Influences of protein kinase C inhibitors on the expression of IL-2 and IFN-γ by human T-lymphocytes

AIM:To investigate the influences of protein kinase C(PKC) inhibitors on the expression of interleukin-2(IL-2) and interferon-γ(IFN-γ) byin vitro activated T-lymphocytes. METHODS:Double fluorescent staining together with flow cytometry was adopted to detect intracellular cytokines and to analyze the effects of H7 and gossypol on IL-2 and IFN-γ expression levels of T-lymphocytes stimulated with phorbol ester (PDB)+ionomycin(I) in the presence of monensin.RESULTS:The expression rates of IL-2 and IFN-γ of CD3⁺ T cells stimulated with PDB+I for 4 h were 16.64±2.04 and 25.81±3.53(x±s), respectively, which were significantly higher than that of control (1.06±0.22 and 3.12±0.77)(P＜0.05). Gossypol was able to inhibit the expression of IL-2 and IFN-γ significantly, with the expression rates of 2.08±0.12 and 9.01±1.90, respectively. At the presence of 50 μmol/L H7, the rates of IL-2⁺ and IFN-γ⁺ CD3⁺ T cells were 0.43±0.06 and 2.40±0.27, respectively. The effect of H7 was stronger than that of gossypol. CONCLUSION:PKC plays an important role in the expression of IL-2 and IFN-γ of CD3⁺T cells and its inhibitors H7 and gossypol exert significant inhibitory effect on the expression of these two cytokines. It is suggested that H7 and gossypol may have modulatory effect on T-cell-dependent specific immune responses by inhibiting PKC activity.     

Inhibitory effect of berberine on the activation and proliferation of T lymphocytes

AIM: To investigate the effect of berberine (Ber) on the activation and proliferation of T lymphocytes and its mechanism of action. METHODS: Whole peripheral blood from normal subjects was stimulated with phytohemagglutinin (PHA) or phorbol ester (PDB) plus ionomycin (Ion) and the expression levels of CD69 and CD25 were evaluated with flow cytometry after the staining with appropriate fluorescent monoclonal antibody. The distribution of cell cycles was analyzed by propidium iodide staining and dead cells by 7-aminoactinomycin live staining. RESULTS: 100 μmol/L and 50 μmol/L of Ber had significant inhibition of the expression of CD69 on T cells stimulated with PDB plus Ion or PHA, while effect of 25 μmol/L Ber was not significant. And as time of action extended, the extent of inhibition decreased. For the expression of CD25, Ber at the concentrations as above all exerted significant inhibitory effect in a dose-dependent manner. Moreover, Ber could block lymphocytes cell cycle progression from G₀/G₁ phase to S and G₂/M phase without phase specificity. Besides, live staining analysis revealed that Ber did not have significant cytotoxicity on lymphocytes. CONCLUSIONS: Ber significantly inhibits the expression of early and mid activation antigens of T cells and also blocks the progression of lymphocytes cell cycles. These results suggest that Ber exerts immunosuppression effect through inhibiting the activation and proliferation of T cells.     

The inhibitory effect of quercetin on in vitro activation of T lymphocytes

AIM:To investigate the inhibitory effect of quercetin on in vitro activation of T lymphocytes by polyclonal activators with CD69 expression as an activation marker.METHODS:After being separated from lymphoid nodes of a C57BL/6 mouse, the lymphocytes were exposed to polyclonal activators (PDB or Con A) with or without quercetin. Then they were harvested at 2 h, 6 h and 24 h, respectively. The expressional rates of CD69 on T lymphocytes were assessed by two-color immunofluorescent staining and flow cytometry, and the inhibitory rates of quercetin at different time points were estimated.RESULTS:Quercetin had no effect on the expressional rate of CD69 on T lymphocytes under resting states. After the stimulation with PDB or Con A, the expressional rates of CD69 on T lymphocytes in the present of quercetin (10 μmol/L) showed significant decrease compared with those of control groups at different time points (P＜0.01). The inhibitory rate of quercetin on CD69 expression stimulated by PDB dropped sharply from 2 h to 24 h, whereas the inhibitory rate of quercetin on Con A action were relatively stable.CONCLUSION:Quercetin has inhibitory effects on the activation of T lymphocytes by Con A or PDB, suggesting that the action site of quercetin may be on PKCθ or its downstream. Furthermore,these inhibitory effect seems to be reversible.     

