Role of Kv1.3 channel of CD4+T lymphocytes in the formation of atherosclerosis in rat spleen

AIM: To investigate the function of voltage-gated potassium channel Kv1.3 and its possible role in CD4⁺ T lymphocytes in the formation of atherosclerosis (AS) in rat spleen. METHODS: The rat atherosclerosis model was established by feeding high-fat diet. The proportion of lymphocytes was determined by flow cytometry. The CD4⁺ T lymphocytes were separated using immunomagnetic bead. The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was detected. The concentrations of intracellular calcium and cytokines were also measured. RESULTS: (1) The proportion of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (74.93%±2.15% vs 67.80%±2.54%, P<0.05). (2) After stimulated with concanavalin A (ConA), the proliferation of CD4⁺ T lymphocytes in AS group was significantly higher than that in control group (1.1321±0.1750 vs 0.7971±0.0955, P<0.05). (3) After stimulated with ConA, the concentration of intracellular calcium in AS group was higher than that in control group. (4) In AS group, the releases of cytokines of IL-2 and TNF-α in AS group were significantly higher when stimulated with ConA for 48 h than that for 24 h. (5) The mRNA expression of Kv1.3 in CD4⁺ T lymphocytes was greatly higher in AS group than that in control group (3.670±1.579 vs 1). CONCLUSION: In AS rats, the increase in CD4⁺ T lymphocytes as well as the augmentation of Kv1.3 mRNA expression in the cells suggest that up-regulation of Kv1.3 mRNA expression in CD4⁺ T lymphocytes may be involved in the mechanism of atherosclerotic formation in rat spleen.     

Correlation between CD4+CD28-T cells and lymphocytic apoptosis in rheumatoid arthritis patients

AIM: To investigate the fraction of CD4⁺CD28^-T cells and its correlation with lymphocytic apoptosis in peripheral blood of rheumatoid arthritis (RA) patients.METHODS: The RA patients and age-matched health controls were selected in the study. The lymphocytes were isolated from peripheral blood. CD4⁺ T cells without CD28 expression (CD4⁺ CD28^-) were analyzed by flow cytometry. The incidence of apoptosis in the cells cultured with or without PHA for 24 h was determined by flow cytometry with Annexin V-FITC/PI di-staining. The correlation between the fraction of CD4⁺CD28^-T cells and lymphocytic apoptosis was also observed.RESULTS: The fraction of CD4⁺CD28^-T cells was significantly higher in RA group than that in healthy control group (7.79%±3.52% vs 1.89%±1.78%, P<0.05). The apoptotic level in PHA cultured lymphocytes was significantly lower in RA group than that in healthy controls (11.38%±5.73% vs 19.46%±6.32％, P<0.05). Spearman analysis showed that there was a negative correlation between the fraction of CD4⁺CD28^-T cells and apoptotic level of activated lymphocytes (r=-0.433,P<0.01).CONCLUSION: The increased CD4⁺CD28^-T cells contribute to prolong the lifespan of activated lymphocytes in peripheral blood of RA patients, and the persistence of activated lymphocytes may contribute to the pathogenesis of RA.     

Effects of intravenous immunoglobulin on IL-4 levels and CD20+ cells in peripheral blood and recurrent rate of infants with wheezing

AIM: To investigate the effects of intravenous immunoglobulin on IL-4 levels and CD20⁺ cells in peripheral blood and the recurrence rate of infants with wheezing. METHODS: IL-4 levels and CD20⁺ cells in peripheral blood of 30 normal infants and 66 infants with wheezing were tested by flow cytometry and ELISA, respectively. The relief time of wheezing and recurrent rate were also recorded. RESULTS: The IL-4 levels and CD20⁺ cells in the wheezing infants were higher than those in controls(P<0.01). The IL-4 levels and CD20⁺ cells in the wheezing infants were decreased after routine treatment but were still higher than those in control infants after treatment. The IL-4 levels and CD20⁺ cells in the wheezing infants were decreased after immunoglobulin treatment and were almost the same as controls after treatment. The relief time of wheezing in the infants with immunoglobulin treatment was shorter than that in the infants with routine treatment(P<0.01), and recurrent rate of immunoglobulin treatment was lower than that of routine treatment(P<0.05). CONCLUSION: The IL-4 levels and CD20⁺ cells in peripheral blood are increased more significantly in infants with wheezing than those in control infants. The mechanisms of wheezing relief and decreasing the recurrent rate by intravenous immunoglobulin are associated with the down-regulation of IL-4 levels and CD20⁺ cells.     

