两种木贼提取物对动脉粥样硬化早期模型大鼠平滑肌凋亡及BCl-2、Caspase-3表达的调控

目的研究木贼正丁醇提取物及提取剩余物对动脉粥样硬化(AS)早期大鼠主动脉平滑肌细胞凋亡率及Bcl-2、Caspase-3表达的影响。方法雄性SD大鼠随机分为正常对照、模型、阳性药、提取物及提取剩余物组。复制食饵性AS早期模型,用木贼提取物、提取剩余物分别预防性给药,以吉非罗齐为阳性药对照。9w后,观察主动脉壁病理学变化,流式细胞术定量检测主动脉壁平滑肌细胞凋亡率及Bcl-2、Caspase-3的表达。结果木贼正丁醇提取物及提取剩余物组的凋亡率明显高于模型组(P<0.01);Bcl-2低于模型组(P<0.01);Caspase-3高于模型组(P<0.01)。结论木贼正丁醇提取物及提取剩余物通过控制AS早期主动脉壁平滑肌细胞Bcl-2、Caspase-3表达,促进平滑肌细胞凋亡,阻止AS的进程。     

玉米苞叶煎剂对高脂血症模型大鼠血管内皮及平滑肌增殖与凋亡的干预作用

目的 观察高脂血症大鼠主动脉内皮和平滑肌细胞增殖与凋亡对玉米苞叶煎剂作用的药效反应.方法 选用雄性SD大鼠,随机分为正常对照、病理模型、阳性对照及玉米苞叶煎剂组,复制食饵性高脂血症,玉米苞叶煎剂预防性给药,以吉非罗齐为阳性药对照.观察血脂及主动脉壁病理变化,流式细胞术定量检测内皮及平滑肌细胞增殖指数及凋亡率.结果 玉米苞叶煎剂组总胆固醇(TC)、甘油三酯(TG)、极低密度脂蛋白(VLDL)较模型组降低明显(均P＜0.05);与模型组相比,内皮凋亡率下降和平滑肌凋亡率升高均有显著性差异(均P＜0.05),内皮增殖指数轻度上升(P＞0.05),平滑肌增殖指数低于模型组(P＜0.05).结论 玉米苞叶煎剂可通过调节脂代解,阻断AS早期的内皮-平滑肌效应.     

两种木贼提取物对高血脂大鼠血管内皮保护作用的扫描电镜观察

目的扫描电镜对比观察两种木贼提取物对高血脂大鼠主动脉内皮细胞损伤的影响。方法50只健康雄性SD大鼠随机分为正常组,模型组,吉非罗齐（阳性对照药）组,木贼正丁醇提取物组,正丁醇提取剩余物组。实验9w后取主动脉用扫描电镜观察血管内皮细胞的结构变化。结果木贼提取物组大鼠血管内皮细胞排列比较整齐,形态结构基本正常,内皮细胞间隙较小,细胞黏附和脂质沉积少见。结论超微结构观察提示两种木贼提取物对高血脂所致主动脉血管内皮细胞损伤有良好的修复和保护作用,正丁醇提取物效果尤为明显。     

木贼正丁醇萃取物对高脂血症大鼠血管内皮黏附分子表达的影响

目的研究木贼正丁醇萃取物对高脂血症大鼠血脂及主动脉内皮细胞黏附功能的影响。方法选用雄性SD大鼠,随机分为正常对照组、病理模型组、吉非罗齐组及木贼正丁醇治疗组。复制食饵性动脉粥样硬化早期动物模型,用木贼萃取物预防性给药,以吉非罗齐为阳性药对照。实验9 w,检测血脂,流式细胞术定量检测主动脉内皮细胞ICAM-1、VCAM-1的表达及凋亡率,光镜下观察主动脉形态学变化。结果木贼萃取物组血清TG、VLDL水平较模型组显著下降(P<0.05),内皮ICAM-1表达较模型组明显下降(P<0.05),VCAM-1有下降趋势(P>0.05),凋亡率显著下降(P<0.05);形态学显示木贼萃取物组主动脉内皮损伤程度较模型组减轻。结论木贼正丁醇萃取物可保护血管内皮,降低内皮细胞黏附分子表达,阻止动脉粥样硬化早期炎症反应的发生。     

