Protective effect of diphenyleneiodonium,a NADPH oxidase inhibitor,on hyperpermeability of endothelial cells exposed to AOPP-HSA in vitro

AIM: To investigate the effect of advanced oxidation protein product-human serum albumin (AOPP-HSA) at different concentrations on the permeability of human umbilical vein endothelial cell (HUVEC) monolayer and the protective effect of NADPH oxidase inhibitor diphenyleneiodonium (DPI) against AOPP-HSA exposure. METHODS: Cultured HUVECs were exposed to 200 mg/L HSA (control) or AOPP-HSA (50, 100 and 200 mg/L). The permeability of the endothelial monolayer was assessed by measuring CMFDA-labeled THP-1 cells across the endothelial cells. The cultured HUVECs were treated with HSA (200 mg/L), AOPP-HSA (200 mg/L), or AOPP-HSA (200 mg/L) + DPI (100 μmol/L), and the activation of NADPH oxidase, endothelial monolayer permeability and cytoskeleton rearrangement were evaluated. RESULTS: AOPP-HSA increased the permeability of the endothelial cell monolayer, and AOPP-HSA at 200 mg/L significantly increased the phosphorylation level of NADPH oxidase in the cells. Treatment with 100 μmol/L DPI obviously attenuated AOPP-HSA-induced NADPH oxidase activation, the increase in the permeability of the cell monolayer and the cytoskeleton rearrangement. CONCLUSION: AOPP-HSA increases the hyperpermeability of HUVEC monolayer via the phosphorylation of NADPH oxidase, and the NADPH oxidase inhibitor DPI reverses such effects.     

ROS mediates regulation of intracellular Ca2+ induced by angiotensin II in primarily cultured medullary neurons

AIM: To investigate the role of reactive oxygen species (ROS) in the regulation of intracellular Ca²⁺ induced by angiotensin II (Ang II) in the primarily cultured medullary neurons. METHODS: Primarily cultured medullary neurons were prepared from 14-day-old embryos of Sprague-Dawley rats in the study. The identification of medullary neurons was assessed by double-labeling immunofluorescence. To explore the role of ROS, mainly the superoxide (O₂^·), the O₂^·generation was measured using the fluorogenic probe dihydroethidium (DHE). To determine intracellular free calcium concentration ([Ca²⁺]_i), the neurons were loaded with the Ca²⁺-specific dye Fura-2/AM. The cell viability after adding Ang II was also examined using CCK-8 assay. RESULTS: Most of the cultured cells were medullary neurons, more than 80% of which were glutamate positive neurons. Ang II (5 μmol/L) increased the level of ROS within 10 min in the medullary neurons. Ang II at 5 μmol/L induced a significant[Ca²⁺]_i increase in the medullary neurons, and the effect of Ang II occurred rapidly and reached a peak within 20 min after administration. The level of[Ca²⁺]_i started to decline after washout. The Ca²⁺ elevation induced by Ang II was significantly decreased by apocynin or TEMPOL. No significant difference in the cell viability between control group and 5 μmol/L Ang II treatment group was observed. CONCLUSION: ROS is involved in the regulation of[Ca²⁺]_i induced by Ang II in the primarily cultured medullary neurons, suggesting a potential intracellular signaling mechanism involved in the Ang II-mediated oxidant regulation of central neural control of blood pressure.     

Oxidized low-density lipoprotein induces autophagy in macrophages via CD36-mediated oxidative stress

