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.     

Erythropoietin inhibits eryptosis induced by reactive oxygen species

AIM: To observe the influence of erythropoietin (EPO) on eryptosis and production of reactive oxygen species (ROS) in erythrocytes under stimulation of hydrogen peroxide (H₂O₂),and to explore its related mechanism. METHODS: The erythrocyte suspension (1%) was cultured in vitro and divided into 3 groups:control group (C group, the culture medium was PBS), H₂O₂ group (H group, the culture medium was PBS containing H₂O₂ at final concentration of 100 μmol/L) and EPO group (E group, the culture medium was PBS containing H₂O₂ at final concentration of 100 μmol/L and EPO at final concentration of 2×10⁴ U/L). The erythrocytes were collected at 24 h and 60 h. The eryptosis was detected by flow cytometry with Annexin V staining. The production of ROS and intracellular calcium ion concentration ([Ca²⁺]_i) were also analyzed by flow cytometry. RESULTS: The eryptosis in C group was increased as the incubating time extended. The eryptosis in H group was higher than that in C group (P<0.01), while that in E group was lower than that in H group (P<0.01). Meanwhile, ROS production and[Ca²⁺]_i were higher in H group than those in C group (P<0.01), but those were lower in E group than those in H group (P<0.05 or P<0.01). CONCLUSION: EPO inhibits eryptosis induced by H₂O₂ and its mechanism may be related to antioxidant effect and change of[Ca²⁺]_i.     

Salidroside increases release of intracellular free Ca2+ from sarcoplasmic reticulum in cultured rat cardiomyocytes

AIM: To observe the effects of salidroside on intracellular free Ca²⁺ concentration in cultured rat cardiomyocytes. METHODS: Primarily cultured cardiomyocytes of neonatal rats were divided into control group, different concentrations of salidroside groups and verapamil pretreatment+different concentrations of salidroside groups. The fluorescent intensity of intercellular free calcium concentration ([Ca²⁺]_i) in cultured cardiomyocytes of newborn rats loaded with fluo-3/AM(5 μmol/L) was measured by laser scanning confocal microscopy. RESULTS: Salidroside at concentrations of 15 mg/L, 30 mg/L and 60 mg/L elevated [Ca²⁺]_i in cultured rat cardiomyocytes with the peak values of 574.08±4.65, 591.86±3.64 and 618.66±4.27, respectively (all P<0.01), indicating that the effect of salidroside on the level of [Ca²⁺]_i was dose-dependent. In the presence of verapamil in D-Hanks solution, salidroside also elevated the fluorescent intensity of [Ca²⁺]_i in cardiomyocytes from 357.74±3.13, 387.17±2.37 and 391.43±1.34 to 480.86±3.98, 496.70±3.08 and 522.18±3.19, respectively (all P<0.01). CONCLUSION: Salidroside increases the release of [Ca²⁺]_i from sarcoplasmic reticulum in cultured rat cardiomyocytes.     

Antagonistic effects of cyproheptadine and anisodamine on [Ca2+]i elevation induced by TNFα in endothelial cell strains

AIM: To study the effects of cyproheptadine (Cyp) and anisodamine (Ani) on the changes of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by tumor necrosis factor (TNFα) in single endothelial cells, and to explore the mechanisms of TNFα mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothelial cell strains (ECV304) were seed in 35 mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [Ca²⁺]_i in single endothelial cell was determined by laser scanning confocal microscopy (LSCM). RESULTS: [Ca²⁺]_i in single endothelial cell after stimulation of TNFα rapidly increased in a dose-dependent manner and approached the peak value within 60 seconds, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca²⁺]_i elevation was more obvious in nuclear than in cytoplasma, and decreased slowly. Cyp (3×10^-5, 6×10^-5 mol/L) and Ani (2×10^-5, 4×10^-5 mol·L^-1) markedly inhibited TNFα (1.2×10^-9 mol·L^-1)-induced [Ca²⁺]_i elevation. CONCLUSIONS: TNFα markedly induces elevation of [Ca²⁺]_i in single endothelial cell, it may be an important mechanism of TNFα-induced shock and tissue injury. Cyp and Ani obviously suppress TNFα-induced [Ca²⁺]_i elevation, which probably is one of the mechanisms of their antishock effects.     