木贼对动脉粥样硬化早期大鼠内皮细胞凋亡及Bax、Bcl-2表达的影响

目的 观察木贼对大鼠动脉粥样硬化早期血管内皮细胞凋亡及bax、bcl—2表达的影响。方法 高脂饲料复制SD大鼠高脂血症和早期动脉粥样硬化模型，同时给以不同剂量木贼水煎刑灌胃，实验10w后，流式细胞术定量检测各组主动脉内皮细胞凋亡率及bax、bcl—2基因蛋白表达。结果 模型组内皮细胞凋亡率及两个基因表达明显高于正常组(P＜0．01)；木贼组内皮细胞凋亡率与bax表达量显著低于模型组(P＜0．01)，bcl—2比较虽无统计学意义，但用药后bax／bcl—2比值差异具有显著性。结论 木贼可从基因水乎调控早期动脉粥样硬化主动脉内皮细胞凋亡，干预动脉粥样硬化始动环节及其发生。     

玉米苞叶对动脉粥样硬化家兔平滑肌细胞凋亡及p53和Fas蛋白表达的影响

目的：探讨玉米苞叶防治动脉粥样硬化（AS）的机制。方法：选用大耳白家兔，复制家兔AS模型，随机分为动脉粥样硬化模型组，玉米苞叶组和正常对照组，成模后给予玉米苞叶煎剂治疗，8w后处死家兔，采用流氏细胞术检测平滑肌细胞的凋亡率以及凋亡相关基因p53和Fas蛋白表达。结果：模型组血管平滑肌细胞的凋亡率明显高于对照组（P＜0．05），p53和Fas蛋白的表达增强（P＜0．05），主动脉壁肉眼观测出现典型斑块。玉米苞叶组平滑肌细胞的凋亡率明显低于模型组（P＜0．05），p53和Fas蛋白表达下调（P＜0．05）；主动脉斑块面积较模型组明显减小，结论：玉米苞叶通过调节p53和Fas蛋白表达而调节AS家兔平滑肌细胞凋亡。     

白黎芦醇对AS模型大鼠主动脉bcl-2及caspase-3表达影响的研究

目的 观察白黎芦醇对动脉粥样硬化(AS)模型大鼠主动脉壁平滑肌细胞bcl-2和caspase-3 mRNA表达的影响.方法 雄性SD大鼠复制食饵性AS早期模型,用药组灌胃白藜芦醇70 mg/(kg·d)共4 w.造模给药用逆转录聚合酶链反应,观察对照组、模型组和用药组主动脉壁平滑肌细胞bcl-2和caspase-3 mRNA的表达.结果 白藜芦醇组通过影响AS早期主动脉壁平滑肌细胞bcl-2和caspase-3表达,阻止AS的进程.结论 白藜芦醇对大鼠动脉粥样硬化具有保护作用,并能提高AS斑块稳定性.     

海洋贝类综合提取物对实验性动脉粥样硬化的治疗作用

目的研究海洋贝类综合提取物对实验性动脉粥样硬化的治疗作用。方法先建立高脂食饵性鹌鹑动脉粥样硬化模型，然后经口服连续给予5、10及20g/kg海洋贝类综合提取物，于用药第2、4周末测定血清及主动脉和心肌中脂质含量，并对主动脉、冠状动脉和肝脏进行肉眼及光镜组织学检查。结果用药2周后5、10及20g/kg海洋贝类综合提取物均可显著降低血清甘油三酯水平（P〈0．05及P〈0．01），20g/kg海洋贝类综合提取物组血清总胆固醇、甘油三酯、低密度脂蛋白胆固醇水平明显降低，高密度脂蛋白胆固醇水平明显升高（P〈0．05）；用药4周后，5、10及20g/kg海洋贝类综合提取物组血清总胆固醇、甘油三酯、低密度脂蛋白胆固醇水平明显降低（P〈0．05及P〈0．01），高密度脂蛋白胆固醇水平明显升高（P〈0．05及P〈0．01）；主动脉壁及心肌组织中总胆固醇、甘油三酯含量也明显降低（P〈0．05及P〈0．01）。病理检测结果发现鹌鹑冠状动脉、主动脉内膜粥样硬化斑块的病变程度较模型组明显减轻。结论海洋贝类综合提取物治疗用药具有降低高血脂，促进动脉粥样硬化消退作用。     

丹参单体对肿瘤坏死因子损伤的血管平滑肌细胞炎症介质IL-1β表达的影响

目的探讨丹参主要单体成分丹酚酸B和丹参酮ⅡA在动脉粥样硬化（AS）发病过程中发挥作用的物质基础和作用靶点。方法通过体外培养大鼠主动脉平滑肌细胞，建立肿瘤坏死因子（TNF—α）损伤的细胞模型，并运用放射免疫方法，检测上清液中IL-1β的表达。结暴高剂量丹参酮IIA对IL-1β表达抑制作用最强（P〈0．01），其余中药组抑制作用次之（P〈0．05）。结论丹酚酸B和丹参酮ⅡA均可以通过抑制AS时炎症介质的表达达到防治AS的目的。     

