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1.
AIM: To investigate the effects of nicotine on activation of PMNs, adhesion of PMNs-HUVEC and expression of ICAM-1 mRNA in HUVEC. METHODS: Activation of PMNs was measured by detecting the activity of β-glucuronidase and lysozym of PMNs. Adhesion of PMNs and HUVEC was observed. Northern blot was conducted for quantitating ICAM-1 mRNA. RESULTS: Nicotine could increase the activity of β-g [(8.76± 1.01)μg/107·h vs(14.87±2.00)μg/107·h,P<0.05]and Lysozym [(20.0±1.5)μg/107·h vs(36.5±4.4)μg/107·h,P<0.05], and also could promote adhesion of PMNs-HUVEC(38.5±9.8 vs 61.0±4.4,P＜0.05). The expression of ICAM-1 mRNA was induced by nicotine in dose-dependent fashion (10-5-10-3mol/L).After a 2 h treatment of HUVEC with nicontine(10-4mol/L), the level of ICAM-1 mRNA is above the control(1.23 vs 1.63) and the highest level (2.03) is at a 12 h treatment. 764-3 can obviously counteract the above effect of nicotine. CONCLUSIONS: Nicotine could activate PMNs, enhance adhesion of PMNs-HUVEC and increase the expression of ICAM-1 mRNA in HUVEC.  相似文献   

2.
AIM:To study the effects of oxidized high-density lipoprotein (oxHDL) on the expression of monocyte chemoattractant protein-1(MCP-1) and intercellular adhesion molecule-1(ICAM-1) and intracellular free calcium concentration ([Ca2+]i) level in cultured human umbilical venous endothelial cells(HUVECs). METHODS:The MCP-1 protein content in the medium of conditioned HUVEC was measured by ELISA, and the ICAM-1 on HUVECs was detected by indirect immunofluorescence, and [Ca2+]i was determined by Fluo-3/AM, the injury of cells was observed by scanning electron microscopy (SEM).RESULTS:oxHDL could induce the expression of MCP-1 and ICAM-1 in HUVECs. In oxHDL group (HUVECs were incubated with 100 mg protein/L oxHDL for 24 h), the levels of MCP-1, ICAM-1 and [Ca2+]i increased by 160%, 60% and 70% respectively compared with the control group (P＜0.01). When HUVECs were incubated with 300 mg protein/L oxHDL for 24 h, cells were injured obviously. CONCLUSION:By inducing the expression of ICAM-1 and MCP-1 in endothelial cells, oxHDL may promote monocyte-endothelium adhesion and monocyte migration to intima, it may promote atherosclerosis as oxidized low-density lipoprotein (oxLDL).  相似文献   

3.
AIM:To study the effect of cholecystokinin octapeptide(CCK-8) on lipopolysaccharide (LPS)-stimulated pulmonary interstitial macrophage(IM)in vitro.METHODS:Pulmonary IM were isolated and cultured in the presence of LPS, CCK-8, proglumide(the antagonist of CCK receptors) and vehicle alone or together. The expression of mCD14 protein was assayed by flow cytometry, and sCD14 in the supernatant was analyzed semi-quantitatively by Western blot, and TNF-α in the supernatant was detected with ELISA.RESULTS:CCK-8, at concentrations from 10-7mol/L to 10-6mol/L inhibited significantly the expression of mCD14, the release of sCD14 and TNF-α to the supernatant up-regulated by LPS(1mg/L). The effect of CCK-8 was inhibited by proglumide. CONCLUSION:CCK-8 modulated negatively several functions of LPS-stimulated pulmonary IM through CCK receptors, which may be one of the mechanisms for CCK-8 to alleviate the inflammation in lung tissues during endotoxemia.  相似文献   

