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。

The Effects of Different Degree Endoplasmic ReticulumStress on theStability of Atherosclerosis Plaque in ApoE -/-mice

Objective:By using the established model of unstable atherosclerosis plaque in ApoE - / - mice, we induced differentdegree of endoplasmic reticulumstress by giving different doses of tunicamycin, and observed the effects on the stablity of atherosclerosisplaque.Methods:Forty 6-8 weeks old ApoE-/- mice were fed with high-fat diet for 1 week and were randomly divided into the controlgroup and the surgery group. Mice in surgery group were subjected to perivascular carotid collar placement (PCCP), after which fat feedingwas continued. After 9 weeks, the animals were sacrificed and H&E staining of carotid artery was performed to observe the formationof atherosclerotic plaque. To observe the effect of different doses tunicamycin on atherosclerotic plaque stability, mice were randomly dividedinto four groups. Saline, saline,low-dose and high-dose tunicamycin were treated (intraperitoneal injection) respectively in mice incontrol, PCCP, low-dose and high-dose tunicamycin groups. Two weeks later, mice were sacrificed. H&E staining was conducted for observationof carotid plaque morphology, oil red O staining for observation of lipid accumulation within plaque, immunohistochemistry fordetectionofmacrophage accumulation withinplaque,Western-blot for the detectionofGRP78,Atg7 andp62 expressionlevels.Results:H&Estaining showed that compared with PCCP only and high-dose tunicamycin group, low-dose tunicamycin group had significantly smallerlipid pool within carotid plaque and the plaque was more stable; Oil Red O staining showed that compared with PCCP only andhigh-dose tunicamycin group, low-dose tunicamycin group had significantly lower lipid content within carotid plaque (P<0.05);macrophages immunohistochemical staining showed compared with PCCP only and high-dose tunicamycin group, there were significantlyfewer macrophages within carotid plaque in low-dose tunicamycin group (P<0.05).Conclusion:PCCP surgery with high lipid diet can successfully establish unstable atherosclerotic plaque model which atherosclerotic plaque was highly instable. Small-dose of tunicamycinintervention can make the carotid artery lumen patches relatively smaller, internal lipid pool significantly smaller, fibrous cap more thickerand plaque structural more integrity, plaque structure more stable, reduced Lipid content in the plaque and significantly decreasedmacrophages, suggesting that moderate endoplasmic reticulum stress caused by small-dose of tunicamycin could protect unstableatherosclerotic plaque.