Endogenous hydrogen sulfide results in increase of reactive oxygen species induced by angiotensin II in neurons

AIM：To determine the effect of endogenous hydrogen sulfide (H2S) on the production of reactive oxygen species (ROS) in medullary neurons induced by angiotensin II (Ang II). METHODS：Primary cultured rat medullary neurons were used in the study. Identification of medullary neurons and the co-expression of cystathionine β-synthetase (CBS) were detected by double-labeling immunofluorescence. Medullary neurons were treated with Ang II in the presence or absence of sodium butyrate (NaBu, a CBS agonist; 100 μmol/L, 250 μmol/L and 500 μmol/L). ROS production was measured by dihydroethidium staining. The activity of total superoxide dismutase (SOD) was detected by ELISA. The mRNA expression of CBS was determined by real-time PCR. RESULTS：The medullary neurons in the cultured cells were over 90%. Ang II (1 μmol/L) significantly increased ROS level in the medullary neurons. Ang II inhibited the activity of total SOD in the medullary neurons. CBS was expressed in the medullary neurons. Ang II decreased the mRNA expression of CBS. NaBu (250 μmol/L and 500 μmol/L) inhibited ROS production induced by Ang II with a dose-dependent manner, while NaBu alone had no influence on the ROS level in the medullary neurons. CONCLUSION：Ang II increases the level of ROS in medullary neurons partly by inhibiting the activity of total SOD and the mRNA expression of CBS. Endogenous H2S inhibits the ROS level increased by Ang II in the medullary neurons.     

Mitochondrial reactive oxygen species and atrial fibrillation

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. Mitochondrial oxidative stress is supposed to contribute to development, progression and self-perpetuation of AF. Reactive oxygen species (ROS) is the major molecule mediating mitochondrial oxidative stress damage. ROS can alter the redox status of various molecular targets, which quite specifically leads to functional alterations of ion channel activity or activation of a variety of redox sensitive signal transduction pathways. Eventually, it leads to atrial electrical remodeling and promotes the development of AF. Therefore, mitochondrial oxidative stress pathways may be a new target for the therapy of atrial fibrillation.     

The effects of calcium and reactive oxygen species in rat kidney during ischemia and reperfusion period

AIM and METHODS: Electron cytochemical methods were used to study the changes of calcium and reactive oxygen species in rat kidney during ischemia and reperfusion period.RESULTS:By the end of 1h ischemia, intra-cellular calcium increased. There were no H₂O₂ generation at this time. In the early reperfusion period, large amount of H₂O₂ generated. At this time, there were no evident changes of intra-cellular calcium compare with 1h ischemia group. In the later reperfusion period, less H₂O₂ generated. Intra-cellular calcium increased continuously.CONCLUSION: Calcium and reactive oxygen species all participated in ischemia-reperfusion injury, but the time they participated and their effects were different.     

PPARγ agonist inhibits high glucose-induced production of reactive oxygen species by UCP2 up-regulation

AIM: To explore the effects of PPARγ on the elevated level of reactive oxygen species (ROS) induced by high glucose and its mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured with DMEM containing high glucose (33 mmol/L D-glucose), and DMEM containing lower glucose (5.5 mmol/L D-glucose) was used as control. Superoxide anion and nitric oxide fluorescence probes were used to observe the effects of PPARγ agonist on ROS and NO productions in the HUVECs. The uncoupling protein 2 (UCP2) protein level in the HUVECs was detected by Western blotting. RESULTS: PPARγ agonist pioglitazone inhibited the ROS generation and prevented the decrease in NO level under high glucose condition, and these effects were reversed by pretreatment with PPARγ antagonist GW9662. The results of Western blotting indicated that PPARγ agonist pioglitazone up-regulated the UCP2 expression under high glucose condition, and this effect was also blocked by GW9662. Inhibition of UCP2 by genipin attenuated the effect of pioglotazone on the ROS production. CONCLUSION: Activation of PPARγ inhibits ROS generation under high glucose condition, and this effect may mediate by up-regulation of UCP2.     

