Polysaccharide extracted from laminaria japonica aresch inhibits platelet activation and protects endothelial cells

AIM: To study the relationship between the effects of polysaccharide L01 extracted from laminaria japonica aresch on platelet activation and endothelial cells. METHODS: A rat model of endothelial injury was established via injecting adrenaline. The percentage of platelet adhesion was evaluated by filtration method, the activation of platelet aggregation was observed on a glass plate with collodion membrane, the content of vWF in rat plasma was measured by ELISA, the damaged degree of aortic vascular endothelial was evaluated by immunity histochemistry. RESULTS: The percentage of platelet adhesion and aggregation in model group were higher than those in NS group from the 3th and 4th day during the model made (P<0.05, P<0.01). The percentage in both L01 high-dose group (50 mg/kg) and low-dose group (10 mg/kg) at the 4th and 5th day was lower than that in model group (P<0.05, P<0.01). The content of vWF in rat plasma in model group was higher than that in NS group and in L01 high-dose group at 4th day (P<0.05). The same results were presented by the comparison among model group and NS group, both L01 high-dose group and low-dose group at the 5th (P<0.05). The measure of intact endodermis lengths (μm) stained by immunohistochemistry demonstrated that the length in model group was shorter than that in NS group (P<0.05), whereas the length in L01 high-dose group and low-dose group was obviously longer than that in model group (P<0.05) at the 4th and 5th day. CONCLUSION: The inhibitory effect of L01 on platelet activation may be related with its protective effect on vascular endothelial cells.     

Effect of CKLF1-C19 polypeptide on differentiation of human lung fibroblast into myofibroblast induced by TGF-β

AIM: To investigate the effect of CKLF1-C19 polypeptide (C19) on differentiation of human lung fibroblast (LFB) into myofibroblast (MFB) induced by TGF-β. METHODS: LFBs were cultured and identified. LFBs were treated with TGF-β (5 μg/L) to establish the cell model of LFB differentiate into MFB. The LFBs were divided into 6 experimental groups including control group, TGF-β group, and TGF-β plus different doses (1, 0.1, 0.01, 0.001 mg/L) C19 groups. The cell morphology, cell proliferation rate, and the expression of α smooth muscle actin (α-SMA) and collagen I were observed. RESULTS: Human primary LFB was successfully cultured and was confirmed by the method of immunofluorescence. TGF-β at 5 μg/L induced proliferation and differentiation of LFB. The mRNA levels of α-SMA and collagen I in TGF-β group were higher than that in control group (P<0.05).The cell proliferation rates, mRNA levels of α-SMA and collagen I, and the protein expression of α-SMA in 0.01 mg/L+TGF-β group and 0.001 mg/L+TGF-β group were markedly lower than those in TGF-β group (P<0.05). CONCLUSION: C19 at 0.01 mg/L and 0.001 mg/L effectively inhibits differentiation of LFB into MFB induced by TGF-β, thus inhibiting the process of airway remodeling and fibrosis to some extent.     

Effect of recombinant MIF on lung fibroblast proliferation and collagen synthesis in vitro

AIM: To investigate the influence and mechanism of recombinant macrophage migration inhibitory factor (rMIF) on fibroblasts. METHODS: MRC-5 fibroblasts were divided into two groups: the treated group was treated with rMIF (25-100 μg/L, 12 h, 24 h or 48 h) and the control was non-rMIF treatment. The activity of proliferation in both groups was investigated and compared by CCK-8 means. Synthesis of collagen in the culture supernatants was detected by the hydroxyproline. The expression of collagen type I mRNA was examined using RT-PCR analysis. The level of collagen type I protein induced by rMIF was quantified by Western blotting. RESULTS: The production of proliferation ratio of fibroblasts treated with 50 μg/L and 100 μg/L rMIF at 24 h or 48 h were increased obviously (P<0.05 or P<0.01). The collagen synthesis significantly increased after stimulation with 100 μg/L rMIF for 48 h (P<0.01). rMIF significantly increased the expression of collagen type I mRNA and protein in a dose dependent manner compared with control (P<0.05 or P<0.01). CONCLUSION: rMIF upregulates the proliferation of fibroblasts and has an effect on the production of collagen. These results suggest that MIF may play important roles in the pathogenesis of airway remodeling.     

