作者：王婷婷，李妍，张明明，蒋国健，张东伟

单位：

710032陕西省西安市，空军军医大学唐都医院心内科 通信作者：张东伟，E-mail：zhdwuu@163.com

Units：

Department of Cardiology, Tangdu Hospital, Air Force Medical University, Xi'an 710032, China Corresponding author: ZHANG Dongwei, E-mail: zhdwuu@163.com

关键词：

动脉粥样硬化; 斑块,动脉粥样硬化; SIRT6; CD36; 巨噬细胞;

Keywords：

Atherosclerosis; Plaque, atherosclerotic; SIRT6; CD36; Macrophages

CLC：

DOI：

10.12114/j.issn.1008-5971.2022.00.080

Funds：

国家自然科学基金资助项目（81900338）

摘要：

目的 通过动物实验和细胞实验分析SIRT6增加动脉粥样硬化斑块稳定性的机制。方法 本实验时间为2019年12月至2021年12月。动物实验:将10只雄性C57BL小鼠作为空白对照组;将20只雄性ApoE-/-小鼠随机分为动脉粥样硬化组和动脉粥样硬化+SIRT6 Tg组,每组10只。采用高脂饲料喂养动脉粥样硬化组和动脉粥样硬化+SIRT6 Tg组小鼠16周以构建动脉粥样硬化小鼠模型。其中动脉粥样硬化+SIRT6 Tg组通过尾静脉注射200μl滴度为1×109 eg/ml的腺相关病毒(AAV)-SIRT6,共注射3次,在1周内完成注射,以过表达SIRT6。干预16周后,采用HE染色检测各组小鼠动脉粥样硬化斑块面积,Masson染色检测各组小鼠动脉粥样硬化斑块中胶原含量,免疫组化染色检测各组小鼠动脉粥样硬化斑块中CD68表达水平。细胞实验:取对数生长期的巨噬细胞,将其分为空白对照组、氧化低密度脂蛋白(ox-LDL)组(采用50μg/ml的ox-LDL干预24 h)、ox-LDL+Ad-SIRT6组(转导腺病毒Ad-SIRT6 24 h,用50μg/ml的ox-LDL干预24 h);采用Western blotting法检测各组巨噬细胞中SIRT6、CD36表达水平。取对数生长期的巨噬细胞,将其分为空白对照组、ox-LDL组(采用50μg/ml的ox-LDL干预24 h)、ox-LDL+Ad-SIRT6组(转导腺病毒AdSIRT6 24 h,用50μg/ml的ox-LDL干预24 h)、ox-LDL+Ad-SIRT6+Ad-CD36组(转导腺病毒Ad-SIRT6 24 h,转导腺病毒Ad-CD36 24 h,用50μg/ml的ox-LDL干预24 h);采用TUNEL染色检测各组巨噬细胞凋亡率。结果 动物实验:空白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高于动脉粥样硬化+SIRT6Tg组(P<0.05)。空白对照组小鼠动脉粥样硬化斑块中胶原含量为0,动脉粥样硬化组小鼠动脉粥样硬化斑块中胶原含量低于动脉粥样硬化+SIRT6 Tg组(P<0.05)。空白对照组小鼠动脉粥样硬化斑块中CD68表达水平为0,动脉粥样硬化组小鼠动脉粥样硬化斑块中CD68表达水平高于动脉粥样硬化+SIRT6 Tg组(P<0.05)。细胞实验:ox-LDL组、ox-LDL+Ad-SIRT6组巨噬细胞中SIRT6表达水平低于空白对照组,CD36表达水平高于空白对照组(P<0.05);ox-LDL+Ad-SIRT6组巨噬细胞中SIRT6表达水平高于ox-LDL组,CD36表达水平低于ox-LDL组(P<0.05)。ox-LDL组、ox-LDL+Ad-SIRT6组、ox-LDL+Ad-SIRT6+Ad-CD36组巨噬细胞凋亡率高于空白对照组,ox-LDL+Ad-SIRT6组、ox-LDL+Ad-SIRT6+Ad-CD36组巨噬细胞凋亡率低于ox-LDL组,ox-LDL+Ad-SIRT6+Ad-CD36组巨噬细胞凋亡率高于ox-LDL+Ad-SIRT6组(P<0.05)。结论 SIRT6能够缩小ApoE-/-小鼠动脉粥样硬化斑块面积,增加动脉粥样硬化斑块中胶原含量,减少动脉粥样硬化斑块中巨噬细胞的浸润,抑制巨噬细胞凋亡,从而增加动脉粥样硬化斑块稳定性,而这一作用是通过下调CD36的表达水平来实现的。

