Role of mitochondrial permeability transition pore in TNFα-induced cardioprotection in isolated rat hearts subjected to hypoxia and reoxygenation

AIM: To investigate whether tumor necrosis factor α (TNFα) pretreatment can inhibit mitochondrial permeability transition pore opening in isolated rat hearts subjected to hypoxia and reoxygenation. METHODS: Isolated perfused rat hearts were subjected to 30 min regional hypoxia (occlusion of left anterior descending artery) and 120 min reoxygenation. The infarct size, lactate dehydrogenase (LDH) release during reoxygenation and ventricular hemodynamic parameters were measured. RESULTS: Pretreatment with TNFα at concentration of 1×104 U/L for 7 min followed by 10 min washout reduced the infarct size and LDH release, and improved the left ventricular performance (left ventricular developed pressure and rate-pressure product) and left ventricular end-diastolic pressure during hypoxia and reoxygenation. Administration of atractyloside (Atr, an opener of mitochondrial permeability transition pore, 20 μmol/L) for 20 min (last 5 min of hypoxia and first 15 min of reoxygenation) and paxilline (Pax, a calcium activated potassium channel antagonist, 1 μmol/L) for 5 min before hypoxia attenuated the reduction of infarct size and LDH release and improved the left ventricular performance induced by TNFα. CONCLUSION: The findings indicate that in the isolated rat heart model, TNFα protects myocardium against hypoxia and reoxygenation injury via inhibiting mitochondrial permeability transition pore opening as well as activating calcium, activated potassium channel.     

Effects of diazoxide preconditioning on mitochondrial respiratory function and enzyme activity in rats

AIM: To investigate the effects of the selective mitochondrial ATP-sensitive K+ channels opener diazoxide on mitochondrial respiratory function and enzyme activity in isolated rat myocardium under ischemia/reperfusion.METHODS: Observation was made on rat hearts perfused with Langendorff apparatus.72 Sprague-Dawley (SD) rats were randomly divided into 4 groups: normal group (NOR),ischemia reperfusion (IR),diazoxide group (DIA) and 5-hydroxydecanoate (5-HD) antagonized diazoxide group (5HD-DIA).Hearts isolated from SD rats were mounted on a Langendorff apparatus and started with a 20 min perfusion for equilibration.NOR went on perfusion for another 100 min after equilibration.IR underwent 40 min global ischemia and followed by 30 min reperfusion after 30 min stabilization.DIA was administered with K-H solution containing diazoxide at concentration of 50 μmol/L for 10 min before ischemia and reperfusion.5HD-DIA was infused with 100 μmol/L 5-HD (a specific mitochondrial ATP sensitive K+ channel blocker) and the same procedure was carried out as DIA group.Hearts were taken down to extract mitochondrial at the end-equation,before ischemia and at the end-reperfusion for determination of mitochondrial respiratory function and the enzyme activity of mitochondria.RESULTS: At the end of reperfusion,mitochondrial respiratory function (mitochondrial respiratory control rate,P/O ratio and state 3 respiration) and mitochondrial enzyme activity (NADH oxidase,succinate oxidase and cytochrome C oxidase) in DIA group were better than those in IR group and 5HD-DIA group (P<0.05),but worse than those NOR group (P<0.01).No significant difference in all parameters was observed between IR and 5HD-DIA (P>0.05).CONCLUSION: Preconditioning with mitochondrial ATP sensitive potassium channel opener,diazoxide,protects rat heart mitochondria against ischemia-reperfusion injury.The mechanisms are involved in the safeguarding of respiratory function and activity of enzymes of respiratory chain.     

Inhibition of hypothermic preservation-induced Smac/DIABLO protein expression by diazoxide in donor rat myocardium

AIM: To investigate the effect of a mitochondrial ATP-sensitive potassium channel (mitoKATP) opener diazoxide (DE) on Smac/DIABLO protein expressions in rat heart suffered from different duration of hypothermic preservation. METHODS: The Langendorff model of isolated rat heart was used. After stored in 4 ℃ Celsior solution with or without DE (30 μmol/L) for different time (0, 3, 6, 9 or 12 h). Cell apoptosis was detected by TUNEL technique. The expression of Smac/DIABLO protein in cytoplasm and total caspase-3 protein in myocardia tissue was also analyzed by Western blotting. RESULTS: (1) Compared to the hypothermic preservation groups, DE reduced the percentage of apoptotic cells and the expression of caspase-3 protein in myocardia tissue. (2) The peak of Smac/DIABLO protein expression level appeared at 6 h after hypothermic preservation, and which was postponed to 9 h by DE. (3) The above effects of DE were attenuated by a mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD). CONCLUSION: The findings indicate that in the isolated rat heart, DE protects myocardium against different duration of hypothermic preservation injury via opening of mitoKATP channel and inhibition of Smac/DIABLO protein expression.     

Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced injury in H9c2 cardiac cells

AIM: To investigate the roles of ATP-sensitive potassium (K_ATP) channels in high glucose-induced cardiac injury and the inhibitory effect of hydrogen sulfide (H₂S) on the cardiomyocyte injury. METHODS: The expression level of K_ATP channel protein was tested by Western blot. The cell viability was measured by CCK-8 assay. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining. Mitochondrial membrane potential (MMP) was examined by JC-1 staining. RESULTS: After the H9c2 cells were treated with 35 mmol/L glucose (high glucose, HG) for 1~24 h, the protein level of K_ATP channel was significantly reduced at 6 h, 9 h, 12 h and 24 h, reaching the minimum level at 12 h and 24 h. Pretreatment of the cells with 400 μmol/L NaHS (a donor of H₂S) prior to exposure to HG for 12 h considerably blocked the down-regulation of K_ATP channels induced by HG. Pretreatment of the cells with 100 μmol/L mitochondrial K_ATP channel opener diazoxide, 50 μmol/L non-selective K_ATP channel opener pinacidil or NaHS obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the number of apoptotic cells and the MMP loss. Pretreatment with 100 μmol/L mitochondrial K_ATP channel antagonist 5-hydroxydecanoic acid or 1 mmol/L non-selective K_ATP channel antagonist glibenclamide attenuated the above cardioprotective effects of NaHS. CONCLUSION: K_ATP channels mediate the inhibitory effect of H₂S on HG-induced cardiac injury.     

Selenium dioxide induces leukemia cell apoptosis and changes of intracellular reactive oxygen species (ROS) and Ca2+ levels

AIM: The effects of selenium dioxide (SeO2) on proliferation, apoptosis, intracellular reactive oxygen species (ROS) and Ca2+ levels in three leukemia cell lines NB4, K562 and HL-60 were investigated. METHODS: Three leukemia cell lines were treated with 3-30 μmol/L SeO2. Flow cytometry was used to detect apoptosis rate, and analyze the changes of ROS and Ca2+ level within cells. RESULTS: SeO2 at 10 and 30 μmol/L inhibited proliferation in three leukemia cell lines. Treatment with 30 μmol/L SeO2 for 48 h induced 54.0%, 46.5%, 49.6% apoptosis in NB4, K562, and HL-60 cells, respectively, and also markedly decreased ROS and Ca2+ levels among three cell lines. The rate of ROS positive cells in NB4 and HL-60 decreased with the increase in SeO2 concentrations. ROS was clearly reduced with 30 μmol/L SeO2 in K562. Ca2+ levels were tardily declined with 10, 30 μmol/L SeO2 in NB4 and HL-60 cells. Ca2+ levels were clearly reduced with 30 μmol/L SeO2 in K562. CONCLUSION: SeO2 induces apoptosis in three leukemia cells. The declines of intracellular ROS and Ca2+ levels are involved in apoptosis induced by SeO2.     

Electrophysiological effects of amiodarone on pacemaker cells in guinea-pig left ventricular outflow tract under conditions of hypoxia,acidosis and treatment with epinephrine

