苯扎贝特对内皮细胞LOX-1及抗凋亡基因Bcl-2影响

目的研究苯扎贝特对氧化型低密度脂蛋白(ox-LDL)作用的人脐静脉内皮细胞(HUVECs)血凝素氧化型低密度脂蛋白受体-1(LOX-1)及抗凋亡基因Bcl-2的影响。方法体外培养HUVECs,反转录聚合酶链反应(RT-PCR)观察各组LOX-1及Bcl-2 mRNA的变化,Western-blot方法观察各组Bcl-2蛋白的变化。结果ox-LDL组与正常对照组比较LOX-1表达显著增加(P<0.05),抗凋亡基因Bcl-2表达降低(P<0.05),苯扎贝特组LOX-1 mRNA表达减少(P<0.05),Bcl-2 mRNA和蛋白表达增加(P<0.05)且呈浓度效应依赖关系。结论苯扎贝特可通过下调LOX-1的表达,上调抗凋亡基因Bcl-2表达从而抑制ox-LDL引起的内皮细胞凋亡。     

瑞舒伐他汀联合氯沙坦对氧化型低密度脂蛋白诱导的人单核巨噬细胞肝X受体表达的影响

目的探讨瑞舒伐他汀、氯沙坦联合对氧化低密度脂蛋白(ox-LDL)诱导的人单核-巨噬细胞肝X受体(LXR)表达的影响及可能的机制。方法分离并培养人单核细胞,转化为巨噬细胞。据实验要求设置空白对照组、ox-LDL模型对照组、氯沙坦组、瑞舒伐他汀组、瑞舒伐他汀联合氯沙坦组,反转录聚合酶链反应(RTPCR)法测定各组LXR的mRNA的表达。结果与空白对照组比较,ox-LDL模型对照组LXRmRNA表达明显降低(P<0.05);氯沙坦组与ox-LDL模型对照组比较,LXRmRNA表达显著增加(P<0.05);瑞舒伐他汀组与OX-LDL模型对照组比较,LXR mRNA表达显著增加(P<0.05);瑞舒伐他汀联合氯沙坦组与ox-LDL模型对照组比较,LXR mRNA表达显著增加(P<0.05),且高于单药组(P<0.05)。结论瑞舒伐他汀、氯沙坦均可上调LXR的表达,而瑞舒伐他汀与氯沙坦以适宜剂量联合后,LXR的表达增高比单独用药更为显著。     

血凝素样氧化低密度脂蛋白对单核巨噬细胞源泡沫细胞基质金属蛋白酶-9的调控作用

目的观察氧化低密度脂蛋白(ox-LDL)对人单核巨噬细胞源泡沫细胞分泌的基质金属蛋白酶-9(MMP-9)的表达和活性的影响,以及此过程中血凝素样氧化低密度脂蛋白受体-1(LOX-1)的调控作用。方法体外原代培养人单核细胞来源的巨噬细胞,传至2～6代用于实验。依不同浓度ox-LDL(20、40、80mg/L)作用24h,以及ox-LDL40mg/L作用1、12、24h分组,另设LOX-1受体阻断剂多聚肌苷酸(PolyI)干预组。反转录-聚合酶链反应(RT-PCR)测定LOX-1和MMP-9mRNA表达,酶谱法分析MMP-9的活性。结果 ox-LDL作用后,LOX-1和MMP-9mRNA表达升高,MMP-9的分泌及活性升高(P<0.05),且诱导作用呈浓度-时间依赖性;多聚肌苷酸抑制后,MMP-9mRNA表达及活性明显下降(P<0.05)。结论 LOX-1作为ox-LDL的特异性受体,介导ox-LDL诱导的上调单核巨噬细胞LOX-1、MMP-9表达及活性的作用。     