活性氧调控下草莓果实衰老过程中活性氧与超微弱发光的关系

【目的】探讨草莓果实衰老过程中超微弱发光(ultraweak luminescence,UWL)与活性氧的关系。【方法】以‘红颜’草莓果实为试材,采用H_2O_2处理和茶多酚处理草莓果实,对比分析促进和清除活性氧条件下草莓果实衰老过程中UWL和活性氧的变化及2者之间的关系。【结果】在草莓果实衰老过程中,H_2O_2处理、茶多酚处理和对照的脂氧合酶(LOX)活性和过氧化氢(H_2O_2)含量均先上升后下降,整体上升;超氧阴离子(O_2~)产生速率、丙二醛(MDA)含量和相对电导率均持续上升;H_2O_2处理和对照的UWL均下降、茶多酚处理的UWL上升。在整个果实衰老过程中,H_2O_2处理的O_2~产生速率、MDA含量和相对电导率均高于对照,茶多酚处理后3者均低于对照;H_2O_2处理的UWL强度均低于对照,茶多酚处理的UWL强度均高于对照。【结论】在草莓果实衰老过程中,UWL强度随着活性氧水平的积累而下降;促进活性氧情况下加剧了草莓果实活性氧上升积累,同时加剧了UWL强度下降;清除活性氧则缓减了活性氧上升积累,同时缓减了UWL强度下降。以上结果暗示活性氧并不是引发UWL的直接来源,而应是通过影响其他细胞结构或物质代谢从而间接影响UWL。     

Roles of reactive oxygen species in pluripotent stem cell functions

Pluripotent stem cells are characterized by the properties of self-renewal and the ability to differentiate into multiple cell types. Reactive oxygen species (ROS) are highly reactive metabolites. High levels of ROS are toxic and involved in stem cell senescence and apoptosis. However, regulation of ROS has an important role in maintaining “stemness” and differentiation of the stem cells. The role of ROS in the stem cells varies among different stem cell types. NADPH oxidase is one of the major sources of ROS in stem cells. Excessive amounts of ROS are produced in various pathophysiological states such as atherosclerosis, heart failure, hypertension, diabetes, and aging. Induced pluripotent stem cells have the potential to be used in modeling of ROS-associated diseases.Understanding the molecular mechanisms how ROS regulate the functions of stem cells will greatly enhance their translational applications. In this review, we summarize the recent progress regarding the roles of ROS in regulating the functions of embryonic and induced pluripotent stem cells.     

Effect of probucol and simvastatin on reactive oxygen species production and HO-1 expression induced by advanced glycation end products in rat renal microvascular endothelial cells

AIM: To investigate the effect of probucol and simvastatin on the production of reactive oxygen species (ROS) and heme oxygenase-1 (HO-1) expression induced by advanced glycation end products (AGEs) in rat renal microvascular endothelial cells (RMECs). METHODS: RMECs isolated and cultured from rat kidney were divided into 4 groups: normal control group, AGEs group, probucol group and simvastatin group. The levels of ROS were determined by the molecular probes of DCFH-DA. The expression of HO-1 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. RESULTS: (1) AGEs up-regulated ROS production and HO-1 expression in RMECs. (2) Probucol up-regulated HO-1 expression in RMECs, and inhibited the increasing level of ROS and expression of HO-1 in RMECs induced by AGEs. (3) Simvastatin also inhibited the increasing level of ROS in RMECs induced by AGEs, but it had no effect on HO-1 expression in RMECs with or without AGEs.CONCLUSION: Protective effect of probucol on the dysfunction of RMECs induced by AGEs may be related with its effect on the expression of HO-1 at mRNA and protein levels. Simvastatin also plays roles in antioxidation and renal protection, but is ineffective in the modulation of HO-1 expression.     

