短链脂肪酸作用下伤寒沙门菌诱导巨噬细胞凋亡机制的探讨

目的了解短链脂肪酸(SCFA)作用伤寒沙门菌诱导巨噬细胞凋亡机制。方法将SCFA作用伤寒沙门菌感染巨噬细胞8 h后,检测TNF-α、caspase3、caspase8、caspase9及NO的产生量,同时检测加入caspase3、caspase8、caspase9抑制剂和TNF-α抗体后的细胞凋亡率。结果作用8 h后caspase3、caspase8及NO、TNF-α的产生量均高于对照组(P0.01)。caspase3、caspase8抑制剂和TNF-α抗体均能不同程度抑制SCFA作用伤寒沙门菌诱导的巨噬细胞凋亡(P0.01)。结论 SCFA作用伤寒沙门菌诱导巨噬细胞凋亡可以通过NO及TNF-α介导,caspase3和caspase8参与的外源性凋亡途迳。     

金葡菌L型垂直感染影响胎儿发育的机制研究

目的探讨金黄色葡萄球菌(金葡菌)L型垂直感染影响胎儿发育的机制。方法采用全胚胎培养方法观察不同浓度(50、100、200和400CFU/ml)金葡菌L型感染体外培养小鼠胚胎48h后对胚胎发育的影响,及用流式细胞术法检测金葡菌L型感染脐静脉内皮细胞0、2、4、6、8与10h后各时间段的细胞凋亡率。结果金葡菌L型可影响体外小鼠胚胎的发育,50CFU/ml组胚胎生长发育指标头长和颅臀长首先受到影响,100CFU/ml及以上组胚胎生长发育各项指标均明显低于对照组。金葡菌L型可诱导脐静脉内皮细胞发生凋亡,与对照组比较差异有统计学意义(P0.05或P0.01)。结论金葡菌L型通过胎盘屏障后垂直感染影响胎儿发育的机制可能是通过直接作用于胚胎和/或是通过破坏脐静脉内皮细胞功能这两种方式而影响胎儿发育的。     

花生四烯酸细胞色素P450表氧化酶对凋亡内皮细胞Bcl-2表达、Caspase-3及MAPK活性的影响

花生四烯酸经细胞色素P450表氧化酶代谢产生的内皮来源超极化因子(EDHF)[表氧化二十烷烯酸(EETs)]对内皮细胞具有保护作用。研究了转染细胞色素P450表氧化酶基因CYPBM3·F87V、CYP2C11OR及CYP2J2产生内源性EETs,通过检测内皮细胞中Bcl-2表达、caspase-3的活性及MAPK磷酸化水平探讨内源性EDHF的内皮细胞保护效应及其抗TNF-α诱导内皮细胞凋亡的作用机制。原代培养的牛主动脉血管内皮细胞转染CYP450表氧化酶基因24h后,加入TNF-α作用一定时间诱导内皮细胞凋亡,用Western印迹方法检测Bcl-2的表达,MAPK磷酸化水平,同时测定caspase-3的活性。结果显示转染表氧化酶基因能抑制TNF-α诱导的时间依赖性Bcl-2下调,抑制Caspase-3的激活。TNF-α使细胞内磷酸化MAPK水平呈时间依赖性减低,转染表氧化酶基因后细胞内的磷酸化MAPK水平较对照组升高。因此,转染表氧化酶基因CYPBM3·F87V、CYP2C11OR以及CYP2J2使内皮细胞产生内源性EETs(EDHF)通过激活MAPK(ERK1/2)途径,抑制抗凋亡基因Bcl-2的降解,抑制caspase-3的激活,从而抑制TNF-α诱导的内皮细胞凋亡,因而具有内皮保护效应。     