Effect of nicotine on systolic and diastolic function of rat aortic smooth muscle cells

AIM:To observe the effects of nicotine on systolic and diastolic function of rat aortic vascular smooth muscle cells (VSMCs). METHODS:The primary rat aortic VSMCs were cultured in vitro. After exposed to nicotine at different concentrations for 24 h, the cytoskeleton of the VSMCs was stained with rhodamine-phalloidin,the photographs of the VSMCs in different experimental groups were taken and the surface area was measured to reflect the cell contractility. Collagen contraction method was also used to determine the effect of nicotine on the contractility of rat aortic VSMCs. RESULTS:The primary rat aortic VSMCs were successfully cultured. After the VSMCs were treated with nicotine (0.1 μmol/L, 1 μmol/L, 10 μmol/L and 100 μmol/L) for 24 h, the skeleton showed a significant contraction, and the cell plate shape was obviously enhanced in a concentration-dependent manner. The results showed that 10 μmol/L was the optimal concentration of nicotine for VSMCs (P<0.01). The collagen contraction method also showed that 10 μmol/L nicotine contracted the rat aortic VSMCs. With the increase in the nicotine action time, the maximum contraction effect was observed at 60 min (P<0.01). CONCLUSION:Nicotine has a strong contractile effect on VSMCs of rat aorta, and its contractile effect is dependent on concentration and time.     

Correlation analysis between visualization of hemorrheologic changes and platelet function in patients with acute coronary syndrome after percutaneous coronary intervention

AIM:To intuitionally observe the characteristics of blood rheology in the patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI) for 1 year to 3 years by micro-channel array flow analyzer (MC-FAN) combined with other platelet function indexes, and to explore the correlations between the test results of MC-FAN and platelet function. METHODS:This study brought 74 patients with ACS after PCI for 1 year to 3 years into test group, and 21 healthy subjects were enrolled as normal group. The levels of platelet aggregation test (PAgT), platelet adhesiveness test (PAdT), P-selectin, platelet-derived growth factor BB (PDGF-BB) and von Willebrand factor (vWF) were detected. MC-FAN HR300 was used to detect the transiting time (MC-FAN TT) of the blood passing through the model body capillaries. The differences of the test results between the 2 groups were compared, and the correlations between the results of MC-FAN and platelet function in the patients with ACS after PCI were also explored. RESULTS:Compared with normal group, the MC-FAN TT in test group was prolonged (P<0.01), the ability of erythrocyte deformation was weakened, and the leukocyte attaching the vascular wall and platelet adhesion and aggregation relatively increased. The levels of PAgT, PAdT, P-selectin and PDGF-BB in test group were all higher than those in normal group (P<0.01). No difference of vWF between the 2 groups was observed. The intergroup correlation analysis showed that there were correlations between MC-FAN TT and platelet function, in which 10 μL MC-FAN TT and 30 μL MC-FAN TT had the most significant correlation with P-selectin (r=0601, P<0.01; r=0334, P<0.01), 60 μL MC-FAN TT had the most significant correlation with PAgT (r=0527, P<0.01), and 100 μL MC-FAN TT had the most significant correlation with PAdT (r=0. 815, P<0.05). CONCLUSION:The visualization of hemorrheologic changes and platelet function in the patients with ACS after PCI are abnormal.There are correlations between MC-FAN TT and platelet function.The results of MC-FAN can objectively evaluate the blood rheology of the patients, and provide the reference for clinical treatment.     

