鸢尾素对载脂蛋白E基因敲除小鼠动脉粥样硬化的影响

目的研究鸢尾素对载脂蛋白E基因敲除(ApoE~(-/-))小鼠动脉粥样硬化的影响。方法将ApoE~(-/-)小鼠随机分为正常对照组、动脉粥样硬化模型组、鸢尾素组,每组10只,分别给予普通饮食+生理盐水、高脂饮食+生理盐水、高脂饮食+鸢尾素,饲养至12周后处死,处死前测体重,用化学法测定总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDLC)、高密度脂蛋白胆固醇(HDLC)、血清超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量。采用HE染色和整体油红O染色分析主动脉粥样斑块面积大小,实时荧光定量(qRT-PCR)检测主动脉CD36、NF-κB p65的mRNA表达,Western blot检测主动脉CD36、NF-κB p65、SOD的蛋白表达。结果动脉粥样硬化模型组主动脉斑块面积明显高于正常对照组,内膜明显增厚,而鸢尾素组较动脉粥样硬化模型组斑块面积减少,内膜增厚程度下降。与正常对照组比较,动脉粥样硬化模型组小鼠体重、TC、TG、LDLC、MDA水平升高,NF-κB p65、CD36mRNA和蛋白表达增加,而血清SOD活性、HDLC水平减低(P0.05)。与动脉粥样硬化模型组比较,鸢尾素组小鼠体重、TC、TG、LDLC、MDA水平降低,NF-κB p65、CD36 mRNA和蛋白表达下调,而血清SOD活性、HDLC水平升高(P0.05)。结论鸢尾素能减轻ApoE~(-/-)小鼠动脉粥样硬化的发生发展,这可能与其降低小鼠体重和血脂、抑制炎症反应、抗氧化应激有关。     

Hypertension and endothelial dysfunction in apolipoprotein E knockout mice

Mice lacking ApoE (Apoe(-/-)) develop initially hypercholesterolemia and lastly atherosclerosis. This study examined hemodynamics and endothelial function in 6-week-old Apoe(-/-) mice with hypercholesterolemia only, 7.5-months-old Apoe(-/-) mice with both hypercholesterolemia and atherosclerosis, and age matched controls. One day after implantation of catheters into the carotid artery, arterial pressure was measured in conscious, unrestrained mice. Compared with the respective controls, there was a significant increase in arterial pressure and the ratio of left ventricular weight to body weight in 7.5-month-old Apoe(-/-) mice but not in 6-week-old Apoe(-/-) mice. Histopathological analysis demonstrated significant renal artery disease in the form of extensive atheromatous plaques only in 7.5-month-old Apoe(-/-) mice, whereas no atherosclerotic lesions were found in 6-week-old Apoe(-/-) mice. For evaluation of endothelial function, a laser Doppler perfusion imager with a computer-controlled optical scanner was used to measure cutaneous blood perfusion on the dorsal side of one hind paw before and after topical application of mustard oil, which is known to induce nitric oxide-mediated vasodilation. The mustard oil treatment elicited a substantial increase in blood perfusion (P<0.01), which was similar between 6-week-old Apoe(-/-) mice and controls but significantly blunted in 7.5-month-old Apoe(-/-) mice versus control mice, suggesting nitric oxide-mediated vasodilation is diminished in 7.5-month-old Apoe(-/-) mice but not in 6-week-old Apoe(-/-) mice. In contrast, the increase in blood perfusion induced by topical administration of cilostazol, which induces vasodilation via cyclic adenosine monophosphate, was not different between 7.5-month-old Apoe(-/-) mice and controls. Thus hypertension and endothelial dysfunction observed in 7.5-month-old Apoe(-/-) mice may be due mainly to atherosclerosis.     