Effects of 2-chloroadenosine and IPP on intracellular reactive oxygen species

AIM:To study the effect of selective A1 adenosine receptor agonist, 2-chloroadenosine (2-CA), and non-peptide phosphoantigen isopentenyl pyrophosphate (IPP) on the intracellular reactive oxygen species (ROS) at the level of signaling event. METHODS:M1 cell line, derived from a subclone of the SV40-transformed xeroderma pigmentosum (XP) cell line, was treated with 2-CA, 8-br-cAMP, IPP at 37 ℃ incubator for 30 min, and intracellular ROS was measured by flow cytometry and CytoFluorometer. RESULTS:Under an appropriate control, 2-CA and IPP reduced intracellular ROS by 20%-30%. CONCLUSION:The data revealed that 2-CA and IPP inhibited cellular oxidant formation at the level of signaling event, suggesting both may have some anti-oxidant properties in vivo.     

Hydrogen sulfide suppresses the increase in reactive oxygen species in neurons induced by angiotensin II via ERK1/2 signal pathway

AIM: To investigate the effect of hydrogen sulfide (H₂S) on the reactive oxygen species (ROS) level in medullary neurons induced by angiotensin II (Ang II). METHODS: Primarily cultured rat medullary neurons were divided into 5 groups as follows: control group, Ang II group, sodium hydrosulfide(NaHS) group, NaHS with Ang II group, and PD98059 (an inhibitor of p-ERK1/2) with Ang II group. ROS production was measured with dihydroethidium (DHE) staining. The expression of p-ERK1/2 and ERK1/2 was determined by Western blotting. The activity of neurons was detected by CCK-8 assay. RESULTS: Ang II at concentration of 100 nmol/L significantly increased ROS level in the neurons, but the effect was inhibited by NaHS at concentrations of 50~200 μmol/L, while NaHS alone had no influence on the ROS level in neurons. Additionally, PD98059 also depressed the ROS level in neurons induced by Ang II. Furthermore, the enhanced expression of p-ERK1/2 in the neurons induced by Ang II was significantly reduced by NaHS. CONCLUSION: H₂S remarkably inhibits the ROS level in the neurons induced by Ang II via activation of MAPK signal pathways, especially p-ERK1/2, indicating that H₂S rescues neurons from oxidative stress through declining the enhanced expression of p-ERK1/2.     

Role of reactive oxygen species in 23-hydroxybetulinic acid-induced apoptosis in LoVo cells

AIM: To investigate the changes of reactive oxygen species (ROS) in apoptosis of LoVo cells induced by 23-hydroxybetulinic acid.METHODS: LoVo cells were treated with 23-hydroxybetulinic acid. The apoptotic morphological change was observed under the light microscope. Intracellular ROS production and the rate of apoptosis were detected by flow cytometry.RESULTS: LoVo cells improved apoptotic morphological changes treated with 23-hydroxybetulinic acid for 48 h. At concentrations of 25, 50, 100, 200 μmol/L of 23-hydroxybetulinic acid, the apoptotic rates of LoVo cells were (7.17±2.31)%, (15.60±4.02)%, (32.47±5.25)% and (52.71±5.93)%, respectively. The results indicated a certain concentration-dependent relationship. 23-hydroxybetulinic acid caused an increase in the ROS production, and the ROS levels were 2.83±0.80, 5.97±1.72, 12.53±2.57 and 16.73±4.58. Compared with the control group (2.13±0.32), the increase in ROS production in LoVo cells at the concentration of 100, 200 μmol/L of 23-hydroxybetulinic acid treatment was significant (P<0.05). CONCLUSION: 23-hydroxybetulinic acid induces LoVo cell apoptosis. The production of ROS may play a crucial role in the process of the LoVo cell apoptosis.     

Effects of EDRV and DIDS on reactive oxygen species levels in acute ischemia-reperfusion injured myocardium