Effect of Aurora protein kinase inhibitor VX-680 on homogeneity adhesion and migration in human hepatoma HepG2 cell

AIM:To study the effect of Aurora protein kinase inhibitor VX-680 on homogeneous adhesion and migration ability in human hepatocellular carcinoma cell line HepG2. METHODS:The HepG2 cell were divided into experimental group and control group, respectively. VX-680 was used in experimental groups at 3 concentrations (3.125 μmol/L group, 6.25 μmol/L group and 12.5 μmol/L group). DMSO was used in the control group. The effects of VX-680 at different concentrations on the adhesion ability of human hepatocellular carcinoma HepG2 cells were observed by cell slow aggregation test and separation experiment. The effects of VX-680 at different concentrations on the migration ability of HepG2 cells was detected by wound healing assay. The expression of E-cadherin in HepG2 cells was detected by Western blot. RESULTS:The results of the slow aggregation test showed that compared with the control group, the number of cell clumps formed in experimental groups was significantly decreased (P<0.01). The results of separation experiment showed that the ratio of N_TC/N_TE gradually decreased with the increased concentration of VX-680. The results of wound healing assay showed that as the concentration of VX-680 increased, the cell scratch healing ability gradually weakened compared with control group. The results of Western blot showed that the protein expression of E-cadherin in the HepG2 cells increased with the increased concentration of VX-680 (P<0.05). CONCLUSION:VX-680 increases the homogeneous adhesion and inhibits the migration of HepG2 cells.     

Bone marrow hematopoietic microenvironment in patients with systemic lupus erythmatosus

AIM: To investigate bone marrow hematopoietic microenvironment in patients with systemic lupus erythmatosus (SLE), and to explore the pathogenesis of SLE.METHODS: Bone marrow stroma cells were collected from SLE patients.Colony forming units of stromal cells, and expression of fibronectin, laminin and type IV collagen, adhesive molecules, and some cytokines were detected by cell culture, immunohistochemistry, flow-cytometry, ELISA, and RT-PCR assay, respectively.RESULTS: The number of the colony forming units of stromal cells and their morphology in SLE group were the same as the control group (P>0.05).We did not find any difference of the expression of fibronectin, laminin and type IV collagen in them.Expression of ICAM and VCAM were (56.4±14.8)% and (55.6±12.2)%, respectively, obviously higher than those in control group (P<0.01).IL-6 in bone marrow stromal cell culture suspension of SLE was higher than that in control group (P<0.01).SCF and SDF-1 were not different between two groups (P<0.05).MIP-1 and IFN-γ in SLE patients were obviously higher than those in control group (P<0.01).TGF-β was on the opposite (P<0.01).The later three cytokines were correlated to SLEDAI score (P<0.01).CONCLUSION: Bone marrow microenvironment of SLE patients was deficient in the expression of cell surface adhesion molecules and cytokine secretion, which contributed to the pathogenesis of SLE.     

Effect of integrin α2 on adhesion of neuroblastoma cells to co llagen

AIM:To study the effect of integrin α2 on adhesion of SK-N-SH neuroblastoma cells to collagen.METHODS:Adhesion of the SK-N-SH cells to immobilized collagen w as tested with various concentration of Mg2+,Ca2+ and with 10 μg/L anti-α2 monoclonal antibody (mAb) 6F1.A570 was detected as adhesio n cell numbers.RESULTS:Mg2+-dependent adhesion of SK-N-SH cells to type I collagen was increased significantly,with peak adhesion at concentration of 1 mmol/L Mg2+.A570 with or without Mg2+ was 0.59±0.03 and 0.25±0.01 respectively (P<0.01).No effect of Ca2+ was found on SK-N-SH cell adhesion.6F1 (10 mg/L) blocked the adhesion completely.CONCLUSION:Integrin α2 regulates neuroblastoma cells adhesion t o collagen and suggest that it may play a role in tumor cell migration,invasion and metastasis.     