阿托伐他汀对ox-LDL诱导的人脐静脉内皮细胞中Wnt信号通路及相关因子表达的影响

目的:研究阿托伐他汀对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)中Wnt信号通路及相关因子Wnt1、β-连环蛋白(β-catenin)及蓬乱蛋白1(dvl-1)表达的影响。方法:将体外培养的HUVECs分为空白对照组、ox-LDL(50 mg/L)组和低、高剂量(ox-LDL 50 mg/L+阿托伐他汀0.5、10μmol/L)组,分别加入相应药物培养24 h后,用硝酸还原酶法测定各组细胞中一氧化氮(NO)含量,用免疫组化法和蛋白质印迹法检测各组细胞中Wnt1、β-catenin及dvl-1蛋白的表达。结果:与空白对照组比较,其他3组细胞中NO含量明显减少(P<0.01),Wnt1、β-catenin、dvl-1蛋白表达均明显增强(P<0.01);与ox-LDL组比较,低、高剂量组细胞中NO含量均明显增加(P<0.01),Wnt1、β-catenin、dvl-1蛋白表达均明显减弱(P<0.01)。结论:阿托伐他汀能抑制ox-LDL诱导的HUVECs中Wnt信号通路及相关因子表达,其可能是阿托伐他汀抗动脉粥样硬化的机制之一。     

阿托伐他汀对ox-LDL诱导的人脐静脉内皮细胞缝隙连接的影响

目的:研究阿托伐他汀对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)连接蛋白(Connexin)43、Connexin40表达的影响。方法:将体外培养的HUVECs分为对照组、ox-LDL组、阳性对照组和阿托伐他汀组,除对照组外,其余组分别先在培养液、加入50μmol/L庚醇的培养液、加入不同剂量(0.01、0.1、1.0、10μmol/L)阿托伐他汀的培养液中处理30min,再分别加入50mg/L的ox-LDL继续培养24h。采用硝酸还原酶法测定各组NO浓度,免疫细胞化学法测定Connexin43蛋白定位表达,蛋白印迹法检测Connexin43、Connexin40蛋白定量表达。结果:与对照组比较,其余各组NO浓度均明显减少、Connexin43和Connexin40蛋白定量表达均明显增加(P均<0.01),其中除阳性对照组和10μmol/L阿托伐他汀组外,剩余各组均有Connexin43蛋白定位表达。与ox-LDL组比较,阳性对照组NO浓度明显增加、Connexin43和Connexin40蛋白定量表达明显减少(P<0.05或P<0.01),阿托伐他汀组随剂量增加NO浓度逐渐增加、Connexin43和Connexin40蛋白定量表达逐渐减少(P<0.05或P<0.01)。与阳性对照组比较,10μmol/L阿托伐他汀组上述指标均无统计学差异(P>0.05)。结论:阿托伐他汀呈剂量依赖性抑制ox-LDL诱导的HUVECs中Connexin43、Connexin40蛋白的增加,保护内皮间缝隙连接,从而发挥他汀类药物调脂外的抗动脉粥样硬化作用。     

吡格列酮对人脐静脉内皮细胞CD40/CD40L表达的影响

目的探讨吡格列酮对氧化低密度脂蛋白(oxLDL)刺激下的人脐静脉内皮细胞(HUVECs)CD40/CD40L表达的影响。方法原代培养人脐静脉内皮细胞,给予oxLDL刺激和不同浓度吡格列酮干预。采用流式细胞技术检测CD40/CD40L在细胞上的表达,采用反转录-聚合酶链反应(RT-PCR)检测LOX-1mRNA的表达。结果OxLDL以浓度和时间依赖的方式刺激HUVECs表达CD40/CD40L,吡格列酮以剂量依赖的方式减轻ox-LDL刺激HUVECs表达CD40/CD40L。同时我们还观察到oxLDL能刺激HUVECs上调LOX-1mRNA的表达,而吡格列酮能明显抑制这种作用。结论吡格列酮能减轻oxLDL刺激下的人脐静脉内皮细胞CD40/CD40L的表达,其作用的机制可能与其抑制了HUVECs的LOX-1受体表达有关。     

