重组水蛭素对载脂蛋白E基因敲除小鼠动脉粥样硬化斑块的影响及机制

Effect and Mechanism of Recombinant Hirudin on Atherosclerotic Plaques in Apolipoprotein E Knockout(ApoE^(- /-) ) Mice

目的探讨重组水蛭素对载脂蛋白E基因敲除(Apo E-/-)小鼠动脉粥样硬化斑块的影响及机制。方法高脂饲喂7~8周龄Apo E-/-小鼠24只,按体重、窝别进行随机配对分组,分为药物组及模型组,每组12只,以12只高脂饲喂的7~8周龄C57BL/6J小鼠为正常对照组。药物组于10周龄起隔日给予重组水蛭素0.25 mg/kg腹腔注射,连续注射5周;模型组给予等量生理盐水注射;正常对照组不进行任何处理,均给予高脂饮食饲喂20周龄时处死。检测血清TC、TG、LDL-C、HDL-C、高敏C-反应蛋白(hs-CRP)、E-选择素(E-selectin)、白细胞介素-6(IL-6)、基质金属蛋白酶2(MMP-2)水平;HE染色及形态计量学测定主动脉根部斑块面积/管腔面积及细胞外脂质面积/斑块面积;Western blot测定主动脉钙库操纵性钙通道信号分子,包括基质交感分子1(STIM1)、Orai1蛋白、瞬时受体电位通道1(TRPC1)变化。结果 20周龄时模型组小鼠主动脉血管壁脂质斑块形成。与正常对照组比较,模型组小鼠TC、TG、LDL-C明显升高(P<0.01),血清炎症因子hs-CRP、IL-6、E-selectin、MMP-2水平亦明显升高(P<0.01,P<0.05);小鼠主动脉STIM1、Orai1及TRPC1蛋白表达增多(P<0.01);与模型组比较,药物组小鼠斑块面积/管腔面积及细胞外脂质面积/斑块面积明显下降(P<0.05,P<0.01);血清TC、LDL-C、hs-CRP、E-selction、IL-6、MMP-2水平亦降低(P<0.05,P<0.01);主动脉根部STIM1、TRPC1、Orai1蛋白表达下调(P<0.05,P<0.01)。结论水蛭素能改善Apo E-/-小鼠的血脂、内皮功能,下调STIM1、Orai1、TRPC1的表达,并通过影响上述环节进而延缓动脉粥样硬化的发生与发展。

Objective To explore the effect and mechanism of hirudin on atherosclerotic plaques in apolipoprotein E knockout （ApoE-^-） mice. Methods Totally 24 ApoE-^- mice, 7 -8 weeks old were fed with high fat diets. They were randomly divided into the recombinant hirudin treatment group （drug group） and the model group according to body weight and different dens, 12 in each group. Twelve C57BL/6J mice, 7 -8 weeks old fed with high fat diet were recruited as the normal control group. Recombinant hirudin （0.25 mg/kg） was intraperitoneally injected to mice in the drug group from the 10th week old once every other day for five successive weeks. Equal volume of normal saline was injected to mice in the model group. Mice in the normal control group received no treatment. All mice were sacrificed after fed with high fat diet until they were 20 weeks old. Serum levels of total cholesterol （TC）, triglyceride （TG）, low-density lipoprotein （LDL）, high-density lipoprotein （HDL）, high-sensitive C-reactive protein （hs-CRP）, E-selectin, interleukin-6 （IL-6）, and stromal metalloproteinase-2 （MMP-2） were detected. The plaque/lumen area and extracellular lipid composition/ plaque area were analyzed by HE staining and morphometry. Changes of signaling molecules in store-operatedcalcium channels, including stromal interacting molecule 1 （STIM1）, Orail protein, and transient receptor potential channel 1 （TRPC1） were determined by Western blot. Results Lipid plaque formed in the aorta vessel wall of 20-week old mice in the model group. Compared with the normal control group, serum levels of TC,TG and LDL increased （P 〈0. 01 ）, hs-CRP, E-selction, IL-6, and MMP-2 obviously increased （P 〈0. 01, P 〈0. 05） in the model group; expression levels of STIM1, TRPC1, and Orail significantly increased （P 〈0. 01 ）. Com- pared with the model group, the plaque/lumen area and the extracellular lipid composition/plaque area significantly decreased in the drug group（P 〈0. 05, P 〈0.01 ） ; serum levels of TC and LDL, hs-CRP, E-selction, IL-6, and MMP-2 obviously decreased （P 〈0. 05, P 〈0. 01 ） ; expression levels of STIM1, TRPC1, and Orail were significantly down-regulated （P 〈0.05, P 〈0. 01 ）. Conclusion Hirudin could significantly improve lipids and endothelial functions of ApoE-/- mice, down-regulate expression levels of STIM1, Orail, and TRPC1, and thus dela- ying the occurrence and development of atherosclerosis.