AIM: To investigate the effect of oxidized low-density lipoprotein (ox-LDL) on autophagy in macrophages and the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with 2 mg/L anti-CD36 monoclonal antibody (anti-CD36 mAb), 5 μmol/L diphenyleneiodonium (DPI), 3 mmol/L 3-methyladenine (3-MA) or 1 μmol/L rapamycin for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The viability of the cells was measured by MTT assay. The activities of lactic dehydrogenase (LDH) in the medium and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase (SOD) in the cells as well as the levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) were determined to characterize the membrane integrity and the oxidative stress, respectively. The protein levels of beclin-1 and microtubule-associated protein 1 light chain 3-II (LC3-II), 2 important molecular markers of autophagy, were examined by Western blotting. RESULTS: ox-LDL induced autophagy in RAW264.7 macrophages as assessed by upregulation of beclin-1 and LC3-II. Similar to 3-MA, an autophagy inhibitor, anti-CD36 mAb significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II. Anti-CD36 mAb suppressed the ox-LDL-induced oxidative stress as revealed by decreased NADPH oxidase activation, ROS and MDA generation as well as increased SOD activity. Similar results were observed in the cells pretreated with DPI, a NADPH oxidase inhibitor. Moreover, DPI significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II. Inaddition, the decrease in the cell viability and increase in LDH release induced by ox-LDL were promoted by 3-MA and blocked by rapamycin (an autophagy inducer). CONCLUSION: ox-LDL induces autophagy in RAW264.7 macrophages, which may be involved in CD36-mediated ox-LDL uptake and subsequent activation of oxidative stress, and moderate activation of autophagy may protect macrophages from ox-LDL-induced injury.     

Effects of nitric oxide on mitochondrial damage caused by exogenous calcium

AIM: To study the effects of nitric oxide (NO) on mitochondrial damage caused by exogenous calcium. METHODS: Normal myocardial mitochondria were divided into three groups; L-arginine control group (CG), Ca^2＋-damaged group (DG) and L-NAME-preserved group (PG). Mitochondria of all groups were incubated at 30 ℃ with reaction medium containing 20 μmol/L EDTA, 100 μmol/L CaCl₂ and 1 μmol/L L-NAME with 100 μmol/L CaCl₂ respectively. Then the NO₂^-/NO₃^- contents, mitochondrial viability and membrane potential were investigated. RESULTS: The NO₂^-/NO₃^- contents of DG was obviously higher than that of CG and PG, meanwhile, there was no obvious difference between CG and PG. Mitochondrial viability and membrane potential of DG were significantly lower than that of CG and PG, and negatively related to NO^-₂/NO^-₃ contents (r=-0.5297, P＜0.01; r=-0.6041, P＜0.01). But, the mitochondrial viability and membrane potential of PG were still lower than that of CG. CONCLUSION: Exogenous calcium could activate mitochondrial nitric oxide synthase resulting in NO production and the latter play an important role in decreasing mitochondrial viability and membrane potential.     

Farrerol inhibits nicotine-induced proliferation of pulmonary vascular smooth muscle cells by enhancing Kv1.5 and Kv2.1 expression

AIM:To study the effect of farrerol (Far) on nicotine-induced proliferation of rat pulmonary smooth muscle cells (PASMCs), and further to explore its relationship with voltage-dependent potassium channels (Kv) 1.5 and Kv2.1. METHODS:Firstly, the effect of nicotine on the proliferation of PASMCs was detected by cell counting method, and the optimal concentration of nicotine was selected. Primary cultured PASMCs were randomly divided into 5 groups:normal control group, nicotine (1 μmol/L)group, nicotine (1 μmol/L) + Far (10^-6 mol/L, 10^-5 mol/L and 10^-4 mol/L) Far group. The activity of caspase-3 was measured by apoptosis kit, the cell viability was measured by CCK-8 assay, the apoptotic rate was analyzed by flow cytometry. The expression of Kv1.5 and Kv2.1, and apoptosis-related factors Bcl-2 and Bax at mRNA and protein levels was determined by RT-qPCR and Western blot respectively. RESULTS:Nicotine at 1 μmol/L increased the number of PASMCs to the maximum extent (P<0.01). Nicotine at 1 μmol/L significantly reduced the caspase-3 activity and enhanced the cell viability of the PASMCs (P<0.01). Farrerol at 10^-6~10^-4 mol/L eliminated the effect of PASMCs induced by nicotine in a concentration dependent manner. Compared with control group, nicotine at 1 μmol/L significantly increased the proliferation and inhibited the apoptotic rate of rat PASMCs (P<0.01). The apoptotic rate of PASMCs in farrerol intervention group was significantly higher than that in nicotine group (P<0.01). Nicotine at 1 μmol/L significantly inhibited the expression of Kv1.5, Kv2.1 and Bax but increased the expression of Bcl-2 in PASMCs (P<0.01). Farrerol at 10^-5 mol/L obviously inhibited the effect of PASMCs induced by nicotine. CONCLUSION:Farrerol eliminates nicotine-induced inhibition of caspase-3 and Bax, and enhancement of Bcl-2 in PASMCs by enhancing Kv1.5 and Kv2.1 expression.     

