不同程度内质网应激对ApoE-/- 小鼠动脉粥样硬化斑块稳定性的影响

目的:采用Apo E-/-小鼠建立不稳定动脉粥样硬化斑块模型,给予不同剂量衣霉素,观察其对动脉粥样硬化斑块稳定性的影响。方法:取40只6-8周的Apo E-/-小鼠随机分为对照组和手术组。对照组小鼠给予正常饮食;手术组小鼠行右侧颈总动脉套管术(Perivascular carotid collar placement,PCCP),同时给予高脂喂养。9周末分别取对照组和手术组小鼠颈动脉,HE染色观察小鼠颈动脉斑块形成情况。成功造模后,将小鼠随机分为正常对照组、单纯PCCP组、小剂量衣霉素组和大剂量衣霉素组;正常对照组和单纯PCCP组给予生理盐水腹腔注射,小剂量衣霉素组和大剂量衣霉素组分别给予小剂量衣霉素、大剂量衣霉素腹腔注射。2周后,处死小鼠,通过HE染色观察颈动脉斑块形态,油红O染色观察斑块内脂质聚集,抗巨噬细胞免疫组化染色观察斑块内巨噬细胞聚集,Western-blot检内质网应激标志蛋白GRP78和自噬标志蛋白Atg7、P62的表达水平。结果:HE染色结果显示:与单纯PCCP组和大剂量衣霉素组相比,小剂量衣霉素组颈动脉腔内的斑块脂质池减少,斑块结构较为完整且相对稳定;油红O染色结果显示:小剂量衣霉素组斑块内脂质含量显著降低(P0.05 vs单纯PCCP组和大剂量衣霉素组);巨噬细胞免疫组化染色显示:与单纯PCCP组和大剂量衣霉素组相比,小剂量衣霉素组斑块内巨噬细胞的含量显著降低(P0.05);Western-blot结果显示:小剂量衣霉素干预诱导的一定程度的内质网应激可以适度上调自噬(P0.05 vs单纯PCCP组和大剂量衣霉素组)。结论:PCCP手术加高脂饮食可以短期成功建立小鼠不稳定动脉粥样硬化斑块模型,其动脉粥样硬化斑块不稳定性较高,而小剂量衣霉素干预可以使得颈动脉管腔内斑块相对较小,内部脂质池明显较小,纤维帽变厚且结构更完整,斑块结构较稳定;斑块内脂质含量降低;巨噬细胞含量明显降低,且小剂量衣霉素组自噬水平适度上调。因此,小剂量衣霉素干预引起的适度的内质网应激一定程度对动脉粥样硬化斑块起到保护作用。     

三七总皂苷通过调节TLR4/SYK信号抑制ApoE 基因敲除小鼠动脉粥样硬化泡沫细胞的形成

三七总皂苷(Panax notoginseng saponins, PNS)是从五加科人参属植物三七根部提取的有效活性成分,临床广泛应用于动脉粥样硬化疾病的治疗,但其相关机制尚不完全清楚,本课题对ApoE基因敲除(ApoE~(-/-))小鼠的体内实验研究发现PNS具有明显的改善血脂的功能,并对小鼠动脉粥样硬化斑块的形成具有抑制作用,其分子机制与TLR4/SYK信号通路相关;进一步通过体外细胞实验发现PNS能够抑制小鼠斑块内巨噬细胞的吞噬能力和CD36的表达,从而抑制泡沫细胞的形成。因此我们认为三七总皂苷可通过调节TLR4/SYK信号抑制ApoE~(-/-)小鼠泡沫细胞的形成从而抑制动脉粥样硬化的进展。     