Relationship between Th1/Th2 balance and CD28/CTLA-4 expression on peripheral blood T cells in patients with systemic lupus erythematosus

AIM: To investigate the pattern of Th1/Th2 balance in systemic lupus erythematosus(SLE) patients and the relationship between CD28/CTLA-4(cytotoxic T-lymphocyte antigen-4) molecule expression and Th1/Th2 balance.METHODS: Eighteen SLE patients met the ARA 1997 updated SLE criteria were selected in the study. According to Bombardier's SLEDAI criteria, all patients were classified into two groups: active group(12 cases) and static group(6 cases). Fourteen normal individuals, matched for age and sex of the patients, served as controls. The peripheral blood mononuclear cells(PBMCs) were isolated by density gradient centrifugation and cultured in RPMI-1640 culture medium. After treated with PMA(5 μg/L) and ionomycin(500 μg/L) for 72 h, the PBMCs were collected, the contents of IFN-γ and IL-10 in the supernatant of cultured PBMCs were detected using enzyme linked immunosorbent assay(ELISA). The expression of CD28 and CTLA-4 molecules on T cells were detected by flow cytometric technique with double staining by FITC or PE labeled monoclonal antibodies. RESULTS: The level of IL-10 was higher in the PBMCs of active and static SLE patients(351.29 ng/L±153.31 ng/L and 319.37 ng/L±153.39 ng/L) than that in controls(254.48 ng/L±120.69 ng/L), but the difference did not reach statistical significance(P>0.05). The level of IFN-γ was significantly lower in the PBMCs of active SLE patients(25.76 ng/L±16.09 ng/L) than that in controls(50.71 ng/L±27.92 ng/L, P＜0.05). The ratio of IL-10/IFN-γ was significantly higher in active SLE patients(18.74±13.77) than that in controls(6.66±4.95, P<0.05). Either before or after culture, the expression of CD28 molecule on CD3⁺and CD8⁺ T cells from all SLE patients was not remarkably different from that in the cells of controls. Before culture, the expression of CTLA-4 molecule on CD3⁺T cells of active SLE patients(0.79%+0.37%) was significantly lower than that in the cells of controls(1.31%+0.61%, P<0.05). After culture, the expression of CTLA-4 molecule on CD3⁺ T cells of SLE patients was still lower than that in the cells of normal controls without statistical significance(P＞0.05).The expression level of CD28 molecule on CD3⁺ or CD8⁺ T cells in active SLE patients and controls was not correlated with the levels of IFN-γ and IL-10 in the supernatants(P＞0.05). The level of CTLA-4 molecule expression on CD3⁺ T cells of active SLE patients was positively correlated with IFN-γ level(r=0.681, P<0.05), while was negatively correlated with IL-10 levels(r=-0.624,P＜0.05) and the ratio of IL-10/IFN-γ(r=-0.738, P<0.01). The level of CTLA-4 molecule expression on CD3⁺ or CD8⁺ T cells of controls showed no correlation with IFN-γ levels, while showed negative correlations with IL-10 level(r=-0.587, P<0.05; r=-0.563, P<0.05, respectively).CONCLUSION: There is a bias in the differentiation of Th0 cells towards Th2 in SLE patients. CTLA-4 probably plays an important role in this mechanism through suppressing the signal transmitted by CD28.     

Effects of dexamethasone on intracellular expression of TH1/TH2 cytokines in experimental asthma mice

AIM: To study the effects of dexamethasone (DXM) on intracellular expression of T_H1/T_H2 cytokines and the mechanism of that during the development of asthma. METHODS: Eighteen BALB/c mice were divided into 3 groups at random: control group, asthma group, and DXM treated group, with 6 mice in each. The expressions of T-box expressed in T cells (T-bet) and GATA-binding protein-3 (GATA-3) in lung tissue were detected by Western blotting. The expressions of intracellular cytokines interleukin-4 and interferon-γ in CD4⁺ T cell were measured by flowcytometry.RESULTS: The results of flow cytometry indicated that the ratio of intracellular cytokines IL-4/IFN-γ in CD4⁺ T cells in asthma group was much higher than that in control group (P<0.01), the ratio of intracellular IL-4/IFN-γ in T cells in DXM group was lower than that in asthma group significantly (P<0.01). The expression of T-bet in lung tissue in asthma group was lower than that in control group significantly (P<0.01), while GATA-3 was higher than that in control group significantly (P<0.01). The expressions of T-bet and GATA-3 in DXM group were much lower than those in asthma group (P<0.01), but the decreased degree of GATA-3 was more than that of T-bet. CONCLUSION: With pathological process of asthma, to reverse the ratio of IL-4/IFN-γ in CD4⁺ T cell by regulating T-bet and GATA-3 expression can improve the inflammatory reaction and may be one of the mechanisms of DXM in treating asthma.     

Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

Effect of G-CSF on dendritic cell subsets in donors mobilized peripheral blood cells

AIM: To explore the effect of granulocyte colony stimulating factor (G-CSF) on donor’s dendritic cell （DC）subsets in peripheral blood cells (PBC). METHODS: The subsets of dendritic cells in PBC were analyzed by CD34^-Lin^- HLA-DR⁺ cells and the levels of IL-12p40, IL-10, IFN-γ, IL-4 in the serum were tested by ELISA before (25 cases) or after G-CSF mobiling. The relationship between the ratio of DC₁/DC₂ (CD11c⁺CD123^-/CD11c^-CD123⁺) and CD34⁺/MNC was explored. RESULTS: CD34⁺/MNC in PBC harvests was above 0.4% in 23 cases, and the ratio of DC₁/DC₂ was lower, the HLA-DR expression of DC₂ was enhanced after G-CSF mobiling than before, but the levels of IL-12p40, IL-10, IFN-γ, IL-4 in donors serum and CD83 expression of DC₂ were not changed by G-CSF. CONCLUSION: DC₂ increased accompanied by the increase in CD34⁺ cells in the PBC harvests. Although the expression of HLA-DR in DC₂ was up-regulated by G-CSF, these DC₂ did not regulate Th2 cells to excrete inhibitor cell factors and kept on the precursor characters.     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

Effects of salvianolic acid B on the energy metabolism and hydrocephalus in mice with cerebral ischemia

AIM: To explore the effects of salvianolic acid B (SalB) on the energy metabolism and hydrocephalus in mice with cerebral ischemia.METHODS: NIH mice were randomly divided into four groups: sham-operated group,cerebral ischemia group,SalB-treated group and nimodipine-treated group.The brain tissue energy charge (EC),phosphocreatine (PCr),the activity of ATPase,excitability amino acid (EAA) content and water content of brain were measured when cerebral ischemia for 30 min.RESULTS: EC (0.520±0.034),PCr content ［(98.344±13.249) μmol/g］,the activity of Na⁺-K⁺-ATPase ［(0.593±0.013)×10³ U/g］ and Ca²⁺-ATPase ［(0.484±0.053)×10³ U/g］ in SalB-treated group were significantly higher than those in cerebral ischemia group {EC (0.465±0.037),PCr content ［(81.614±9.919) μmol/g］ ,the activity of Na⁺-K⁺-ATPase ［(0.244±0.065)×10³ U/g］,the activity of Ca²⁺-ATPase ［(0.321±0.086)×10³ U/g］} (P<0.01).The glutamate (Glu) content ［(0.405±0.110) μmol/g］,aspartate (Asp) content ［(0.141±0.020) μmol/g］ and water content of brain ［(38.1±0.1)%］ in SalB-treated group were markedly lower than those in cerebral ischemia group ［ Glu content (0.550±0.140) μmol/g,Asp content (0.287±0.050) μmol/g,water content of brain (44.1±0.1)%］ (P<0.05,P<0.01).CONCLUSION: The increase in cerebral energy metabolism and the activity of ATPase,and decrease in EAA content in brain tissue are the mechanism of SalB alleviating hydrocephalus at the early stage of cerebral ischemia in mice.     