Effects of tetrandrine and fructose-1, 6-diphosphate on the elevated intrasynaptosomal [Ca2+]i induced by excitatory amino acids

AIM: To study the effects of tetrandrine(Tet) and fructose-1, 6-diphosphate(FDP) on the elevated intrasynaptosomal [Ca²⁺]_i induced by excitatory amino acids(EAA). METHODS: A rapid method for preparing synaptosomes was used, and intrasynaptosomal free calcium([Ca²⁺]_i) was measured by using the fluorescent indicator quin-2. RESULTS: L-glutamate(Glu, 100 μmol/L), aspartate(Asp, 100 μmol·L^-1), N-methy1-D-aspartate(100 μmol/L) and Glu(50 μmol/L) plus Asp(50 μmol/L) all elevated intrasynaptosomal [Ca²⁺]_i in a dose-dependent manner. Pretreatment with Tet(10, 30, 60 μmol/L), FDP(15, 30, 75, 150 μmol/L), MK-801(10, 20 μmol/L) and Tet(15, 30 μmol/L) plus FDP(15, 30 μmol/L) all attenuated the increase in intrasynaptosomal [Ca²⁺]_i induced by EAAs mentioned as above in a dose-dependent manner, and the effect of Tet plus FDP was most significant. CONCLUSION: Both Tet and FDP inhibited a rise in intrasynaptosomal [Ca²⁺]_i induced by EAAs, which may be one of mechanisms that Tet and FDP pretect cerebral tissues against ischemia injury.     

Effects of leptin on BSEP protein expression and signaling pathway in HepG2 cells

AIM: To investigate the effects of leptin on the expression of bile salt export pump (BSEP) and signaling pathway in human hepatocellular carcinoma cell line HepG2. METHODS: HepG2 cells were cultured in vitro. Leptin at concentrations of 10^-8, 10^-7 and 10^-6 mol/L was used as a stimulating factor. The protein levels of adenosine monophosphate-activated protein kinase alpha subunit (AMPKa), phosphorylated AMPKa (p-AMPKa) and BSEP in the HepG2 cells at 24 h, 48 h and 72 h were detected by Western blotting. The optimal culture time and leptin concentration were selected, and compound C at concentration of 10 μmol/L was added to this group. The protein expression of BSEP was detected by Western blotting. RESULTS: Intervention of HepG2 cells with leptin for 72 h increased the protein expression of AMPKa gradually in a concentration-dependent manner, and leptin at concentration of 10^-6 mol/L induced the strongest AMPKa expression (P<0.01). Intervention of HepG2 cells with leptin for 24 h increased the phosphorylation level of AMPKa gradually in a dose-dependent manner (P<0.01). The effect of leptin on the increase in the protein expression of p-AMPKa was also in a time-dependent manner (P<0.01). After intervention with different concentrations of leptin for 24 h, the protein expression of BSEP in the HepG2 cells was gradually increased by the stimulation of leptin in a concentration- and time-dependent manner (P<0.01). Compared with NC group, the protein expression of BSEP in 10^-6 mol/L leptin group and 10^-6 mol/L leptin+10 μmol/L compound C group was increased at 72 h (P<0.01), and that in 10^-6 mol/L leptin+10 μmol/L compound C group was lower than that in 10^-6 mol/L leptin group at 72 h (P<0.01). CONCLUSION: Leptin promotes the protein expression of BSEP in HepG2 cells by leptin-AMPK-BSEP signaling pathway. Leptin promotes the increases in AMPKa protein and the level of phosphorylation of AMPKa in HepG2 cells.     