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5.
AIM: To explore the role of basic-fibroblast growth factor (bFGF) in the development of pulmonary hypertension induced by hypoxia. METHODS: 1) The pulmonary arteries of SD rats with hypoxia for one and two weeks were isolated, from which the total RNA were extracted by acid guanidinium thiocyanate-phenol-chlorform .Then the levels of mRNA were measured by RT-PCR. 2) About 3mm-long arterial rings cut from SD rat pulmonary arterial stem were suspended between stainless steelhooks in chamber with warmed (37℃) Kreb's solution. Different concentrations of bFGF were added in a cumulative fashion into the chamber where the rings were suspended. The cumulative concentration response curve was obtained. RESULTS: 1)The levels of bFGF mRNA in pulmonary artery of rats with hypoxia were increased significantly compared with those that without hypoxia (2578±384 counts·min-1 (control) vs 5303±756 (hypoxia) for 1 week and 4054±547 (hypoxia) for 2 weeks, P all ＜0.05). 2) bFGF at concentrations ranged from 5.56×10-10~2.78×10-7mol/L caused dose-dependent contraction of vessel rings of rat pulmonary artery (r=0.695,P＜0.05), with EC50 being 2.62×10-7mol/L. CONCLUSION: bFGF may play an important role in the hypoxic pulmonary hypertension.  相似文献   

6.
AIM:To investigate the effect of platelet inhibitor from Agkistrodon halys venom (AHV-PI) on lipopolysaccharide (LPS) induced human umbilical vein endothelial cells (HUVECs) injury in vitro, and to explore its mechanism. METHODS:Cultured HUVECs were induced inflammatory injury by LPS (1 mg/L). The experiment was divided into blank control group, LPS group, AHV-PI group and AHV-PI+LPS group. The viability of HUVECs was measured by MTT assay. The morphological changes of HUVECs were observed under inverted microscope. The optimum concentration of AHV-PI at 5 mg/L was selected. Flow cytometry was used to detect apoptosis of HUVECs. Immunohistochemical method was used to observe the expression of tissue type plasminogen activator (t-PA) and plasminogen activator indhibitor-1 (PAI-1) of HUVECs. ELISA was used to detect the concentrations of intercellular adhesion molecule-1 (ICAM-1) and tissue factor (TF) in the supernatant. The activation and translocation of NF-κB subunit p65 were observed by immunofluorescence staining. RESULTS:The HUVECs were spindle shaped, the ratio of length to width was increased, the cells were fibroblast-like, and granular substance appeared in the cytoplasm in LPS group. The viability and morphological changes of HUVECs were not significantly affected as treated with AHV-PI at concentration of 0~5 mg/L, but the viability of HUVECs induced by LPS was inhibited and the morphological changes were alleviated. Compared with the blank control group, the levels of TF and ICAM-1 in the supernatant increased, and the expression of t-PA and PAI-1 in the HUVECs was decreased in LPS group. Compared with the LPS group, the contents of TF and ICAM-1 in the supernatant were significantly decreased, the expression of t-PA and PAI-1 in the HUVECs was increased and the expression of nucleus NF-κB p65 was decreased in AHV-PI+LPS group (P<0.05). CONCLUSION:AHV-PI reduces HUVECs damage. The protective mechanism is related to the inhibition of cytokine secretion and NF-κB activation.  相似文献   

7.
AIM:To observe the effects of angiopoietin 4 (Ang-4) on lipopolysaccharide (LPS)-induced injury of human umbilical vein endothelial cells (HUVECs). METHODS:The EnVision immunohistochemical method was used to identify the HUVECs. After pre-treated with different doses of Ang-4 for 0.5 h, HUVECs was exposed to LPS at concentration of 10 mg/L for 24 h. The cell viability was evaluated by MTT assay. The content of tumor necrosis factor-alpha (TNF-α) in the supernatant and the concentrations of intracellular and supernatant von Willebrand factor (vWF) were detected by ELISA. The mRNA levels of Toll-like receptor 4 (TLR4), NF-κB p65 and TNF-α were determined by real-time PCR. RESULTS:Factor Ⅷ in the cytoplasm was positive in the HUVECs.Compared with normal group, LPS reduced the cell viability (P<0.01), and significantly increased the secretion of TNF-α and vWF (P<0.01). The mRNA expression of TLR4, NF-κB p65 and TNF-α also increased (P<0.01). Ang-4 at concentration of 100 μg/L enhanced the cell viability (P<0.01), reduced the content of vWF and TNF-α, and inhibited the LPS-induced increases in the mRNA levels of TLR4, NF-κB p65 and TNF-α (P<0.01). CONCLUSION: Ang-4 antagonizes LPS-induced damage in HUVECs by inhibiting TLR4-NF-κB p65-TNF-α signaling pathways.  相似文献   