Effects of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1α) and stromal cell-derived factor 1 (SDF-1) in pulmonary artery endothelial cells (PAECs) of SD rats and to investigate the role of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells to PAECs. METHODS: Immunomagnetic beads were used to separate and purify the CD34⁺/CXCR4⁺ progenitor cells derived from the peripheral circulation of SD rats. The expression of HIF-1α and SDF-1 in PAECs exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) was detected by immunofluorescence, Western blotting and ELISA. The migration index and adhesion rate were measured in the progenitor cells, which were subjected to the following different treatments: (1) normoxia (21% O₂); (2) hypoxia 12 h; (3) hypoxia 12 h +HIF-1α inhibitor (2ME2); (4) hypoxia 12 h+SDF-1 neutralizing antibody; (5) hypoxia 12 h+2ME2+SDF-1 neutralizing antibody.RESULTS: The expression of HIF-1α and SDF-1 in PAECs was effectively induced by the hypoxic exposure, and both of them reached the peak levels after 12 h of hypoxic treatment (P<0.01), while administration of 2ME2 decreased the hypoxia-induced SDF-1 expression (P<0.05). Treatment of the PAECs with 2ME2 or SDF-1 neutralizing antibody attenuated the migration index and adhesion rate of progenitor cells to the PAECs (P<0.05). CONCLUSION: There is a HIF-1α/SDF-1 signaling axis in hypoxia-exposed PAECs, which may play a crucial role in the migration and adhesion of progenitor cells to PAECs.     

Deficiency in Na-K-2Cl co-transporter impaired hearing and balance in mice

AIM: We generated transgenic mice of NKCC1^-/- (homozygous mutant),NKCC1^+/- (heterozygous) and NKCC1^+/+ (wild-type) that have a targeted disruption in the NKCC1 gene to investigate the role of Na-K-2Cl (NKCC1) channel in auditory function of the inner ear.METHODS: Hearing threshold and endocochlear potential (EP) were measured in the NKCC1^-/-,NKCC1^+/- and NKCC1^+/+ mice by auditory brainstem response (ABR) and EP recordings,respectively.The inner ears of the mice were removed and examined morphologically with the light microscope.RESULTS: The auditory function of NKCC1^+/+ mice was normal,the mean value for ABR thresholds in response to click sound was ［（23.13±3.78）dB,SPL］,EP was （98±16）mV.The mean value for ABR thresholds to click sound was elevated in NKCC1^+/- mice ［（38.49±12.29） dB,SPL］,relative to that significantly increased in NKCC1^+/+ mice (P<0.01).EP in NKCC1^+/- mice was about （78±7） mV,significantly decreased than that in NKCC1^+/+ mice (P<0.05).NKCC1^-/- mice were completely deaf,the ABR waveform was not observed for even 100 dB SPL sound stimuli used and EP was nearly disappeared (EP,4 mV±6 mV).NKCC1^-/- mice were deaf and demonstrated difficulties in maintaining their balance.NKCC1^-/- mice exhibited a marked atrophy of the stria vascularis,contraction of the endolymphatic compartments and collapse.CONCLUSION: NKCC1 channel plays a critical role in potassium homeostasis of endolymph in the inner ear.Mice lacking of NKCC1 can lead to changing K⁺ concentration in endolymph and influence auditory function subsequently.NKCC1 knockout mice exhibit marked structural abnormalities of the cochlea as well.     

Effect of fluvastatin on left ventricular remodeling and caspase-3 expression after myocardial infarction in rats

AIM: To observe the effect of fluvastatin (FV) on left ventricular remodeling and expression of caspase-3 after myocardial infarction (MI) in rats. METHODS: Rats were divided into 4 groups: group Ⅰ (sham), group Ⅱ (sham+FV), group Ⅲ (MI) and group Ⅳ (MI+FV). group Ⅱ and Ⅳ were treated with FV (10 mg·kg^-1·d^-1) for 4 weeks. The left ventricular structure, echocardiography and hydroxyproline were observed. The expression of caspase-3 was measured by immunohistochemistry and RT-PCR. RESULTS: Compared with MI group, there was a improvement of ultrastructure and index of left ventricular remodeling, and decrease in hydroxyproline in MI+FV group (all P<0.05). The number of caspase-3 positive cells also decreased in MI+FV group, and RT-PCR showed the level of caspase-3 mRNA expression was lower than that in MI group (P<0.05). CONCLUSION: Fluvastatin improves left ventricular remodeling after myocardial infarction, decreases the expression of caspase-3 and inhibits apoptosis.     