Effects of hydrogen peroxide on sodium current in acutely isolated rat hippocampal CA1 neurons

AIM and METHODS: The effects of hydrogen peroxide on Na⁺ currents were studied in freshly dissociated rat hippocampal CA1 neurons using the whole-cell patch-clamp techinique. RESULTS: ①H₂O₂ caused a dose-dependent and voltage-dependent increase in the voltage-activated Na⁺ currents. The amplitudes of Na⁺ currents were increased (48.0±4.2)% and (88. 2±5. 1)% (n=10) by H₂O₂ at 10 μmol/L and 100 μmol/L, respectively. ②H₂O₂ (10 μmol/L) did not affect the activation process, but changed the inactivation process significantly. Before and after application of 10 μmol/L of H₂O₂, the half-inactivation voltage was (-64.58±1.22)mV and (-53.55±0.94)mV (n=10, P<0.01), but the slope factor was not changed. CONCLUSION: As a product of oxidation metabolism, H₂O₂ is related to some diseases in the central nervous system.     

Mechanism of coronary artery constriction induced by 5-HT

AIM:To investigate the possible mechanism of coronary artery contraction induced by 5-hydroxytryptamine (5-HT). METHODS:Isolated coronary artery rings were obtained from male Wistar rats, and the vascular tension meter was used to determine the tension of the coronary artery rings. The effects of inhibitors of different signaling pathway on vascular contraction tension induced by 5-HT were observed. RESULTS:Firstly, we found that 5-HT_2A receptor antagonist sarpogrelate (1 μmol/L) completely eliminated the coronary artery contraction induced by 5-HT. Phospholipase C_β (PLC_β) inhibitor U73122 (10 μmol/L and 50 μmol/L), Rho-related protein kinase inhibitor Y-27632 (3 μmol/L and 10 μmol/L) and protein kinase C δ subunit (PKCδ) inhibitor rottlerin (3 μmol/L and 10 μmol/L) significantly inhibited the contraction of coronary artery ring caused by 5-HT (P<0.05). In addition, compared with the untreated group, vascular contraction tension induced by 5-HT was also decreased significantly by L-type calcium channel (Cav1.2) blocker nifedipine (1 μmol/L), store-operated Ca²⁺ entry (SOCE) inhibitor SKF96365 (10 μmol/L and 30 μmol/L) and 2-aminoethoxydiphenyl borate (2-APB, 50 μmol/L and 100 μmol/L) (P<0.05). At the same time, 5-HT also induced vasoconstriction after treated with nifedipine (1 μmol/L) Kerbs-Henseleit (K-H) liquid without calcium (P<0.05). CONCLUSION:5-HT activates 5-HT_2A receptor induced coronary artery contraction, possibly related to the PKC/Rho kinase signaling pathway and calcium regulation.     

Protective effect of hydrogen sulfide on PC12 cells injured by ATP

AIM: To prove the purinergic signaling mechanism of the neuroprotective action of hydrogen sulfide by observing the effects of sodium hydrosulfide (NaHS), a donor of hydrogen sulfide, on the cell viability, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the change of membrane permeability in the PC12 cells injured by adenosine triphosphate (ATP). METHODS: PC12 cells in logarithmic growth phase were randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treated with ATP after cultured for 24 h. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before treated with ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, and the other treatments were as the same as those in NaHS+ATP group. The cell viability was assessed by MTT assay. The [Ca²⁺]_i was detected by Fura-2/AM staining. The membrane permeability was observed by staining with fluorescent dye YO-PRO-1.RESULTS: ATP at concentration of 0.3 mmol/L showed no injury effect on the cells. However, the cell viability was dropped gradually in a dose-dependent manner as the ATP at doses of 1, 3, 5 and 10 mmol/L. The decline of cell viability by ATP was obviously reversed by 200 μmol/L of NaHS in the PC12 cells (P<0.05), but exasperated by 800 μmol/L of NaHS (P<0.05). At the same time, ATP evoked the increase in [Ca²⁺]_i in a dose-dependent manner, which was inhibited by NaHS (P<0.05). Furthermore, the YO-PRO-1 uptake induced by ATP in a dose-dependent and time-dependent manner was also reduced by NaHS (P<0.05). CONCLUSION: Hydrogen sulfide has protective effect on the PC12 cells injured by ATP. The mechanism may be related to the reverse of the increased [Ca²⁺]_i and YO-PRO-1 uptake.     

