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.     

Effects of different group B streptococci strains on platelet activation

AIM: To explore the ability of different group B streptococci (GBS) strains on inducing platelet activation. METHODS: Six strains of GBS, separated from the septic patients with thrombocytopenia, were used as the inducers. Light transmission aggregometry was used to measure platelet aggregation. Scanning electron microscopy (SEM) was performed to investigate the interaction of platelets with bacteria. The expression of platelet CD62P, Toll-like receptor 2 (TLR2) and TLR4 was determined by flow cytometry and Western blotting. Furthermore, the activity of platelet TLR2 (or TLR4) was blocked by anti-TLR2 (or anti-TLR4) monoclonal antibody, and the platelet aggregation induced by GBS was detected. RESULTS: Only 3 of 6 GBS strains isolated from the septic patients induced platelet aggregation and up-regulated the expression of CD62P and TLR2 in the platelets (P < 0.05), but not TLR4. Incubation with anti-TLR2 antibody, but not anti-TLR4 antibody, significantly blocked platelet aggregation induced by GBS.CONCLUSION: Some GBS strains from the patients are able to trigger platelet activation in vitro, and platelet TLR2 may play an important role in the interaction between GBS and platelets.     

Establishment and assessment of a method for acid elution of platelet HLA class I antigens

AIM: To establish an effective method for acid elution of platelet HLA class I antigens, and to evaluate the optimal condition and the feasibility of clinical application of the acid-elution technique. METHODS: Platelets were treated with citric acid buffer at different pH levels (pH=2, 3, 5, 7). Expression of HLA class I antigens and P-selectin (CD62P) on the platelet surface was analyzed by multicolor flow cytometry. The proportion of early apoptotic platelets was detected by Annexin V staining. The maximum platelet aggregation rate was determined by electrical impedance aggregometry.RESULTS: With the decrease in the pH levels of the citric acid buffer (from pH=7 to pH=2), the expression of HLA class I antigens on the platelet surface was remarkably decreased. However, the rates of platelets activation (CD62P expression) and early apoptosis (Annexin V expression) were both significantly increased. Compared with PBS, treatment of the platelets with citric acid buffer at pH 3.0 remarkably reduced the expression of platelet HLA-class I antigens (P<0.05). Although the rates of the platelet activation and apoptosis were also significantly increased (P<0.05), the aggregation of platelet was not remarkably reduced (P>0.05).CONCLUSION: Acid elution of platelet HLA-class I antigens with citric acid buffer at pH 3.0 at 0 ℃ can be use as an attempt to produce HLA-eluted platelets. This technique of acid-elution needs further improvement and standardization before clinical use.     

Effects of Tongxinluo on activation of platelet in rabbit model of atherosclerosis

AIM: To study the effects of Tongxinluo on the activation of platelets in a rabbit model of atherosclerosis. METHODS: New Zealand rabbits were randomly divided into 7 groups: normal group, model group, the groups treated with high, medium and low doses of Tongxinluo micropowder (0.15, 0.3 and 0.6 g·kg^-1·d^-1), atorvastatin group (2.5 mg·kg^-1·d^-1), and aspirin group (12.5 mg·kg^-1·d^-1). The rabbits in normal group was fed with common diet for 12 weeks, and the rabbits in model group were fed with high-fat diet for 12 weeks to establish atherosclerosis model. The rabbits in the rest groups were treated with the corresponding drugs, at the same time to give high-fat diet. Fasting for 12 h after the last treatment, whole blood was collected to perform the blood routine test, and to measure serum and plasma levels of lipids, platelet factor 4 (PF4) and soluble CD62P (sCD62P). Flow cytometry was used to analyze platelet calcium ion concentration. Electron microscopy was used for platelet superfine observations, and light microscopy for observing the pathological changes. RESULTS: Compared with normal group, the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), platelet counts, and mean platelet volume in model group were significantly elevated, and the levels of PF4, sCD62P and calcium were also significantly increased (P < 0.05). Compared with model group, except aspirin group, the levels of TG, TC and LDL-C in high, medium and low doses of Tongxinluo groups and atorvastatin group were effectively decreased. The platelet counts and mean platelet volume in all treatment groups were markedly decreased, and the serum levels of PF4, sCD62P and Ca²⁺ in platelet (P < 0.05) were reduced. In electron microscopic observation, the shape of platelet was regular and organelles distributed uniform in normal group. However, in model group, the shape of platelet was irregular, pseudopodia forming was obviously observed, and α particles and dense granules decreased, indicating that the platelet was activated. To a different extent, the platelet shape, increase in the number of α particles and dense granules were improved in treatment groups and the damage of the cytoplasm was attenuated. Through histopathological observation, the intimal was smooth and complete in normal group. In the model group, the intimal thickness markedly increased, foam cell aggregated, and plaque was formed. Compared with model group, the intimal thickening and the number of foam cells were significantly decreased, and plaque formation was not obvious in atorvastatin group and high dose of Tongxinluo group. The pathological damages in the other treatment groups were alleviated in different degrees. CONCLUSION: Tongxinluo significantly inhibits the activation of platelets in the process of atherosclerosis, and has important clinical value to delay the atherosclerotic thrombosis.     

