NARC-1蛋白在新西兰兔动脉粥样硬化病变中的表达

Investigation of NARC-1 Protein Expression in Atherosclerotic Lesion of New Zealand Rabbit

目的探讨NARC-1在新西兰兔动脉粥样硬化病变血管壁中的表达。方法健康纯种雄性新西兰白兔16只,适应性喂养1周后,随机分为对照组和高胆固醇组,每组8只;对照组给予普通颗粒饲料喂养,高胆固醇组给予高胆固醇饲料(2%胆固醇)喂养;两组动物于8周末安乐处死。全自动生化分析仪测定兔血浆中甘油三酯、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇含量,计算动脉粥样硬化指数、高密度脂蛋白胆固醇/低密度脂蛋白胆固醇比值。苏丹Ⅳ染色测量兔主动脉粥样硬化病变面积。油红O染色及HE染色观察兔主动脉病理组织学改变,并进行病理形态学定量分析。免疫组织化学法与免疫印迹法检测兔主动脉组织NARC-1蛋白的分布及表达水平。结果8周末,高胆固醇组兔血浆总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇和、动脉粥样硬化指数水平明显升高,而高密度脂蛋白胆固醇/低密度脂蛋白胆固醇比值明显降低。高胆固醇组出现了明显的动脉粥样硬化病变,主动脉内膜面粥样硬化病变面积为27.41%±2.12%,与对照组(0.51%±0.20%)相比差异显著(P<0.05)。病理组织学显示高胆固醇组兔主动脉内膜明显增厚,内膜/中膜厚度比明显增大;油红O染色光镜下显示,高胆固醇组兔主动脉内皮下斑块内泡沫细胞呈橘红色,对照组主动脉内膜未见明显着色;HE染色光镜下显示,高胆固醇组主动脉内膜显著增生,形成明显斑块,斑块中纤维组织增生,可见大量泡沫细胞,胞核较小,胞浆内含有大量脂质空泡,对照组内膜薄且结构完整。免疫组织化学检测发现高胆固醇组兔主动脉NARC-1蛋白表达明显,染色呈强阳性,且群集于内膜斑块处;在高倍镜下,NARC-1蛋白主要分布在泡沫细胞的胞浆和胞膜,而在对照组兔血管壁组织中未见表达。免疫印迹检测发现高胆固醇组兔主动脉NARC-1蛋白呈高表达,而对照组兔血管壁组织未见表达。结论新西兰兔的主动脉粥样硬化病变处NARC-1蛋白表达明显,其主要分布在泡沫细胞的胞浆和胞膜中,正常对照组兔主动脉血管壁组织未检测到NARC-1蛋白。提示NARC-1/PCSK9是影响动脉粥样硬化病变形成与发展的重要因素。

Aim To examine the expression of NARC-1 protein in atherosclerotic lesion of New Zealand rabbits. Methods Male purebred New Zealand rabbits with adaptation feeding for 1 week, were randomly divided into control group （n = 8） and high-cholesterol group （n = 8）. The control group was given a normal control diet and the hlgh-fat group was fed a high-cholesterol diet （ 2% cholesterol）. All rabbits were euthanized at the end of the 8-week experiment. Plasma triglyceride （ TG）, total cholesterol （ TC）, high density lipoprotein cholesterol （ HDLC ） and low density lipoprotein cholesterol （LDLC） were determined by commercially enzymatic methods. Atheresclerosis index （AI） and the HDLC/LDLC ratio were calculated. Sudan IV staining was used to examine the aortic atheresclerotic lesions. The pathological changes of rabbit aortic preparations was quantified by computer-assisted image analysis of oil red O and HE staining using Image-Pro Plus image analysis software. NARC-1 protein distribution and expression was analyzed by immunohistochemistry and Western blotting. Results Plasma TC, HDLC and LDLC concentrations, and AI were markedly increased, but the HDLC/LDLC ratio was markedly decreased in high-cholesterol group. High-cholesterol diet induced significant aortic atherosclerotlc lesions. Histopathology analysis showed that aortic intima was obviously thickened and the intima/media thickness ratio was significantly increased in high-cholesterol group rabbits. Oil red O staining indicated that the number of foam cells in artery endothelium were markedly increased in the high-cholesterol group compared with the control group. HE staining revealed the aortic intima in control rabbits was intact and thin, compared to thickened aortic intima and remarkable atherosclerotic plaques in high-fat group rabbits. Immunohistochemical detection showed NARC-1 protein was found in the intima plaque and located in cytoplasma and plasma membrane of foam cells in high-fat group. Western blotting analysis also indicated NARC-1 protein expression was significantly elevated in the aortic atherosclerotie plaque of high-fat group rabbits. Conclusion The high-cholesterol diet induced aortic atherosclerotic lesion in New Zealand rabbits. NARC-1 protein was remarkably expressed in aortic atherosclerotic plaque and located in the cytoplasma and plasma membrane of the foam cells in high-fat group rabbits. The results indicate that NARC-1/PCSK9 is one of the important factors involved in atherosclerosis development.