作者：王婷婷，程锦，任何，李文杰，赵晓洁，薛玉刚

单位：

空军军医大学唐都医院心内科

Units：

Department of Cardiology, Tangdu Hospital, Air Force Medical University, Xi'an 710032, China

关键词：

动脉粥样硬化; SIRT6; C57BL小鼠; 巨噬细胞;

Keywords：

Atherosclerosis; SIRT6; C57BL mouse; Macrophages

CLC：

DOI：

10.12114/j.issn.1008-5971.2023.00.099

Funds：

国家自然科学基金青年科学基金项目（82200404）；

摘要：

目的 分析SIRT6延缓动脉粥样硬化病程进展的机制。方法 本实验时间为2022年1—10月。动物实验：将10只SPF级野生型雄性C57BL小鼠作为对照组，随机选取10只雄性ApoE-/-小鼠作为动脉粥样硬化组，剩余10只雄性ApoE-/-小鼠作为动脉粥样硬化+SIRT6-/-组。对照组小鼠采用普通饲料喂养16周。动脉粥样硬化组和动脉粥样硬化+SIRT6-/-组小鼠采用高脂饲料喂养16周以构建动脉粥样硬化模型。此外，动脉粥样硬化+SIRT6-/-组小鼠采用CreLoxP方法构建巨噬细胞特异性SIRT6缺陷模型。采用HE染色检测各组小鼠动脉粥样硬化斑块面积，Western blot法检测各组小鼠主动脉组织中SIRT6、IL-32、细胞间黏附分子1(ICAM-1)表达水平，免疫荧光染色检测各组小鼠主动脉组织中CD31表达情况。细胞实验：取对数生长期的巨噬细胞RAW 264.7，将其随机分为对照组（不进行干预）、动脉粥样硬化组[采用50μg/ml的氧化型低密度脂蛋白（ox-LDL）干预24 h]、动脉粥样硬化+Ad-SIRT6组（转染腺病毒Ad-SIRT6,24 h后用50μg/ml的ox-LDL干预24 h）、动脉粥样硬化+Ad-SIRT6+Ad-IL-32组（转染腺病毒Ad-SIRT6,24 h后转染腺病毒Ad-IL-32,24 h后用50μg/ml的ox-LDL干预24 h）。采用油红O染色检测各组巨噬细胞中脂质含量。结果 HE染色结果显示，对照组小鼠动脉粥样硬化斑块面积为0；动脉粥样硬化组小鼠动脉粥样硬化斑块面积小于动脉粥样硬化+SIRT6-/-组（P<0.05）。动脉粥样硬化组、动脉粥样硬化+SIRT6-/-组小鼠主动脉组织中SIRT6表达水平低于对照组，IL-32、ICAM-1表达水平高于对照组（P<0.05）；动脉粥样硬化+SIRT6-/-组小鼠主动脉组织中SIRT6表达水平低于动脉粥样硬化组，IL-32、ICAM-1表达水平高于动脉粥样硬化组（P<0.05）。免疫荧光染色结果显示，对照组小鼠血管内膜表面光滑完整，无增厚现象；动脉粥样硬化组小鼠可见粥样硬化斑块形成，血管内膜表面欠光滑且不完整；相较于动脉粥样硬化组，动脉粥样硬化+SIRT6-/-组小鼠血管内膜下可见明显的脂质沉积，血管内膜表面欠光滑且不完整。油红O染色结果显示，对照组巨噬细胞中并未发现脂质堆积，动脉粥样硬化组巨噬细胞中脂质含量较对照组明显增多，动脉粥样硬化+Ad-SIRT6组巨噬细胞中脂质含量较动脉粥样硬化组明显减少，动脉粥样硬化+AdSIRT6+Ad-IL-32组巨噬细胞中脂质含量较动脉粥样硬化+Ad-SIRT6组明显增多。结论 SIRT6可通过下调IL-32的表达来抑制炎症反应，从而改善内皮细胞功能，减少巨噬细胞中脂质沉积，从而减少泡沫细胞的形成，进而抑制动脉粥样硬化斑块的形成，最终延缓动脉粥样硬化病情进展。