Study on the activation of blood platelets by propylene-acidamide grafted polypropylene membrane in vitro

AIM: To evaluate the blood compatibility of a new bioartificial reactor membranous material (propylene-acidamide grafted polypropylene membrane, PP-g-AAm) in vitro. METHODS: Contacted PP-g-AAm membrane and PP (polypropylene) membrane with platelet-rich plasma in a swing bed, 37 ℃, to simulate the conditions in vivo, and another group of PRP without any membranes was set as control group. ELISA was used to study the expression of β-thromboglobulin, and flow cytometry was used to study CD62P and CD63 expression of the activated blood platelets after contacting the two kinds of membranes with PRP. Scanning electrical microscopy was used to study the configuration and numbers of platelet cells adhered on the membranes. RESULTS: After contacting with PRP 30 min, β-TG expression showed marked difference between the two kinds of material groups and the control group (P<0.01, P<0.05), and the difference between the two kinds of membrane groups was also significant (P<0.05). There were obviously differences on the expression of CD62P and CD63 between the two kinds of membranes after contacting with PRP for 30 min (P<0.05，P<0.01). When enlarged 10 000 times, the disfiguration of the platelet cells adhered on the two kinds of membranes after one hour were found by scanning electrical microscopy, and the numbers of platelets on the PP membrane were more than the PP-g-AAm membrane markedly. CONCLUSION: The PP-g-AAm membrane has better blood compatibility than the PP membrane.     

The clinical application of fluorescently-labeled monoclonal antibody against P-selectin

AIM: To investigate the clinical significance in determination of the P-selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). METHODS: The P-selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, were analyzed using fluorescently-labeled SZ-51 by direct FCM comparing with indirect FCM and enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of P-selectin on platelet membrane is higher in DM (23.92%±15.83%), in hyperlipidemia (18.34%±9.46%) and in cerebral infarction (19.32%±10.38%) than normal subjects (3.38%±1.11%) (P<0.01). In addition, similar results on P-selectin were obtained by indirect FCM and ELISA in patients with DM and cerebral infarction. CONCLUSION: FITC-labeled SZ-51-IgG can be used in FCM, and it would be a new and sensitive method in detecting platelet activation.     

