Effect of pioglitazone on pre-adipocytes differentiation and GILZ expression

AIM: To investigate the roles of pioglitazone on differentiation and expression of GILZ in 3T3-L1 pre-adipocytes. METHODS: The morphological changes during 3T3-L1 cell differentiation were observed. The cells were treated with pioglitazone at concentrations of 1×10^-4～1×10^-2 mmol/L for 48 h, then the relative content of triglyceride were analyzed by oil red O staining at 2nd, 4th and 6th day during adipogenesis. The mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) was measured by real-time PCR. GILZ protein expression was determined by Western blot after the cells were treated with pioglitazone at concentrations of 1×10^-4 ～1×10^-2 mmol/L for 48 h.RESULTS: Oil-red O staining showed that the relative contents of triglyceride in adipocytes were increased with the increase in the pioglitazone concentration. Compared with the control, the relative contents of triglyceride in group 1×10^-3 mmol/L and group 1×10^-2 mmol/L were significantly increased (P < 0.05). The mRNA expression of PPARγ2 and LPL was also increased with the increase in the pioglitazone concentration. When pioglitazone concentration was more than 1×10^-3 mmol/L, compared with the control, the mRNA expression of PPARγ2 and LPL significantly increased (P < 0.01). The protein expression of GILZ was decreased with the increase in the pioglitazone concentration.CONCLUSION: Pioglitazone down-regulates GILZ expression, and up-regulate PPARγ2 expression and the downstream functional factor such as LPL.     

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.     

中国园艺学会2012年学术年会暨庆祝《园艺学报》创刊50周年—园艺学进展论坛征文通知

"中国园艺学会2012年学术年会暨庆祝《园艺学报》创刊50周年—园艺学进展论坛"将于2012年10月在西安举行,即日起征集:①研究论文摘要,②有关园艺学进展的综述文章。经审查合格的摘要和综述将分别收入"中国园艺学会2012年学术年会论文摘要集"和"庆祝《园艺学报》创刊50周年—园艺学进展论坛专辑",并于会前以     

PPARγ agonist inhibits high glucose-induced production of reactive oxygen species by UCP2 up-regulation

AIM: To explore the effects of PPARγ on the elevated level of reactive oxygen species (ROS) induced by high glucose and its mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured with DMEM containing high glucose (33 mmol/L D-glucose), and DMEM containing lower glucose (5.5 mmol/L D-glucose) was used as control. Superoxide anion and nitric oxide fluorescence probes were used to observe the effects of PPARγ agonist on ROS and NO productions in the HUVECs. The uncoupling protein 2 (UCP2) protein level in the HUVECs was detected by Western blotting. RESULTS: PPARγ agonist pioglitazone inhibited the ROS generation and prevented the decrease in NO level under high glucose condition, and these effects were reversed by pretreatment with PPARγ antagonist GW9662. The results of Western blotting indicated that PPARγ agonist pioglitazone up-regulated the UCP2 expression under high glucose condition, and this effect was also blocked by GW9662. Inhibition of UCP2 by genipin attenuated the effect of pioglotazone on the ROS production. CONCLUSION: Activation of PPARγ inhibits ROS generation under high glucose condition, and this effect may mediate by up-regulation of UCP2.     

Effect of pachyman polysaccharides on Th17/Treg balance in systemic lupus erythematosus patients

AIM: To investigate the immunomodulatory effect of pachyman polysaccharides (PPS) on T helper 17 cell (Th17)/regulatory T cell (Treg) balance in the peripheral blood of systemic lupus erythematosus (SLE) patients. METHODS: The CD4⁺ T cells were isolated from the peripheral blood samples obtained from 45 SLE patients and 35 healthy controls enrolled in our study using magnetic bead separation method. The proportions of Th17 and Treg cells were measured by flow cytometry. The CD4⁺ T cells from SLE patients and healthy controls were treated with PPS. The cytoto-xicity of PPS was evaluated by detecting cell viability with MTT assay. The contents of interleukin-17 (IL-17), IL-6, IL-10 and transforming growth factor-β (TGF-β) were measured by ELISA. The expression of retinoid-related orphan receptor γt (RORγt) and forkhead box protein P3 (Foxp3) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTS: The Th17 cells were significantly elevated, while Treg cells were obviously decreased in the SLE patients compared with the healthy control group (P<0.05). Compare with control group, the contents of IL-17 and IL-6 were decreased, while the contents of IL-10 and TGF-β were increased (P<0.05). The expression of RORγt at mRNA and protein levels was down-regulated and the expression of Foxp3 was up-regulated (P<0.05). The ratio of Th17/Treg was decreased in 100 μg/L nontoxic PPS-treated CD4⁺ T cells isolated from the SLE patients (P<0.05). CONCLUSION: PPS treatment inhibits Th17 cells and elevates Treg cells in the CD4⁺ T cells isolated from SLE patients, which may have a therapeutic effect on SLE patients.     