Influence of histone deacetylase inhibitor romidepsin on phenotype and function of T lymphocytes in vitro

AIM: To explore the effects of romidepsin (FK228), a novel histone deacetylase inhibitor, on the effector and regulatory T cells in vitro.METHODS: As the reactive cells, lymphocytes, CD4⁺ T cells and CD8⁺ T cells were labelled with CFSE, and stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group), or PBS (placebo group).After 72 h, the proliferation of the cells was detected in different groups. The lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group),or PBS (placebo group). After 72 h, the percentage of CD4⁺ Foxp3⁺ T cells and the levels of related cytokines were detected in different groups. RESULTS: The proliferation of CFSE-labelled lymphocytes, CD4⁺ T cells and CD8⁺ T cells triggered by anti-CD3 and anti-CD28 mAbs all were inhibited when cultured with romidepsin at concentrations of 1 μmol/L, 3 μmol/L and 5 μmol/L in a dose-dependent manner (P<0.05). Compared with placebo group, in the presence of anti-CD3 and anti-CD28 mAbs, 1 μmol/L romidepsin did not increase the percentage of CD4⁺ Foxp3⁺ T cells (P>0.05). When cultured with romidepsin at concentrations of 3 μmol/L and 5 μmol/L, the percentage of CD4⁺ Foxp3⁺ T cells was enhanced markedly (P<0.05). The levels of IL-10 and TNF-α in the supernatant were markedly increased in positive control group and 3 experimental groups (P<0.05), and the levels of cytokines in different experimental groups were gradually decreased with the elevation of FK228 concentration (P<0.05). The level of TGF-β was slightly increased in positive control group with no significant difference compared with placebo group (P>0.05). With the increase in the concentration of FK228 in different experimental groups, the TGF-β level was increased in a dose-dependent manner and there were significant differences in the 3 experimental groups. Meanwhile, significant differences existed between experimental groups and placebo group and between experimental groups and positive control group (P<0.05). CONCLUSION: Romidepsin inhibits the proliferation of CD4⁺ and CD8⁺ effector T cells and increases the percentage of CD4⁺ Foxp3⁺ regulatory T cells. It may be related to the increased level of TGF-β, but independent of IL-10.     

Changes of myocardial nuclear membrane Ca2+-ATPase function in ischemia/reperfusion injury

AIM: The changes of myocardial nuclear membrane Ca²⁺ -ATPase function was investigated in ischemia/reperfusion injury. METHODS: The model of myocardial ischemia/reperfusion injury was established in rats. Myocardial nuclei were purified with sucrose density centrifugation, the activity of Ca²⁺ -ATPase was measured and calcium uptake was assayed with [⁴⁵ Ca²⁺ ] . RESULTS: Plasma levels of malondialdehyde (MDA) and free fatty acid (FFA) in myocardial ischemia/reperfusion injury increased significantly( P<0.01 vs control). Ca²⁺ -ATPase activity and [⁴⁵ Ca²⁺ ] uptake was lower than normal at below 10 μmol/L, while higher at 50 μmol/L. CONCLUSION:These data indicate dysfunction of nuclear menbrane calcium pump and [⁴⁵ Ca²⁺ ] uptake function in myocardial ischemia/reperfusion injury.     

