULTS:Compared with high glucose group, shikonin reversed high glucose-induced decrease in cell viability and increase in apoptosis in a concentration-dependent manner. High concentration of glucose induced high levels of MDA and ROS, while decreased the levels of SOD and GSH-Px. However, after treatment with shikonin, the contents of MDA and ROS were decreased, while the activities of SOD and GSH-Px were increased as compared with high glucose group. Furthermore, the high concentration of glucose up-regulated the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear). Compared with high glucose group, the protein levels of cleaved caspase-3, HO-1 and Nrf2 (nuclear) were partly decreased after treatment with shikonin. CONCLUSION:Shikonin alleviates high glucose-induced vascular endothelial cell apoptosis. Its mechanism may be related to activation of Nrf2/HO-1 signaling pathway and down-regulation of oxidative stress in vascular endothelial cells.     

Effects of oxidative stress and inflammatory response on kidney injury in mice with hyperthyroidism

AIM To explore the effects of oxidative stress and inflammatory response on kidney injury induced by hyperthyroidism in mice. METHODS Forty male Kunming mice were randomly divided into control group （n=20） and L-thyroxine （T4） group （n=20）. The mice in T4 group were intraperitoneally injected with T4 diluent at a dose of 1 mg/kg to induce hyperthyroidism, and those in control group were injected with normal saline of the same volume. After 7 weeks, the mice were weighed and dissected, the kidneys were removed and weighed, and the length of tibia was also measured. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the kidney tissues were detected. The pathological changes of the kidney tissues were observed by HE staining. The levels of 4-hydroxynonenal （4-HNE）-modified proteins, interleukin-1 receptor-associated kinase 1 （IRAK1） and tumor necrosis factor receptor-related factor 6 （TRAF6） were determined by Western blot and immunohistochemistry. RESULTS Compared with control group, the body weight of the mice was decreased, while the kidney size and weight were increased significantly in T4 group. In addition, the ratios of kidney weight/body weight and kidney weight/tibia length were also increased （P<0.05）. In T4 group, the renal tubules were enlarged, and the epithelial cells of renal tubules were swollen and exfoliated, with vacuolar degeneration. Furthermore, reduced SOD activity, and increased MDA content and 4-HNE-modified proteins were found in T4 group, all of which were related to oxidative stress （P<0.05）. The levels of inflammation-related proteins IRAK1 and TRAF6 were significantly increased in T4 group （P<0.05）. CONCLUSION Excessive T4 may lead to kidney hypertrophy and injury in mice, and the mechanism may be related to oxidative stress and inflammatory response.     

Inhibitory effect of salidroside on liver oxidative stress in rats with non-alcoholic steatohepatitis

AIM：To explore the effects of salidroside (SDS) on the oxidative stress in liver tissues from rats with non-alcoholic steatohepatitis (NASH). METHODS：The experimental animal model of NASH was established in SD rats fed on high-fat and high-cholesterol diet (HFHCD) for 14 weeks. SDS (300 mg·kg-1·d-1) was administered via gavage daily from the 8th week after HFHCD feeding. At the end of the 14th week, serum samples were taken for detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC). Liver tissues were taken for TG, TC, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) detection. The content of 8-isoprostaglandin F2α (8-iso-PGF2α) in liver tissues was determined by ELISA. The liver histopathological changes were observed under microscope with HE staining. The expression of 8-hydroxydeoxyguanosine (8-OHdG) in liver tissues was determined by immunohistochemical staining. RESULTS：At the end of the 14th week, ALT, AST, TG and TC in serum, and TG, TC, MDA and 8-iso-PGF2α in liver homogenate in NASH model group were significantly increased compared with control group, while SOD and GSH in liver tissues were significantly decreased. The liver expression of 8-OHdG in NASH model group was higher than that in control group. Compared with NASH model group, SDS significantly inhibited the elevation of serum ALT, AST, TG and TC, and liver TG, TC, MDA and 8-iso-PGF2α, but increased the levels of SOD and GSH in liver tissues. Meanwhile, the liver histopathological score and 8-OHdG expression were decreased in SDS treatment group. CONCLUSION：Salidroside can effectively inhibit steatohepatitis induced by HFHCD, and its antioxidant effect may be one of the mechanisms.     

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.     