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.     

Role of calcium ion in the cyotoxicity and DNA fragment induction in HL-60 cells by 6F isolated from Pteris semipinnata L.

AIM: To investigate the changes of cytosolic free calcium concentration([Ca²⁺]_i) and expression of Bcl-2 in HL-60 cells treated by 6F isolated from Pteris semipinnata L.(PSL), and to discuss the relations between calcium ion and cytotoxicity and DNA fragment induction effects of 6F. METHODS: HL-60 cells were used as in vitro model. [Ca²⁺]_i was measured on fluorescent spectrophotometry using Fura-2/AM as Ca²⁺ indicator. Bcl-2 expressing level was measured by flow cytometry. Tetrazolium salt(MTT) and diphenylamine staining methods were applied for cytotoxicity assay and DNA fragmentation detection, respectively. RESULTS: [Ca²⁺]_i increased obviously in a dose and time dependent manner after treated HL-60 cells with 6F. 6F decreased the expressing level of Bcl-2. Adding 2 mmol/L Ca²⁺ to the medium, or 1 mmol/L EDTA to chelate Ca²⁺, or 4 μmol/L calcium ionophore A 23187 to increase the concentration of cytosolic Ca²⁺, the DNA fragment induction by 6F was not affected, whereas the cytotoxicity of 6F was enhanced. 250 μmol/L Zn²⁺ attenuated the DNA fragment induction, and the cytotoxicity of 6F against HL-60 cells was enhanced significantly. CONCLUSION: It was speculated that the decreased expressing of Bcl-2 by compound 6F was related to increased [Ca²⁺]_i in HL-60 cells, and DNA fragment induction was possibly catalyzed by Ca²⁺ - independent DNase.     

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.     

Effect of interleukin-2 on intracellular calcium levels in rat ventricular myocytes during anoxia and reoxygenation

AIM: To investigate the effect of interleukin-2(IL-2) on the intracellular calcium in electrically stimulated adult rat ventricular myocytes during anoxia and reoxygenation. METHODS: The isolated cardiac ventricular myocytes were exposed to 5 min anoxia followed by 10 min reoxygenation. Chemical anoxia was introduced by Krebs-Henseleit(K-H) solution containing 10^-3 mol/L sodium dithionite. The spectrofluorometric method was used to verify intracellular calcium transient with fura-2/AM as calcium fluorescence probe. RESULTS: It was shown that during anoxia, the amplitude of Ca²⁺ transient was decreased, diastolic [Ca²⁺]_i, time to peak and time to relaxation of Ca²⁺ transient were increased. All the parameters were got back but did not returned to the pre-anoxia level during reoxygenation. IL-2 at 2×10⁵ U/L administrated during anoxia aggravated the effect of rexoxygenation on [Ca²⁺]_i transient. Pretreatment with a specific κ opioid antagonist, nor-BNI(10^-8 mol/L), abolished the effect induced by IL-2 during anoxia on the [Ca²⁺]_i transients, whereas specific δ opioid antagonist, naltrindole(10^-6 mol/L), did not cancel the effect. CONCLUSION: It is concluded that administration of IL-2 during anoxia aggravated the effect of reoxygenation on the [Ca²⁺]_i transients of isolated ventricular myocytes, which was mediated by cardiac κ opioid receptor pathway.     