Effect of selenium compounds on ROS and NO production and NOS activity in human umbilical cord mesenchymal stem cells

AIM: To investigate the effects of selenium (Se) compounds on the generation of nitric oxide (NO) and reactive oxygen species (ROS), and the activity of nitric oxide synthase (NOS) in umbilical cord mesenchymal stem cells (MSCs). METHODS: MSCs were cultured in the medium of DMEM/F12 containing 10% FBS and exposed to the compounds of selenium , selenocysteine (Se-Cys) or nano-selenium (Nano-Se) at concentrations of 0.1 μmol/L to 0.5 μmol/L. The concentration of NO and the activity of NOS in treated MSCs were detected by the method of nitrate reductase assay. The fluorescent intensity of ROS was determined by DCFH-DA probe. RESULTS: The production of NO was (18.13±6.80) μmol/L in the control MSCs,(20.93±5.68) μmol/L in the MSCs treated with Se(Ⅳ) at concentration of 0.1 μmol/L and (16.73±5.03) μmol/L in the MSCs treated with Se(Ⅳ) at concentration of 0.5 μmol/L for 24 h. The production of NO was (17.20±9.11) μmol/L (P<0.05) in the MSCs treated with Se(IV) at concentration of 0.1 μmol/L and (9.98±4.35) μmol/L (P<0.01) in the MSCs treated with Se(IV) at concentration of 0.5 μmol/L for 48 h, which was significantly lower than that in the control MSCs . No inhibitory effect of Nano-Se and Se-Cys on the production of NO in MSCs was observed. Compared with the control MSCs, Se(Ⅳ) at concentrations of 0.1 μmol/L and 0.5 μmol/L significantly inhibited the generation of ROS and the activity of NOS in MSCs at 24 h and 48 h. CONCLUSION: Se(Ⅳ) inhibits NO/ROS production and NOS activity in a dose-dependent manner in MSCs.     

Effect of inhaled nitric oxide on adhesion molecule CD11b expression in lung neutrophils of meconium aspiration rabbits

AIM:To evaluate effects of inhaled nitric oxide(iNO) on adhesion molecule CD11b expression on lung neutrophils in experimental meconium aspiration syndrome(MAS) rabbits treated with conventional mechanical ventilation under room air or 100%O₂. METHODS:Animals were randomly allocated to 8 groups(n=48) of 6 each: two MAS model groups(under room air or 100%O₂ without iNO treatment), 6 treatment groups were treated with continuous NO inhalation at a dose of 0.2×10^-6mol/L, 0.33×10^-6mol/L or 0.67×10^-6mol/L respectively for 12 hours under room air or 100%O₂. Mean systemic arterial pressure(SAP) and methemoglobin (MeHb) were performed at basement time, 0, 2, 4, 12 hours. Expression of CD11b on neutrophils in the bronchoalveolar lavage fluid(BALF) was detected with flow cytometry. RESULTS:SAP, MeHb at different time among different groups were within the normal scale. CD11b expression on the neutrophils in the BALF significantly decreased in groups of inhalation 0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO, compared with the two MAS model groups. (x±s: under 21%O₂, 0.33×10^-6 mol/L NO, 121±20 υs 392±204; 0.67×10^-6 mol/L NO, 112±30 υs 392±204;under 100%O₂, 0.33×10^-6 mol/L NO, 113±24υs293±65; 0.67×10^-6 mol/L 102±114 υs293±65, P<0.05). 0.2×10^-6mol/L NO inhalation did no effect on CD11b expression. (x±s:21%O₂, 190±101 υs 392±204; 100%O₂, 222±85 υs 293±65; P>0.05). No statistic difference was observed between groups inhaled 0.33×10^-6 mol/L NO and 0.67×10^-6mol/L NO. CONCLUSION:0.33×10^-6 mol/L or 0.67×10^-6 mol/L NO inhalation down-regulated the CD11b expression on the neutrophils in BALF to reduce the sequestration of neutrophils in rabbit lung.     