瑞舒伐他汀对载脂蛋白E基因敲除小鼠动脉粥样硬化的影响

目的 探讨瑞舒伐他汀对载脂蛋白E基因敲除(apoE-/-)小鼠主动脉粥样硬化的影响.方法 取apoE-/-小鼠18只建立动脉粥样硬化模型,C57BL/6小鼠12只为对照组.apoE-/-小鼠予高脂饲料,C57BL/6小鼠予普通饲料.12周后,随机抽取C57BL/6小鼠和apoE-/-小鼠各6只,判断是否成模.将成模后余下的12只apoE-/-小鼠随机分为模型组和瑞舒伐他汀治疗组(瑞舒伐他汀10 mg·kg-1·d-1灌胃),每组6只.余下的6只C57BL/6小鼠作为对照组.再过12周后处死小鼠,行血脂及主动脉HE、Masson、油红O染色观察动脉斑块,免疫组织化学方法检测主动脉组织平滑肌肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)、巨噬细胞表面分子-3(Mac-3)表达.结果 模型组小鼠胆固醇、低密度脂蛋白水平均高于对照组(P均＜0.01),甘油三酯水平与对照组比较差异无统计学意义.模型组小鼠主动脉组织内可见明显动脉粥样硬化斑块形成,α-SMA、TGF-β1和Mac-3表达均较高于对照组(P均＜0.01).瑞舒伐他汀治疗组小鼠胆固醇、低密度脂蛋白、甘油三酯水平与模型组比较差异无统计学意义,但斑块内脂肪含量少于模型组,胶原含量多于模型组.治疗组α-SMA表达与模型组比较差异无统计学意义,治疗组TGF-β1、Mac-3表达均低于较模型组(P均＜0.01).结论 瑞舒伐他汀可以减轻apoE-/-小鼠动脉粥样硬化模型中的脂质沉积和炎症反应,可以增加其胶原含量,利于斑块的稳定,具有抗动脉粥样硬化的作用,对血脂无影响.

Abstract：

Objective To investigate the effect of rosuvastatin on atherosclerosis in apoE-knockout ( apoE - / - ) mice. Methods Eighteen 6-week-old apoE - / - mice fed with high fat diet were used as atherosclerosis models, twelve 6-week-old C57BL/6 mice fed with normal diet were used as control. After twelve weeks, six apoE -/ - mice were used to observe the formation of atherosclerosis. Another 12 apoE -/- mice were divided into placebo treated group (n =6) and rosuvastatin group (n =6,10 mg· kg-1 ·d -1 per gavage) and treated for 12 weeks. Then, blood was collected for measuring lipid, aorta was prepared for morphologic study (HE, Oil red O, Masson) and immunohistochemical analysis (α-smooth activor protein, transforming growth factor β1, macrophage surface molecule-3 ). Results Serum cholesterol and low density lipoprotein levels were significantly higher in apoE -/- mice fed with high fat diet than in C57/ BL6 mice( all P ＜0. 01 )while triglyceride level was similar between the two groups, these were not affected by rosuvastatin. Similarly, atherosclerotic lesion area in apoE -/ - mice fed with high fat diet was also not significantly reduced by rosuvastatin, while lipid deposition could be significantly reduced and collagen deposition could be significantly increased in the aortic atherosclerotic lesions by treatment with rosuvastatin.Upregulated TGF-β1 and Mac-3 expression in the aortic atherosclerotic lesions in apoE -/- mice fed with high fat diet could also be significantly reduced by rosuvastatin (all P ＜ 0. 01 ), suggesting reduce inflammatory responses in the atherosclerotic lesion and stable atherosclerotic plaque post rosuvastatin treatment. Conclusion Reducing inflammatory responses and stabilizing plaque properties might contribute to the anti-atherosclerosis effects of rosuvastatin in mice high fat diet fed apoE -/- mice.     

Hydrogen decreases athero-susceptibility in apolipoprotein B-containing lipoproteins and aorta of apolipoprotein E knockout mice