PPARγ mediates effects of diosgenin on proliferation and apoptosis in human glioblastoma U87MG cells

AIM:To investigate the effect of diosgenin (Dio) on the proliferation, apoptosis and expression of peroxisome proliferator-activated receptor γ (PPARγ) in human glioblastoma U87MG cells and its possible mechanism. METHODS:Human astrocytes (HA) and U87MG cells were cultured in vitro and treated with Dio (0, 10, 20, 30, 40 and 50 μmol/L) and GW9662 (5 μmol/L) for 48 h, and then the cell viability was detected by CCK-8 assay. Cell colony formation assay was used to assess the proliferation potential. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. The mRNA expression level of PPARγ was measured by RT-PCR. Western blot was used to determine the protein levels of PPARγ, cyclin D1, cyclin E1, Bcl-2 and Bax. RESULTS:Dio had no significant influence on the viabi-lity of HA (P>0.05). However, Dio remarkably reduced the viability of U87MG cells in a dose-dependent manner (P<0.05) with IC₅₀ of 24.31 μmol/L. Meanwhile, Dio remarkably diminished colony formation ability (P<0.05), induced G₀/G₁ phase arrest of the cell cycle and apoptosis (P<0.05), up-regulated the expression of PPARγ at mRNA and protein levels, increased the protein level of Bax (P<0.05), and down-regulated the protein levels of cyclin D1, cyclin E1 and Bcl-2 (P<0.05) in a dose-dependent manner. However, these effects induced by Dio were inhibited by GW9662 (P<0.05), a specific inhibitor of PPARγ. CONCLUSION:Dio may inhibit proliferation and induce apoptosis in human glioblastoma U87MG cells most likely via up-regulating the expression of PPARγ, and then down-regulating the protein levels of cyclin D1, cyclin E1 and Bcl-2, and up-regulating the protein level of Bax.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

Expression of Foxp3+ Tregs and PD1 in gastric cancer tissues and their correlation with clinicopathological factors and prognosis

AIM: To investigate the expression of Foxp3⁺ regulatory T cells (Foxp3⁺ Tregs) and programmed death receptor 1 (PD1) in gastric cancer tissues and their association with clinicopathological factors and prognosis of the patients. The correlation between the 2 molecules was also analyzed at the same time. METHODS: The tumor sections from 111 gastric cancer patients were stained for Foxp3 and PD1 by the method of immunohistochemistry. The associations of the expression levels of these 2 molecules with clinicopathological factors involved in the disease progression and prognosis were statistically analyzed. The relationship of their expression was detected. RESULTS: Foxp3⁺ Tregs and PD1 were expressed in the gastric cancer tissues, and PD1 was expressed in the tumor infiltrating lymphocytes (TILs). The expression of Foxp3 and PD1 was correlated with lymph node metastasis, clinicopathological stage and prognosis of gastric cancer patients. The expression of these 2 determinants in the patients with lymph node metastasis and an advanced clinicopathological stage was distinctly higher (P <0.05). The patients with positive expression of the 2 indexes presented a lower overall survival rate and worse prognosis (P <0.05). A significantly positive correlation between the infiltration of Foxp3⁺ Tregs and the expression of PD1⁺ TILs was also observed (P <0.01).CONCLUSION: Foxp3⁺ Tregs and PD1⁺ TILs co-infiltrate in the gastric cancer tissues, which can be used as biological markers to predict the disease progression and prognosis.     

Calreticulin expression is necessary for protective immune effect of T-cell vaccine in EAE mice

AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG_35-55-specific CALR⁺ and CALR^- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.     

Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Effects of pioglitazone on cognitive impairments induced by isoflurane anesthesia in aged mice