花生四烯酸细胞色素P450表氧化酶对凋亡内皮细胞Bc1-2表达、Caspase-3及MAPK活性的影响

花生四烯酸经细胞色素P450表氧化酶代谢产生的内皮来源超极化因子(EDHF)[表氧化二十烷烯酸(EETs)]对内皮细胞具有保护作用.研究了转染细胞色素P450表氧化酶基因CYPBM3·F87V、CYP2C110R及CYP2J2产生内源性EETs,通过检测内皮细胞中Bc1-2表达、caspase-3的活性及MAPK磷酸化水平探讨内源性EDHF的内皮细胞保护效应及其抗TNF-α诱导内皮细胞凋亡的作用机制.原代培养的牛主动脉血管内皮细胞转染CYP450表氧化酶基因24h后,加入TNF-α作用一定时间诱导内皮细胞凋亡,用Western印迹方法检测Bcl-2的表达,MAPK磷酸化水平,同时测定caspase-3的活性.结果显示转染表氧化酶基因能抑制TNF-α诱导的时间依赖性Bcl-2下调,抑制Caspase-3的激活.TNF-α使细胞内磷酸化MAPK水平呈时间依赖性减低,转染表氧化酶基因后细胞内的磷酸化MAPK水平较对照组升高.因此,转染表氧化酶基因CYPBM3·F87V、CYP2C11OR以及CYP2J2使内皮细胞产生内源性EETs(EDHF)通过激活MAPK(ERK1/2)途径,抑制抗凋亡基因Bcl-2的降解,抑制caspase-3的激活,从而抑制TNF-α诱导的内皮细胞凋亡,因而具有内皮保护效应.     

球状脂联素在波动性高血糖诱导人脐静脉内皮细胞凋亡中的作用

目的球状脂联素在波动性高血糖诱导人脐静脉内皮细胞凋亡中的作用。方法在不同条件下体外培养人脐静脉内皮细胞,分别或联合加入球状脂联素(gAD)、单磷酸腺苷激活蛋白激酶(AMPK)的激活剂AICAR和AMPK的阻滞剂araA。采用MTT比色法测定细胞活性,流式细胞仪检测细胞凋亡率,Western blot检测AMPKα和磷酸化AMPKα蛋白表达。结果分别与对照组和恒定高血糖组比较,波动性高血糖显著抑制细胞活性和增加细胞凋亡率。gAD明显抑制波动性高血糖诱导的细胞凋亡。AICAR和gAD可明显激活AMPK的表达。araA可明显抑制gAD诱导的AMPK蛋白表达。结论波动性高血糖比恒定性高血糖更易促进内皮细胞凋亡,gAD明显抑制波动性高血糖诱导内皮细胞凋亡,其机制可能与激活AMPK有关。     

二苯乙烯苷对氧化诱导内皮细胞凋亡及Caspase-3和PARP表达的影响

为研究二苯乙烯苷对过氧化氢(H2O2)诱导人脐静脉内皮细胞凋亡的影响并初步探讨二苯乙烯苷抗凋亡的可能机制,采用不同浓度H2O2和二苯乙烯苷处理内皮细胞24 h,以MTT法检测细胞生长活力、Hoechst33258染色观察细胞形态、流式细胞仪检测凋亡率等方法筛选造模内皮细胞凋亡的H2O2浓度和二苯乙烯苷最佳的抗内皮细胞凋亡作用浓度.RT-PCR、Western-blot分别检测Caspase-3、PARP mRNA及蛋白质的表达.结果发现,与空白对照组相比,300μmol/L H2O2作用后,内皮细胞增殖明显受到抑制,Hoechst33258染色可见大量凋亡细胞,细胞凋亡率显著增加,流式细胞仪检测出明显的凋亡峰,Caspase-3和多聚腺苷酸二磷酸核糖基聚合酶(PARP)的表达量显著增加.经二苯乙烯苷处理后,随着二苯乙烯苷浓度增加,细胞的增殖率随之增加,凋亡细胞数减少,凋亡率逐渐降低,与H2O2组比较,10μmol/L的二苯乙烯苷能够显著提高细胞增殖率,降低细胞凋亡率,并显著减少Caspase-3和PARP表达.以上结果表明,二苯乙烯苷能够抑制由H2O2诱导的人脐静脉内皮细胞凋亡,增加细胞生长活性,降低细胞凋亡率,其作用机制可能与下调Caspase-3和多聚腺苷酸二磷酸核糖基聚合酶(PARP)的表达有关.     