核因子-κB调控老年大鼠脾淋巴细胞凋亡及淫羊藿总黄酮对其影响

目的探讨核因子-κB（nuclearfactor—kappaB，NF-κB）对老年大鼠脾淋巴细胞凋亡的调控作用及淫羊藿总黄酮（epimedium total flaonoids，EF）对老年大鼠脾淋巴细胞凋亡的调控作用是否通过NF—κB实现。方法分别给予老年大鼠模型NF-κB抑制剂二硫代氨基甲酸吡咯烷（PDTC）和EF干预，提取大鼠脾淋巴细胞，用流式细胞仪检测细胞凋亡，Westem blot检测P65蛋白表达，电泳迁移率（electrophoretic mobility shift assay，EMSA）检测NF-κB的活性。结果老年大鼠脾淋巴细胞凋亡率比青年大鼠高（P〈0．01），PDTC使老年大鼠的凋亡率更高，而EF使老年大鼠的凋亡率降低，与老年组相比有显着性差异（P〈0．05），且EF能拮抗PDTC诱导老年大鼠胆淋巴细胞凋亡。与青年大鼠相比老年大鼠脾淋巴细胞P65蛋白表达显著下调（P〈0．01），而使用EF的老年大鼠组P65蛋白表达明显上调，表明EF诱导P65高表达。老年大鼠脾淋巴细胞NF-κB的活性弱于青年大鼠（P〈0．01），而PDTC处理组的NF—κB活性最低与老年大鼠组相比差异明显（P〈0．01），EF干预组的NF-κB的活性最强，与老年大鼠组相比差异非常显著（P〈0．01）。相关分析表明，淋巴细胞NF—κB活性与细胞凋亡率呈负相关（r=-0．57，P〈0．01）。结论NF-κB通过抑制淋巴细胞凋亡而参与老年大鼠免疫衰老调控过程；EF可能通过提高NF-κB的活性和诱导P65高表达来抑制淋巴细胞凋亡从而延缓免疫衰老进程。     

木贼对大鼠高脂血症及主动脉内膜早期粥样硬化病变的影响

目的　观察木贼对食饵性高脂血症大鼠血清学指标及主动脉内膜早期粥样硬化病变的影响。方法　采用SD大鼠 ,随机分为对照组、模型组和木贼给药组。高脂饲料造模 ,同时给木贼煎剂灌胃 ,实验 1 0w后 ,检测血清脂质水平 ,光镜和电镜观察主动脉内膜形态变化。结果　给药两组血清总胆固醇和低密度脂蛋白明显降低 ,高密度脂蛋白明显升高 ,与模型组相比均有显著性差异 (P <0 0 1 )。形态学观察发现 ,模型组主动脉内膜出现典型粥样硬化病变 ,木贼煎剂给药后能明显减轻主动脉内膜受损程度。结论　木贼可通过降血脂保护主动脉内膜 ,干预动脉粥样硬化的早期病变发生     

c-Myc antisense oligonucleotides preserve smooth muscle differentiation and reduce negative remodelling following rat carotid arteriotomy

OBJECTIVES: The vascular biology of restenosis is complex and not fully understood, thus explaining the lack of effective therapy for its prevention in clinical settings. The role of c-Myc in arteriotomy-induced stenosis, smooth muscle cell (SMC) differentiation and apoptosis was investigated in rat carotids applying full phosphorothioate antisense (AS) oligonucleotides (ODNs). METHODS: Carotid arteries from WKY rats were submitted to arteriotomy and to local application of ODNs through pluronic gel. Apoptosis (deoxynucleotidyl transferase-mediated dUTP nick end-labelling), SMC differentiation (SM22 immunofluorescence) and vessel morphology and morphometry (image analysis) were determined 2, 5 and 30 days after injury, respectively. RESULTS: AS ODNs induced a 60% decrease of target c-Myc mRNA 4 h after surgery in comparison to control sense (S) and scrambled ODN-treated carotids (p < 0.05). A significant 37 and 50% decrease in SM22 protein in the media of S ODN-treated and untreated carotids was detected when compared to uninjured contralateral arteries (p < 0.05). This reduction in SM22 expression was prevented in AS ODN-treated carotids. Stenosis was mainly due to adventitial constrictive remodelling. Lumen area in AS ODN-treated carotids was 35% greater than in control arteries 30 days after surgery (p < 0.05). TUNEL assay revealed increased apoptosis in AS ODN-treated carotids (p < 0.05). CONCLUSIONS: c-Myc AS ODNs reduce arteriotomy-induced negative remodelling. This is accompanied by maintained SMC differentiation and greater apoptosis. The combination of reduced c-Myc-induced proliferation and increased apoptosis may thus underlie the less severe remodelling upon treatment with c-Myc mRNA AS ODN.     