Immunosuppressive effect of dihydroartemisinin on murine T lymphocytes

AIM: To investigate the effect of dihydroartemisinin (DHA) on the proliferation of murine T lymphocytes stimulated by Con A in vitro and its related immunosuppressive mechanism. METHODS: Murine T lymphocytes were stimulated by Con A and treated with different concentrations of DHA. Cell proliferation was measured by carboxyl fluoresce in diacetate succinmidyl ester (CFDA-SE) staining. The expression of CD69, CD25 and CD71,which was the marker of early, middle, later activation of CD3+ T lymphocytes, was measured by flow cytometry (FCM) combined with two-color immunofluorescent staining of cell surface antigen. Fluorescence calcium indicator fluo-4/AM was used to measure the change of the intracellular calcium concentration (［Ca2+］i) of murine T lymphocytes. The distribution of the cell cycle was analyzed by PI staining. The expression of CD69, the early activation antigen on CD4+CD25high Treg was also measured by FCM combined with three-color immunofluorescent staining. RESULTS: The result of CFDA-SE staining showed that DHA efficiently inhibited the Con A-induced proliferation of T-lymphocytes in a time-and dose-dependent manners. DHA showed modestly increased proportions of CD69 and CD25 on Con A-stimulated CD3+T cells, but inhibited the expression of CD25 in a dose dependent manner. DHA with Con A, but not DHA alone, caused an increase in intracellular calcium concentration of T cells. The results of FCM analysis with PI staining showed that DHA imposed a total cell cycle arrest in G0/G1 and prevented cells entering S phase and G2/M phase. Furthermore, DHA reduced the expression of CD69 on CD4+CD25high Treg. CONCLUSION: DHA, which exhibits immunosuppressive effect on the proliferation of murine T-lymphocytes, is promising to be developed as an immunosuppressive reagent.     

Influence of maxadilan on human adipose-derived stem cells

AIM: To investigate the effect of maxadilan, which specifically activates pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1 receptor), on the proliferation, apoptosis and differentiation potential of human adipose-derived stem cells (ASCs). METHODS: ASCs from human adipose tissue were isolated by enzymatic digestion and cultured. ASCs were confirmed by the analysis of the markers for cell phenotypes by flow cytometry (FCM) and adipogenic/osteogenic induction. The effect of maxadilan on ASCs viability was analyzed by CCK-8 assay and FCM. ASCs were irradiated by ultraviolet C (UVC) at 254 nm and the absorbance of apoptotic ASCs induced by various doses of UVC was measured by CCK-8 assay. ASCs were exposed to 702 J/m² UVC for 24 h to induce apoptosis. The effect of maxadilan on ASC apoptosis was analyzed by FCM and the determination of caspase 3 and caspase 9 levels. RESULTS: Adipose-derived stem cells were confirmed by the detection of the positive expression of cell phenotypes including CD29, CD44, CD59 and CD105 by FCM. The data of CCK-8 assay revealed that ASCs treated with maxadilan (80 nmol/L) had the strongest ability of proliferation. The data of FCM also demonstrated that the addition of 80 nmol/L maxadilan to ASCs in experimental group markedly improved the proliferation capacity of the cells compared with control group (P<0.05). The apoptosis of ASCs exposed to 702 J/m² UVC was dramatically inhibited by the treatment with maxadilan (80 nmol/L). Such process involved the caspase signaling pathway including caspase 3 and caspase 9. There was statistical significance (P<0.05) between experiment group (ASCs irradiated by UVC and supplemented with maxadilan) and control group (ASCs only irradiated by UVC). Meanwhile, adipogenic and osteogenic differentiation potentials were both positive in experiment group and control group. CONCLUSION: Maxadilan promotes proliferation and inhibits apoptosis of the ASCs. The differentiation potential of ASCs toward adipogenic and osteogenic lineages wouldn't be altered by maxadilan. Maxadilan would benefit to growth and expansion of ASCs in vitro.     

TGF β1 inhibits the maturation of dendritic cells and down-regulates TLR4 expression

AIM: To investigate the effects of transforming growth factor β1 (TGF-β1) on murine-derived dendritic cells (DC). METHODS: Murine bone marrow cells were cultured with GM-CSF and TGF-β1 to develop TGF β-DC. Then they were stimulated by lipopolysaccharide (LPS). Their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using BrdU ELISA method. IL-12 p70 protein was detected by ELISA and the expressions of Toll like receptor 4 (TLR4) on DCs were measured by semi-quantitative RT-PCR and FCM. RESULTS: Compared to immature DC (imDC) cultured with GM-CSF alone, the expressions of CD80, CD86, I-Ab and CD40 in TGF β-DC were lower. The TGF β-DC was resistant to maturation by LPS. Maturation resistance was evident from a failure to up-regulate CMs, to stimulate larger T cell proliferation and to increase secretion of IL-12 p70. Down-regulation of TLR4 expression on TGF β-DC was also found. CONCLUSION: TGF-β1 inhibits the expression of co-stimulatory molecules on DC. It is resistant to maturation stimulus (LPS) and might be linked with TLR4 down-regulation.     