肝细胞生长因子抑制糖基化终产物诱导人脐静脉内皮细胞的凋亡作用

目的探讨肝细胞生长因子(HGF)抑制糖基化终产物(AGEs)诱导人脐静脉内皮细胞凋亡的作用及其相关分子机制。方法体外培养ECV-304人脐静脉内皮细胞,采用噻唑蓝(MTT)法测定HGF对AGEs作用后ECV-304细胞生长抑制率的影响;通过Hoechst33258荧光染色观察细胞形态学改变、流式细胞术测定AnnexinV-FITC/PI双染标记的细胞凋亡率,检测HGF对AGEs诱导ECV-304细胞凋亡的影响;Western印迹法检测Bax、Bcl-2蛋白的表达。结果HGF能明显降低AGEs对ECV-304细胞生长的抑制作用;AGE诱导培养的ECV-304细胞出现明显的凋亡形态学改变,在一定浓度范围内,ECV-304细胞凋亡率与AGEs的浓度和作用时间呈依赖关系,加入HGF处理后可显著降低不同时间的内皮细胞凋亡率;HGF作用ECV-304细胞后Bcl-2蛋白表达明显升高,而Bax蛋白表达无明显变化。结论AGEs能诱导内皮细胞凋亡,而HGF能部分抑制AGEs诱导的内皮细胞凋亡,其作用机制可能与上调Bcl-2蛋白的表达水平有关。     

Caspase-9抑制剂对SD大鼠椎间盘软骨终板细胞凋亡的影响

目的探讨caspase-9抑制剂对低胎牛血清培养诱导的大鼠椎间盘软骨终板细胞凋亡影响的研究。方法取3月龄SD大鼠椎间盘软骨终板,序贯消化法获取细胞原代培养,以1%FBS培养48 h为诱导凋亡条件。实验分为1%FBS凋亡组、caspase-9抑制剂组（Z-LEHD-FMK）及DMSO对照组,分别处理细胞48 h,后经流式细胞仪检测细胞凋亡率、Western blot检测procaspase-9,active caspase-9及active caspase-3的表达。结果流式细胞仪检测显示,caspases-9抑制剂组细胞凋亡率（26.3±2.56）%与1%FBS组（40.8±0.84）%及DMSO组（40.2±1.56）%相比凋亡率较低,有显著统计学差异（P〈0.05）;Western blot检测caspases-9抑制剂组active caspase-9及active caspase-3较1%FBS凋亡组及DMSO对照组表达均明显减少,有显著统计学意义（P〈0.05）。结论 Caspase-9抑制剂能明显抑制低胎牛血清培养诱导的大鼠椎间盘软骨终板细胞凋亡,有望成为治疗椎间盘退变的新型药物。     

铜绿假单胞菌L型诱导血管内皮细胞凋亡机制的探讨

目的探讨铜绿假单胞菌L型诱导血管内皮细胞凋亡机制。方法 Annexin V-FITC/PI双染荧光法和流式细胞术法检测铜绿假单胞菌L型感染血管内皮细胞8 h时的凋亡率,分光光度法检测细胞Caspase-9活化情况。结果铜绿假单胞菌L型和血管内皮细胞共培养8 h时细胞凋亡率、Caspase-9活化程度较对照组增高（P〈0.05）。结论铜绿假单胞菌L型可通过活化Caspase-9的线粒体途径诱导血管内皮细胞凋亡。     

凋亡诱导因子在姜黄素诱导的大鼠肺成纤维细胞凋亡中的作用

目的：研究姜黄素对肺纤维化大鼠肺成纤维细胞增殖、凋亡的影响,探讨凋亡诱导因子（AIF）在肺成纤维细胞凋亡中的作用。方法：将体外培养的肺纤维化大鼠成纤维细胞,分别于不同浓度的姜黄素（5、10、20、40μM）和caspase-3抑制剂Z-DEVD-fmk（20μM）孵育,观测细胞生长状态变化。MTT检测成纤维细胞增殖抑制率;流式细胞仪检测细胞凋亡率;Western-Blot测定凋亡诱导因子（AIF）蛋白表达及核转位结果：流式细胞术检测细胞凋亡,5～40μM姜黄素处理12 h,其凋亡率呈浓度依赖,对照组相比,差异显著;而抑制caspase-3并不能完全阻止细胞凋亡。Western-Blot结果显示,姜黄素处理组出现凋亡诱导因子（AIF）蛋白表达与核转位,抑制caspase-3活性后未检测出AIF表达结论：姜黄素可抑制肺成纤维细胞增殖,其诱导大鼠肺成纤维细胞凋亡,可能与线粒体释放AIF有关。     

Caspase pathway of elaidic acid (9t-C18:1)-induced apoptosis in human umbilical vein endothelial cells