瑞舒伐他汀对载脂蛋白E 基因敲除小鼠动脉粥样硬化中 调节性T 细胞的影响

目的:探讨瑞舒伐他汀对载脂蛋白E基因敲除(ApoEKO)小鼠动脉粥样硬化中调节性T细胞的影响。方法:首先将30只ApoEKO小鼠建立动脉粥样硬化模型,随机分为高胆固醇饮食组(对照组)、瑞舒伐他汀低剂量组和瑞舒伐他汀高剂量组,各组分别给予蒸馏水或瑞舒伐他汀进行干预8周;将主动脉根部行冰冻切片油红染色,评估粥样硬化斑块面积大小;免疫组织化学法检测主动脉根部粥样硬化斑块处调节性T细胞(Treg)的表达。结果:各组小鼠均有动脉粥样硬化斑块形成,采用瑞舒伐他汀治疗的小鼠动脉粥样硬化斑块的面积明显小于未经治疗的小鼠(P<0.01),同时瑞舒伐他汀能明显增加粥样硬化病变处调节性T细胞的表达,且呈现剂效关系。结论:本实验观察到瑞舒伐他汀不仅能减小ApoEKO小鼠的主动脉粥样硬化斑块,且能使调节性T细胞的表达增多,推测瑞舒伐他汀可以通过促进调节性T细胞的生成而起到抑制动脉粥样硬化的作用。     

C/EBP同源蛋白促进动脉移植片和动脉粥样硬化斑块中平滑肌细胞的增殖

<正>C/EBP同源蛋白(CHOP)是介导内质网应激(ER stress)引起的未折叠蛋白反应(unfolded protein response,UPR)的主要效应分子,它促进动脉粥样硬化斑块局部的巨噬细胞凋亡,从而加速动脉粥样硬化进程,但CHOP不依赖于髓系细胞而促动脉粥样硬化的作用机制尚不明确。最近美国哥伦比亚大学Ira Tabas的研究小组利用血管平滑肌细胞特异性CHOP敲除的APOE-/-小鼠揭开了上述谜底。他们发现,经过12周的高脂饮食,平滑肌细胞特异性敲除CHOP的APOE-/-小鼠动脉粥样     

大蒜素通过上调ABCA1表达减少脂质在THP1巨噬细胞源性泡沫细胞中蓄积

泡沫细胞形成是动脉粥样硬化(atherosclerosis,As)发生、发展的核心环节,抑制泡沫细胞脂质蓄积是预防As的关键.本研究以人源性THP1单核巨噬细胞为研究对象,通过用豆蔻酸佛波乙酯(phorbol myristate acetate,PMA)(160 nmol/L)诱导细胞24 h,ox-LDL(50 mg/m L)处理细胞48 h,构建泡沫细胞模型,探讨大蒜素(allicin)对THP1细胞泡沫化及腺苷三磷酸结合盒转运体A1(ATPbinding cassette transporter A1,ABCA1)的影响.RT-PCR、Western印迹分析显示,大蒜素可上调巨噬细胞ABCA1表达.油红O染色、高效液相色谱及液体闪烁计数揭示,大蒜素促进细胞内胆固醇流出,降低THP1巨噬细胞内总胆固醇(total cholesterol,TC)、游离胆固醇(free cholesterol,FC)、胆固醇酯(cholesterol ester,CE)含量,抑制泡沫细胞形成.此外,RNA干扰证明沉默ABCA1减少细胞的胆固醇流出,增加脂质蓄积.本研究结果提示,大蒜素可上调THP1巨噬细胞中ABCA1的表达,从而促进细胞内胆固醇的流出,降低泡沫细胞内胆固醇蓄积的作用.我们的结果为解释大蒜素预防As提供了新的证据.     

Krüppel样因子4通过支持PPARγ信号通路维持细胞稳态以保护血管平滑肌细胞免受泡沫细胞样表型转化的损害（英文）

动脉粥样硬化(AS)被普遍认为是一种血管壁细胞(包括内皮细胞和血管平滑肌细胞)、循环细胞以及固有免疫原性细胞(例如单核细胞/巨噬细胞)等多种细胞综合作用引起的炎症性疾病。其中血管平滑肌细胞(VSMCs)胆固醇超负荷形成的泡沫细胞可能在动脉粥样硬化的进展中发挥重要作用。Krüppel样因子4(KLF4)是一种关键的抗炎转录因子,尤其在心血管疾病方面,已被证实发挥了重要的血管功能保护作用。然而,目前尚不清楚KLF4是否在AS过程胆固醇对VSMCs的损伤中发挥保护作用。该研究旨在探讨KLF4在AS进展过程中VSMCs泡沫细胞样表型转化的作用及其分子机制。小鼠AS造模结果显示,KLF4缺失增加动脉粥样硬化斑块面积(P<0.05),并增加动脉壁脂质蓄积(P<0.05)及血清胆固醇含量(P<0.05),加速AS进展。细胞内油红O染色及胆固醇含量测定研究证实,KLF4缺失促进VSMCs内胆固醇蓄积(P<0.05)。QRT-PCR和Western印迹结果证实,KLF4缺失促进VSMCs胆固醇摄取、合成、促炎因子分泌及巨噬细胞黏附和胆固醇损伤诱导的巨噬细胞标志物的表达(P<...     