Changes in cell adhesion molecules and composition of complement activation in patients with acute myocardial infarction

AIM: To explore the possible changes in cell adhesion molecules and composition of complement activation in patients with acute myocardial infarction (AMI). METHODS: The expression of leukocyte CD18, soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular-cell adhesion molecule-1 (sVCAM-1) and composition of complement activation (sC5b-9) concentrations of patients with AMI (67 cases), old myocardial infarction (OMI, 42 cases) and 38 healthy volunteers were measured using enzyme-linked immunosorbent assay(ELISA). RESULTS: The expression of leukocyte CD18, sICAM-1,sVCAM -1 and sC5b-9 were significantly higher in AMI patients than that in normal controls and OMI patients(P＜0.01). Interestingly, it was also found that the expression of leukocyte CD18, sICAM-1,sVCAM-1 and sC5b-9 concentrations were much higher in patients with ventricular arrhythemia (VA) and the died than that in patients without VA and survivals (P＜0.01). Furthermore, the leukocyte CD18 expression, sICAM-1 and sVCAM-1 were positively correlated to sC5b-9 in AMI patients (r=0.648,0.652,0.668,0.698,0.914,0.725,0.737,0.752,0.792,P＜0.01),and leukocyte CD18 expression was positively correlated to sICAM-1 and sVCAM-1(r=0.662,0.683,0.695,0.738,0.744,0.745, P＜0.01).CONCLUSION:The interaction of cell adhesion molecules and composition of complement activation might participate in the occurance and development of AMI,and closely related to the seriousness of patients'condition and prognosis.     

The expression of β2 integrins and L-selectin on acute lymophocytic leukemic cells and its clinical implications

AIM and METHODS: To investigate the expression of adhesion molecule β₂ integrins (CD11a、CD11b) and L-selectin (CD62L )on acute lymophocyte leukemia (ALL) cells and its clinical implications. Adhesion molecules CD11a, CD11b and CD62L of 45 ALL patients and 25 health people were measured by flow-cytometric analysis. RESULTS: ①CD11a and CD11b expression were lower on ALL cells than the normal hematopoietic cells. The rate of low expression was 100% for CD11b, 50% for CD11a, respectively. CD62L expression were higher on ALL cells than the normal hematopoietic cells. ②The CD11a was lower expressed on B-ALL than T-ALL. CD62L was higher on T-ALL than B-ALL. ③The expression of CD11a in the invasion group was much higher than that in the non-invasive group (P<0.05). ④The levels of CD11a, CD11b were returned to normal levels at remission. CONCLUSION: These results suggest that there are abnormalities in the expression of cell adhesion molecules in ALL which may help identify ALL subtypes and the treatment effect.     

Injury effect of adenosine triphosphate on N9 microglia

AIM:To investigate the injury effect of adenosine triphosphate(ATP) on N9 microglia. METHODS:N9 microglia in logarithmic growth phase was randomly divided into 3 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treatment with ATP after cultured for 24 h. In KN-62 intervention group, after pretreatment with KN-62 for 30 min, ATP was added in the cells. The cell viability was assessed by XTT assay. Cellular morphological changes were observed under phase-contrast microscope. The cell cycle and apoptosis were detected by flow cytometry. The expression of P2X₇ receptor was examined by immunofluorescence staining. The protein levels of P2X₇ receptor were measured by Western blotting. The concentration of IL-1β in the culture supernatant was detected by ELISA. RESULTS:ATP at dose of 500 μmol/L and 1 mmol/L only caused small damage to the cell viability of N9 microglia. The cell viability was 88.5%±5.5% and 88.2%±8.4% after treated with ATP for 24 h,respectively. The cell viability dropped rapidly and cell shrinkage occurred when the concentration of ATP increased to 2 mmol/L or higher. With the extension of experiment time, the cell viability and cell density decreased further and cell shrinkage was getting worse. KN-62 intervention improved the viability of N9 microglia injured by ATP. The morphology and density of N9 microglia in KN-62 intervention group were much better than those in ATP group. ATP arrested N9 microglia at S phase and increased cell apoptosis significantly(P<0.01 vs control group). KN-62 intervention obviously relieved the cell cycle arrest and decreased the cell apoptosis caused by ATP(P<0.01). ATP and KN-62 intervention had no effect on the distribution of P2X₇ receptor. The protein levels of P2X₇ receptor had no significant difference among the 3 groups(P>0.05). ATP and KN-62 intervention had no effect on the release of IL-1β. CONCLUSION:High dose of ATP damages N9 microglia and its mechanism may be related to cell cycle arrest and apoptosis mediated by P2X₇ receptor but not to inflammatory response caused by microglia.     