辛伐他汀对氧化型低密度脂蛋白及葡萄糖诱导的LOX-1表达的不同作用

【摘要】目的研究辛伐他汀干预对于氧化型低密度脂蛋白（ox-LDL）及葡萄糖诱导的巨噬细胞表面血清凝集素样氧化型低密度脂蛋白受体（LOX）-1表达的影响,以及核因子（NF）-κB的作用。方法U937细胞经佛波酯诱导分化后,将含有50mg/L ox-LDL和（或）25mmol/L葡萄糖的培养液与上述细胞共同孵育,应用吡咯烷二硫氨基甲酸（PDTC）及不同浓度辛伐他汀（1、10μmol/L）干预上述细胞,干预前后用ELISA检测LOX-1蛋白的表达及NF-κB的活性,RT-PCR检测LOX-1mRNA含量。结果ox-LDL、高糖及联合组均上调LOX-1表达,NF-κB的抑制剂PDTC可以抑制其作用,辛伐他汀可以下调ox-LDL诱导的LOX-1表达增加,且高浓度下调明显;但辛伐他汀对于高糖诱导LOX-1表达无明显影响。各组间NF-κB活性改变与LOX-1表达基本一致。结论辛伐他汀可以下调ox-LDL诱导的巨噬细胞LOX-1表达,但对葡萄糖诱导的LOX-1表达无明显影响,LOX-1表达调控与NF-κB信号途径有关。     

血管紧张素-Ⅱ对血管内皮细胞内皮素-1 mRNA表达水平的影响及阿托伐他汀的保护作用

目的观察血管紧张素(Ang)-Ⅱ对血管内皮细胞内皮素(ET)-1 mRNA表达水平的影响及阿托伐他汀的保护作用。方法将培养的血管内皮细胞随机分为4组:空白对照组(仅给予细胞培养液)、单纯Ang-Ⅱ组(细胞培养液中加入Ang-Ⅱ,使其终浓度为10-7mol/L)、Ang-Ⅱ+小剂量阿托伐他汀组(在单纯Ang-Ⅱ组的基础上加入阿托伐他汀,使阿托伐他汀的终浓度为0.1μmol/L)、Ang-Ⅱ+大剂量阿托伐他汀组(在单纯Ang-Ⅱ组的基础上加入阿托伐他汀,使阿托伐他汀的终浓度为1μmol/L)。以反转录-聚合酶链反应(RT-PCR)方法检测血管内皮细胞的ET-1mRNA表达水平。结果①与空白对照组相比,单纯Ang-Ⅱ组血管内皮细胞的ET-1 mRNA表达水平显著增高(P<0.01);②与单纯Ang-Ⅱ组相比,两个Ang-Ⅱ+阿托伐他汀组大鼠的心肌ET-1 mRNA表达水平显著降低(P<0.01);③Ang-Ⅱ+高浓度阿托伐他汀组的ET-1 mRNA表达水平显著低于Ang-Ⅱ+低浓度阿托伐他汀组(P<0.05)。结论Ang-Ⅱ可使血管内皮细胞ET-1 mRNA表达水平显著增高,阿托伐他汀可逆转这一作用,且其作用呈剂量依赖性。     

同型半胱氨酸对血管内皮细胞功能损害的体外研究

目的：探讨高同型半胱氨酸血症对血管内皮细胞功能损伤的机制和改善其损伤的方法。方法：体外培养脐静脉血管内皮细胞，用RT-PCR方法检测血管内皮细胞在不同浓度的同型半胱氨酸或同时加入氟伐他汀孵育24h后血凝素样氧化低密度脂蛋白受体-1（LOX-1）和内皮型一氧化氮合酶（eNOS）的mRNA表达。结果：同型半胱氨酸实验组的LOX-1mRNA表达增多，其中100μmol／L组显著增多（P〈0．05）；各组之间eNOS的mRNA表达差异无统计学意义（P〉0．05）。同时加入氟伐他汀后，LOX-1和eNOS的mRNA表达均无显著改变（P〉0．05）。结论：高同型半胱氨酸血症可能通过提高血管内皮细胞的LOX-1水平加速动脉粥样硬化的进程，如何防治尚需进一步研究。     