核因子кB与动脉粥样硬化新进展

动脉粥样硬化(atherosclerosis,As)是一种炎症性病变,它以血管壁上巨噬细胞源性的泡沫细胞和大量趋化因子、细胞因子和生长因子堆积为主要特征.这些因手调节着固有细胞的迁移、分化和转归,最终影响动脉粥样硬化斑块形成,其中一个关键的调节因子就是转隶因子核因子-кB(NF-кB).过去它都被一直认为是一个促进动脉粥样硬化的因子,主要是由于它调节许多与动脉粥样硬化有关促炎基因的表达.最近有文献报道说NF-кB有可能在促炎、抗炎、细胞的生存和增值方面巧妙地监护着动脉粥样硬化过程的平衡.因此,本文就NF-кB与动脉粥样硬化病变有关的启动、泡沫细胞的形成、炎症、免疫、平滑肌细胞增值、纤维帽形成、细胞凋亡关系的新进展作一综述.     

马尾松树皮提取物预防apoE基因敲除小鼠肝脏脂肪变性

目的 探究马尾松树皮提取物(Pinus massoniana bark extract,PMBE)对载脂蛋白E(ApoE)基因敲除小鼠肝脏脂肪变性的影响.方法 给8只ApoE KO雄性小鼠每天口服PMBE(30 mg/kg)2周,然后喂养高胆固醇及高脂饮食8周后,与普通饮食(NC)组及高脂饮食(HCD)组进行对比,取各...     

番茄皂苷A对小鼠血脂及肝脏脂肪的作用

该研究以ApoE基因缺陷小鼠和高脂饲料诱导的高血脂症模型小鼠为对象,采用药理学方法研究了番茄皂苷A对血脂及肝脏脂肪的调节作用。在ApoE基因缺陷小鼠和高脂饲料诱导的高血脂症模型小鼠中,通过灌胃给予番茄皂苷A:取血,测定血清中总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDLC)、低密度脂蛋白胆固醇(LDLC)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、尿素氮(BUN)、肌酐(Cr)、葡萄糖(Glu)的含量和活性;处死小鼠后,取肝脏称重,计算肝脏指数;精确称取一部分肝脏,测定肝脏脂质的含量。结果表明:番茄皂苷A对ApoE基因缺陷小鼠可以降低血清TC、HDLC、LDLC的含量,对ALT、AST、BUN、Cr、Glu没有影响,说明番茄皂苷A可以降低ApoE基因缺陷小鼠血中胆固醇含量,对血糖没有影响,对肝肾功能无影响;对高脂饲料诱导的高血脂症模型小鼠,可以降低血清TC、HDLC的含量,可以降低肝脏TC的含量,对ALT、AST、BUN、Cr、Glu没有影响,说明番茄皂苷A可以改善高脂饲料诱导的高血脂症模型小鼠的脂质代谢,且对肝肾功能无影响。该研究结果表明番茄皂苷A具有一定的降低胆固醇的作用,且不影响肝肾功能。     