Expression and significance of thrombospondin-1 in myocardium of type 2 diabetic cardiomyopathy rats

AIM: To investigate the expression and significance of thrombospondin-1 (TSP-1) in left ventricular myocardium of type 2 diabetic cardiomyopathy (DCM).METHODS: The rat model of DCM was established by eating a high-fat diet together with injection of low dose streptozocin (30 mg/kg) intrapertoneally.After 12 weeks,the content of collagen was quantified by Masson staining.The mRNA level of TSP-1 was determined by quantification real-time RT-PCR,while the protein level of TSP-1 was analyzed by Western blotting and immunohistochemistry.RESULTS: Compared with the control group,the content of collagen in the DCM group was increased greatly (11.01±3.05 vs 16.92±3.18,P<0.01).The mRNA and protein expressions of TSP-1 were significantly higher than those in control group (0.0089±0.0034 vs 0.0141±0.0037,P<0.05；96.38±16.80 vs 129.98±16.96,P<0.05).In DCM group,the mRNA and protein expressions of TSP-1 showed significantly positive correlations with the levels of fasting blood glucose and collagen (r=0.762,P<0.01; r=0.717,P<0.05; r=0.735,P<0.01; r=0.750,P<0.01).There was a significantly positive correlation of TSP-1 mRNA level with LVEDP (r=0.658,P<0.05).In contrast,there was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.605,P<0.05; r=-0.694,P<0.05).There was a significantly positive correlation of TSP-1 protein with LVEDP (r=0.716,P<0.05).There was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.633,P<0.05; r=-0.669,P<0.05).CONCLUSION: The increased expression of TSP-1 may play an important role in the development of myocardial interstitial fibrosis in DCM.     

Expression and significance of angiopoietin-2 and Tie-2 receptors in a rat model of acute lung injury

AIM: To explore the expressions and significance of angiopoietin-2 (Ang-2) and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie-2) receptors in a rat model of acute lung injury (ALI). METHODS: Wistar rats (n=42) were divided into control group (n=12) and cecal ligation and puncture (CLP) group (n=30). Control group underwent sham operation, and CLP group underwent cecal ligation and puncture to make the model of ALI. 12 h after sham operation or CLP, 6 rats in each group were killed, and arterial blood gas analysis and lung coefficient were tested. The expressions of Ang-2 and Tie-2 receptors in lung tissue were observed by immunohistochemical method. Blood samples of the rest rats were collected from vena caudalis, and Ang-2 levels were measured by enzyme linked immunosorbent assay (ELISA). The mortality rate in each group within 36 h was compared. The lung architecture was observed under microscope. RESULTS: The lung architecture in control group was clear and intact. Alveolar septum was thicker, blood capillary was congested, and neutrophils and macrophages were infiltrated in the lung tissue in CLP group. Tie-2 receptors were expressed in bronchial epithelial cells, smooth muscle cells and endothelial cells in control group. Besides the similar expression as control group, high expression of Tie-2 on neutrophils and macrophages in CLP group was observed. In the adhesion location of Tie-2 receptors positive inflammatory cells, there was stronger staining in endothelial cells. Ang-2 was expressed in smooth muscle cells, bronchial epithelial cells and endothelial cells in control group. The Ang-2 level in CLP group were higher than that in control group ［(8.14±1.74) μg/L vs (4.63±0.49) μg/L, P<0.01］, and the Ang-2 level of dead rats was higher than that of survival rats within 36 h in CLP group ［(8.95±1.61)μg/L vs (6.80±0.96)μg/L, P<0.01］. Oxygen partial pressure in control group was lower (P<0.01) and lung coefficient was higher (P<0.01) than that in CLP group. CONCLUSION: Ang-2 and Tie-2 receptors may participate in the pathophysiology of ALI, and Ang-2 level is correlated with mortality.     

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.     