ObjectiveIt is to characterize the underlying molecular mechanisms of the anti-atherosclerotic effects of hydrogen (dihydrogen; H₂), a novel antioxidant. In particular, to examine the effects of hydrogen on athero-susceptibility in lipoproteins and aorta of apolipoprotein E knockout (apoE?/?) mice.Methods and resultsPlasma analysis by enzymatic method and spectrophotometric measurement showed that eight weeks intraperitoneally injection of hydrogen-saturated saline remarkably decreased plasma total and non-high-density lipoprotein (non-HDL) cholesterol, and malondialdehyde in apoE?/? mice fed either chow or high fat diet. Western blot analysis showed hydrogen treatment reduced the contents of apolipoprotein B (apoB), a major protein constituent of non-HDL in either plasma or hepatic tissues. Moreover, ELISA assay revealed that the production of tumor necrosis factor-α and interleukin-6 were significantly suppressed by hydrogen in RAW264.7 macrophages, after stimulation with the isolated non-HDL from treated or untreated mice. Immunohistochemistry of aortic valve sections revealed that hydrogen suppressed the expression of several proinflammatory factors and decreased vessel wall infiltration of macrophages. Besides, real-time PCR and Western blot analysis disclosed that hepatic scavenger receptor class B type I (SR-BI), ATP-binding cassette (ABC) transporters ABCG8, ABCB4, ABCB11, and macrophage SR-BI, were all induced by hydrogen treatment. Finally arterial wall lipid disposition displayed by oil red O staining was reduced significantly in aortic root and whole aorta en face in hydrogen administrated mice. In addition, hydrogen significantly improved HDL functionality in C57BL/6J mice assessed in two independent ways, namely (i) stimulation of cholesterol efflux from macrophage foam cells by measuring HDL-induced [³H]cholesterol efflux, and (ii) protection against LDL oxidation as a measure of Cu²⁺-induced TBARS formation.ConclusionThese results reveal that administration of hydrogen-saturated saline decreases athero-susceptibility in apoB-containing lipoprotein and aortic atherosclerosis in apoE?/? mice and improves HDL functionality in C57BL/6J mice.     

高棕榈酸饮食对ApoE基因敲除小鼠动脉粥样硬化的影响

1目的：观察高棕榈酸饮食对载脂蛋白E（ApoE）基因敲除小鼠的血脂、血浆游离脂肪酸水平、动脉粥样硬化斑块面积、斑块中胶原含量和基质金属蛋白酶2表达的影响。方法：将20只6～8周龄雄性ApoE基因敲除小鼠随机分为对照组和高棕榈酸饮食组,每组10只。分别给予普通小鼠饲料和含5％棕榈酸的饮食,连续喂养12周。用比色法检测血脂和血浆游离脂肪酸水平;主动脉根部连续石蜡切片,Masson染色检测斑块内胶原含量,免疫组化法检测主动脉基质金属蛋白酶2的表达。结果：两组血脂水平无明显差异。与对照组相比,高棕榈酸饮食组血浆游离脂肪酸水平显著升高,主动脉斑块内胶原含量显著降低,主动脉基质金属蛋白酶2表达明显增加（P均〈0．05）。结论：高棕榈酸饮食能够升高血浆游离脂肪酸水平,降低斑块内胶原含量,从而降低动脉粥样硬化斑块稳定性,其机制可能与其上调基质金属蛋白酶2的表达有关。     

Selective effect of conjugated linoleic acid isomers on atherosclerotic lesion development in apolipoprotein E knockout mice

Research suggests that conjugated linoleic acid (CLA) may inhibit atherosclerosis, but there are contradictory results in different animal models fed heterogeneous mixtures of CLA isomers. This study addressed the hypothesis that the individual CLA isomers may exert different atherogenic properties. ApoE(-/-) mice were fed isocaloric, isonitrogenous westernized diets containing 0.15% cholesterol and enriched with 1% (w/w) cis-9,trans-11-CLA (c9,t11-CLA), trans-10,cis-12-CLA (t10,c12-CLA) or linoleic acid (control diet) for 12 weeks. At the end of the dietary intervention, the effects of CLA isomers on the development of atherosclerotic vascular lesions, lipid metabolism, inflammation and oxidative stress were assessed. The t10,c12-CLA diet had a profound pro-atherogenic effect, whereas c9,t11-CLA impeded the development of atherosclerosis. En face aortic lesion assessment showed more dorsal and lumbar extensions presenting atherosclerotic foci after the t10,c12-CLA diet. Furthermore, animals fed t10,c12-CLA had pronounced hyperlipidemia, higher 8-iso-prostaglandin F(2alpha) levels, higher vulnerable atherosclerotic plaque with a lower smooth muscle and fibre contents and higher macrophage content and activation, assayed as plasma chitotriosidase compared to the control or c9,t11-CLA dietary groups. Plasma chitotriosidase activity was more closely associated with the extent of the plaque than with MOMA staining or than monocyte chemoattractant protein-1 levels. Our results demonstrate that CLA isomers differentially modulate the development of atherosclerosis, c9,t11-CLA impedes, whereas t10,c12-CLA promotes atherosclerosis. These opposing effects may be ascribed to divergent effects on lipid, oxidative, inflammatory and fibro muscular components of this pathology. Plasma chitotriosidase is a better indicator of dietary fat interventions that alter plaque monocyte activity in this murine model.     