Effects of chronic hypoxia on intracellular calcium concentration in pulmonary arterial smooth muscle cells

AIM:To study the effect of chronic hypoxia (CH) on the intracellular calcium (［²⁺］_i) in pulmonary artery smooth muscle cells (PASMCs) and the role of L-type calcium channel and calcium store. METHODS:The rat chronic hypoxia model was set up and intervene the PASMCs with normal PSS, calcium-free PSS, nifedipine, and heparine respectively. The resting ［Ca²⁺］_i was determined with the Fura-2/AM calcium imaging technique. RESULTS:(1) The ［Ca²⁺］_i in CH group in normal PSS was higher than that in control group in normal PSS (P<0.05). The ［Ca²⁺］_i in CH group in normal PSS was higher than that in calcium-free PSS (P<0.05). (2) No obvious change of ［Ca²⁺］_i before and after application of nifedipine in PASMCs of CH groups was observed. (3) No difference of ［Ca²⁺］_i before and after application of heparine in PASMCs of CH groups was detected. CONCLUSION:Chronic hypoxia increased the ［Ca²⁺］_i in PASMCs. Chronic hypoxia induced increase in ［Ca²⁺］_i may relate to the influx of extracellular calcium, but not due to the L-type calcium channel or the IP3R modulation only.     

The Effect and mechanism of gestational estrogen and progesterone level on H-Y mismatched skin graft survival in murine model

AIM: To investigate the effect of estrogen(E₂) and progesterone(P₄) alone or applied together to H-Y skin graft and the potential mechanism.METHODS: The female C57BL/6 mice were ovariectomized and divided into four groups(n=12 in each). The mice were treated consecutively for 14 d with subcutaneous injection of saline, E₂ and P₄ alone or in combination, respectively. Before and after H-Y skin grafting, half mice of each group were sacrificed, and the CD4⁺CD25⁺Foxp3⁺ regulatory T cells of peripheral blood and the serum cytokines were detected by flow cytometry and ELISA, respectively. The skin graft survivals of the other half were observed.RESULTS: E₂ alone could significantly augment the proportion of regulatory T cells. In the presence of H-Y antigen, this effect was further enhanced(P<0.05). By contrast, P₄ had no effect on the expression of Foxp3, regardless of the presence of H-Y antigen or not(P>0.05). The effect of E₂ in combination with P₄ was similar to that of E₂ alone(P>0.05). The administration of sex hormone regardless of E₂ and P₄ alone or in combination, significantly decreased production of pro-inflammatory cytokines, but increased production of anti-inflammatory cytokines(P<0.05). The skin graft survivals were significantly prolonged in the different experimental groups compared to vehicle control group. E₂ and P₄ had a synergistic effect to prolong the skin graft survivals(P<0.05). CONCLUSION: E₂ and P₄ suppress the inflammatory response and enhance the regulatory response to exogenous antigen, through influencing the levels of cytokines and/or the proportion of regulatory T cells, which may contribute to induce the transplant tolerance.     

Chronic exposure to house dust mite induces airway remodeling related to Th1 inflammation in mice

AIM: To investigate the inflammatory characteristics in the airway of mice with chronic exposure to dust mite. METHODS: The α-SMA-Cre/R26R transgenic reporter mice were intranasally exposed to dust mite extract for 60 d (DME group), and then subjected to the measurement of lung resistance. The performance of bronchoalveolar lavage, pathological changes of the lung tissues and splenocytes isolation 24 h after the last challenge were observed. The protein extracts from the lungs were subjected to the detection of α-smooth muscle actin (α-SMA) by Western blotting. The supernatants of the lung homogenate were collected for testing the levels of interleukin-4 (IL-4) and interferon-γ (IFN-γ) with enzyme-linked immunosorbent assay. CD4⁺ T-cell subsets of the splenocytes were analyzed by flow cytometry.RESULTS: The mice chronically exposed to dust mite extract demonstrated severe airway hyperresponsiveness. The pulmonary pathological sections with HE staining manifested strong evidence of airway remodeling in DME group, corresponding to an enhanced X-gal staining that is related to α-SMA activation in the subepithelial basement membrane of bronchia. Total cell and lymphocyte counts were increased in the lungs of DME group compared to control group. No difference was found in eosinophil count of mice between DME and control groups. There was an elevated level of IFN-γ in the lungs of DME challenged mice coordinated with an increased proportion of IFN-γ-producing CD4⁺ T cells in the splenocytes.CONCLUSION: Chronic exposure to dust mite in the mice induces Th1-dominant inflammation with an airway hyperresponsiveness and the development of airway remodeling.     