8.
AIM:To observe the effect of adrenomedullin(ADM)on proliferation of vascular smooth muscle cells(VSMC) induced by urotensin Ⅱ(UⅡ). METHODS:DNA synthesis of cultured rat aortic VSMC was measured by [3H]-TdR incorporation. The activities of mitogen activated protein kinase(MAPK) were determined by isotope tagged with [γ-32P]-ATP. RESULTS:UⅡ(10-8mol/L) significantly increased [3H]-TdR incorporation of VSMC and MAPK activities by 38%(P＜0.05) and 260%(P＜0.01) respectively compared with control group. Compared with UⅡ group, 10-10,10-9,10-8mol/L ADM decreased [3H]-TdR incorporation of VSMC by 7%(P＞0.05), 32%(P＜0.05)and 41%(P＜0.01),respectively, and diminished MAPK activities by 24%(P＞0.05), 32%(P＜0.05)and 36%(P＜0.05),respectively. CONCLUSION:ADM inhibits proliferation of VSMC induced by urotensin Ⅱ through inhibiting MAPK activation.  相似文献   

9.
AIM:To purify human yolk sac mesenchymal stem cells (hYS-MSC) and investigate its adipogenic differentiation potential. METHODS:hYS-MSC were separated from yolk sac and purified via passaged culture. Flow cytometric analysis was used to identify the phenotype of hYS-MSC and the alkaline phosphatase(AKP) expression of hYS-MSC was also tested. Adipogenic differentiation of hYS-MSCs was induced by 10 mg/L insulin, 10-5mol/L indomethacin and 10-6mol/L dexamethasone. Oil Red O was used for fat staining. RESULTS:hYS-MSCs were purified at passages 2 or 3. Flow cytometric analysis showed the phenotype of purified YS-MSCs was uniformly positive for CD29, CD44, CD105, and CD166, and negative for reactivity to antigens CD34, CD45, or CD86. hYS-MSCs were weakly but clearly positive in AKP. Adipogenic differentiation of YS-MSCs was induced by 10 mg/L insulin, 10-5mol/L indomethacin and 10-6mol/L dexamethasone. Accumulation of lipid-rich vacuoles positive in oil red O staining within the cells were appeared and nuclears were pushed to one side of the cells during the period of induction. CONCLUSION:The phenotype of hYS-MSC is coincident with adult human mesenchymal stem cells. hYS-MSC can be induced to differentiate into adipocytes in vitro.  相似文献   

10.
AIM:To study the effect of Aima recipe (AM), a traditional Chinese medicine, on the protein and mRNA expression of TNFα and ICAM-1 in the bronchus of rats with chronic bronchitis(CB).METHODS:15 male Wistar rats were randomly divided into three groups: control group, chronic bronchitis (CB) group, CB plus AM group. The protein and mRNA expression of TNFα and ICAM-1 were assayed by immunohistochemistry and in situ hybridization methods.RESULTS:TNFα, ICAM-1 protein and mRNA were more strongly expressed in the area of bronchial epithelium in the CB group compared with control group (P＜0.01), treatment with AM significantly decreased their expressions(P＜0.05). CONCLUSION:Downregulating the expressions of TNFα and ICAM-1 mRNA and protein in the area of bronchial epithelium may be one of the mechanisms underlying therapeutic effect of AM on chronic bronchitis.  相似文献   