Fcgamma receptor deficiency confers protection against atherosclerosis in apolipoprotein E knockout mice

IgG Fc receptors (FcgammaRs) play a role in activating the immune system and in maintaining peripheral tolerance, but their role in atherosclerosis is unknown. We generated double-knockout (DKO) mice by crossing apolipoprotein E-deficient mice (apoE(-/-)) with FcgammaR gamma chain-deficient mice (gamma(-/-)). The size of atherosclerotic lesions along the aorta was approximately 50% lower in DKO compared with apoE(-/-) control mice, without differences in serum lipid levels. The macrophage and T-cell content of lesions in the DKO were reduced by 49+/-6% and 56+/-8%, respectively, compared with the content in apoE(-/-) lesions. Furthermore, the expression of monocyte chemoattractant protein-1 (MCP-1), RANTES (Regulated on Activated Normal T-cell Expressed and Secreted), and intercellular adhesion molecule-1 (ICAM-1) and the activation of nuclear factor-kappaB (NF-kappaB) were significantly reduced in aortic lesions from DKO mice. In vitro, vascular smooth muscle cells (VSMCs) from both gamma(-/-) and DKO mice failed to respond to immune complexes, as shown by impaired chemokine expression and NF-kappaB activation. ApoE(-/-) mice have higher levels of activating FcgammaRI and FcgammaRIIIA, and inhibitory FcgammaRIIB, compared with wild-type mice. The DKO mice express only the inhibitory FcgammaRIIB receptor. We conclude that FcgammaR deficiency limits development and progression of atherosclerosis. In addition to leukocytes, FcgammaR activation in VSMCs contributes to the inflammatory process, in part, by regulating chemokine expression and leukocyte invasion of the vessel wall. These results underscore the critical role of FcgammaRs in atherogenesis and support the use of immunotherapy in the treatment of this disease.     

Propagermanium reduces atherosclerosis in apolipoprotein E knockout mice via inhibition of macrophage infiltration

Monocyte chemoattractant protein-1 (MCP-1), which binds to C-C chemokine receptor 2, has been implicated as the primary source of monocyte chemoattractant function in the early stages of atherosclerosis. Recently, propagermanium, a drug used clinically for the treatment of chronic hepatitis in Japan, has been shown to inhibit C-C chemokine receptor 2 function and suppress monocyte/macrophage infiltration in vitro and in vivo. Given the importance of monocyte infiltration in atherogenesis, the inhibition of it by propagermanium might prevent atherosclerosis. Apolipoprotein E knockout (apoE-KO) mice were fed an atherogenic high cholesterol diet with or without 0.005% propagermanium for 8 or 12 weeks. Although the plasma lipid levels were unchanged by the drug treatment, atherosclerotic lesion area in the aortic root was reduced by 50% in the drug-treated apoE-KO mice compared with the nontreated apoE-KO mice after 8 weeks of cholesterol feeding (0.62+/-0.12 versus 1.27+/-0.07 mm2, respectively; P<0.01). Moreover, the accumulation of macrophages in the lesions was markedly reduced in the drug-treated group (macrophage positive area, 0.23+/-0.06 mm2 [drug-treated group] versus 0.67+/-0.07 mm2 [control group]; P<0.01). After 12 weeks of cholesterol feeding, atherosclerotic lesion formation in the aortic root and in the descending thoracic aorta was significantly reduced in the drug-treated group. Inhibition of macrophage infiltration by propagermanium prevented the formation of atherosclerotic lesions in apoE-KO mice. This drug may serve as a therapeutic tool for the treatment of atherosclerosis.     