MSC-conditioned medium activates Nrf2/ARE pathway to protect H9c2 cells against oxidative stress

AIM: To investigate the protective effect of mesenchymal stem cell (MSC)-conditioned medium (MSCCM) on myocardial cell line H9c2 and its mechanism. METHODS: Verification of MSC was performed by flow cytometry analysis, followed by MTT assay to determine the optimal incubation time of MSCCM with myocardial cells. The cells were divided into 4 groups: normal (N) group, model (M) group, M+MSCCM group and MSCCM group. The cells in M+MSCCM group and MSCCM group were pre-incubated with MSCCM for 24 h. The cells in M group and M+MSCCM group were treated with 300 μmol/L H₂O₂ for 4 h to imitate oxidative injury of myocardial cells. Mitochondrial membrane potential and apoptotic rate of injured myocardial cells were detected by flow cytometry. The ROS production was measured by fluorescence microscopy. The nuclear translocation of Nrf2 and expression of HO-1 was examined by Western blot. RESULTS: No difference of mitochondrial membrane potential, apoptotic rate or ROS production between MSCCM group and N group was observed (P>0.05). The mitochondrial membrane potential depolarization, apoptotic rate and ROS production in M+MSCCM group were significantly lower than those in M group (P<0.01). The nuclear translocation of Nrf2 and expression of HO-1 in the myocardial cells were increased with MSCCM incubation time prolonged. CONCLUSION: MSCCM protects the myocardial cells against oxidative injury induced by H₂O₂. The anti-oxidative mechanism would be associated with the activation of Nrf2/ARE pathway.     

Docosahexaenoic acid protects human retinal pigment epithelial cells against oxidative stress-induced apoptosis

AIM: To observe the effect of docosahexaenoic acid(DHA) on H₂O₂-induced apoptosis in human retinal pigment epithelium cells and its molecular mechanism. METHODS: Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro, and 12.5 mmol/L H₂O₂ was used to mimic the oxidative stress condition. The cells were treated with 30~100μmol/L DHA for 4~24 h. The expression of heme oxygenase-1(HO-1) at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The enzymic activity of HO-1 was measured by colorimetry. Production of reactive oxygen species(ROS) was determined by fluorescent probe. Activation of NF-E2-related factor 2(Nrf2) was examined by immunofluorescence method. Apoptosis of ARPE-19 cells was analyzed by flow cytometry. RESULTS: The mRNA and protein expression and the enzymic activity of HO-1 were significantly increased in the ARPE-19 cells after DHA treatment. Meanwhile, nuclear translocation of Nrf2 was also observed. Apoptosis appeared and ROS was produced upon H₂O₂ incubation. In contrast, DHA at 100μmol/L significantly abrogated H₂O₂-induced apoptosis and ROS production. Furthermore, silencing of HO-1 by specific siRNA, or treatment with ZnPP, an inhibitor of HO-1, partly counteracted the protective effect against H₂O₂-induced apoptosis and ROS production. CONCLUSION: DHA protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase-1 expression after Nrf2 activation.     

Hypoxic preconditioning increases tolerant ability of old human bone marrow mesenchymal stem cells to oxidative stress injury through AKT pathway

AIM: To investigate the protective effect of hypoxic preconditioning on human bone marrow mesenchymal stem cells (hBM-MSCs), and to provide basic experimental support for more effective autologous stem cell transplantation in aged patients. METHODS: The old hBM-MSCs were subjected to hypoxic preconditioning using a hypoxia incubator chamber for 24 h. The cells were divided into young group, old group and old+hypoxia group (with 24 h hypoxic preconditioning). Hydrogen peroxide (H₂O₂, 300 μmol/L) was applied to simulate the oxidative stress. The cells were treated with 50 μmol/L LY294002 for 2 h to inhibit PI3K/AKT pathway. BrdU incorporation and CCK-8 assay were used for analyzing the cell proliferation and viability. The protein levels of Bax, Bcl-2 and p-AKT were measured by Western blot. RESULTS: BrdU-positive cells, which represented the cell proliferation, and the cell viability were significantly increased in old+hypoxia group compared with old group (P<0.05). The protein level of Bax decreased (P<0.05) and Bcl-2 increased (P<0.05) in old+hypoxia group compared with old group after using 300 μmol/L H₂O₂ simulate. the oxidative stress. The phosphorylation of AKT was enhanced by hypoxic preconditioning in old group (P<0.05). The protective effect of hypoxic preconditioning on the cell survival was decreased after treated with LY294002 (inhibitor of the PI3K/AKT pathway) (P<0.05). CONCLUSION: Hypoxic preconditioning increases the survival and proliferation of old hBM-MSCs by activation of AKT pathway.     

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.