Effects of psychological stress on in vitro expression of activated surface molecules on T cells of peripheral blood from healthy persons

AIM: To study cellular and molecular mechanism involved in increasing susceptibility of infection in psychological stress persons. METHODS: Comparative studies were performed with double staining and flow cytometry analysis on immunophenotyping and in vitro expression of early activating surface molecule CD69 in response to mitogens on T cells from peripheral blood of 20 healthy college student volunteers before and after psychological stress. A series of term final examinations was defined as psychological stress. RESULTS: Immunophenotyping analysis showed no statistically significant difference in the percentage of CD2, CD3, CD4, CD8, CD19, CD20, CD16 and CD56 positive lymphocyte populations before and after psychological stress. There was a statistically significant decrease in the in vitro expression of CD69 in response to polyclonal stimulators on the T cells from persons after psychological stress than those before psychological stress. The percentage of CD69 expression (CD69⁺CD3⁺/CD3⁺%) in response to PHA and PDB in the whole blood culture for 72 hours decreased respectively from 28.1±4.1 and 80.7±6.8 on the T cells obtained before psychological stress to 17.6±3.8 and 65.8±7.9 on those obtained after psychological stress, while there was no statistically significant difference between the CD69 expression rates without stimulators on the T cells obtained before and after psychological stress. CONCLUSIONS: The effects of psychological stress to immune system is not on the level of changing proportions of the sub-populations within peripheral blood lymphocytes. Psychological stress can decrease the activating response of T cells in healthy persons, which may be responsible for the increase of susceptibility to infection in the psychological stress persons.     

The characteristics of TCRVα24+ NKT cells in response to in vitro stimulation

AIM: To investigate the amount and patterns of expressing CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells, and compare with that of CD3⁺ T cells from human peripheral blood in response to in vitro stimulation. METHODS: The whole blood was stained with three-color immunofluorescence directly or after cultured with PDB+ionomycin (Ion) for 6 h, then the mononuclear cells were separated by lysing red blood cells. The expression rates of CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells and CD3⁺ T cells were estimated by flow cytometer. RESULTS: As a proportion of mature T cells, the ratio of TCRVα24⁺ NKT cells to CD3⁺ T cells was about (1.34±0.42)%. The expression rates of CD69 on TCRVα24⁺ NKT cells and CD3⁺T cells in response to PDB+Ion for 6 h were (96.71±1.33)% and (98.60±0.47)%, respectively, while the ratio were (11.47±2.86)% and (1.07±0.45)% in the unstimulated group, and there were significant difference between them. The expression rates of IL-4 and IFN-γ on TCRVα24⁺ NKT cells stimulated with PDB+Ion for 6 h were (48.62±2.44)% and (46.65±8.91)%, respectively, which were significantly higher than that of unstimulated group [(31.57±3.31)%, (13.45±6.29)%] and that of stimulated CD3⁺ T cells, though the expression rates on stimulated CD3⁺ T cells were significantly higher than that of unstimulated CD3⁺ T cells. CONCLUSION: There is small amount of NKT cells in adult human peripheral blood. The expression rates of IFN-γ and IL-4 on these lymphocytes are higher than CD3⁺ T cells, suggesting that NKT cells are important immunomodulatory cells in special microvironments.     

Study on polarized Th1/Th2 cell in vitro from adult human peripheral blood

AIM: To investigate the details of CD4⁺ T cell polarized to Th1/Th2 in vitro. METHODS: After isolated the PBMCs and blood-plasma from adult human peripheral blood by Ficoll-Hypaque centrifugation, the PBMC culture procedure with or without the self-blood -plasma was applied to polarize T cells in vitro, these cells were polarized by PHA(20 mg/L),non-PHA respectively. The polarized rates of Th cell after 24 h,48 h,72 h were estimated respectively by flow cytometry following two-color immunofluorescent staining. RESULTS: CD4⁺T cell would polarize to Th1/Th2 two subsets after self-cytokines and PHA activation in vitro. The polarized rates of T cell after cultured for 24 h,48 h and 72 h were (13.28%±1.59%)/(12.70%±1.65%),(17.19%±1.03%)/(17.50%±1.30%),(19.49%±2.87%)/(18.58%±1.49%) respectively, but the polarized rates of T cell were very low if without self-blood-plasma. The difference between them was significant. The ratios of Th1/Th2 cells were about 1. CONCLUSION: CD4⁺T cell from adult human peripheral blood would polarize to Th1/Th2 two subsets in the presence of self-blood-plasma and PHA(20 mg/L) in vitro, and the cell number of Th1 and Th2 would be in balance.     