Heterogeneity of basal intracellular calcium concentration and its relations to the reactivity in mouse peritoneal macrophages

AIM: To investigate the heterogeneity of basal intracellular free calcium concentration([Ca²⁺]_i) in peritoneal macrophages(PM) and whether it is relative to the reactivity of PM at the single cell level. METHODS:[Ca²⁺]_iimplicated stimulated were measured by fluorescent microscopic imaging system after loading with fluorescent probe fura-2/AM. Superoxide(O₂^-)produced by single PM was determined by modified NBT test. RESULTS: The values of basal[Ca²⁺]_idetermined in 392 PMs of 7 mice showed normal distribution [(54±24) nmol/L, n=392] with wide range(less than 20 nmol/L to more than 100 nmol/L), among which about 50% were in the range of 40-60 nmol/L. When stimulated with PMA or fMLP,[Ca²⁺]_iwas increased, the peak values were positively correlated with the basal[Ca²⁺]_iin one mouse(PMA stimulated cells: r=0.52, P<0.01, n=58; fMLP stimulated cells:r=0.59, P<0.01, n=44. Both of the experiments were repeated in 3 mice, the results in the other 2 mice were similar). The O₂^- in PMA stimulated PMs were also positively correlated with the basal i(r=0.42, P<0.01, n=43, repeated in 4 mice, the results in the other 3 mice were similar). CONCLUSION: Basal[Ca²⁺]_iin murine PM is heterogeneous, and the value of basal[Ca²⁺]_iis tightly correlated to the reactivity of PM stimulated by proinflammatory factors.     

Relaxation effect of isoliensinine on isolated mouse airway smooth muscle

AIM: To study the relaxation effect of isoliensinine on high K⁺-induced isolated mouse airway smooth muscle (ASM) and the underlying mechanism. METHODS: The muscle tension transducer was used to detect the effects of isoliensinine on high K⁺-induced precontraction and Ca²⁺ influx in ASM. The technique of patch-clamp and calcium imaging system were respectively used to examine the effects of isoliensinine on LVDCC currents and[Ca²⁺]_i of the ASM cells (ASMCs). RESULTS: Isoliensinine significantly relaxed precontracted ASM induced by high K⁺ in a concentration-dependent manner. The maximum relaxation ratio was(95.3±3.9)% by isoliensinine at 100 μmol/L. In addition, LVDCC currents were measured using the whole-cell patch-clamp technique, which were abolished by isoliensinine. High K⁺-induced 340/380 nm fluorescence ratio of Fura-2 was 0.63±0.10 in ASMCs, while it decreased to 0.36±0.05 after the addition of isoliensinine (P<0.01). When isoliensinine was added at the peak point of[Ca²⁺]_i, the ratio rapidly decreased from 0.74±0.02 to 0.42±0.05 (P<0.01). Moreover, isoliensinine inhibited high K⁺-induced Ca²⁺ influx-mediated contraction of ASM. CONCLUSION: Isoliensinine inhibits LVDCC currents, terminates Ca²⁺ influx and reduces[Ca²⁺]_i, eventually resulting in relaxation of the ASM, indicating isoliensinine might be a potential bronchodilator.     

Role of potassium channels in the regulation of intracellular free Ca2+ concentration of pulmonary artery smooth muscle cells in rats

AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca²⁺]_i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca²⁺]_i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K⁺-channel antagonist 4-aminopyridine (4AP), but not the Ca²⁺-activated K⁺-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K⁺-channel antagonist glibenclamide (Glib) increased [Ca²⁺]_i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca²⁺]_i , but Glib had no effect on [Ca²⁺]_i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: K_V plays an important role in the regulation of [Ca²⁺]_i and proliferation of PASMCs. K_Ca serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. K_ATP has no effect on [Ca²⁺]_i and proliferation of PASMCs in normoxic and hypoxic conditions.     

Calcium-sensing receptor modulates pulmonary artery tension through G-protein-PLC-IP3 pathways

AIM: To observe the role of calcium-sensing receptor (CaSR) in the regulation of pulmonary artery tension. METHODS: The intracellular calcium concentration ([Ca²⁺]_i) was detected by laser-scanning confocal microscopy, and the pulmonary artery tension was determined by the pulmonary arterial ring technique. RESULTS: Increased levels of [Ca²⁺]_o or Gd³⁺ (an agonist of CaSR) induced the increase in [Ca²⁺]_i and pulmonary artery constriction in a concentration-dependent manner. Additionally, the effects of Ca²⁺ and Gd³⁺ were inhibited by U73122 and D609 (specific inhibitor of PLC), and 2-APB and heparin (specific antagonist of IP₃ receptor). However, U73343 (U73122 inactive analogue) did not take effect. CONCLUSION: CaSR may be involved in the regulation of pulmonary artery tension by increasing [Ca²⁺]_i through G-protein-PLC-IP₃ pathway.     