Native incretins prevent the development of atherosclerotic lesions in apolipoprotein E knockout mice

Aims/hypothesis  

Several lines of evidence suggest that incretin-based therapies suppress the development of cardiovascular disease in type 2 diabetes. We investigated the possibility that glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) can prevent the development of atherosclerosis in Apoe ^−/− mice.     

Evaluation of vascular function in apolipoprotein E knockout mice with angiotensin-dependent renovascular hypertension

It is known that the endothelial function is compromised in atherosclerosis and arterial hypertension and that angiotensin is an important factor contributing to both pathophysiologies. The aim of this study was to evaluate the vascular function in a hypercholesterolemia/atherosclerosis model, in the angiotensin II-dependent 2-kidney 1-clip (2K1C) hypertension model and when both conditions coexist. Eight-week-old apolipoprotein E knockout (apoE; n=20) and C57BL/6 (C57; n=20) mice underwent a 2K1C or sham operation and were studied 28 days later. Mean arterial pressure was higher in apoE-2K1C and C57-2K1C (126+/-3 and 128+/-3 mm Hg) when compared with the apoE-Sham and C57-Sham (103+/-2 and 104+/-2 mm Hg, respectively; P<0.05). The vascular reactivity to norepinephrine (NE; 10(-9) to 2 x 10(-3) mol/L), acetylcholine (ACh), and sodium nitroprusside (SNP; 10(-10) to 10(-3) mol/L) was evaluated in the mesenteric arteriolar bed through concentration-effect curves. NE caused vascular hyper-reactivity in apoE-Sham, apoE-2K1C, and C57-2K1C (maximal response 146+/-5, 144+/-5, and 159+/-4 mm Hg, respectively) compared with C57-Sham (122+/-7 mm Hg; P<0.05). The ACh-induced relaxation was smaller (P<0.05) in apoE-2K1C and C57-2K1C (maximal response 53+/-3% and 46+/-3%) than in apoE-Sham and C57-Sham mice (78+/-5% and 73+/-4%). SNP-induced vascular relaxation showed similar concentration-effect curves in all groups. We conclude that in C57-2K1C mice, the increased reactivity to NE and the decreased endothelium-dependent relaxation contribute to the maintenance of hypertension. The apoE mouse, at early stages of atherosclerosis, shows hyper-reactivity to NE but does not have endothelium dysfunction yet. However, the concurrence of both pathophysiologies does not result in additive effects on the vascular function.     

培哚普利和依那普利对ApoE基因敲除小鼠动脉粥样硬化进展的影响

目的 探讨血管紧张素转换酶抑制剂(ACEI)对ApoE基因敲除小鼠动脉粥样硬化病变进程的影响,特别是对斑块成分的影响,并比较培哚普利与依那普利的疗效.方法 ApoE基因敲除小鼠随机分为培哚普利组、依那普利组及对照组3组.对主动脉根部斑块进行定量分析,并评估斑块胶原含量及脂核面积.以冰冻切片进行免疫荧光检查,观察斑块内单核细胞/巨噬细胞-2(MOMA-2)、细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子(VCAM-1)、基质金属蛋白酶-9(MMP-9)的表达.结果 各实验组之间的血压、血脂差异无统计学意义.与对照组比较,培哚普利组与依那普利组的斑块面积分别少了25.33%和22.86%(P均＜0.01),但是两组ACEI的斑块面积差异无统计学意义.培哚普利组与依那普利组减小脂核面积(分别为52.98%及38.98%,P均＜0.01)及MOMA-2(分别为88.38%及52.16%,P均＜0.01)、ICAM-1(分别为80.87%及49.59%,P均＜0.01)、VCAM-1(分别为77.56%及56.44%,P均＜0.01)、MMP-9(分别为86.93%及55.56%,P均＜0.01)的表达,并增加斑块胶原含量(分别为298.36%及168.14%,P均＜0.01),而且培哚普利组在这些方面均显著优于依那普利组(P均＜0.05).结论 ACEI在不影响血脂和血压的情况下可以抑制ApoE基因敲除小鼠动脉粥样硬化斑块的炎症并延缓动脉粥样硬化的进展.尽管培哚普利和依那普利在减少斑块面积方面差异无统计学意义,但是培哚普利在稳定斑块方面优于依那普利.     