LOX-1对氧化低密度脂蛋白所诱导的PAI-1基因表达的影响

目的探讨血凝素样氧化型低密度脂蛋白受体(LOX)-1对氧化低密度脂蛋白(ox-LDL)所诱导1型纤溶酶原激活物抑制剂-1(PAI-1)表达的影响。方法不同浓度ox-LDL培养人脐静脉内皮细胞(HUVECs),采用倒置显微镜观察内皮细胞形态变化,通过反转录-聚合酶链反应(RT-PCR)测定LOX-1、PAI-1mRNA表达,Westernblot、酶联免疫吸附试验(ELISA)分别测定LOX-1、PAI-1蛋白的表达。结果不同浓度ox-LDL组,LOX-1和PAI-1mRNA和蛋白的表达明显增加,且呈浓度依赖性(P<0.01);用250μg/mLPoly(Ⅰ)与HUVECs预先作用2h后,再加入50μg/ml的ox-LDL培养24h,与未加Poly(Ⅰ)相比,LOX-1和PAI-1mRNA和蛋白的表达明显减少(P<0.05)。结论ox-LDL可以调节培养的脐静脉内皮细胞LOX-1和PAI-1表达;LOX-1作为ox-LDL特异性受体,介导了ox-LDL诱导脐静脉内皮细胞表达LOX-1。同时该实验提示了ox-LDL诱导脐静脉内皮细胞表达PAI-1是通过LOX-1介导的。     

熊果酸抑制HepG2中C-反应蛋白的异常表达及其对HUVECs的损伤

目的:研究不同剂量的熊果酸(UA)对IL-6诱导的HepG2细胞中C-反应蛋白(CRP)异常表达的抑制作用以及对CRP所致人脐静脉血管内皮细胞(HUVECs)损伤的保护作用.方法:IL-6(30 ng/mL)、UA(6.5、12.5、25μmol/L)与IL-6(30 ng/mL)共同作用于HepG2细胞48 h,分别用MTT法、Western blot法及RT-PCR法检测各组细胞活力、CRP蛋白及mRNA表达情况.CRP(25 μg/mL)、UA(5,10,20 μmol/L)与CRP(25 μg/mL)共同作用于HUVECs 24 h,分别用MTT法、Western blot法及RT-PCR法检测各组细胞增殖、VCAM-1和LOX-1的蛋白及mRNA表达.结果:UA能显著抑制IL-6诱导的HUVECs细胞活力下降及细胞中CRP蛋白与mRNA的表达升高;UA显著抑制CRP引起的内皮细胞增殖,并且在mRNA及蛋白水平均能显著抑制CRP诱导的HUVECs异常高表达VCAM-1及LOX-1.结论:UA可通过抑制肝脏合成炎症因子CRP而降低血液中CRP浓度,并降低CRP等炎症因子对内皮细胞的损伤等途径从而发挥抗心肌缺血及动脉粥样硬化等心血管疾病的作用.     

Nifedipine prevents apoptosis of endothelial cells induced by oxidized low-density lipoproteins

Calcium channel blockade has been shown to inhibit experimental atherosclerosis, and early clinical trials suggest that it also reduces atherosclerosis in humans. However, the mechanisms underlying the direct protective effect of calcium channel blockade on endothelial cell injury are not fully understood. The apoptosis of endothelial cells induced by oxidized low-density lipoproteins (oxLDL) may provide a mechanistic clue to the "response-to-injury" hypothesis of atherogenesis. Here we report that the calcium channel blocker, nifedipine, prevents the apoptosis of human umbilical venous endothelial cells (HUVECs) induced by oxLDL via downregulation of the endothelial receptor for oxidized LDL (LOX-1) and inhibition of CPP32-like protease activity. The incubation of HUVEC with oxLDL increased LOX-1 mRNA levels and CPP32-like protease activity, and induced apoptosis. Preincubation of HUVEC with nifedipine before incubation with oxLDL significantly suppressed the increase in LOX-1 mRNA levels and CPP32-like protease activity, preventing apoptosis in a dose-dependent manner. These results suggest that nifedipine blocks the suicide pathway leading to the apoptosis of endothelial cells by decreasing LOX-1 mRNA levels and CPP32-like protease activity. Thus, nifedipine seems to play a protective role against the "response-to-injury" hypothesis of atherogenesis.     