Apoptosis induced by 7-ketocholesterol is enhanced in smooth muscle cells derived from OLETF rats

To clarify whether an increased proliferative potential of vascular smooth muscle cells (SMC) under diabetic conditions augments the susceptibility of the cells to 7-ketocholesterol-induced apoptosis, we investigated the difference in sensitivity to 7-ketocholesterol between SMC obtained from diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and the control Long-Evans Tokushima Otsuka (LETO) rats. The outgrowth rate from aortic wall explants and cell proliferation were higher in SMC derived from OLETF rats (OLETF-derived SMC) compared to those from LETO rats (LETO-derived SMC). When 7-ketocholesterol was added to SMC, the amount of fragmented DNA increased significantly in OLETF-derived compared to LETO-derived SMC. The amount of fragmented DNA induced by 7-ketocholesterol decreased significantly in both OLETF- and LETO-derived SMC when they were incubated without fetal bovine serum. By adding PDGF-BB to LETO-derived SMC, the amount of fragmented DNA induced by 7-ketocholesterol increased significantly. These results suggest that apoptosis of SMC induced by 7-ketocholesterol may be accelerated when SMC acquire a high proliferative potential by prolonged exposure to diabetic condition.     

HMG-CoA reductase inhibitors induce apoptosis in neointima-derived vascular smooth muscle cells

In the context of atherogenesis and restenosis, vascular smooth muscle cell (SMC) proliferation and apoptosis play a crucial role. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) have been shown to inhibit the migration and proliferation of SMC, and to induce apoptosis in different cell types including SMC. However, it is not known whether these agents induce apoptosis in neointimal SMC. We investigated the effects of statin treatment on neointimal SMC as compared to medial cells by using trypan blue counting, MTT test, Annexin V staining, cell cycle analysis and a co-culture model. The incubation of neointimal or medial SMC with lovastatin reduced the MTT activity as well as the total cell number, and increased the amount of trypan blue positive cells, indicative of cell death. We tested by staining with Annexin V/propidium iodide, specific antibodies to active caspase-3, TUNEL reaction, and by the appearance of a sub-G1 peak, whether the observed increase in cell death was due to apoptosis. After treatment with lovastatin, programmed cell death was slightly increased in medial SMC, while neointimal cells showed a pronounced rate of apoptosis. In an attempt to mimic early phases of restenosis in vitro by seeding low density neointimal cells onto high density medial cells, we found that statin treatment induced cell death preferentially in the neointimal SMC. Our results suggest that statins enhance the rate of apoptosis in neointimal SMC, which may be an interesting feature to reduce restenosis after successful angioplasty.     

Differences in the glutathione system of cultured aortic smooth muscle cells from young and aged rats

We studied the relation between the glutathione (GSH) system and cell proliferation in a model of smooth muscle cells (SMC) derived from the thoracic aorta of 4–6-week-old (young) and 15-month-old (aged) rats. SMC from aged rats showed greater levels of total non-protein thiol compounds (T-SH), increased glutathione transferase (GST) and increased glutathione reductase (GSSG-Red) activities compared with cells from young rats. These changes were associated with an increased proliferation rate of SMC from aged rats. To evaluate the role of GSH on cell proliferation better, a specific inhibitor of gamma-glutamyl-cystein synthetase, -buthionine-SR-sulphoximine (BSO) was used. BSO showed a dose-dependent inhibition of cell growth, with an IC₅₀ of 10⁻⁴ M, after 48–72 h of incubation. Removal of BSO restored cell growth, further suggesting a link between GSH levels and vascular cell proliferation. The inhibitory effect of BSO was about two times greater on SMC from young than on SMC from aged rats. BSO showed 56% inhibition on the proliferation of SMC from young rats and 32% inhibition on SMC from aged rats (10⁻⁴ M, 72 h of incubation). A parallel reduction of GSH levels of 38% and 19% for SMC from young and aged rats, respectively, was observed, suggesting that age-related factors may influence the involvement of GSH system in cell proliferation.     