AIM: To investigate the effects of chloride channel inhibitor 4,4- diisothiocyanostilbene-2,2- disulfonic acid (DIDS) and free radical scavenger edaravone(EDRV) on the production of reactive oxygen species in acute ischemia-reperfusion injured (I/RI) myocardium. METHODS: Male Sprague-Dawley rats, subjected to myocardial ischemia for 30 min and reperfusifor for 4 h, were divided into 5 groups: sham group, I/RI group, DIDS group, EDRV group and DIDS+EDRV group. The rats were treated with EDRV (10 mg/kg for 5 min) before reperfusion or/and DIDS (14 mg/kg,4 mL·kg^-1·h^-1 for 2 h) at the beginning of reperfusion by a program-controlled injection micropump . The myocardiac tissues were collected immediately at the end of reperfusion. The levels of ROS, OH· and O₂^- were determined by the methods of spectrofluorophotometry and colorimetry. Myocardial apoptosis was detected by TUNEL method. Serum superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentration were measured by colorimetry. RESULTS: Compared with I/RI group, myocardial apoptotic index, the levels of ROS, O₂^-, OH· and MDA were significantly reduced, and SOD activity was significantly increased in DIDS group, EDRV group and DIDS+EDRV group (P<0.05). Compared with DIDS group, the levels of ROS,O₂^-, OH· and MDA were significantly decreased, and SOD activity was significantly increased in EDRV group (P<0.05).No statistical difference of myocardial apoptotic index between these two groups was observed (P>0.05). Compared with DIDS+ EDRV group, myocardial apoptotic index in DIDS group and EDRV group was significantly increased (P<0.05), and no significant difference of ROS, O₂^-, OH·, MDA and SOD between the two groups was found (P>0.05).CONCLUSION: DIDS and EDRV protect myocardial cells from apoptosis by inhibition of ROS activity. Combinative use of the two reagents has stronger cardioprotectiue effect, suggesting that they have different regulatory pathways.     

Increased central reactive oxygen species mediate the attenuated baroreflex function in heart failure

AIM: To determine the effect of reactive oxygen species on the baroreflex and to investigate the intracellular mechanism responsible for baroreflex dysfunction in the heart failure state.METHODS: In the rat model of cardiomyocytes infarct induced heart failure, baroreflex function was evaluated by measuring the relationship between renal sympathetic nerve activity(RSNA)responses and change of blood pressure by intravenous injection of nitroglycerin and phenylephrine. Alteration in baroreflex function was measured under the different reactive oxygen species(ROS)level induced by intracerebroventricular administration of several chemicals. RESULTS: (1)The range of RSNA response, average slope and maximum gain of baroreflex function curve were(92.2±9.9) mmHg,(0.07%±0.01%)/mmHg and(1.20%±0.10%)/mmHg, respectively, in CHF rats, which were significantly lower than those in sham rats(65.6±7.4) mmHg,(0.13%±0.02%)/mmHg and(3.00%± 0.20%)/mmHg(P<0.01).The minimum RSNA of baroreflex curve was higher in CHF rats than that in sham rat［(21.6%±4.8%)vs(7.5%±2.1%), P<0.01］.(2)Intracerebroventricular(icv)infusion of superoxide scavenger tempol and NADPH oxidase inhibitor apocynin significantly improved the blunted baroreflex in CHF rats. On contrast, icv administration of superoxide dismutase inhibitor diethyldithiocarbamate(DETC)decreased baroreflex function in sham rats.(3)The superoxide production in the hypothalamus of CHF rats was higher than that in sham rats［(73.9±9.8)RLU·5min-1·mg-1vs(40.6±7.1)RLU·5min-1·mg-1, P<0.01］.(4)Protein expression of NADPH oxidase subunits gp91phox in the paraventricular nucleus of the hypothalamus were increased by 1.3 fold in CHF rats than that in sham rats. CONCLUSION: Elevated intracellular ROS in the hypothalamus plays an important role in the attenuation of baroreflex function in the heart failure state and results from upregulation of NADPH oxidase protein expression.     

Role of reactive oxygen species and ERK1/2 signaling pathway in proliferation and apoptosis of hypoxic rat pulmonary arterial smooth muscle cells