XJP-1 protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by inhibiting NADPH oxidase subunit expression and modulating the PI3K/Akt/eNOS pathway

Endothelial apoptosis triggered by oxidized low-density lipoprotein (ox-LDL) can accelerate the progression of endothelial dysfunction in atherosclerosis. (±)7,8-Dihydroxy-3-methyl-isochromanone-4 (XJP-1) is a natural phenolic compound derived from banana peel. In the present study, we investigated the anti-apoptotic effect of XJP-1 in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL and explored underlying mechanisms. Our results showed that in the presence of ox-LDL, XJP-1 significantly attenuated ox-LDL-mediated cytotoxicity, apoptosis, caspase-3 activation, reactive oxygen species (ROS) generation, and NADPH oxidase subunit (p22phox and p47phox) expression in HUVECs. In addition, the anticytotoxic and anti-apoptotic effect of XJP-1 was partially inhibited by a PI3K inhibitor (LY294002), an Akt inhibitor (SH-6), a specific eNOS inhibitor (l-NAME) and a NADPH oxidase inhibitor (DPI). In exploring the underlying mechanisms of XJP-1 action, we found that XJP-1 eliminated ox-LDL-induced dephosphorylation of Akt and eNOS in a dose-dependent manner. However, XJP-1 alone upregulation of Akt and eNOS phosphorylation were blocked by LY294002 and SH-6. Moreover, XJP-1 increased NO production, but this effect was abolished by LY294002, SH-6 and l-NAME. The inhibition of ox-LDL-induced endothelial dysfunction by XJP-1 is due at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.     

Cholesterol-lowering drugs inhibit lectin-like oxidized low-density lipoprotein-1 receptor function by membrane raft disruption

Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is up-regulated in atherosclerotic lesions. Statins are the principal therapeutic agents for cardiovascular diseases and are known to down-regulate LOX-1 expression. Whether the effect on the LOX-1 receptor is related to statin-mediated cholesterol-lowering activity is unknown. We investigate the requirement of cholesterol for LOX-1-mediated lipid particle internalization, trafficking, and processing and the role of statins as inhibitors of LOX-1 function. Disruption of cholesterol-rich membrane microdomains by acute exposure of cells to methyl-β-cyclodextrin or chronic exposure to different statins (lovastatin and atorvastatin) led to a spatial disorganization of LOX-1 in plasma membranes and a marked loss of specific LOX-1 function in terms of ox-LDL binding and internalization. Subcellular fractionation and immunochemical studies indicate that LOX-1 is naturally present in caveolae-enriched lipid rafts and, by cholesterol reduction, the amount of LOX-1 in this fraction is highly decreased (≥60%). In contrast, isoprenylation inhibition had no effect on the distribution and function of LOX-1 receptors. Furthermore, in primary cultures from atherosclerotic human aorta lesions, we confirm the presence of LOX-1 in caveolae-enriched lipid rafts and demonstrate that lovastatin treatment led to down-regulation of LOX-1 in lipid rafts and rescue of the ox-LDL-induced apoptotic phenotype. Taken together, our data reveal a previously unrecognized essential role of membrane cholesterol for LOX-1 receptor activity and suggest that statins protect vascular endothelium against the adverse effect of ox-LDL by disruption of membrane rafts and impairment of LOX-1 receptor function.