11.
AIM:To explore the effects of hydrogen sulfide (H2S) on proliferation of vascular smooth muscle cells (VSMC) stimulated by endothelin (ET-1, 10-7mol/L) and mitrogen-activated protein kinase (MAPK) activity in VSMCs.METHODS:Cultured VSMCs were divided into six groups: (1) control group, (2) serum group, (3) endothelin group, (4) NaHS groups, (5) serum+NaHS group, and (6) endothelin+NaHS group. VSMC proliferation was measured by[3H]-TdR incorporation and MAPK activity in VSMC was determined by radioactivity assay.RESULTS:ET-1 increased VSMC[3H]-TdR incorporation by 2.39 times (P＜0.01) and MAPK activity by 1.62 times(P＜0.01), as compared with control. H2S (5×10-5-5×10-4mol/L) decreased VSMC[3H]-TdR incorporation and MAPK activity by 16.8%-37.4% and 7.4%-33.6%, respectively (P<0.05 or P＜0.01).CONCLUSION:This study demonstrates that H2S inhibits ET-1-induced proliferation of VSMC, which might be mediated by the inhibition of MAPK.  相似文献   

12.
AIM: To investigate the role of MEK1/2, a subfamily of mitogen activated protein kinase-kinase (MAPKK), in expression of intercellular adhesion molecule-1 (ICAM-1) induced by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVECs). METHODS: Expression levels of ICAM-1 mRNA and its protein were assayed using RT-PCR and Western blotting, respectively, in HUVECs pretreated with different concentrations of LPS for different times with or without PD98059, a specific inhibitor of MEK1/2. RESULTS: LPS up-regulated the expression of ICAM-1 mRNA and its protein in HUVECs in a concentration- and time-dependent manners. The expression levels of ICAM-1 mRNA and its protein began to elevate at 2 h after LPS treatment, and reached nearly a peak value at 6 h after LPS (100 μg·L-1) treatment. PD98059 (10 μg·L-1) significantly inhibited LPS-induced expression of ICAM-1 mRNA and protein, the expression inhibitory rates of which were 54.4% and 44.9%, respectively (P<0.01). CONCLUSION: Modulation of MEK1/2 signaling pathway might be a new and useful strategy for the prevention and treatment of vascular endothelial injury induced by LPS.  相似文献   

13.
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14.
AIM:To investigate the role of nuclear factor κB (NFκB) in the induction of iNOS gene by TNFα and LPS in endothelial cells and the effect of antioxidant on the induction of iNOS. METHODS:Nitrite was determined based on Griess reaction. iNOS mRNA was analyzed using Northern blot. NFκB in the cell nucleole was detected with electrophoretic mobility shift assay.RESULTS: (1)NO production and iNOS mRNA expression induced by LPS and TNFα was blocked by pyrrolidine dithiocarbamate(PDTC) or N-acetylcysteine(NAC). (2)LPS and TNFα triggered the activation and translocation of NFκB, and PDTC or NAC inhibited the activation of NFκB induced by LPS and TNFα. CONCLUSIONS:(1)The induction of iNOS gene by TNFα and LPS is dependent on the activation of NFκB.(2)Antioxidants may inhibit the induction of iNOS gene through the inhibition of NFκB activation.  相似文献   