Characteristics of intact and ruptured atherosclerotic plaques in brachiocephalic arteries of apolipoprotein E knockout mice

The brachiocephalic arteries of fat-fed apolipoprotein E knockout mice develop plaques that frequently rupture and form luminal thromboses. The morphological characteristics of plaques without evidence of instability or with healed previous ruptures (intact) and vessels with acutely ruptured plaques (ruptured) have now been defined, to understand the process of plaque destabilization in more detail. Ninety-eight apolipoprotein E knockout mice were fed a diet supplemented with 21% lard and 0.15% cholesterol, for 5 to 59 weeks. Of these 98 mice, 51 had an acutely ruptured plaque in the brachiocephalic artery. Ruptured and intact plaques differed in terms of plaque cross-sectional area (intact, 0.109+/-0.016 mm2; ruptured, 0.192+/-0.009 mm2; P=0.0005), luminal occlusion (intact, 35.3+/-3.3%; ruptured, 57.7+/-1.9%; P<0.0001), the number of buried caps within the lesion (intact, 1.06+/-0.12; ruptured, 2.66+/-0.16; P<0.0001), fibrous cap thickness (intact, 4.7+/-0.6 microm; ruptured, 2.0+/-0.3 microm; P=0.0004), and lipid fractional volume (intact, 35.9+/-3.0%; ruptured, 50.7+/-2.2%; P=0.0019). This study confirms that plaque rupture is a frequent occurrence in the brachiocephalic arteries of apolipoprotein E knockout mice on a high-fat diet. The data also show that ruptured plaques in these mice show many of the characteristics of vulnerable plaques in humans. This supports the use of this model in studies of the mechanisms and therapy of plaque rupture.     

Reduction of atherosclerosis in apolipoprotein E knockout mice by activation of the retinoid X receptor

A common feature of many metabolic pathways is their control by retinoid X receptor (RXR) heterodimers. Dysregulation of such metabolic pathways can lead to the development of atherosclerosis, a disease influenced by both systemic and local factors. Here we analyzed the effects of activation of RXR and some of its heterodimers in apolipoprotein E -/- mice, a well established animal model of atherosclerosis. An RXR agonist drastically reduced the development of atherosclerosis. In addition, a ligand for the peroxisome proliferator-activated receptor (PPAR)gamma and a dual agonist of both PPARalpha and PPARgamma had moderate inhibitory effects. Both RXR and liver X receptor (LXR) agonists induced ATP-binding cassette protein 1 (ABC-1) expression and stimulated ABC-1-mediated cholesterol efflux from macrophages from wild-type, but not from LXRalpha and beta double -/-, mice. Hence, activation of ABC-1-mediated cholesterol efflux by the RXR/LXR heterodimer might contribute to the beneficial effects of rexinoids on atherosclerosis and warrant further evaluation of RXR/LXR agonists in prevention and treatment of atherosclerosis.     

老龄载脂蛋白E基因敲除小鼠血脂及病理组织学观察

目的:观察用普食喂养而非以往研究中的高脂饮食喂养的载脂蛋白E基因敲除小鼠(Apo E-/-)的血脂及病理组织学变化特点。方法:各选取20只8周龄雄性小鼠和同龄同性C57BL/6J小鼠为对照,普食喂养6~8个月,测定血清中总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL)的含量。常规制备主动脉的病理切片,进行HE染色,观察病理学变化。结果:Apo E-/-小鼠血清中TC、TG、LDL含量明显高于对照组(P0.05),且Apo E-/-小鼠主动脉根部病理切片明显脂质斑块形成。结论:普食喂养8月龄Apo E-/-小鼠是研究动脉粥样硬化(AS)的理想动物模型,为AS性心血管疾病研究者提供了参考资料。