AIM：To investigate the effects of pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, on the cognitive impairments and inflammatory cytokine production induced by isoflurane in aged mice. METHODS：Male C57BL/6J mice (11-month-old, n=136) were assigned randomly into 5 groups: control group (Con), isoflurane group (Iso), 10 mg/kg pioglitazone + isoflurane group (Pi10+Iso), 20 mg/kg pioglitazone + isoflurane group (Pi20+Iso) and 20 mg/kg pioglitazone alone group (Pi20). The mice in all isoflurane-treated groups were exposed to oxygen mixed with 1.4% isoflurane for 2 h. The mice in Con group and in Pi20 group were exposed to oxygen only for 2 h. Pioglitazone was suspended in 1% carboxymethyl cellulose (CMC). Pioglitazone (10 mg/kg or 20 mg/kg) was gavaged 2 h prior to the exposure of isoflurane or oxygen alone. The same volume of 1% CMC was gavaged in Con group and in Iso group. Fear conditioning tests were performed to determine the learning and memory abilities 48 h after isoflurane exposure. Fresh cerebral cortice and hippocampi were dissected to measure the protein expression of PPARγ by Western blotting, and the contents of IL-1β and TNF-α were analyzed by ELISA 6 h after isoflurane exposure. RESULTS：Compared with Con group, the response of freezing behavior decreased (P<0.05) and IL-1β content in the hippocampus increased (P<0.05) in Iso group. Compared with Iso group, the response of freezing behavior and PPARγ protein expression level had no significant change (P>0.05) in Pi10+Iso group, but the response of freezing behavior and PPARγ protein expression level increased significantly (P<0.05) and IL-1β content in the hippocampus decreased (P<0.05) in Pi20+Iso group. IL-1β content in the cerebral cortex and TNF-α levels both in the cerebral cortex and the hippocampus showed no significant difference among all groups (P>0.05). CONCLUSION：Pioglitazone attenuates cognitive impairments and the elevates the level of IL-1β in the hippocampus induced by isoflurane in aged mice.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

CD4+CD25+FOXP3+ Tregs and HBV-specific CTLs in peripheral blood from chronic hepatitis B patients

AIM: To investigate the roles of CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs) and HBV-specific cytotoxic T-lymphocytes (CTLs) in peripheral blood from the patients with chronic hepatitis B (CHB).METHODS: Peripheral blood mononuclear cells from 28 patients with CHB and 15 healthy controls were analyzed for Treg frequency using flow cytometry and for HBV-specific CTLs using enzyme-linked immunospot assay (ELISPOT).The clinical data of HBV-infected patients were considered.RESULTS: The frequency of CD4⁺CD25⁺FOXP3⁺Tregs was higher in the patients with CHB than that in the patients of healthy controls (3.14%±0.97% vs 1.95%±0.68%, P<0.05), and a positive correlation was found between Tregs and the DNA levels of HBV (r=0.831, P<0.01).HBV-specific CTLs were detected by ELISPOT in CHB patients and a negative correlation was observed between Tregs and CTLs (r=-0.540, P<0.01).CONCLUSION: Peripheral blood CD4⁺CD25⁺FOXP3⁺ Tregs in CHB patients are increased and closely correlated with the DNA replication of HBV and CTLs, suggesting that the clearance of HBV can be influenced by the inhibition of cellular immunoreaction through Tregs.     

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.     

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.     

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.     

Role of B cells in CD45RB antibody-induced transplantation immune tolerance

AIM: To investigate the role of B cells in CD45RB antibody-induced transplantation immune tolerance. METHODS: Single cell suspension was made from the spleen of BALB/c nude mice disposed by CD45RB antibody, then mixed cultured with T cells of BALB/c mice and spleen cells of C57BL/6 mice. The Th1, Th2, Treg and Tm cells were monitored by flow cytometry during the culture process. The skin graft model was set up with B6.μMT^-/- mice as receptors and BALB/c mice as donors. CD45RB antibody was intraperitoneally injected into the receptors after transplantation and then CD3⁺CD45RB^hi cells were detected by flow cytometry. In another mixed lymphocyte culture, CD45RB antibody was added, and then B cells were isolated and injected into B6.μMT^-/- mice through the tail vein. The heart transplantation model was established with B6.μMT^-/- mice as receptors and BALB/c mice as donors, and then the survival and the migration of B cells to the thymus were observed. RESULTS: When T lymphocytes were co-cultured with B lymphocytes treated with anti-CD45RB monoclonal antibody(mAb) in vivo, the percentages of Th2 and Treg cells were up-regulated and Th1 cells were down-regulated, but Tm cells were not altered as compared with the control. In vivo without B lymphocytes, anti-CD45RB mAb also down-regulated the expression of CD45RB in T lymphocytes. The reduction was faster and the percentage of CD3⁺CD45RB^hi T cells was not altered as compared with the control. The B lymphocytes treated with anti-CD45RB mAb in vitro prolonged the lifetime of receptor in heart transplantation model but failed to induce complete tolerance. After recieving B cells treated with anti-CD45RB mAb and allogeneic heart transplantation, B cells migrated to the thymus in B6.μMT^-/- mice. CONCLUSION: B lymphocytes play a definite role in the transplantation immune tolerance induced by anti-CD45RB mAb through their affection on T-cell subgroups and also in the central tolerance. However, the induction of immune tolerance can not only rely on B cells.