CD45+CD86+ cells isolated from para-aortic lymph nodes in a murine abortion-prone model

AIM: To address whether the analysis of CD45+CD86+ cells isolated from para-aortic lymph nodes (PLNs) is valuable in assessment of the status of local immunity at the feto-maternal interface. METHODS: CBA/J×DBA/2, virgin CBA/J, and CBA/J×BALB/c mice were used as an abortion-prone model (group A), non-pregnant controls (group N), and fertile controls (group F), respectively. The percentage of CD45+CD86+ cell in the CD45+ cell group (CD45+CD86+ percentage for short) and the absolute number of these cells were determined with flow cytometry (FCM), using mononuclear cells isolated from PLNs collected on day 5.5, 9.5, and 13.5 of gestation, respectively, and mononuclear cells from placentas on day 13.5 of gestation. To clarify the identity of these CD86+ cells, FCM was also performed with CD3, CD19 and DX5 as markers for T cells, B cells, and NK cells, respectively. RESULTS: Both resorption rate and absolute number of resorption were significantly higher in group A (29.3%, 1.8±1.0) than those in group F (4.8%, 0.3±0.5, P<0.01, respectively). Similarly, both cell percentage and absolute number of CD45+CD86+ cells in PLNs collected on day 13.5 of gestation were significantly higher in group A than that in group F (27.5%±14.0% vs 12.3%±7.1%, and 1 362±687 vs 615±353, P<0.01, respectively). The CD45+CD86+ percentage was around 7.5% in virgin CBA/J mice, similar to the 10.6% in CBA/J×DBA/2 mice on day 5.5 of gestation, but increased dramatically to 23.9% by day 9.5 (P<0.01 vs virgin mice and P<0.05 vs CBA/J×DBA/2 mice on day 5.5), and remained at a higher level (27.5%) until day 13.5. However, this trend was not observed in group F during pregnancy. CONCLUSIONS: The increased CD45+CD86+ percentage on day 9.5, when resorption begins, may support the assumption that CD45+CD86+ cells play a role in the course of embryo resorption. Lymphocyte phenotypic analysis in the lymph nodes that drain the pregnant uterus may be helpful to assess the status of local immunity at the feto-maternal interface.     

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.     

High expression of CD34, Sca-1, Bcl-2 and desmin proteins in mouse muscle-derived stem cells

AIM: To explore the characteristics and phenotype of the muscle-derived stem cells(MDSCs) of mouse.METHODS: Skeletal muscle specimens were harvested from C57BL/6 mice of 2 weeks old. Preplate technique was applied to isolate MDSCs. The cellular growth status and morphology of the primary MDSCs were observed. The sphere-forming, proliferation and differentiation assays were performed and flow cytometry(FCM), immunocytochemistry and Western blotting were used to characterize MDSCs.RESULTS: The isolating process contained 6 consecutive days of differentiated attachment. MDSCs were round or short spindle-shaped cells. The growth curve of MDSCs showed logarithmic growth, and the doubling time of MDSCs was(9.69±2.08) h. Cloning efficiency of MDSCs was(13.35±2.54)%. When the cells at high confluence(>50%) or cultured with low concentration of serum(2% FBS), they tended to fuse to form myotubes. The observations of FCM and immunofluorescence showed that the phenotypic characteristics of MDSCs were antibody-positive for CD34, Sca-1, Bcl-2 and desmin(>90%). With increasing the level of cell purification, the upregulation of desmin expression and the downregulation of α-SMA expression from preplate 1 cells(PP1) to preplate 6 cells(PP6) were observed by Western blotting.CONCLUSION: The preplate technique can effectively isolate MDSCs. MDSCs express the antigens of CD34, Sca-1, Bcl-2 and desmin at high levels, and the 4 proteins can be used to identify MDSCs. With a high proliferating ability in vitro, MDSCs are ideal seed cells for tissue engineering.     