15.
AIM:To evaluate effects of inhaled nitric oxide(iNO) on adhesion molecule CD11b expression on lung neutrophils in experimental meconium aspiration syndrome(MAS) rabbits treated with conventional mechanical ventilation under room air or 100%O2. METHODS:Animals were randomly allocated to 8 groups(n=48) of 6 each: two MAS model groups(under room air or 100%O2 without iNO treatment), 6 treatment groups were treated with continuous NO inhalation at a dose of 0.2×10-6mol/L, 0.33×10-6mol/L or 0.67×10-6mol/L respectively for 12 hours under room air or 100%O2. Mean systemic arterial pressure(SAP) and methemoglobin (MeHb) were performed at basement time, 0, 2, 4, 12 hours. Expression of CD11b on neutrophils in the bronchoalveolar lavage fluid(BALF) was detected with flow cytometry. RESULTS:SAP, MeHb at different time among different groups were within the normal scale. CD11b expression on the neutrophils in the BALF significantly decreased in groups of inhalation 0.33×10-6 mol/L or 0.67×10-6 mol/L NO, compared with the two MAS model groups. (x±s: under 21%O2, 0.33×10-6 mol/L NO, 121±20 υs 392±204; 0.67×10-6 mol/L NO, 112±30 υs 392±204;under 100%O2, 0.33×10-6 mol/L NO, 113±24υs293±65; 0.67×10-6 mol/L 102±114 υs293±65, P<0.05). 0.2×10-6mol/L NO inhalation did no effect on CD11b expression. (x±s:21%O2, 190±101 υs 392±204; 100%O2, 222±85 υs 293±65; P>0.05). No statistic difference was observed between groups inhaled 0.33×10-6 mol/L NO and 0.67×10-6mol/L NO. CONCLUSION:0.33×10-6 mol/L or 0.67×10-6 mol/L NO inhalation down-regulated the CD11b expression on the neutrophils in BALF to reduce the sequestration of neutrophils in rabbit lung.  相似文献   

16.
AIM:To investigate the effect of endothelin(ET) on taurine transportation in rat cardiac myocytes in vitro.METHODS: In cultured cardiac myocytes of neonatal rats, taurine transportation velocity was measured by radio-ligand method. RESULTS: ET(10-10-10-8 mol/L) could inhibit taurine transportation in a dose-dependent manner.10-10,10-9 and 10-8mol/L of ET significantly decreased taurine transpotation by 13%, 38% and 71%, respectively (P＜0.01), compared with control group. H7,BQ123 and Pre-PMA can reverse the inhibition of ET on taurine transportation dramatically(P＜0.01).CONCLUSION:The binding of ET and ET-A receptor might activate protein kinase C,which inhibits taurine transportation in cultured myocytes of rats.  相似文献   

17.
AIM: To study the effects of cyproheptadine (Cyp) and anisodamine (Ani) on the changes of intracellular free Ca2+ concentration ([Ca2+]i) induced by tumor necrosis factor (TNFα) in single endothelial cells, and to explore the mechanisms of TNFα mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothelial cell strains (ECV304) were seed in 35 mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [Ca2+]i in single endothelial cell was determined by laser scanning confocal microscopy (LSCM). RESULTS: [Ca2+]i in single endothelial cell after stimulation of TNFα rapidly increased in a dose-dependent manner and approached the peak value within 60 seconds, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca2+]i elevation was more obvious in nuclear than in cytoplasma, and decreased slowly. Cyp (3×10-5, 6×10-5 mol/L) and Ani (2×10-5, 4×10-5 mol·L-1) markedly inhibited TNFα (1.2×10-9 mol·L-1)-induced [Ca2+]i elevation. CONCLUSIONS: TNFα markedly induces elevation of [Ca2+]i in single endothelial cell, it may be an important mechanism of TNFα-induced shock and tissue injury. Cyp and Ani obviously suppress TNFα-induced [Ca2+]i elevation, which probably is one of the mechanisms of their antishock effects.  相似文献   

18.
AIM: To study the effect of meglumine cyclic adenylate (MCA) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes in vitro. METHODS: The whole bone marrow adherent culture method was used to isolate, culture and amplify the BMSCs. The surface markers of BMSCs were determined by flow cytometry analysis. MCA at concentrations of 10-2 mol/L, 10-3 mol/L, 10-4 mol/L, 10-5 mol/L, 10-6 mol/L and 10-7 mol/L was added to the culture medium containing the second generation of BMSCs.5-Azacytidine(5-Aza) was used as a positive control. The cell viability was measured by MTT method.The cAMP content in BMSCs was detected by ELISA. The mRNA expression of GATA-4, Cx43 and β-MHC in MCA group and MCA+H89 (a PKA inhibitor) group was measured by SYBR-RT-PCR. The differentiation effects of MCA and 5-Aza were compared by flow cytometry. RESULTS: Most of the BMSCs expressed CD44 and CD71, and did not express CD45. MCA inhibited the viability of BMSCs in a time-and dose-dependent manner, and MCA atthe concentration of 10-2 mol/L showed particularly remarkable effect. MCA significantly increased intracellular cAMP level in BMSCs in a concentration-dependent manner. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group were significantly higher than that in blank group (P<0.05), and the highest effect was under the condition of MCA induction at the concentration of 10-3 mol/L for 3 days. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group was higher than that in 5-Aza group and H89+MCA group (both P<0.05). Differentiation rate in MCA group was slightly higher than that in 5-Aza group (20.24%±1.02% vs 18.39%±0.58%, P<0.05). CONCLUSION: MCA stimulates BMSCs to increase intracellular cAMP production and inhibits the viability of BMSCs, thus promoting the mRNA expression of GATA-4, β-MHC and Cx43 through the cAMP/PKA signaling pathway.  相似文献   