Effects of crocetin on the apoptosis and the changes of its related regulating proteins caspase-3 and Bcl-2 induced by H2O2 in myocardial cells

AIM: To observe the effect of crocetin on the apoptosis and the changes of its related regulating proteins caspase-3 and Bcl-2 expression induced by hydrogen peroxide (H₂O₂) in cultured cardiomyocytes. METHODS: Changes of cellular morphology were detected under microscope. Apoptosis rates of the cells were analyzed by PI staining with flow cytometry. Expressions of caspase-3 and Bcl-2 proteins in the cells were determined by immunofluorescence with flow cytometry. RESULTS: In the concentrations used, more severe morphological changes with higher apoptosis rate of the cultured myocardial cells were seen in each H₂O₂ group than that in control group. When treated with 1×10-4 mol·L^－1 H₂O₂, the caspase-3 was increased and Bcl-2 protein decreased remarkably in the cells. But each dosage of crocetin, especially the highest one (5×10^－5 mol·L^－1, P<005 compared with 5×10^－7 mol·L^－1 group), seemed efficient in maintaining the cell morphology, reducing the cell apoptosis rate and improving the changes in caspase-3 and Bcl-2 protein expression in the cells exposed to 1×10^－4 mol·L^－1 H₂O₂. CONCLUSION: Crocetin obviously inhibits the apoptosis induced by H₂O₂ in the cultured myocardial cells. The mechanisms may involve the balance of the functions of the apoptosis-related regulating proteins, caspase-3 and Bcl-2 protein.     

Neuropeptide Y induced redistribution of intracellular free calcium in rat cardiomyocytes

AIM: To investigate the effect of neuropeptide Y (NPY) on intracellular free calcium(［Ca²⁺］_i) and Ca²⁺ sarcoplasmic reticulum of cardiomyocytes in rats.METHODS: Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY at concentration of 100 nmol/L for 24 h. Fluorescent indicator Fluo-4 AM was used to detect ［Ca²⁺］_i and Fluo-5N AM was used to detect Ca²⁺ in sarcoplasmic reticulum (SR). Calcium image was recorded by laser scanning confocal microscope. The SR Ca²⁺ load was estimated by caffeine-induced Ca2+ transient (CCT). RESULTS: 24 h after incubation with NPY, compared with control group, the concentration of ［Ca²⁺］_i was significantly elevated (P<0.05), and the concentration of free Ca2+ in SR (［Ca²⁺］_SR) was significantly decreased (P<0.05), and the peak of CCT was attenuated.CONCLUSION: Stimulation with NPY for 24 h causes redistribution of free calcium in rat cardiomyocytes, namely the elevation in ［Ca²⁺］_i and decline in ［Ca²⁺］_SR.     

Combination of Treg inhibition and intratumoral transfection of Ad.GM-CSF enhances the chemotherapeutic effect of doxorubicin against hepatocellular carcinoma

AIM: To explore the possibility that combination of regulatory T-cell (Treg) inhibition and intratumoral transfection of Ad.GM-CSF enhances the chemotherapeutic effect of doxorubicin against hepatocellular carcinoma (HCC). METHODS: C57BL/6J subcutaneous HCC model was established. The mice were randomly divided into 6 groups with 12 mice in each group when the tumor volume reached to 100-150 mm³: control group, cyclophosphamide(CTX) group, doxorubicin (DOX) group, Ad.GM-CSF group, CTX+DOX group and CTX+DOX+Ad.GM-CSF group. Four mice in each group were sacrificed for spleen cytotoxic T-cell(CTL) activity assay and tumor tissue histological examination 5 d after the final therapy. Eight mice in each group were examined for tumor growth and survival. RESULTS: CTX enhanced the chemotherapeutic effect of doxorubicin against HCC by inhibiting the tumor growth (P<0.05) and prolonging the mice survival (62.13 d±4.21 d vs 79.88 d±9.00 d, P<0.05), which was significantly strengthened by the combined use of Ad.GM-CSF (79.88 d±9.00 d vs 106.13 d±5.23 d, P<0.01). Compared with other groups, more tumor necrosis in tumor specimens and more infiltration of CD8⁺ T-lymphocytes in CTX+DOX+Ad.GM-CSF group were observed. Furthermore, the spleen cytotoxic T-cell(CTL) activity was significantly improved (P<0.05). CONCLUSION: Doxorubicin-based chemotherapy and immunotherapy by the method of Treg inhibition combined with intratumoral transfection of Ad.GM-CSF have synergistic effect against HCC.