AIM:To investigate the change of reactive oxygen species (ROS) production in hypoxic pulmonary arterial smooth muscle cells (PASMCs) of rats, the effect of ROS on the expression of extracellular signal-regulated kinase (ERK)1/2 protein, and the role of ROS and ERK1/2 in the imbalance between proliferation and apoptosis of PASMCs.METHODS: Primary cultures of PASMCs were established and cells between passages 2 to 3 were used for experiments. PASMCs were treated with tiron, a membrane permeable ROS scavenger, and PD98059, an ERK1/2 inhibitor, under normoxia or hypoxia condition. The ROS production was measured by DCFH-DA and NBT reduction. The expression of phosphorylated-ERK1/2 (p-ERK1/2) protein was detected by immunofluorescence. Cell proliferation was examined by MTT colorimetric assay and the expression of PCNA. Cell apoptosis was detected by TUNEL.RESULTS: (1)Compared with control group, the ROS levels in hypoxia group were significantly increased (P<0.01). (2) In hypoxia group, the proliferative capacity was higher and the apoptosis index was lower than those in control group (P<0.01). Tiron significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). (3) The expression of p-ERK1/2 in hypoxia group were higher than that in control group (P<0.01), which were significantly suppressed by tiron (P<0.01)．(4) PD98059 significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). The proliferative capacity and apoptosis index was similar in hypoxia+tiron+PD98059 group to those in hypoxia+tiron group (P>0.05).CONCLUSION:The hypoxia-mediated increase in PASMCs proliferation and the decrease in PASMCs apoptosis are related to the overproduction of intracellular ROS through downstream activation of ERK1/2. ROS and ERK1/2 play important roles in the hypoxic remodeling of pulmonary artery.     

Regulatory effects of reactive oxygen species on the production of PAI-1 in 3T3-L1 adipocytes and its related possible mechanisms

AIM: To investigate the regulatory effects of reactive oxygen species (ROS) on the production of plasminogen activator inhibitor 1 (PAI-1), and try to determine the signaling cascades involved in it. METHODS: 3T3-L1 cells were cultured and differentiated into mature adipocytes. Cell viability was measured by MTT. The PAI-1 mRNA expression levels were evaluated by quantitative real-time PCR. Quantification of the PAI-1 protein levels secreted into conditioned medium was performed by multiplex immunoassay and sandwich ELISA. The phosphorylation status of protein kinases was determined by Bio-Plex phosphoprotein assays. RESULTS: In 3T3-L1 adipocytes, H2O2 significantly augmented the expression of PAI-1. Also, H2O2 activated several signaling pathways including ERK1/2, JNK, Akt, p70 S6K and JAK/STAT. Verified by protein kinase inhibitors, Akt, JAK/STAT and ERK1/2 may participate in the H2O2-induced increase in PAI-1. CONCLUSION: H2O2 markedly up-regulates the production of PAI-1 in 3T3-L1 adipocytes via some intracellular signaling pathways such as Akt, JAK/STAT and ERK1/2.     

一氧化氮对猕猴桃果实营养品质和活性氧代谢的影响

分别用10、20和30μL.L-1一氧化氮(NO)气体熏蒸猕猴桃果实,研究NO对采后徐香猕猴桃(Actinidia chi-nensis Planch.cv.Xuxiang)的营养品质变化和活性氧代谢的影响。结果表明,20μL.L-1 NO处理后的猕猴桃果实含有较低的可溶性固形物和较高的可滴定酸、维生素C,且果实中丙二醛和超氧自由基质量分数低于其他体积分数的NO熏蒸处理(10和30μL.L-1 NO)。20μL.L-1 NO处理降低了猕猴桃果实中LOX活性,延缓了猕猴桃果实采后期间CAT活性的降低,显著提高了猕猴桃果实SOD和POD活性,且显著降低了猕猴桃果实中过氧化氢的质量分数。20μL.L-1 NO气体熏蒸保持了猕猴桃中较高的叶绿素、类胡萝卜素以及维生素E的质量分数。     

Effects of platelet-activating factor on cultured neurons and astrocytes

AIM: To observe the effect of platelet-activating factor (PAF) on cultured neuronal viability and glial fibrillary acidic protein (GFAP) expression in cultured astrocytes. METHODS: Neurons and astrocytes obtained from the brain cortex of the embryo and newborn mice respectively were cultured and purified, and they were divided into the control and experimental groups. PAF was added into the experimental groups at concentrations of 4, 8 and 16 μmol/L. Each group was cultured for 4 h, 24 h and 72 h, respectively. MTT method and immunohistochemistry were used to observe the neuronal viability and GFAP expression in astrocytes, respectively. RESULTS: During different time after adding PAF at different concentrations into cultured neurons and astrocytes, respectively, neuronal viability declined, and the number of astrocytes decreased, but GFAP expression in survival astrocytes increased. The effects were shown to be in a concentration-dependent manner. CONCLUSION: PAF decreases the neuronal viability directly and influences the neuronal survival indirectly by astrocytes.     