19.
AIM: To explore the expression of CD14 in rat Kupffer cells (KCs). METHODS: In rat KCs induced by LPS or the mediators from KCs induced by LPS, the changes of CD14 expression were measured by RT-PCR and immunohistochemistry.The expressions of TNFα mRNA、IL-6 mRNA or the concentrations of TNFα、IL-6 were estimated by in situ hybridization and radioimmunoassay, respectively. RESULTS: LPS increased the expression of CD14 in KCs in a dose-dependent fashion (LPS, 1 μg/L-10 mg/L) and in a time-dependent fashion(0.5 h-24 h, peaked at 3-6 hours). While the expression of CD14 in KCs stimulated by the active mediators from KCs which had been exposed to LPS 1 hour were obviously increased. CONCLUSIONS: There was a close relationship between LPS or the active mediators from KCs induced by LPS and the expressions of CD14. It is implied that the increase in CD14 expression may be induced by LPS and the cytokines produced by KCs, it also reveals that there is a auto-regulated loop in CD14 expression.  相似文献   

20.
AIM:To study the effect of farrerol (Far) on nicotine-induced proliferation of rat pulmonary smooth muscle cells (PASMCs), and further to explore its relationship with voltage-dependent potassium channels (Kv) 1.5 and Kv2.1. METHODS:Firstly, the effect of nicotine on the proliferation of PASMCs was detected by cell counting method, and the optimal concentration of nicotine was selected. Primary cultured PASMCs were randomly divided into 5 groups:normal control group, nicotine (1 μmol/L)group, nicotine (1 μmol/L) + Far (10-6 mol/L, 10-5 mol/L and 10-4 mol/L) Far group. The activity of caspase-3 was measured by apoptosis kit, the cell viability was measured by CCK-8 assay, the apoptotic rate was analyzed by flow cytometry. The expression of Kv1.5 and Kv2.1, and apoptosis-related factors Bcl-2 and Bax at mRNA and protein levels was determined by RT-qPCR and Western blot respectively. RESULTS:Nicotine at 1 μmol/L increased the number of PASMCs to the maximum extent (P<0.01). Nicotine at 1 μmol/L significantly reduced the caspase-3 activity and enhanced the cell viability of the PASMCs (P<0.01). Farrerol at 10-6~10-4 mol/L eliminated the effect of PASMCs induced by nicotine in a concentration dependent manner. Compared with control group, nicotine at 1 μmol/L significantly increased the proliferation and inhibited the apoptotic rate of rat PASMCs (P<0.01). The apoptotic rate of PASMCs in farrerol intervention group was significantly higher than that in nicotine group (P<0.01). Nicotine at 1 μmol/L significantly inhibited the expression of Kv1.5, Kv2.1 and Bax but increased the expression of Bcl-2 in PASMCs (P<0.01). Farrerol at 10-5 mol/L obviously inhibited the effect of PASMCs induced by nicotine. CONCLUSION:Farrerol eliminates nicotine-induced inhibition of caspase-3 and Bax, and enhancement of Bcl-2 in PASMCs by enhancing Kv1.5 and Kv2.1 expression.  相似文献   

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