AIM: To study the electrophysiological effects of amiodarone on the pacemaker cells in guinea-pig left ventricular outflow tract under the conditions of hypoxia, acidosis and treatment with epinephrine.METHODS: The action potentials of the pacemaker cells in guinea-pig left ventricular outflow tract were recorded by conventional intracellular microelectrode technique. The effects of amiodarone on the spontaneous slow response potentials were investigated under the conditions of hypoxia, acidosis and treatment with epinephrine.RESULTS: (1) Amiodarone at concentration of 0.1 μmol/L markedly decreased the rate of pacemaker firing (RPF) and maximal diastolic potential (MDP), lengthened 80% of the duration of action potential (APD80). Amiodarone at concentration of 1 μmol/L significantly decreased the velocity of diastolic depolarization (VDD) and RPF, the maximal rate of depolarization (Vmax), MDP and amplitude of action potential (APA), lengthened 50% of the duration of action potential (APD50) and APD80. Amiodarone at concentration of 10 μmol/L led to a significant decrease in VDD and RPF, Vmax, MDP and APA, a notable lengthening in APD50 and APD80 was also observed. (2) Under the condition of hypoxia and perfusion with deprived glucose content for 15 min, VDD, RPF, MDP, Vmax and APA decreased significantly, APD50 was shortened notably. Under the condition of hypoxia, amiodarone at concentration of 1 μmol/L significantly decreased VDD, RPF and Vmax, increased MDP, lengthened APD50 and APD80 as compared to the cells treated with hypoxia only. (3) Perfusion with pH 6.8 solution for 10 min, VDD and RPF significantly decreased, Vmax and APA notably reduced, APD80 was markedly shortened. Under the condition of acidosis for 10 min, amiodarone significantly decreased VDD, RPF, MDP and APA, lengthened APD50 and APD80 as compared to the cells under the condition of acidosis only. (4) Perfusion of epinephrine at concentration of 10 μmol/L for 10 min resulted in a significant increase in VDD, RPF, Vmax, MDP and APA, a notable shorting in APD50 and APD80 was also observed. Compared to 10 μmol/L epinephrine group, 1 μmol/L amiodarone+10 μmol/L epinephrine significantly reduced VDD, RPF, Vmax, MDP and APA, lengthened APD50 and APD80.CONCLUSION: Amiodarone markedly decreases the autorhythmicity of the pacemaker cells in guinea-pig left ventricular outflow tract. This electrophysiological effects were significantly influenced by hypoxia, acidosis and epinephrine.     

Effects of hypoxia on endothelial nitric oxide synthase expression in cerebral artery endothelial cells

AIM: To investigate the molecular mechanism by which hypoxia affect the endothelial nitric oxide synthase (eNOS) expression in cerebral artery endothelial cells (CAECs). METHODS: Primary cultured porcine CAECs were exposed to hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h. The eNOS mRNA level was determined by RT-PCR. The level of eNOS protein was detected by Western blotting. After specific PKC inhibitors BIM Ⅰ(1 μmol/L) and G6983 (1 μmol/L) were added, CAECs were exposed to hypoxia for 24 h. The effect of hypoxia on eNOS mRNA stability was analyzed after actinomycin D was added. RESULTS: After exposure to hypoxia for 2 h, the levels of eNOS mRNA and protein in CAECs were increased. The levels of eNOS mRNA and protein reached peak after 12 h of hypoxia (about 2.5 fold and 2.0 fold, respectively, compared to control), and remained at higher level even after 48 h of hypoxia. Moreover, hypoxia did not change the stability of eNOS mRNA. The specific PKC inhibitors BIM Ⅰ and G6983 attenuated significantly the effects of hypoxia on eNOS gene expression. CONCLUSION: These results suggest that hypoxia may enhance the expression of eNOS gene in CAECs through PKC signaling pathway, which might be one of the mechanisms of cerebral artery dilation and neuroprotection during cerebral hypoxia.     

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.     

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 glucagon-like peptide-1 on myocardial ischemia-reperfusion/hypoxia-reoxygenation injury in rats

AIM: To investigate the effects of glucagon-like peptide-1 (GLP-1) on myocardial ischemia-reperfusion (IR)/hypoxia-reoxygenation (HR) injury in rats. METHODS: Sprague-Dawley rats were randomly divided into 5 groups: sham group, IR group and IR+GLP-1 (0.03 nmol/L, 0.16 nmol/L and 0.30 nmol/L) groups. IR group and IR+GLP-1 group were subject to 30 min of ischemia and 3 h of reperfusion. The myocardial infarct size, the ultrastructural changes of the myocardial tissues, the apoptosis of the cardiomyocytes, the activity of superoxide dismutase (SOD) and the concentration of malondialdehyde (MDA) were detected. Primarily cultured cardiomyocytes were divided into 5 groups at random: control group, HR group and HR+GLP-1 (1 μmol/L, 5 μmol/L and 10 μmol/L) groups. The morphology and apoptosis of the cardiomyocytes were observed. The levels of lactate dehydrogenase (LDH),MDA,SOD,reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in different groups were detected. RESULTS: Compared with IR group, the myocardial infarct size and cardiomyocyte apoptosis were remarkably reduced, mitochondrial ultrastructures were improved, the activity of SOD was increased and the concentration of MDA was decreased in IR+GLP-1 (0.03 nmol/L, 0.16 nmol/L and 0.30 nmol/L) groups. Compared with HR group, GLP-1 (1 μmol/L, 5 μmol/L and 10 μmol/L) preconditioning significantly decreased the myocardial injury, increased SOD activity, decreased MDA concentration and ROS production, and heightened MMP in a dose-dependent manner. CONCLUSION: GLP-1 protects cardiomyocytes from IR/HR injury, which may be partially due to the effects of anti-oxidative mechanism and the function of mitochondrial protection.     