HFJ大鼠与Wistar大鼠用于动脉粥样硬化造模的比较研究

目的通过高脂喂养和免疫损伤结合的方法,建立HFJ近交系大鼠和Wistar封闭群大鼠动脉粥样硬化动物模型并进行比较分析。方法选择HFJ近交系和Wistar封闭群大鼠,分别随机分为模型组和正常组,正常组给予基础饲料饲喂,模型组给予高脂饲料饲喂,并采用牛血清白蛋白(40mg/kg)和卵清白蛋白(2.5mg/kg)进行免疫损伤,并辅以维生素D3(25万U/kg)灌胃,饲养90d后测定血脂水平、血液生化指标、观察病理变化和血管内皮生长因子(VEGF)免疫组化情况。结果 (1)HFJ和Wistar大鼠正常组相比较,前者TG、TC和LDL-C水平高于后者(P<0.05),HFJ大鼠模型组LDL-C含量明显高于Wistar大鼠(P<0.05);(2)心肌损伤指标,HFJ和Wistar大鼠模型组均较正常组心肌激酶(CK)、心肌激酶同工酶(CK-Mb)明显升高;(3)HE染色发现与Wistar大鼠模型组相比,HFJ近交系大鼠斑块形成更为明显,明显处于动脉粥样硬化病理Ⅲ期,可见纤维帽形成,纤维帽下具有典型的胆固醇结晶裂隙和泡沫细胞,中层平滑肌排列紊乱;(4)免疫组化法测定主动脉弓VEGF蛋白的表达,HFJ大鼠模型组较Wistar大鼠表达升高(P<0.05)。结论成功建立了HFJ大鼠动脉粥样硬化疾病动物模型,与Wistar大鼠相比HFJ大鼠模型特点更为显著,可为动脉粥样硬化研究提供一新的实验动物品系。     

短链脂肪酸丁酸抑制动脉粥样硬化形成及其分子机制

本研究通过观察丁酸对动脉粥样硬化斑块形成以及肠道组织结构和功能的影响,探讨丁酸防治动脉粥样硬化的效应及可能机制.选取8周龄雄性载脂蛋白E基因敲除(apolipoprotein E-knockout,ApoE-/-)小鼠,随机分成对照组(高脂高胆固醇饲料+饮水中给予200 mmol/L氯化钠,n = 10)和丁酸组(高脂...     

Isorhamnetin Attenuates Atherosclerosis by Inhibiting Macrophage Apoptosis via PI3K/AKT Activation and HO-1 Induction

Background and Purpose

Isorhamnetin (Iso) is a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L. Previous studies have revealed its anti-cancer, anti-inflammatory, and anti-oxidant activities. This study investigated the ability of Iso to inhibit oxidized low-density lipoprotein (ox-LDL)-induced cell apoptosis in THP-1-derived macrophages. The effects of Iso on atherosclerosis in vivo were also evaluated in apolipoprotein E knockout (ApoE-/-) mice fed a high fat diet.

Methods and Results

Iso showed significant inhibitory effects on ox-LDL-induced THP-1-derived macrophage injuries via decreasing reactive oxygen species levels, lipid deposition, and caspase-3 activation, restoring mitochondrial membrane potential, reducing the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells, and regulating apoptosis-related proteins. We also determined the protective effects of Iso by PI3K/AKT activation and HO-1 induction. Iso reduced the atherosclerotic plaque size in vivo in ApoE-/- mice as assessed by oil red O, Sudan IV staining, and CD68-positive cells, and reduced macrophage apoptosis as assessed by caspase-3 and TUNEL assays in lesions.

Conclusion

In conclusion, our results show that Iso inhibited atherosclerotic plaque development in ApoE-/- mice by PI3K/AKT activation and HO-1 induction.     

Aliskiren单用及联用氟伐他汀对动脉粥样硬化斑块稳定性的影响

目的:观察和比较肾素抑制剂aliskiren单用或与氟伐他汀(fluvastatin)联用对动脉粥样硬化斑块稳定性的影响。方法:选择4周龄雄性ApoE-/-小鼠通过喂以高脂饮食8周建立动脉粥样硬化模型,将其随机分为5组:模型对照组、aliskiren组、肼屈嗪组、氟伐他汀组、aliskiren与氟伐他汀联合用药组,所有组别均治疗12周。取主动脉根部组织评估斑块面积(HE染色)、斑块内新生血管数量(CD31染色)及斑块稳定性指标(胶原蛋白染色、弹力纤维染色、Mac-3染色、MCP-1染色)。结果:与模型对照组比较,aliskiren单用显著降低动脉粥样硬化斑块面积,减少斑块内新生血管数量以及巨噬细胞浸润、炎症因子表达,增加斑块内弹力纤维及胶原蛋白含量(P0.05或P0.01)。与aliskiren单用组比较,aliskiren与氟伐他汀联用进一步降低斑块面积,改善斑块的稳定性(P0.05或P0.01)。与aliskiren组比较,肼屈嗪组降压幅度相似(P0.05)。与模型对照组比较,肼屈嗪没有明显抑制斑块进展以及改善斑块的稳定性(P0.05)。结论:Aliskiren能够抑制动脉粥样硬化斑块的进展,减少斑块内新生血管形成,改善斑块的稳定性,而其与氟伐他汀联用的治疗效果更佳。     