Effect of pioglitazone on balance of regulatory and effector T cells of uremic apolipoprotein E knockout mice in vitro

AIM: To investigate the effects of pioglitazone on the quantity and function-related factors of regulatory and effector T cells (Treg and Teff ) of uremic apolipoprotein E knockout mice in vitro with or without the stimulation of atherosclerotic plaque-specific antigen oxidized low-density lipoprotein (oxLDL). METHODS: Uremic apolipoprotein E knockout mouse model was established by 2-step surgical procedure. After intervention with different concentrations (2 μmol/L and 20 μmol/L) of pioglitazone and PPARγ antagonist GW9662 (5 μmol/L) on splenocytes of uremic mice for 12 h in the presence or absence of oxLDL (2 mg/L), the levels of CD4⁺ CD25⁺ Foxp3⁺ Treg and IFNγ⁺ CD4⁺ Teff were determined by flow cytometry. The mRNA expressions of Foxp3 and IFNγ was detected by real-time fluorescent quantitative PCR. RESULTS: In vitro, oxLDL induced a Treg/Teff imbalance in splenocytes from the uremic mice. Pioglitazone upregulated the level of Treg and mRNA expression of Foxp3 in the presence of oxLDL, which was not antagonized by GW9662. Meanwhile, pioglitazone downregulated the level of Teff and mRNA expression of IFNγ in the presence or absence of oxLDL, which was reversed by GW9662. CONCLUSION: oxLDL induces a Treg/Teff imbalance in uremic apolipoprotein E knockout mice. Pioglitazone modulates the Treg/Teff imbalance probably through PPARγ-independent and-dependent mechanisms.     

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.     

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.     

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.     

Effect of isoflavone genistein on activation and proliferation of mouse T cells in vitro

AIM: To study the effect of genistein on activation and proliferation of T cells, and explore the molecular mechanism of genistein. METHODS: Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the express of CD69 and CD25 by activated T cells in vitro in response to Concanavalin (ConA )and Phorbol 12, 13-dibutyrate(PDB) or T cell proliferation stained by CFSE in response to PDB / Ionomycin or ConA. RESULTS: Genistein inhibited the expression of CD69 and CD25 in activated T cells in response to Con A in a concentration-dependent manner and in response to PDB in a high concentration. Genistein inhibited proliferation of T cells in both groups in a concentration-dependent manner. CONCLUSION: Genistein inhibited activation and proliferation of T cells in vitro in response to polyclonal stimulus, and it may hold potential as a new immunosuppressant.     

Effects of Bene Jones protein and TGF-β1 on the proliferation of rat renal proximal tubular cells in vitro

AIM:To study the effect of Bene Jones protein (BJP) from multiple myeloma(MM) patient and TGF-β₁ on cultured renal proximal tubular cell(PTC) proliferation.METHODS:[H³]TdR incorporation was used to study the effect of λBJP and TGF-β₁ on cultured rat NRK.52E PTC proliferation, the expression of TGF-β₁ in the supernatant of PTC cultured with BJP was assessed with ELISA.RESULTS:① [H³]TdR incorporation of PTC was inhibited by BJP in a dose-dependent manner, when co-cultured with 100-800 μmol/L BJP and 2.0 μg/L TGF-β₁, the [H³]TdR incorporation was lower than that of BJP alone, especially when BJP≥400 μmol/L;②The expression of TGF-β₁ in the supernatant of PTC cultured with BJP was increased, especially when BJP≥400 μmol/L(P＜0.05);③ The [H³]TdR incorporation of PTC was also inhibited by exogenous TGF-β₁ in a dose-dependent manner.CONCLUSION:λBJP has antiproliferative effect on rat PTC in vitro, The effect is related with stimulating the PTC to produce excessive TGF-β₁, which also has antiproliferative effect on PTC in some degree.     