Abstract：

Objective To analyze the mechanism of SIRT6 delaying the progress of atherosclerosis. MethodsThe experiment was conducted from January to October 2022. Animal experiment: 10 SPF wild-type male C57BL mice wereselected as control group, 10 male ApoE-/- mice were randomly selected as atherosclerosis group, and the remaining 10 maleApoE-/- mice were selected as atherosclerosis+SIRT6-/- group. Mice in control group were fed with normal diet for 16 weeks. Micein atherosclerosis group and atherosclerosis+SIRT6-/- group were fed with high fat diet for 16 weeks to establish atherosclerosismodel. In addition, the macrophage-specific SIRT6 deficiency model was established in atherosclerosis+SIRT6-/- group miceby Cre-LoxP method. The atherosclerotic plaque area of mice in each group was detected by HE staining, the expression levelsof SIRT6, IL-32 and intercellular cell adhesion molecule-1 (ICAM-1) in the aorta tissue of mice in each group were detectedby Western blot, and the expression of CD31 in the aorta tissue of mice in each group was detected by immunofluorescencestaining. Cell experiment: macrophage RAW 264.7 of logarithmic growth stage was selected and randomly divided into control group (no intervention) , atherosclerosis group [treated with 50 μg/ml oxidized low-density lipoprotein (ox-LDL) for 24 h] ,atherosclerosis+Ad-SIRT6 group (transfected adenovirus Ad-SIRT6, after 24 h treated with 50 μg/ml ox-LDL for 24 h) andatherosclerosis+Ad-SIRT6+Ad-IL-32 group (transfected adenovirus Ad-SIRT6, after 24 h transfected adenovirus Ad-IL-32,after 24 h treated with 50 μg/ml ox-LDL for 24 h) . Lipid content in macrophages of each group was detected by oil red Ostaining. Results HE staining showed that the atherosclerotic plaque area of the control group was 0; the atherosclerotic plaquearea of mice in atherosclerosis group was smaller than that in atherosclerosis+SIRT6-/- group (P < 0.05) . The expression levels ofSIRT6 in aorta tissue of mice in atherosclerosis group and atherosclerosis+SIRT6-/- group were lower than those in control group,while the expression levels of IL-32 and ICAM-1 in aorta tissue of mice were higher than those in control group (P < 0.05) .The expression levels of SIRT6 in aorta tissue of mice in atherosclerosis+SIRT6-/- group were lower than those in atherosclerosisgroup, while the expression levels of IL-32 and ICAM-1 in aorta tissue of mice were higher than those in atherosclerosis group(P < 0.05) . Immunofluorescence staining showed that the intima of blood vessels in the control group was smooth and completewithout thickening; atherosclerotic plaque was formed and the intima surface was not smooth and incomplete in the atheroscleroticgroup; compared with the atherosclerosis group, obvious lipid deposition was observed under the intima of the blood vessels inthe atherosclerosis+SIRT6-/- group, and the intima surface was less smooth and incomplete. Oil red O staining showed that nolipid accumulation was found in macrophages of the control group; the lipid content of macrophages in the atherosclerosis groupwas significantly increased compared with that in the control group; the lipid content in macrophages of atherosclerosis+AdSIRT6 group was significantly reduced compared with that of atherosclerosis group; the lipid content in macrophages ofatherosclerosis+Ad-SIRT6+Ad-IL-32 group was significantly increased compared with that of atherosclerosis+Ad-SIRT6 group.Conclusion SIRT6 can inhibit the inflammatory response by down-regulating the expression of IL-32, thereby improvingthe function of endothelial cells, reducing lipid deposition in macrophages, and thus reducing the formation of foam cells, thusinhibiting the formation of atherosclerotic plaques, and ultimately delaying the progress of atherosclerosis.

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