Sodium Ferulate protects human aortic smooth muscle cells against oxidized Lipoprotein(a)

AIM: To investigate the influences of native and oxidized lipoprotein(a) on human arterial smooth muscle cell (SMC) proliferation, change of intracellular free calcium concentration ( [Ca²⁺]_i) and the protective effect of sodium ferulate(SF). METHODS: Lp(a) was oxidized by Cu²⁺ and the extent of oxidation was assessed by the MDA content.Human SMC were incubated in culture media with SF for 12 h, then exposed to Lp(a) and oxidized-Lp(a) , respectively. MTT colorimetry and flow cytometry were used to evaluated the proliferation of SMC and flurorescent indicator Fura-2/AM was used to determined [Ca²⁺]_i. RESULTS: ox-Lp(a) significantly promoted proliferation of SMC and increased [Ca²⁺]_i compared with Lp(a). SF(40,80 mg/L) remarkedly inhibited SMC proliferation and decreased the rising of [Ca²⁺]_i induced by ox-Lp(a) in a dose-dependent manner, but no effect on SMC proliferation and the increase in [Ca²⁺]_i induced by Lp(a).CONCLUSION: ox-Lp(a) induces the strong growth-promoting effect in SMC through increasing in [Ca²⁺]_i, which might be one of the cellular mechanisms responsible for the higher atherogenic potential of ox-Lp(a) compared with Lp(a), and this process can be prevented by inhibiting of oxidation by SF.     

Effects of glucose and Mg2+ in the neurons damaged by glutamate

AIM and METHODS: To observe the effects of glucose-free and Mg²⁺-free in the extracellular fluid on the changes of [Ca ²⁺]_i in the cerebro-cortical neurons damaged by 1mmol/L glutamate using laser confocal scanning microscope. RESULTS: Both frequency and amplitude of neuronal calcium oscillation induced by glutamate were lowered in glucose-free and Mg²⁺-free buffers. The basic [Ca²⁺]_i concentration was lowered in the former case , but it was elevated in the latter case. CONCLUSION: Mg²⁺-free aggravates [Ca²⁺]_i overload induced by 1mmol/L glutamate ,under certain conditions the glucose-free might resist damage role of glutamate and Mg²⁺-free.     

Salvia miltiorrhiza antagonized the alterations of contraction and intracellular calcium of rat ventricular cardiomyocytes induced by anoxia and reoxygenation

AIM:To study the effect of salvia miltiorrhiza (SM) on cell contraction and intracellular calcium of enzymatically isolated rat ventricular myocytes during normoxia and anoxia/reoxygenation.METHODS:Contraction and intracel ular calcium were determined with video tracking system and spectrofluorometric method,and the chemical anoxic method was employed. RESULTS:The ±dL/dt_max, dL of cell contraction and the amplitude of [Ca²⁺]_i in the cardiomyocytes following SM treatment were decreased in a dose-dependent manner. During anoxia, the ±dL/dt_max, dL and amplitude of [Ca²⁺]_i were decreased, while the diastolic Ca²⁺ level was elevated compared with control group. All the contractile parameters and the diastolic Ca²⁺ level were back toward pretreatment values during reoxygenation, but could not return to control level. After the treatment with SM (3 g/L), ±dL/dt_max and dL of cell contraction and the amplitude of [Ca²⁺]_i were higher and the diastolic Ca²⁺ level was lower than those in anoxia/reoxygenation group. CONCLUSION:SM antagonized effects of anoxia and reoxygenation on cell contraction and intracellular calcium in isolated ventricular myocytes.     