In vitro anti-platelet aggregative effect of procyanidins isolated from grape seeds

AIM: To study the possible anti-platelet aggregative mechanisms of procyanidins (PC) isolated from grape seeds in vitro. METHODS: Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from the blood of healthy volunteers. PC,diphenylene iodonium(DPI,a nonspecific NADPH oxidase inhibitor) and apocynin (a specific NADPH oxidase inhibitor) were used to observe the effects on collagen-induced platelet maximum aggregation rate using platelet aggregometer. The influences of PC on platelet NADPH oxidase activity, NO content and superoxide anion (O₂^-·) level were evaluated by chemiluminescence spectrometer. The role of PC in the expression of activated platelet markers (PAC-1 and CD62P) was observed by flow cytometry. RESULTS: PC (100 μmol/L), apocynin (10 μmol/L) and DPI (100 μmol/L) significantly inhibited collagen-induced maximum platelet aggregation rate (P<0.01). In collagen-activated platelets, NO content reduced and O₂^-· level increased,both of which were recovered by PC at concentration of 100 μmol/L (P<0.05). PC also obviously inhibited NADPH oxidase activity (P<0.01), and significantly down-regulated PAC-1 and CD62P expression (P< 0.05) in platelets. CONCLUSION: Procyanidins isolated from grape seeds have the anti-platelet aggregation function through inhibiting NADPH oxidase activity, further influencing platelet NO and O₂^-· levels.     

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 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.     

Effect of TMB-8 on the activation,proliferation and cell-cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of ［8-(diethylamino) octyl-3, 4, 5 -trimethoxybenzoate］ (TMB-8), an intracellular Ca2+ antagonist, on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (Con A) in vitro. METHODS: After stimulated with Con A, T cells were treated with different concentrations of TMB-8 alone and its combination with cyclosporine A (CsA). The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation-related index was determined by carboxyl fluorescin diacetate succinmidyl ester (CFDA-SE) flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining.RESULTS: After 6 h culture, the activation rate of CD69+ T cell in Con A group was (74.88±1.88)%. 10, 20 and 40 μmol/L of TMB-8 inhibited the expression of CD69 (P<0.01), especially in 40 μmol/L (52.55%±1.54%). After 48 h and 72 h culture, the PI of Con A group was 1.24±0.01, 2.05±0.07, respectively. TMB-8 with the concentration up to 5 μmol/L exerted a definite inhibitory effect on the proliferation with a maximal inhibition in 40 μmol/L(P<0.01). In the combination of 10 μmol/L of TMB-8 with 25 μg/L of CsA, an evident synergistic effect was observed (P<0.01). Moreover, the cell-cycle distribution analysis showed that after 48 h culture, the concentration of TMB-8 over 10 μmol/L showed an evident suppression in S phase.CONCLUSION: TMB-8 significantly inhibites the early steps of the Con A-induced T cell activation and proliferation, as well as the progression of T lymphocytes in S phase.     

Effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro

AIM: To observe the effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro. METHODS: The lymphocytes from the lymphoid nodes of BALB/c mice were isolated and primarily cultured. The viability of T cells was assessed by MTT assay. Fluorescence-conjugated monoclonal antibody and flow cytometry (FCM) were used to analyze the expression of T-cell activation marker CD69 in response to concanavalin A (Con A) in vitro. Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining was used to detect the proliferation of T cells in vitro. FCM analysis was used to determine the production of reactive oxygen species (ROS) in the T cells by staining with 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA). The mean fluorescence intensity of DiOC₆(3) staining in the T cells was detected by FCM in order to analyze the effects of salidroside on the activity of the mitochondrial and the mitochondrial membrane potential in the T cells induced by dexamethasone (DEX). The thymus T cells from BALB/c mice were isolated and primarily cultured, and then FCM was also used to analyze the apoptosis of the thymus T cells treated with DEX. RESULTS: Salidroside increased the expression of T-cell activation marker CD69 at the final concentration of 80, 160 and 320 μmol/L (P<0.05). Salidroside promoted the proliferation of T cells induced by Con A for 72 h in vitro (P<0.01). Salidroside reduced the production of ROS (P<0.05) and protected the mitochondrial membrane potential of T cells from the injury of DEX (P<0.01). Salidroside also decreased the apoptosis rate of the thymus T cells induced by DEX in vitro (P<0.01). CONCLUSION: Salidroside promotes the activation and proliferation of T cells induced by Con A, reduces the production of ROS, maintains the mitochondrial membrane potential and protects thymus T cells against apoptosis induced by DEX in vitro.     