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.     

Roles of heat shock protein 90 in the blockage of H2S against cardiomyocyte injuries induced by chemical hypoxia

AIM: To explore the roles of heat shock protein 90 (HSP90) in the blockage of hydrogen sulfide (H2S) against chemical hypoxia-mimetic agent (cobalt chloride, CoCl2)-induced oxidative stress injuries in H9c2 cardiac cell. METHODS: H9c2 cells were treated with CoCl2 to set up the chemical hypoxia-induced the model of cardiomyocyte injury. Sodium hydrosulfide (NaHS, a H2S donor) was added into medium for 30 min before CoCl2 treatment. ATP content was detected by high performance liquid chromatogram (HPLC). Mitochondrial membrane potential (MMP) was measured by rhodamine123 (Rh123) staining and photofluorography. The activity of superoxide dismutase (SOD) was observed using a SOD kit. The expression of heme oxygenase-1 (HO-1) was evaluated by Western blotting. RESULTS: CoCl2 at concentration of 600 μmol/L significantly reduced SOD activity, ATP level and MMP, and enhanced the expression of HO-1 in H9c2 cells. Pretreatment with 400 μmol/L NaHS dramatically inhibited the cytotoxicity induced by CoCl2, increased SOD activity, ATP level and MMP, decreased HO-1 expression. 17-allylamino-17 demethoxygeldanamycine(17AAG), an inhibitor of HSP90, obviously blocked the inhibitory effect of H2S on the CoCl2-induced cytotoxicity, reduced the levels of ATP and MMP, increased HO-1 expression. However, no significantly influence on SOD activity was observed. CONCLUSION: HSP90 may mediate the cardioprotection of H2S via inhibiting the oxidative stress induced by chemical hypoxia.     

Nitric oxide-mediated the cardioprotection of tumor necrosis factor-alpha on cultured neonatal rat cardiomyocytes during hypoxia/reoxygenation

AIM: To investigate the role of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and protein kinase C (PKC) signaling in tumor necrosis factor-α (TNF-α)-induced cardioprotection against hypoxia/reoxygenation (H/R) injury. METHODS: Neonatal rat ventricular myocytes were pretreated with TNF-α or sodium nitroprusside (SNP) or L-arginine (L-Arg), respectively, for 12 h and then subjected to continuous hypoxia for 12 h, followed by reoxygenation for 6 h. The manganese superoxide dismutase (Mn-SOD) activity of the cells was measured after H/R. Myocyte injury was determined by the release of lactic dehydrogenase (LDH). RESULTS: TNF-α (10⁵ U/L) significantly increased the Mn-SOD activity and decreased release of LDH from ventricular myocytes. The cardioprotection against H/R injury was induced by the pretreatment with SNP (5 μmol/L) or L-Arg (5 mmol/L), which was blocked by ODQ (10 μmol/L), the specific sGC inhibitor, and Chel (5 μmol/L), the specific PKC inhibitor. Pretreatment with L-NAME (100 μmol/L), ODQ, Chel, antoxidant 2-MPG (400 μmol/L) or tyrosine kinase inhibitor genistein (50 μmol/L) attenuated the increased Mn-SOD activity and reduced LDH level induced by TNF-α. CONCLUSION: The results suggest that NO may play a role in TNF-α-induced cardioprotection, which is mediated by sGC and PKC.     