Effects of hypertriglyceridemia and fenofibrate on CD40L expression in platelets

AIM: To observe the effects of hypertriglyceridemia and fenofibrate on CD40L expression in platelets in vitro and in vivo. METHODS: In vivo experiments, according to its own strict standards, 20 patients were respectively selected for hypertriglyceridemia group and control group, before and after treatment of fenofibrate for hypertriglyceridemia patients. The CD40 ligand positive rates of platelets by flow cytometry and plasma soluble CD40 ligand by ELISA were examined under the same conditions as control group. The CD40L and sCD40L in each group were compared. In in vitro experiments, all 6 objects plasma was chosen in the same condition except for triglyceridemia, after the co-incubation of these plasma with the same healthy platelets was performed and the interference with wy14643, the CD40 ligand positive rate of platelets by flow cytometry and total platelets CD40 ligand protein content by Western blotting were examined under the same conditions in all objects. The CD40L positive rate and total CD40L content in each group were compared, respectively. RESULTS: The platelet CD40L positive rate and plasma sCD40L concentration in hypertriglyceridemia group were significant higher than those in control group (P<0.01). Followed the TG concentration decreased, the platelet CD40L positive rate and plasma sCD40L concentration decreased after the treatment of fenofibrate, the same as the total platelets CD40L content which was significant higher in hypertriglyceridemia group than that in control group in vitro (P<0.05). No effect of wy14643 on the total CD40L content expression was observed in vitro. CONCLUSION: Hypertriglyceridemia plasma stimulates immune-activation of platelets both in vitro and in vivo. sCD40L may mainly come from CD40L on platelet membrane. PPARα activator of fenofibrate may inhibit the immune-activation of platelets by reducing the concentration of plasma TG, but PPARα activator WY14643 cant inhibit the expression of CD40L and CD40L in vitro.     

Effects of Chlamydia pneumoniae infection on adhesion and migration of vascular smooth muscle cells

AIM: To investigate the effects of Chlamydia pneumoniae (C.pn) infection on the adhesion and migration of vascular smooth muscle cells (VSMCs). METHODS: Primary VSMCs, isolated from the aorta of SD rats, was infected with C.pn after the culture and propagation of C.pn in the HEp-2 cells. The morphological characteristics of C.pn inclusions in VSMCs were examined under the fluorescence microscope with acridine orange (AO) staining. The specific DNA fragment of C.pn was identified by polymerase chain reaction (PCR). Cell adhesion assay was performed to investigate the effect of C.pn infection on the adhesion of VSMCs to collagen I. Wound-healing assay and Transwell assay were performed to detect the effects of C.pn infection on VSMCs migration. RESULTS: The C.pn inclusions and infection spots were observed in VSMCs under fluorescence microscope with AO staining. The inclusions were larger than infection spots in volume, but were much lower in count. A 437 bp-specific fragment of C.pn DNA was detected in C.pn-infected VSMCs by PCR. In the cell adhesion assay, the absorbance values in C.pn infection group were far higher than those in control group 2 h after infection (P<0.01). The cell adhesion ratio in C.pn infection group was 134.38%. The migration distance of VSMCs infected with C.pn was significantly longer than that of the control cells 24 h after infection by a wound-healing assay (P<0.05). More migratory VSMCs infected with C.pn for 24 h were found in a Transwell assay than those in control group (P<0.01). CONCLUSION: C.pn infection significantly promotes the adhesion and migration of VSMCs.     

Effect of recombinant interleukin-13 on 3T3 fibroblasts in vitro

AIM: To investigate the influence and mechanism of recombinant interleukin-13 (rIL-13) on fibroblasts. METHODS: 3T3 fibroblasts were divided into two groups: the treated group was treated with rIL-13 (80 μg/L, 24 h or 48 h) and the control was without rIL-13 treatment. Transmission electron microscope and Hoechst kit were used to observe morphology of 3T3 fibroblasts in both groups. The activity of proliferation in both groups was investigated and compared by MTT means. Western blot was used to analyze the level of collagen type I induced by rIL-13 in fibroblasts. The levels of IL-6 and IL-8 in the culture supernatants were determined by radioimmunoassay. RESULTS: The more ribosomes and mitochondrions, as well as bigger nuclei were found in the treated group. The production of IL-6 and IL-8, and proliferation ratio of fibroblasts treated with rIL-13 for 24 h or 48 h were increased obviously, compared with the control (P<0.01). The expression of collagen type I protein in treated groups was also significantly higher than that in control (P<0.01). CONCLUSION: rIL-13 upregulates the proliferation of fibroblasts. rIL-13 also has an effect on the production of proinflammatory factors and collagen type I. Taken together, these results suggest that IL-13 may play important roles in the pathogenesis of tissue fibrosis.     

