生长素对载脂蛋白E基因敲除小鼠动脉粥样硬化斑块与炎症因子的影响

The effect of ghrelin on the atherosclerosis plaque and inflammatory factors in apolipoprotein E knockout mice

目的观察生长素对载脂蛋白E基因敲除(ApoE^(-/-))小鼠血浆白细胞介素8(IL-8)、单核细胞趋化因子1(MCP-1)、肿瘤坏死因子α(TNF-α)水平和血管壁核转录因子κBp65(NFκBp65)表达及动脉粥样斑块的影响。方法8周龄雄性ApoE^(-/-)小鼠12只饲以西方类型膳食12周建立动脉粥样硬化模型,遗传背景相同的6只8周龄雄性C57BL/6J小鼠饲以同类型膳食作对照。第8周时模型组分为腹腔内注射生长素(100μg/kg)组(n=6)和注射生理盐水(0.1mL)组(n=6),C57BL/6J小鼠亦给予生理盐水(0.1mL)腹腔内注射。第12周时眼眶取血,分离血浆,酶联免疫吸附试验(ELISA)法检测IL-8、MCP-1、TNF-α水平。取小鼠主动脉进行苏丹Ⅳ染色,观察主动脉粥样硬化病变面积占主动脉内膜面积的比例,行主动脉窦HE及油红O染色,观察主动脉窦动脉粥样斑块面积占管腔总面积的比例,行主动脉免疫组化染色,观察NFκBp65的表达。结果与C57BL/6J小鼠比较,ApoE^(-/-)小鼠和ApoE^(-/-)+生长素小鼠总胆固醇和低密度脂蛋白胆固醇高[(6.7±0.5),(7.6±2.0)比(5.5±0.2)mmol/L,(5.6±0.3),(6.0±0.5)比(2.2±0.1)mmol/L,均P<0.05],ApoE^(-/-)+生长素小鼠高密度脂蛋白胆固醇比ApoE^(-/-)组高[(0.5±0.1)比(0.3±0.1)mmol/L,P<0.05]。与C57BL/6J小鼠比较,ApoE^(-/-)小鼠主动脉粥样硬化病变面积占主动脉内膜面积比例增加[(15.1±1.7)%比0],ApoE^(-/-)+生长素小鼠主动脉粥样病变面积占主动脉内膜面积比例较ApoE^(-/-)小鼠降低[(10.1±0.5)%比(15.1±1.7)%,P<0.05];C56BL/6J小鼠主动脉窦无动脉斑块形成(0%),ApoE^(-/-)组和ApoE^(-/-)+生长素组均有动脉斑块形成,但ApoE^(-/-)+生长素组小鼠主动脉窦斑块面积占管腔总面积的比例较ApoE^(-/-)组低[(22.6±2.2)%比(32.4±3.2)%,P<0.01]。ApoE^(-/-)小鼠TNF-α、IL-8和MCP-1水平较C57BL/6J小鼠增高[分别为(24.5±1.7)比(10.1±0.5)ng/L,(33.5±16.7)比(16.8±8.8)ng/L,(78.0±5.6)比(13.5±1.8)ng/L;均P<0.05],ApoE^(-/-)+生长素组TNF-α、IL-8和MCP-1水平较ApoE^(-/-)组降低[分别为(15.5±1.0)比(24.5±1.7)ng/L,(22.0±1.2)比(33.5±16.7)ng/L,(45.5±4.5)比(78.0±5.6)ng/L;均P<0.05]。ApoE^(-/-)小鼠血管壁NF-κBp65表达较C57BL/6J增高,ApoE^(-/-)小鼠+生长素组血管壁NF-κBp65表达较ApoE^(-/-)小鼠组降低(P<0.05)。结论生长素通过抑制炎症反应减少ApoE^(-/-)小鼠动脉粥样斑块形成。

Objective To observe the effect of ghrelin on plasma interleukin-8 （IL-8）, monocyte chemotactic pro tein-1 （MCP-1）, tumor necrosis factor-α（TNF-α） levels, nuclear factor-kappaB p65 （NFκBp65） expression in vascular wall and the regression of atherosclerotic plaque in apolipoprotein E knockout （ ApoE^-/-） mice. Methods Twelve 8 week-old ApoE^-/- mice fed with Western type diet to establish atherosclerosis model, and six 8 week-old C57BL/6J mice fed with the same diet as control. In the eighth week, ApoE^-/- mice were randomly intraperitoneal injected of ghrelin （100 μg/kg） as ghrelin group （ApoE^-/-＋ghrelin group, n= 6） or saline （0.1 mL） as saline group （ApoE^-/- group, n=6）. C57BL/6J mice were also given saline （0.1 mL） intraperitoneal injection. At the end of the 12th week, blood samples were drawn from orbit to detect plasma levels of IL-8, MCP-1 and TNF a using enzyme linked immunosorbent adsorption assay （ELISA）. The aortas were seperated and stained with Sudan IV to calculate the proportion of atherosclerotic lesion area. Aortic sinus HE staining and oil red O staining were used to observe the proportion of atheromatous plaque area accounted for the total lumen area and immunohistochemical staining was used to observe the expression of NFκBp65. Results Compared with C57BL/6J mice, the total cholesterol and low density lipoprotein cholesterol in ApoE / and ApoE^-/- ghrelin group were higher [ （ 6.7± 0.5 ）, （7.6±2.0） vs （5.5±0.2） mmol/L, （5.6±0.3）, （6.0±0.5） vs （2.2±0.1） mmol/L, respectively, all P〈 0. 05]. The levels of high density lipoprotein cholesterol in ApoE^-/-＋ghrelin group were higher than ApoE^-/-group[（0. 5±0. 1 ） vs （0. 3±0.1 ）mmol/L, P〈0. 05]. Compared with C57BL/6J mice, the proportions of arota atherosclerosis lesion increased in ApoE^-/- group [（ 15. 1 ± 1.7）% vs 0], which were also higher than that in ApoE^-/-＋ghrelin group [（15.1±1.7）0/00 vs （10.1±0.5）%, P〈0.05]. C57BL/6J mice had no atherosclerosis plaque formation in aortic sinus, which occured both in ApoE^-/- group and ApoE^-/-＋ghrelin group, and the pro portion of aortic sinus plaque area in ApoE^-/-＋ghrelin group was lower than that in ApoE / group [（22.6 ± 2.2）% vs （32.4±3.2）%, P〈0.01]. The levels of TNF-α, IL-8 and MCP-1 in C57BL/6J andapoE^-/-＋ghrelin mice were lower than that inApoE^-/- mice[（10.1±0.5）, （15.5±1.0） vs （24. 5±1. 7）ng/L, （16.8±8.8）, （22.0±1.2） vs （33.5±16.7）ng/L, （13.5±1.8）, 145.5±4.5） vs （78.0±5.6）ng/L, respectively; all P〈0.05]. NF-κBp65 expression in the vascular wall of ApoE / mice was higher than that of C57BL/6J mice, and which was lower in ApoE^-/- ＋ghrelin group than in ApoE^-/- group （all P〈0.05）. Conclusion Ghrelin can decrease ather osclerosis plaque formation in ApoE^-/- mice by inhibiting inflammatory response.