Receptor and signaling mechanisms involved in zacopride-induced potentiation of IK1

AIM: To investigate the receptor and signaling mechanisms involved in the potentiation of inward rectifier K⁺ current (I_K1) induced by zacopride, a potent 5-HT₃ receptor antagonist and 5-HT₄ receptor agonist. METHODS: The whole-cell patch clamp technique was used to record I_K1. 5-HT₄-receptor antagonist RS23597-190, 5-HT₃-receptor agonist m-chlorophenylbiguanide (m-CPBG), PKA inhibitor KT5720, PKC inhibitor GF109203X and PKG inhibitor KT5823 were applied respectively to determine the regulatory mechanism of I_K1. RESULTS: In the presence of RS23597-190 at concentration of 10 μmol/L which inhibited I_K1, zacopride at concentration of 1 μmol/L still increased I_K1 with the mean increment of 32.5% in inward current (at -100 mV, P<0.05). The I_K1 increment induced by zacopride was not inhibited by m-CPBG at concentration of 10 μmol/L (P>0.05). Furthermore, PKA inhibitor KT5720 at concentration of 5 μmol/L reversed the effect of zacopride (P<0.05), while PKC inhibitor GF109203X and PKG inhibitor KT5823 both at concentration of 5 μmol/L didn't influence the effect (P>0.05). CONCLUSION: Zacopride potentiates I_K1 via a PKA-mediated signaling pathway, which is independent on 5-HT₄ and 5-HT₃ receptors.     

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.     

p38 MAPK-iNOS-NO pathway mediates chemical hypoxia-induced injury in PC12 cells

AIM: To investigate the possibility that p38 MAPK-iNOS-NO pathway mediates the chemical hypoxia-induced injury in PC12 cells. METHODS: PC12 cells were treated with cobalt chloride (CoCl₂, 600 μmol/L) to set up a chemical hypoxia-induced cellular injury model. Cell viability was tested by cell counter kit-8(CCK-8). The morphological changes of the apoptotic cells were detected by Hochest33258 staining. The expression of iNOS was determined by Western blotting. Nitrite accumulation, an indicator of NO production, was measured in cell culture supernatants by Griess reagent assay. RESULTS: Exposure of PC12 cells to CoCl₂ for 24 h significantly enhanced iNOS expression. Exposure of PC12 cells to CoCl₂ for 24 h and 48 h significantly enhanced nitrite accumulation. Pretreatment with L-canavanine (an inhibitor of iNOS,5-20 μmol/L) for 60 min prior to exposure of PC12 cells to CoCl₂ protected PC12 cells against the injuries induced by CoCl₂ with enhanced cell viability and decreased amount of apoptotic cells. Pretreatment with SB203580 (an inhibitor of p38 MAPK) for 60 min prior to exposure of PC12 cells to CoCl₂ down-regulated the expression of iNOS induced by CoCl₂. CONCLUSION: p38 MAPK-iNOS-NO pathway mediates CoCl₂-induced injuries in PC12 cells.     

Celecoxib inhibits viability,induces apoptosis and inhibits autophagy in acute myeloid leukemia cell lines HL-60 and HL-60A