Role and regulation of calcineurin in angiotensin Ⅱ-stimulated cardiac myocyte hypertrophy of rats

AIM: To study the role and regulation of calcineurin(CaN) in angiotensin II(AngⅡ)-stimulated cardiacmyocyte hypertrophy of rats. METHODS: Using AngⅡ to induce the cultured cardiac myocyte hypertrophy of rats, and investigating the effect of CaN inhibitor on [³H]-leucine incorporation of AngⅡ-stimulated cardiomyocytes and the regulation of various factors on CaN activity in cardiomyocytes.RESULTS: AngⅡ can stimulate the CaN activity in cultured neonatal rat cardiomyocytes in a dose- and time-dependent manner. In cardiac myocytes incubated with 10, 100, 1000 nmol·L^-1 of AngⅡ for 12h, the CaN activities increased respectively by 13%,57%(P＜0.05) and 228%(P＜0.01) compared with that in non-stimulated cardiomyocytes. The CaN activities in AngⅡ-stimulated cardiomyocytes were significantly inhibited by losartan(50 μmol·L^-1), H₇(50 μmol·L^-1)and Fura-2/AM(4 μmol·L^-1),while no effect was observed with PD98059(50 μmol·L^-1).The [³H]-leucine incorporation in AngⅡ-stimulated cardiomyocytes increased by 46%(P＜0.01) compared with that in control group, which was dramatically inhibited by cyclosporin A(0.5~5μg/mL). CONCLUSIONS: Calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, may play an important role in AngⅡ-induced cardiac myocyte hypertrophy. The activation of CaN may dependent on the sustained increases of [Ca²⁺]i and be regulated by some protein kinases (such as PKC,etc.).     

Effects of cimetidine on platelet function and thrombosis

AIM:To observe the effects of cimetidine(Cim) on platelet function and thrombosis. METHODS:After incubated with Cimin vitro, rat platelets were activated with ADP or thrombin. The platelet aggregation, platelet malondialdehyde(MDA) formation, platelet intracellular free calcium( [Ca²⁺]_i), and thromboxane B₂ (TXB₂) were measured. The effects of Cim on electric-induced thrombosis in rat carotid artery were examined. RESULTS:Cim potentiated ADP induced platelet aggregation, increased the thrombin induced [Ca²⁺]_i and MDA formation, decreased TXB₂. Also, Cim shortened the duration of electric-stimulated occlution time in rat carotid artery. CONCLUSION:Cim increased platelet function and accelerated thrombosis.     

Salinomycin inhibits proliferation and induces apoptosis of Gleevec-resistant chronic myeloid leukemic cell line K562/Glv

AIM: To study the effect of salinomycin on inhibiting proliferation and inducing apoptosis of Gleevec-resistant chronic myeloid leukemia cell line K562/Glv. METHODS: The inhibitory effect of salinomycin on the growth of K562/Glv cells was detected by CCK-8 assay in vitro. Flow cytometry was used to observe apoptosis, mitochondria membrane potential (ΔΨ_m), reactive oxygen species (ROS) and the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in K562/Glv cells. The activity of caspase-3, -8 and -9 was measured by the method of colorimetry. The levels of cytochrome C, Bcl-2, Bax, β-catenin and phosphorylated low-density lipoprotein receptor-related protein 6 (p-LRP6) were determined by Western blotting. RESULTS: Salinomycin inhibited the growth of K562/Glv cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L inhibited the growth of the cells with the inhibitory rate of (36.70±2.31)%. The cell apoptotic rate was (19.66±2.23)%. Salinomycin at concentration of 0.2 μmol/L decreased the level of ΔΨ_m, and increased the levels of ROS, cytochrome C and[Ca²⁺]_i in the cells. Salinomycin also increased the activity of caspase-3, -8 and -9 in the cells, reduced the ratio of Bcl-2/Bax, and attenuated the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin induces the apoptosis of Gleevec-resistant myeloid leukemia cell line K562/Glv via Bcl-2/Bax and mitochondria-dependent pathways, and inhibits the cell growth through Wnt signal pathway.     

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.