Although TFAs (trans fatty acids) do have effects on many endothelial functions, systemic inflammation and immune disorders, only limited experimental evidence is available that TFAs participate in the pathogenesis of endothelial cell apoptosis. HUVEC (human umbilical vein endothelial cells) were grown in medium with elaidic acid (9t-C18:1) at 50, 100, 200 and 400 μmol/l for 24 h. Apoptosis was measured by flow cytometry, and caspase 3, 8 and 9 activities by colorimetric assay and their mRNA expression by qRT-PCR (quantitative real-time PCR). Results showed that 9t-C18:1 induced apoptosis of HUVEC in a dose-dependent manner. The activities and mRNA expression of caspases 8, 9 and 3 were significantly increased compared with that of the control. Z-IETD-FMK and Z-LEHD-FMK inhibited the activation of caspase 3 and apoptosis induced by 9t-C18:1. Also Z-IETD-FMK inhibited the activation of caspase 9. mRNA expressions of Bid and Smac (second mitochondria-derived activator of caspase)/DIABLO [direct IAP (inhibitor of apoptosis)-binding protein with low pI] were also significantly elevated. We conclude that 9t-C18:1 induces apoptosis of HUVEC through activating caspases 8, 9 and 3. The death receptor pathway and the mitochondrial pathway both participated in the apoptosis course induced by 9t-C18:1.     

Ginsenoside metabolite compound K differentially antagonizing tumor necrosis factor-α-induced monocyte-endothelial trafficking

Human leukocyte endothelial adhesion and transmigration occur in the early stage of the pathogenesis of atherosclerosis. Vascular endothelial cells are targeted by pro-inflammatory cytokines modulating many gene proteins responsible for cell adhesion, thrombosis and inflammatory responses. This study examined the potential of compound K to inhibit the pro-inflammatory cytokine TNF-α induction of monocyte adhesion onto TNF-α-activated human umbilical vein endothelial cells (HUVEC). HUVEC were cultured with 10 ng/ml TNF-α with individual ginsenosides of Rb1, Rc, Re, Rh1 and compound K (CK). Ginsenosides at doses of ?50 μM did not show any cytotoxicity. TNF-α induced THP-1 monocyte adhesion to HUVEC, and such induction was attenuated by Rh1 and CK. Consistently, CK suppressed TNF-α-induced expression of HUVEC adhesion molecules of VCAM-1, ICAM-1 and E-selectin, and also Rh1 showed a substantial inhibition. Rh1 and CK dampened induction of counter-receptors, α4/β1 integrin VLA-4 and αL/β2 integrin LFA-1 in TNF-α-treated THP-1 cells. Additionally, CK diminished THP-1 secretion of MMP-9 required during transmigration, inhibiting transendothelial migration of THP-1 cells. CK blunted TNF-α-promoted IL-8 secretion of HUVEC and CXCR1 expression of THP-1 monocytes. Furthermore, TNF-α-activated endothelial IκB phosphorylation and NF-κB nuclear translocation were disturbed by CK, and TNF-α induction of α4/β1 integrin was abrogated by the NF-κB inhibitor SN50. These results demonstrate that CK exerts anti-atherogenic activity with blocking leukocyte endothelial interaction and transmigration through negatively mediating NF-κB signaling.     

葡萄糖和肿瘤坏死因子-α对内皮细胞中早期生长反应基因-1表达的影响

目的探讨葡萄糖和肿瘤坏死因子-α对内皮细胞中早期生长反应基因-1表达的影响。方法利用人脐静脉内皮细胞体外培养,予以25mmol/L葡萄糖和/或10ng/ml肿瘤坏死因子-α与内皮细胞共同孵育,运用蛋白免疫印迹方法检测细胞中早期生长反应基因-1蛋白的表达,运用酶联免疫吸附法检测纤溶酶原激活抑制物-1的表达。结果葡萄糖和肿瘤坏死因子-α均可增加早期生长反应基因-1的表达,两种因素共同作用产生协同作用。而且,葡萄糖和肿瘤坏死因子-α也可促进纤溶酶原激活抑制物-1的表达。细胞外调节蛋白激酶1/2的抑制剂(PD98059)可下调肿瘤坏死因子-α所诱导的早期生长反应基因-1、纤溶酶原激活抑制物-1的表达,而对葡萄糖所诱导的早期生长反应基因-1的表达无明显影响。结论肿瘤坏死因子-α可能通过细胞外调节蛋白激酶1/2路径促进早期生长反应基因-1和纤溶酶原激活抑制物-1表达。肿瘤坏死因子-α和葡萄糖可能通过不同的信号通路调节早期生长反应基因-1、纤溶酶原激活抑制物-1表达,在肥胖、糖尿病等代谢紊乱所致的血管并发症的发生中起到重要的作用。     

Involvement of Mst1 in tumor necrosis factor-alpha-induced apoptosis of endothelial cells

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-α-induced apoptosis of ECs. Western blot analysis revealed that TNF-α induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-α-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting of propidium iodide-stained cells showed that TNF-α induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-α-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-α-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-α-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.     