Role of fibrinogen activity in the progress of coronary artery disease

AIM:To detect the role of fibrinogen activity (Fa) in the progress of coronary artery disease (CHD).METHODS:Fa was measured with hemorheology methods in patients with CHD stable phase (n=30) and angina pectoris (n=27).RESULTS: (1) Levels of plasmatic fibrinogen and plasmatic viscosity in patients with CHD were higher than that of control group (P＜0.01,P＜0.05).(2)Fa and platelet aggregation activity (Pt max, Pt H, Pt K) in patients with CHD angina pectoris were very much higher than that of control group (P＜0.01, respectively).(3)There was a negative correlation between PT max, Pt H and Fa(r=-0.8379,P＜0.01;r=-0.8784,P＜0.01 respectively) in patients with CHD angina pectoris.CONCLUSION: Fa may play a role in the progress of CHD.     

he effects of constant magnetic field on apoptosis,adhesion molecule expression in endothelial cells and their adhesion rates with monocytes

AIM: To investigate the effects of constant magnetic field on apoptosis, secretion and expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC), and their adhesion rates with THP-1 monocytes induced by angiotensin Ⅱ (AngⅡ).METHODS: The third passage of cultured HUVEC was used.There were six groups: control group, Ang Ⅱ (10^-6 mol/L) group, Ang Ⅱ with 1, 5, 10 or 20 gausses of constant magnetic field group.Samples were collected 24 h after incubation with or without magnetic field.Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and propidinm iodide staining with flow cytometry.Secretion and expression of ICAM-1 and VCAM-1 were detected by ELISA and immunocytochemistry, respectively.Adhesion rate between HUVEC and THP-1 was measured by counting method.RESULTS: Ang Ⅱ at concentration of 10^-6mol/L induced apoptosis in HUVECs (P<0.05 vs control), whereas in 1, 5, 10 and 20 gausses group, apoptosis of HUVECs was significantly lower than that in Ang Ⅱ group (P<0.05).Ang Ⅱ at concentration of 10-6 mol/L significantly increased secretion and expression of ICAM-1 and VCAM-1 (P<0.05 vs control), whereas secretion and expression of ICAM-1 and VCAM-1 in 1, 5, 10 and 20 gausses group significantly decreased, compared with Ang Ⅱ group (P<0.05).The adhesion rates between HUVEC and THP-1 significantly increased 24 h after incubation of HUVEC with Ang Ⅱ (P<0.05 vs control), in contrast, the adhesion rates between HUVEC and THP-1 in 1, 5, 10 and 20 gausses group significantly decreased, compaed with Ang Ⅱ group (P<0.05).CONCLUSIONS: One gauss to 20 gausses of constant magnetic field antagonizes the effects of Ang Ⅱ on HUVEC, decreases apoptosis and expression of ICAM-1 and VCAM-1 in HUVEC, and also decreases the adhesion rates between HUVEC and monocytes induced by Ang Ⅱ.     

Effect of nicotine against apoptosis of rat cortical neurons induced by colchicines

AIM:To explore the mechanism of nicotine against the apoptosis induced by colchicines in rat cortical neurons. METHODS:Cortical neurons were cultured from newborn Sprague-Dawley (SD) rats (less than 12 h). The rate of apoptosis was measured by Hoechst33258 fluorescence staining in the neurons, and the activity of Akt473 was analyzed by assay kit Akt473. RESULTS:The apoptosis of cortical neurons can be induced by 0.1 μmol/L colchicine. The phosphorlation of Akt 473 decreased significantly (1/3 times of the control group, P<0.01). However, when cortical neurons pretreated with 10 μmol/L nicotine for 2 h were cultured with 0.1 μmol/L colchicine for 24 h, the rate of apoptosis decreased from 62% to 38%. The phosphorlation of Akt473 increased significantly in a bell-shape time-dependent manner, which was respectively 1.3, 3.7, 2.4, 2.1 and 1.9 times compared with the control group (P<0.01). CONCLUSION:By activating the signal pathway of Akt473, nicotine may attenuate the apoptosis of cortical neurons induced by colchicines.     