Perilipin1 Deficiency in Whole Body or Bone Marrow-Derived Cells Attenuates Lesions in Atherosclerosis-Prone Mice

Aims

The objective of this study is to determine the role of perilipin 1 (Plin1) in whole body or bone marrow-derived cells on atherogenesis.

Methods and Results

Accumulated evidence have indicated the role of Plin1 in atherosclerosis, however, these findings are controversial. In this study, we showed that Plin1 was assembled and colocalized with CD68 in macrophages in atherosclerotic plaques of ApoE-/- mice. We further found 39% reduction of plaque size in the aortic roots of Plin1 and ApoE double knockout (Plin1-/-ApoE-/-) females compared with ApoE-/- female littermates. In order to verify whether this reduction was macrophage-specific, the bone marrow cells from wild-type or Plin1 deficient mice (Plin1-/-) were transplanted into LDL receptor deficient mice (LDLR-/-). Mice receiving Plin1-/- bone marrow cells showed also 49% reduction in aortic atherosclerotic lesions compared with LDLR-/- mice received wild-type bone marrow cells. In vitro experiments showed that Plin1-/- macrophages had decreased protein expression of CD36 translocase and an enhanced cholesterol ester hydrolysis upon aggregated-LDL loading, with unaltered expression of many other regulators of cholesterol metabolism, such as cellular lipases, and Plin2 and 3. Given the fundamental role of Plin1 in protecting LD lipids from lipase hydrolysis, it is reasonably speculated that the assembly of Plin1 in microphages might function to reduce lipolysis and hence increase lipid retention in ApoE-/- plaques, but this pro-atherosclerotic property would be abrogated on inactivation of Plin1.

Conclusion

Plin1 deficiency in bone marrow-derived cells may be responsible for reduced atherosclerotic lesions in the mice.     

Multicolor fluorescence technique to detect apoptotic cells in advanced coronary atherosclerotic plaques

Apoptosis occurring in atherosclerotic lesions has been suggested to be involved in the evolution and the structural stability of the plaques. It is still a matter of debate whether apoptosis mainly involves vascular smooth muscle cells (vSMCs) in the fibrous tissue or inflammatory (namely foam) cells, thus preferentially affecting the cell-poor lipid core of the atherosclerotic plaques. The aim of the present investigation was to detect the presence of apoptotic cells and to estimate their percentage in a series of atherosclerotic plaques obtained either by autopsy or during surgical atherectomy. Apoptotic cells were identified on paraffin-embedded sections on the basis of cell nuclear morphology after DNA staining and/or by cytochemical reactions (TUNEL assay, immunodetection of the proteolytic poly (ADP-ribose) polymerase-1 [PARP-1] fragment); biochemical procedures (identifying DNA fragmentation or PARP-1 proteolysis) were also used. Indirect immunofluorescence techniques were performed to label specific antigens for either vSMCs or macrophages (i.e., the cells which are most likely prone to apoptosis in atherosclerotic lesions): the proper selection of fluorochrome labeling allowed the simultaneous detection of the cell phenotype and the apoptotic characteristics, by multicolor fluorescence techniques. Apoptotic cells proved to be less than 5% of the whole cell population, in atherosclerotic plaque sections: this is, in fact, a too low cell fraction to be detected by widely used biochemical methods, such as agarose gel electrophoresis of low-molecular-weight DNA or Western-blot analysis of PARP-1 degradation. Most apoptotic cells were of macrophage origin, and clustered in the tunica media, near or within the lipid-rich core; only a few TUNEL-positive cells were labeled for antigens specific for vSMCs. These results confirm that, among the cell populations in atherosclerotic plaques, macrophage foam-cells are preferentially involved in apoptosis. Their death may decrease the cell number in the lipid core and generate a possibly defective apoptotic clearance: the resulting release of matrix-degrading enzymes could contribute to weakening the fibrous cap and promote the plaque rupture with the risk of acute ischemic events, while increasing the thrombogenic pultaceous pool of the plaque core.     