Effect of progesterone on SH-SY5Y cells injured by ATP

AIM: To investigate the neuroprotective effect of progesterone against adenosine triphosphate (ATP)-injured human neuroblastoma SH-SY5Y cells.METHODS: The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations. The cell viability was measured by CCK-8 assay. The membrane permeability was detected using fluorescent dye YO-PRO-1. Cytosolic Ca²⁺ concentration was measured with fluorescent dye Fluo-3/AM. The expression of purinergic P2X₇ receptor was assessed by Western blot.RESULTS: The viability of the SH-SY5Y cells was significantly decreased (P<0.05) and YO-PRO-1 uptake was obviously increased (P<0.05) in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1, 3, 5 and 7 mmol/L for 2 h. The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3, 10 and 30 nmol/L for 30 min (P<0.05) as compared with ATP group. YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced (P<0.05) by progesterone (30 nmol/L) or P2X₇ receptor antagonist KN-62 (500 nmol/L) pretreatment for 30 min, and no obvious difference between treatments with progesterone and KN-62 was observed. Cytosolic Ca²⁺ fluorescence intensity in normal group was a little, but that in ATP group was increased (P<0.05). Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca²⁺ induced by ATP (P<0.05). However, no obvious difference between treatments with progesterone and KN-62 was found. The expression of P2X₇ receptor in ATP group was significantly higher than that in control group (P<0.05), and progesterone inhibited ATP-induced P2X₇ receptor expression (P<0.05).CONCLUSION: Progesterone inhibits P2X₇ receptor expression, membrane pore formation, intracellular Ca²⁺ increase and cell death induced by ATP, so progesterone may protect SH-SY5Y cells against ATP-induced injuries.     

Effect of nitric oxide on adiponectin-induced inhibition of platelet aggregation in hyperlipidemia rats

AIM: To investigate the mechanism that adiponectin inhibits platelet aggregation via nitric oxide (NO) signaling pathway. METHODS: Adult rats were fed with normal or high-fat diet for 14 weeks. Their platelets were immediately isolated and treated with or without recombinant full-length adiponectin (rAPN). The platelet aggregation, NO and superoxide production, endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) expression, and antioxidant capacity were determined. RESULTS: Treatment with rAPN inhibited platelet aggregation induced by hyperlipidemia (P<0.05). Interestingly, total NO, a crucial molecule depressing platelet aggregate and thrombus formation, was significantly reduced, rather than increased in rAPN-treated platelets. Treatment with rAPN significantly decreased superoxide production by 62% (P<0.05) and increased antioxidant capacity by 38% (P<0.05) in hyperlipidemic platelets. Importantly, hyperlipidemia-induced reduction of eNOS phosphorylation and increase in iNOS expression were markedly reversed by rAPN treatment (P<0.05 and P<0.01, respectively). CONCLUSION: Adiponectin is an adipokine that inhibits platelet aggregation by enhancing eNOS activation and attenuating oxidative/nitrative stress including blockage of iNOS expression and superoxide production.     