Application of anti-CD20 antibody in hematopoietic stem cell transplantation for sensitized recipients

AIM: To find a strategy for enhancing engraftment of hematopoietic stem cell in sensitized recipients and to study the effects of anti-CD20 antibody in hematopoietic stem cell transplantation. METHODS: BALB/c mice were sensitized by transfusions of allogeneic spleen cells on 14 d and 7 d. Anti-CD20 antibody (2 mg/mouse) was intravenously injected into sensitized recipients on 11 d. The recipients were used as experimental group, while RPMI-1640 medium (0.2 mL/mouse) was used as control. The sera and splenocytes obtained from the recipients were tested for donor reactive antibody and CD19+ B cells on 0 d. In addition, the recipients were transplanted with 1×107 C57BL/6 bone marrow cells after lethal irradiation on 0 d. The survival rates were observed and blood counts were studied post transplantation. RESULTS: The cytotoxic index in the experimental group and control group were (37.00±3.46)% and (51.80±3.49)%, respectively, and the differences were significant (P<0.01). The percentages of CD19+ B cells in experimental group and control group were (17.32±3.02)% and (34.26±2.87)%, respectively, and the differences were significant (P<0.01). All the recipients in both experimental group and control group died about 14 d post transplantation. The median time was 13 d and 11 d in experimental group and control group, respectively, and no significant difference was found between these two groups (P>0.05). Moreover, a rapid disappearance was observed in the white blood counts, hemoglobin, and platelet of dying animals, indicating the animals died from hematopoietic failure. CONCLUSION: Anti-CD20 antibody is able to deplete B cells and reduce the level of antibody in sensitized recipients, but it can’t enhance the engraftment of allogeneic hematopoietic stem cells in the sensitized recipients.     

Quercetin inhibits endothelin-1-induced T-type Ca2+ channel expression in human umbilical arterial smooth muscle cells

AIM: To investigate the effect of quercetin on endothelin-1-induced T-type calcium channel(TCC) expression in primary cultured human umbilical arterial smooth muscle cells for exploring the protective role of quercetin in cardiovascular system. METHODS: Human umbilical arterial smooth muscle cells were verified by immunocytochemistry. The cells in 2-3 passages were used and randomly divided into control group, quercetin alone group, model group and experimental group. The cells in control group were cultured without any drugs for 24 h. The cells in quercetin alone group were cultured with 80 μmol/L quercetin for 24 h. The cells in model group were cultured with ET-1 at the concentration of 100 nmol/L for 24 h. The cells in experimental groups were pretreated with quercetin for 1 h, then coincubated with 100 nmol/L ET-1 for 24 h. The concentrations of quercetin used in this study were 20, 40and 80 μmol/L, respectively. The expression of α_1G, a TCC major subunit, was assayed at mRNA and protein levels by RT-PCR and Western blotting, respectively. The TCC currents(I_caT) were detected by the technique of whole-cell patch-clamp. RESULTS: Compared with control and experimental group, I_CaT density (P<0.01) and the expression of α_1G at mRNA (P<0.05) and protein (P<0.01) levels in model group were significantly increased. No significant difference in the results of quercetin alone group and control group was observed. CONCLUSION: The protective roles of quercetin in cardiovascular functions are related to the depressive effects of quercetin on ET-1-induced increase in both I_CaT density and the expression of α_1G at mRNA and protein levels in cultured human vascular smooth muscle cells.