The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism.

Smooth muscle cell (SMC) proliferation is thought to play a major role in vascular restenosis after angioplasty and is a serious complication of the procedure. Developing antisense (AS) oligonucleotides as therapeutics is attractive because of the potentially high specificity of binding to their targets, and several investigators have reported inhibition of SMC proliferation in vitro and in vivo by using AS strategies. We report here the results of our experiments on vascular SMCs using AS oligonucleotides directed toward c-myb and c-myc. We found that significant inhibition of SMC proliferation occurred with these specific AS sequences but that this inhibition was clearly not via a hybridization-dependent AS mechanism. Rather, inhibition was due to the presence of four contiguous guanosine residues in the oligonucleotide sequence. This was demonstrated in vitro in primary cultures of SMCs and in arteries ex vivo. The ex vivo model developed here provides a rapid and effective system in which to screen potential oligonucleotide drugs for restenosis. We have further explored the sequence requirements of this non-AS effect and determined that phosphorothioate oligonucleotides containing at least two sets of three or four consecutive guanosine residues inhibit SMC proliferation in vitro and ex vivo. These results suggest that previous AS data obtained using these and similar, contiguous guanosine-containing AS sequences be reevaluated and that there may be an additional class of nucleic acid compounds that have potential as antirestenosis therapeutics.     

人巨细胞病毒对培养人脐静脉内皮细胞增殖和凋亡的影响

目的 :探讨人巨细胞病毒 (CMV)在动脉粥样硬化发生和发展中的作用。　　方法 :采用流式细胞计数仪观察了人 CMV对离体培养的人脐静脉内皮细胞 (HUVEC)增殖和凋亡的影响。本实验分为 5组 ,分别为正常对照组 ,肿瘤坏死因子细胞凋亡组 ,无血清培养细胞凋亡组 ,CMV感染 肿瘤坏死因子细胞凋亡组 ,CMV感染 无血清培养细胞凋亡组。　　结果 :实验发现无血清培养和肿瘤坏死因子诱导的细胞凋亡率较正常对照组升高 ,而 CMV感染的细胞凋亡率明显降低 ;细胞计数的结果表明 CMV感染后 3天的 HUVEC细胞数是未感染的 5倍。流式细胞仪的检测结果表明 :CMV感染细胞的增殖指数明显高于正常对照组 ,CMV可使细胞耐受无血清培养条件 ,保持细胞增殖。　　结论 :人 CMV感染 HUVEC后能够改变 HUVEC的细胞增殖过程 ,抑制细胞凋亡 ,HUVEC的过度增生及 HUVEC感染引起的炎性反应可能影响内皮细胞的功能 ,甚至破坏内皮细胞 ,导致血栓形成、脂质代谢紊乱 ,最终形成动脉粥样硬化。     

基质细胞衍生因子-1及其受体对大鼠主动脉血管平滑肌细胞增殖与凋亡的影响

目的研究基质细胞衍生因子-1(SDF-1)对氧化低密度脂蛋白(ox-LDL)诱导的血管平滑肌细胞(VSMC)增殖与凋亡的影响。方法选用体外培养的大鼠主动脉VSMC,并分为正常对照组、ox-LDL组[动脉粥样硬化(AS)模型]、SDF-1组(AS模型+SDF-1)、SDF-1+12G5组(AS模型+SDF-1+CXCR4单克隆抗体)和12G5组(AS模型+CXCR4单克隆抗体),应用MTT法测VSMC细胞增殖,TUNEL法测细胞凋亡率,RT-PCR法测凋亡基因bax、bcl-2mRNA的表达。结果ox-LDL组的增殖率0.38±0.01,明显高于正常对照组0.28±0.02(P<0.01),明显低于SDF-1组0.44±0.02(P<0.01),与SDF-1+12G5组和12G5组比较差异无显著性(P>0.05);ox-LDL组的凋亡率24.2±2.4,高于正常对照组19.8±2.7和SDF-1组20.7±2.8(P<0.05),而与SDF-1+12G5组和12G5组比较差异无显著性(P>0.05);ox-LDL组bcl-2和bax的比值明显低于正常对照组和SDF-1组(P<0.01),而与SDF-1+12G5组和12G5组差异无显著性(P>0.05)。结论SDF-1可明显促进ox-LDL诱导的VSMC增殖,并抑制细胞凋亡;SDF-1及其受体CXCR4构成的生物学轴可能通过bcl-2/bax途径影响VSMC凋亡。