Response of mitochondrial reactive oxygen species in pulmonary arterial smooth muscle cells under acute hypoxic condition

AIM:To observe the response of mitochondrial reactive oxygen species (ROS) in rat pulmonary artery smooth muscle cells (PASMCs) under acute hypoxic condition. METHODS:The cultured PASMCs were under normoxic (35 ℃, 5% CO2, 21% O2, 74% N2) or acute hypoxic (35℃, 5% CO2, 1% O2, 94% N2) condition. The cells were incubated with molecular probes chloromethyl dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) and RedoxSensor Red CC-1 to detect the ROS generation by laser scanning confocal microscopy. The mitochondria were isolated and mitochondrial inhibitors were used to detect the ROS generation functional unit sites by spectrophotometry under acute hypoxic condition. RESULTS:Under acute hypoxic condition, the intracellular ROS was significantly increased in hypoxia group with 3.35 folds higher of H2O2 than that in normoxia group. The contents of H2O2 and O-·2 in hypoxia group were 1.61 folds higher than those in normoxia group. Compare with hypoxia goup, pretreatment with the mitochondrial electron transport chain (ETC) complex I inhibitor MPP, the complex II inhibitors NPA and TTFA as well as the complex III pre-ubisemiquinone site inhibitor myxothiazol all remarkably reduced hypoxia-induced increase in ROS generation in PASMCs (reduced by 60%, 73%, 75% and 61%, respectively, P<0.01), whereas the complex III postubisemiquinone site inhibitor antimycin A and the complex IV inhibitor NaN3 had no effect on hypoxia-induced increase in ROS generation (increased by 13% and 9.1%, respectively, P>0.05). Direct detection of mitochondrial ROS showed the same results as the intracellular ROS. CONCLUSION: The intracellular ROS increases significantly in rat PASMCs under acute hypoxic condition. The mitochondrial ETC complex I, complex II and complex III pre-ubisemiquinone sites increase ROS generation, whereas the complex III postubisemiquinone site and complex IV do not produce this effect under acute hypoxic condition.     

Production and cytotoxicity of the reactive oxygen species induced by diallyl trisulfide in human myeloid leukemia HL-60 cells

AIM: To explore the production and cytotoxicity of the reactive oxygen species (ROS) induced by diallyl trisulfide (DATS) in HL-60 cells.METHODS: HL-60 cells were either treated with various doses of DATS alone, or DATS combination with apocynin, a specific NADPH oxidase inhibitor, or with antioxidant N-acetyl-L-cysteine (NAC) for 0, 1, 3, 6, 12 and 24 h, respectively. The intracellular ROS level was measured by flow cytometry. The activity of NADPH oxidase was evaluated by NBT reduction experiment. The content of both malondialdehyde (MDA) and the protein carbonyl were analyzed by spectrophotometer. RESULTS: The results from flow cytometry indicated that DATS significantly increased the intracellular ROS level in HL-60 cells (P<0.05), which was dose-and time-dependent. The fluorescence intensities of ROS reached at maximum when HL-60 cells were incubated with 150 μmol/L DATS for 3 h. The NBT reduction experiment showed that DATS activated NADPH oxidase, the highest activity was observed when the cells were exposed to 150 μmol/L DATS for 3 h. DATS induced MDA and protein carbonyl production in HL-60 cells. Furthermore, both MDA and protein carbonyl reached the highest level in the cells exposed to 150 μmol/L DATS for 3 h. Apocynin and NAC attenuated the production of MDA and protein carbonyl, suggesting that ROS induced by DATS was involved in the toxicity to the cells. CONCLUSION: DATS induces ROS production through activating NADPH oxidase in HL-60 cells. ROS increases the oxidation of membrane lipid and protein in HL-60 cells.