Manumycin inhibits the activity of breast cancer cell line SK-BR-3 via inducing apoptosis

AIM: To investigate the anticancer effect of manumycin on abdominal metastatic breast cancer cell line - SK-BR-3 and its relationship with p38 MAPK. METHODS: The test of anticancer effect was performed by the method of MTT, apoptosis induced by manumycin and affected by SB203580, a specific p38 MAPK inhibitor, were examined by caspase-3 activity assay kit, and the protein expression was detected by immunoblotting assay. RESULTS: The inhibition rates at 24 h after treatment with manumycin of 6 μmol/L, 18 μmol/L, 54 μmol/L were (7.4±3.9)%, (21.0±4.4)% and (64.7±4.1)%, respectively and showed dosage-effect relationship. Compared with the control group, the survival rates of the last two treatment groups were decreased significantly (P<0.01). The value of IC₅₀ 24 h after treatment with manumycin was 42.5 μmol/L. Manumycin simultaneously activated caspase-3 protein, which was partly blocked by p38 MAPK inhibitor, SB203580. The results of immunoblotting showed that manumycin increased p38 MAPK protein phosphorylation. CONCLUSION: Manumycin exerts anticancer effect on SK-BR-3 cell line via inducing cell apoptosis, which is partly regulated by p38 MAPK.     

Ag85B regulates myeloid dendritic cell maturation and suppresses expression of TSLPR and OX40L mediated by TSLP in vitro

AIM: To investigate the maturation of mice immature myeloid dendritic cells(mDCs) induced by antigen(Ag)85B of mycobacterium tuberculosis, and the expression of TSLPR and OX40L mediated by TSLP in vitro.METHODS: Recombinant mouse GM-CSF(rmGM-CSF) and rmIL-4 were used to induce bone marrow precursor cells of C57BL/6 mice to differentiate into immature mDCs in vitro. mDCs were identified followed by purification using CD11c binding magnetic beads. The morphological characteristic of mDCs was observed under inverted phase-contrast microscope and scanning electron microscope. The surface phenotypes of mDCs were determined by flow cytometry. To obtain the optimal concentrations of Ag85B and TSLP, immature mDCs were cultured with different concentrations of Ag85B or TSLP at 0(control group), 50, 100 and 200 μg/L for 24 h, and the expression of cell surface molecules CD80, CD86, TSLPR and OX40L was detected by flow cytometry. In addition, the expression of TSLPR and OX40L in Ag85B and TSLP-co-stimulated mDCs was determined by flow cytometry.RESULTS: After 7 d of culture in vitro, the cells showed irregular dendritic protrusions under the inverted-phase contrast microscope, and had wrinkles and dendritic splits under scanning electron microscope, conformed to the morphological characteristics of immature mDCs. The mDCs cells expressed higher level of specific marker CD11c, lower level of co-stimulatory molecules CD80 and CD86, which conformed to the phenotype of immature mDCs. The CD80⁺ and CD86⁺ cell ratios of mDCs displayed significant increases in 50, 100 and 200 μg/L Ag85B or TSLP groups compared with control group(P<0.05). The ratios of TSLPR⁺ and OX40L⁺ cells did not differ among different concentrations of Ag85B groups. The ratios of TSLPR⁺ and OX40L⁺ cells were significantly increased in 100 μg/L and 200 μg/L TSLP groups compared with control group and 50 μg/L TSLP group(P<0.05). Under the circumstance of optimal Ag85B or TSLP treatment concentration at 200 μg/L, there was significantly decreased in TSLPR and OX40L cell ratio of mDCs in Ag85B group or Ag85B combined with TSLP group when compared with TSLP group(P<0.05), and no significant difference among Ag85B group, Ag85B combined with TSLP group and control group was observed.CONCLUSION: Ag85B enhances mDCs maturation by up-regulating the expression of co-stimulatory molecules CD80 and CD86, and inhibit the expression of pro-inflammatory specific molecules TSLPR and OX40L on TSLP-activated mDCs, indicating that Ag85B modifies the development of asthmatic airway inflammation through the pathway of TSLP-activated mDCs.     

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.