Oxidative stress plays an important role in acetaldehyde-induced cardiomyocytes apoptosis

AIM: To elucidate the mechanism of alcoholic myocardiopathy (AHMD) by exploring the role of ROS mediated oxidative stress in acetaldehyde-induced cardiomyocytes apoptosis. METHODS: Cultured rat cardiomyocytes were stimulated with acetaldehyde (100 μmol/L) for 24 h, then the cell apoptotic index were examined and compared to that with alcohol (100 μmol/L) stimulation. The changes of ROS levels and SOD activities were explored by a time-dependent model in acetaldehyde-induced cardiomyocytes. Meanwhile, the phosphorylation changes of ROS mediated MAPK signaling pathway correlated proteins were also detected, with which compared that changes both in pre-adding NAC acetaldehyde-induced cardiomyocytes, and in H2O2 (100 μmol/L) induced cardiomyocytes, respectively. RESULTS: Acetaldehyde had more obvious apoptotic effect than alcohol through caspase 3 activating (P<0.05, vs control), both cellular ROS level and SOD activity increased in a time-dependent way, and reached the peak value of (78.84%±4.33%) for ROS and (0.55±0.02) for SOD among 18 to 24 h, respectively. Meanwhile, JNK and ERK protein phosphorylation upregulated in acetaldehyde-induced cardiomyocytes, and was reversed by NAC anti-oxidative effect. However all the phosphorylation levels were weaker than that in H2O2-induced group. CONCLUSION: Acetaldehyde causes oxidative damage in cardiomyocytes through enhancing cellular ROS level, and induces cardiocytes apoptosis by activating ROS mediated JNK pathway. The novel way of depressing cellular ROS level or blocking the special apoptotic pathway may have effects on AHMD preservation and therapy.     

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.     

Effect of cyclosporin A on isolated mitochondria from normal and ischemic rat brain

AIM: The purpose of this study was to investigate the effect of cyclosporin A on the isolated mitochondria from normal and ischemic rat brains and to observe the possible effect of mitochondrial ATP sensitive potassium channel on mitochondrial permeability transition. METHODS: The swelling of mitochondria isolated from normal and ischemic rat brain was evaluated by pectrophotometry. RESULTS: Cyclosporin A at concentrations of 0.5 μmol/L or 1 μmol/L and diazoxide at concentration of 30 μmol/L significantly decreased the swelling of the normal brain mitochondria induced by 200 μmol/L calcium, which was abolished by atractyloside at 100 μmol/L. However, cyclosporin A at concentration of 5 μmol/L did not affect the mitochondrial swelling. On the mitochondria isolated from ischemic brain, cyclosporin A at 0.5 μmol/L but not 1 μmol/L significantly decreased the mitochondrial swelling, which was cancelled by atractyloside at concentration of 100 μmol/L. Diazoxide at concentration of 30 mol/L had the similar effect with cyclosporin A at 0.5 μmol/L, which was locked by atractyloside at 100 μmol/L or 5-hydroxydecanoate at 100 μmol/L and 200 μmol/L. CONCLUSIONS: Compared with the mitochondria isolated from normal brain, mitochondria from ischemic brain are more sensitive to the inhibition of mitochondria permeability transition pore opening. Activation of mitochondrial ATP potassium channel may be one of the mechanisms by which the opening of mitochondrial permeability transition pore is inhibited.     

AngiotensinⅡ activates autophagy pathway through elevating ROS level in vascular endothelial cells

AIM: To evaluate the effects of angiotensinⅡ (AngⅡ) on autophagy induction in vascular endothelial cells. METHODS: Human vascular endothelial EA.hy926 cells were used in the study. Intracellular reactive oxygen species (ROS) levels were detected by a microplate reader after the cells were treated with AngⅡ (10^-7 mol/L) or AngⅡ combined with antioxidant N-acetyl-L-cysteine (NAC,50 μmol/L) for 24 h. The protein levels of LC3-Ⅱ was detected by Western blotting after the cells were stimulated by different concentrations (10^-8, 10^-7, 10^-6 mol/L) of AngⅡ for 24 h or by AngⅡ (10^-7mol/L) for different time (0 h, 6 h, 12 h, 24 h, 36 h). The number of autophagosomes was evaluated by fluorescence microscopy after stained with acridine orange. Similarly, the protein level of LC3-Ⅱ and the number of autophagosomes were detected after treated with AngⅡ(10^-7mol/L), AngⅡ combined with autophagy inhibitor 3-methyladenine (3-MA) at concentration of 2 mmol/L or AngⅡ combined with NAC at concentration of 50 μmol/L. RESULTS: Intracellular ROS level and LC3-Ⅱprotein level were significantly increased (P<0.05) after the cells were treated with AngⅡ, accompanied by the significant increase in the number of autophagosomes. AngⅡ-induced autophagy (as showed both in LC3-Ⅱprotein level and autophagosomes) was dramatically down-regulated by the treatment with 3-MA or NAC in EA.hy926 cells (P<0.05). CONCLUSION: AngⅡ induces autophagy through elevating ROS levels in EA.hy926 cells.