基于生物芯片探讨高脂饮食对动脉粥样硬化小鼠基因组DNA甲基化的影响

目的:采用DNA甲基化芯片技术探讨高脂饮食对Apo E-/-小鼠动脉粥样硬化模型全基因组DNA甲基化的影响。方法:30只雄性Apo E-/-小鼠随机分为正常组与高脂组,每组15只,正常组给予正常饲料喂养,高脂组给予高脂饲料喂养。16周后,测其血脂、血清同型半胱氨酸水平(Hcy)水平、血清DNA甲基化与血清DNA甲基化转移酶(DNMTs)水平;采用DNA甲基化芯片检测两组小鼠主动脉组织全基因组甲基化情况。结果:与正常组相比,高脂组小鼠血清CHOL、TG、LDL-C均显著升高,HDL-C显著下降;血清DNA甲基化水平与血清DNA甲基化转移酶(DNMTs)水平均显著升高。甲基化芯片结果显示:与正常组相比,高脂组主动脉全基因组中共有875个基因甲基化发生改变,差异具有统计学意义(P0.05),其中高甲基化基因数目496,占总数56.69%;低甲基化基因数目379,占总数的43.31%。结论:高脂饲料可升高主动脉基因组甲基化水平,降低基因组的表达,可能是Apo E-/-小鼠容易形成动脉粥样硬化的机制之一。     

Elucidating atherosclerotic vulnerable plaque rupture by modeling cross substitution of ApoE?/? mouse and human plaque components stiffnesses

The structure of mouse atherosclerotic lesions may differ from that of humans, and mouse atherosclerotic plaques do not rupture except in some specific locations such as the brachiocephalic artery. Recently, our group was the first to observe that the amplitudes of in vivo stresses in ApoE-/- mouse aortic atherosclerotic lesions were much lower and differed from those found in a previous work performed on human lesions. In this previous preliminary work, we hypothesized that the plaque mechanical properties (MP) may in turn be responsible for such species differences. However, the limited number of human samples used in our previous comparative study was relevant but not sufficient to broadly validate such hypothesis. Therefore, in this study, we propose an original finite element strategy that reconstructs the in vivo stress/strain (IVS/S) distributions in ApoE-/- artherosclerotic vessels based on cross substitution of ApoE-/- mouse and human plaque components stiffnesses and including residual stress/strain (RS/S). Our results: (1) showed that including RS/S decreases by a factor 2 the amplitude of maximal IVS/S, and more importantly, (2) demonstrated that the MP of the ApoE-/- plaque constituents are mainly responsible for the low level-compared with human-of intraplaque stress in ApoE-/- mouse aortic atherosclerotic lesions (8.36 ± 2.63 kPa vs. 182.25 ± 55.88 kPa for human). Our study highlights that such differences in the distribution and amplitude of vessel wall stress might be one key feature for explaining for the difference in lesion stability between human coronary and mouse aortic lesions.     

Perforin and Granzyme B Have Separate and Distinct Roles during Atherosclerotic Plaque Development in Apolipoprotein E Knockout Mice

The granzyme B/perforincytotoxic pathway is a well established mechanism of initiating target cell apoptosis. Previous studies have suggested a role for the granzyme B/perforin cytotoxic pathway in vulnerable atherosclerotic plaque formation. In the present study, granzyme B deficiency resulted in reduced atherosclerotic plaque development in the descending aortas of apolipoprotein E knockout mice fed a high fat diet for 30 weeks while perforindeficiency resulted in greater reduction in plaque development with significantly less plaque area than granzyme Bdeficient mice. In contrast to the descending aorta, no significant change in plaque size was observed in aortic roots from either granzyme Bdeficient or perforindeficient apolipoprotein E knockout mice. However, atherosclerotic plaques in the aortic roots did exhibit significantly more collagen in granzyme B, but not perforin deficient mice. Together these results suggest significant, yet separate roles for granzyme B and perforin in the pathogenesis of atherosclerosis that go beyond the traditional apoptotic pathway with additional implications in plaque development, stability and remodelling of extracellular matrix.