AIM: To investigate the effects of celecoxib on viability, apoptosis and autophagy in acute myeloid leukemia (AML) cell lines HL-60 and HL-60A. METHODS: The HL-60 cells and HL-60A cells were cultured with various concentrations (0, 20, 40, 60, 80 and 100 μmol/L) of celecoxib. The inhibitory effect of celecoxib on the cell viability was evaluated by MTT assay. Apoptosis was analyzed by Annexin-V/PI staining. Apoptosis-related and autophagy-related proteins were determined by Western blot. RESULTS: IC₅₀ of celecoxib were 49.4 μmol/L, 32.0 μmol/L and 25.1 μmol/L for HL-60 cells treated with celecoxib for 24 h, 48 h and 72 h, respectively. For HL-60A cells, the corresponding IC₅₀ were 69.1 μmol/L, 42.5 μmol/L and 29.6 μmol/L, respectively. The results of flow cytometry analysis showed the proportions of Annexin-Ⅴ⁺ PI^-, Annexin-Ⅴ⁺ PI⁺ and Annexin-Ⅴ^-PI⁺ cells were increased in the HL-60 cells, and those of Annexin-Ⅴ⁺PI^- and Annexin-Ⅴ⁺ PI⁺ cells were increased in the HL-60A cells treated with celecoxib for 24 h. After treated with celecoxib, the induction of apoptosis was observed, the apoptosis-related proteins cleaved caspase-3 and cleaved PARP were upregulated, the autophagy-related proteins LC3 II and P62 were both increased, and mTOR, p-mTOR, 4-EBP and p-4-EBP were not changed, indicating that celecoxib inhibited autophagy in the AML cells without the mTOR pathway involvement. CONCLUSION: Celecoxib inhibits the viability of HL-60 cells and HL-60A cells in a time-and dose-dependent manner by its effects of inducing apoptosis and necrosis. Celecoxib inhibits mTOR-independent autophagy in AML cells, indicating a possible way of using celecoxib for enhancing the antitumor activity of therapeutic agents to induce cytoprotective autophagy in the AML cells.     

Dynamic changes of the concentration of nitric oxide in ischemic myocardium and its mechanism

AIM: The study was undertaken to explore the dynamic changes of the concentration of nitric oxide(NO) in ischemic myocardium and its mechanism.METHODS: In vivo myocardial ischemia of mice and in vitro perfused isolated heart of rat were used in the experiment. The effects of severity and time of ischemia on NO production, NOS activity and mRNA were examined, respectively. RESULTS: There was a considerable difference (P<0.01) in the concentration of NO between ischemia group [(9.12±1.40) μmol/L] and control group [(20.16±1.67) μmol/L] after Pit(30 U/kg) administration, and the concentration of NO of ischemic group significantly decreased [(9.17±1.33) μmol/L] compared with control group [(19.90±1.95) μmol/L] after 30 minutes of ischemia. Also, the concentration of NO after Pit(20 U/L) administration in K-H and 15 min of ischemia was (15.41±2.00) μmol/L and (15.09±2.00) μmol/L respectively in vitro, significantly lower than control group [(23.83±2.33) μmol/L and (23.63±2.52) μmol/L]. In addition, compared with control group, the number of NOS positive cells, NOS activity as well as mRNA expression in atrial muscle and ventricular muscle of ischemic group were markedly reduced, respectively. CONCLUSION: Myocardial ischemia could reduced the NO level in myocardium, down-regulation of NOS mRNA could be the possible mechanism.     

Apolipoprotein A-I mimetic peptide D4F protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting caspase-12

AIM: To investigate the effect of D4F, an apolipoprotein A-I mimetic peptide, on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and activation of caspase-12, a key molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with D4F (12.5, 25 and 50 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium (DPI, 5 μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin (TM, 4 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and TUNEL detection, respectively. The levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in the cells and the activities of superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were determined. The protein level of caspase-12 was examined by Western blot analysis. RESULTS: Similar to the ERS inhibitor PBA, D4F protected RAW264.7 macrophages from ox-LDL or TM (an ERS inducer)-induced decrease in the viability and increase in apoptotic rate in a dose-dependent manner. Like DPI (an oxidative stress inhibitor), D4F significantly inhibited ox-LDL-induced oxidative stress, as expressed by the decreased generation of ROS and MDA (P <0.01), the increased activity of SOD and the decreased activity of NADPH oxidase (P <0.05). Moreover, similar to PBA and DPI, D4F significantly suppressed ox-LDL-induced activation of caspase-12 in a concentration-dependent manner (P <0.05). Furthermore, D4F also inhibited the caspase-12 activation induced by TM (P <0.05). CONCLUSION: D4F inhibits macrophage apoptosis induced by ox-LDL, and the mechanism is at least partially by reducing oxidative stress and inhibiting the activation of caspase-12.