Martentoxin对TNF-α引起脐静脉内皮细胞释放一氧化氮的影响

目的：研究东亚钳蝎毒素Martentoxin对脐静脉内皮细胞释放NO的影响。方法：人脐带经胶原酶消化分离后获得脐静脉内皮细胞,经抗血管性血友病8因子鉴定后,用TNF-α建立NO释放模型,观察Martentoxin对脐静脉内皮细胞释放NO的影响。结果：①经6,12,24h作用后,10μM、100μM Martentoxin能降低TNF-α引起的NO释放增加（P〈0．05）。②Martentoxin降低TNF-α引起的iNOS活性增强并对TNF—α引起的内皮源性一氧化氮合酶mRNA下调具有一定保护作用（P〈0．05）。结论：Martentoxin抑制TNF-α引起的脐静脉内皮细胞释放NO增加。     

Indoxyl sulfate induces leukocyte-endothelial interactions through up-regulation of E-selectin

Despite a positive correlation between chronic kidney disease and atherosclerosis, the causative role of uremic toxins in leukocyte-endothelial interactions has not been reported. We thus examined the effects of indoxyl sulfate, a uremic toxin, on leukocyte adhesion to activated endothelial cells and the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with indoxyl sulfate significantly enhanced the adhesion of human monocytic cells (THP-1 cell line) to TNF-α-activated HUVEC under physiological flow conditions. Treatment with indoxyl sulfate enhanced the expression level of E-selectin, but not that of ICAM-1 or VCAM-1, in HUVEC. Indoxyl sulfate treatment enhanced the activation of JNK, p38 MAPK, and NF-κB in TNF-α-activated HUVEC. Inhibitors of JNK and NF-κB attenuated indoxyl sulfate-induced E-selectin expression in HUVEC and subsequent THP-1 adhesion. Furthermore, treatment with the NAD(P)H oxidase inhibitor apocynin and the glutathione donor N-acetylcysteine inhibited indoxyl sulfate-induced enhancement of THP-1 adhesion to HUVEC. Next, we examined the in vivo effect of indoxyl sulfate in nephrectomized chronic kidney disease model mice. Indoxyl sulfate-induced leukocyte adhesion to the femoral artery was significantly reduced by anti-E-selectin antibody treatment. These findings suggest that indoxyl sulfate enhances leukocyte-endothelial interactions through up-regulation of E-selectin, presumably via the JNK- and NF-κB-dependent pathway.     

Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin

Background

Elevated plasma counts of endothelial microparticles (MP) have been demonstrated in various diseases with a vascular injury component. We used flow cytometry to study the MP-release from cultured human umbilical vein endothelial cells (HUVEC) stimulated by various agonists. MP-release by a topoisomerase I inhibitor camptothecin has been studied in detail.

Results

Overnight stimulation of HUVEC with either LPS or TNFα, or 30 min stimulation with thrombin, phorbol-myristate-acetate, tissue plasminogen activator, or angiotensin-II did not cause a significant release of annexin V-binding MP. In contrast, induction of apoptosis with 5 μM camptothecin, documented by 60–70% desquamation of HUVEC culture, annexin V-binding to the cells and DNA-fragmentation, led to a release of annexin V-binding microparticles (~80,000 MP/10³ cells). This microparticle-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone (ZVAD). Lower concentration of camptothecin (500 nM) induced comparable microparticle-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed microparticles were free of nucleic acids and 95% of microparticles were 0.3–1 μm in size. Double-labeling flow cytometry assay showed that all annexin V-binding Microparticles expressed CD59 but only approximately 50% of these also expressed CD105.

Conclusions

Camptothecin treated HUVEC released different populations of annexin V-binding membrane microparticles at early stage after proapoptotic stimulation before detection of phosphatidylserine exposure on the cells or DNA fragmentation. The microparticle-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into microparticle-release as a marker of endothelial stimulation and injury.