Effects of autophagy inhibitor bafilomycin A1 on macrophages polarization

AIM:To investigate the effect of bafilomycin A1 (Baf A1) on polarization in mouse macrophages RAW264.7. METHODS:The macrophages RAW264.7 were treated with Baf A1, the concentration of M1/M2 polarization related cytokines were determined by ELISA. The markers of M1/M2 polarization in the macrophages were determined by flow cytometry. The formation of autophagicbody was observed by immunofluorescence. Western blot was used to detect the expression of autophagy related protein levels. RESULTS:The concentration of M1 related proinflammatory cytokine tumor necrosis factor-α (TNF-α) was increased significantly after Baf A1 intervention (P<0.01). However, the concentration of M2 related anti-inflammatory cytokines IL-10 and IL-13 showed no significant difference. The double positive rate of CD197 and HLA-DR (M1 marker) positive cells in Baf A1 treated group were significant higher than that in control group (P<0.05), indicated that Baf A1 induced polarization of macrophage to M1. The results of immunofluoresence showed that the autophagosomes formation was notable increased in Baf A1 group (P<0.05), meanwhile Western blot results showed the expression of autophay related protein LC3-Ⅱ but not P62 was marked up-regulated (P<0.05). For autophagy activator rapamycin (Rapa) treated group, autophagosome formation was also significant increased (P<0.05), but the expression of P62 was notable down-regulated (P<0.05). CONCLUSION:Baf A1 induces polarization of mouse macrophage RAW264.7 to M1, which may be related to the inhibitory effect on the formation of autolysosome.     

The biological characteristics of cytokine-induced killer cells

AIM:To investigate the biological characteristics of cytokine-induced killer (CIK) cellsin vitro.METHODS:The non-adhere peripheral blood monoclear cells from healthy donors were induced into CIK cells in the presence of IFN-γ, IL-1β, IL-2 and anti-CD3 antibody. LAK (lymphokine activated killer) cells were prepared as a control. The cellular phenotype were detected by FCM and immunocytochemistry and the cytotoxicity was measured by LDH release assay.RESULTS:After 2 weeks of induction, the proliferation rate of CIK cells reached a peak and the proportion of CD3⁺ population was above 95%, and then the cells growth entered to plateau phase at week 3. The proportion of CD3⁺CD56⁺ NKT subset cells was 16.5% on day 15 and it had no obvious variety between 2 and 4 weeks. Correspondingly, LAK cells grew slowly and had lower proliferation rate compared with the CIK cells (P<0.01). CIK cells showed higher specific lysis rates to BeL-7402 hepatoma cells than those of LAK cells at different effector to target ratio (P<0.01). Immunocytochemical staining showed the CIK cells highly co-expressed HLA-DR and CD54 antigens. The NKT cells were slightly bigger than CD3⁺CD56^- cells and a large quantity of pseudopodia were observed on their surface.CONCLUSION:The CIK cells have higher proliferation potency and stronger cytotoxicity to lyse tumor cells than LAK cellsin vitro.Within the span of time from 14 to 21 days, the proliferation rate and the proportion of CD3⁺CD56⁺ subset of CIK cells all reach peaks. Therefore, CIK cells in this period are suitable for clinical application.     

Phagocytosis of viable apoptotic cells inhibits the activation of T lymphocytes

AIM: To investigate whether viable apoptotic cells and phagocytosis of them affect the activation of T lymphocytes. METHODS: Ultraviolet irradiation was used to induce apoptotic cells in vitro and the model of phagocytosis of these cells was established. Cytokine TGF_β1 was detected by ELISA. The rate of apoptotic cells and phagocytosis of them were assessed by flow cytometry. Furthermore, flow cytometry was also employed to examine the expression of activation signs, such as CD69, CD25, CD71, of T lymphocytes under the intervention of apoptotic cells and macrophage which ingested apoptotic cells, to reflect whether the apoptotic cells and the phagocytosis of these cells could influence the activation of lymphocytes stimulated by Con A. RESULTS: Ingestion of apoptotic cells increased TGF_β1 secretion. Only the macrophages that had ingested apoptotic cells could suppress the activation of lymphocytes. The expression of the markers of lymphocytes activation such as CD69, CD25, CD71 had been restrained. These inhibition effects were abolished by monoclonal anti-TGF_β1 antibody. CONCLUSION: The macrophages that have ingested apoptotic cells inhibit expression of CD69, CD25 and CD71 of T lymphocytes stimulated by ConA. This effect is dependent on the increase in TGF_β1 secretion in local site.