Abstract：

【Abstract】　Objective To analyze the mechanism of SIRT6 increasing atherosclerotic plaque stability by animalexperiment and cell experiment. Methods The experiment time was from December 2019 to December 2021. Animalexperiment: 10 male C57BL mice were used as blank control group; 20 male ApoE-/- mice were randomly divided intoatherosclerosis group and atherosclerosis+SIRT6 Tg group, 10 mice in each group. The mice in the atherosclerosis group and theatherosclerosis+SIRT6 Tg group were fed with high-fat diet for 16 weeks to establish atherosclerosis model. Among them, theatherosclerosis+SIRT6 Tg group was injected with 200 μl of adeno-associated virus (AAV) -SIRT6 with a titer of 1×109 eg/mlthrough the tail vein for a total of 3 injections, and the injection was completed within 1 week to overexpress SIRT6. After 16weeks of intervention, HE staining was used to detect the area of atherosclerotic plaque, Masson staining was used to detect thecollagen content in the atherosclerotic plaque, and immunohistochemical staining was used to detect the expression level of CD68in the atherosclerotic plaque of mice in each group. Cell experiment: the macrophages in the logarithmic growth phase were takenand divided into blank control group, oxidized low density lipoprotein (ox-LDL) group (intervened with 50 μg/ml ox-LDL for24 h) , ox-LDL+Ad-SIRT6 group (transduced adenovirus Ad-SIRT6 for 24 h, and intervened with 50 μg/ml ox-LDL for 24 h) ;Western blotting was used to detect the expression levels of SIRT6 and CD36 in macrophages in each group. The macrophagesin the logarithmic growth phase were taken and divided into blank control group, ox-LDL group (intervened with 50 μg/mlox-LDL for 24 h) , ox-LDL+Ad-SIRT6 group (transduced adenovirus Ad-SIRT6 for 24 h, and intervened with 50 μg/mlox-LDL for 24 h) , and ox-LDL+Ad-SIRT6+Ad-CD36 group (transduced adenovirus Ad-SIRT6 for 24 h, transduced adenovirusAd-CD36 for 24 h, and intervened with 50μg/ml ox-LDL for 24 h) ; TUNEL staining was used to detect the apoptosis rate ofmacrophages in each group.Results Animal experiment: the area of atherosclerotic plaque in the blank control group was 0; thearea of atherosclerotic plaque in the atherosclerosis group was higher than that in the atherosclerosis+SIRT6 Tg group (P <0.05) .The content of collagen in the atherosclerotic plaque in the blank control group was 0; the content of collagen in the atheroscleroticplaque in the atherosclerosis group was lower than that in the atherosclerosis+SIRT6 Tg group (P <0.05) . The level of CD68expression in the atherosclerotic plaque in the control group was 0; the level of CD68 expression in the atherosclerotic plaque inthe atherosclerosis group was higher than that in the atherosclerosis+SIRT6 Tg group (P <0.05) . Cell experiment: the level ofSIRT6 expression in macrophages in the ox-LDL group and ox-LDL+Ad-SIRT6 group was lower than that in the blank controlgroup, and the level of CD36 expression was higher than that in the blank control group (P <0.05) ; the level of SIRT6 expressionin macrophages in the ox-LDL+Ad-SIRT6 group was higher than that in the ox-LDL group, and the level of CD36 expression waslower than that in the ox-LDL group (P <0.05) . The apoptosis rate of macrophages in ox-LDL group, ox-LDL+Ad-SIRT6 group,ox-LDL+Ad-SIRT6+Ad-CD36 group was higher than that in blank control group (P <0.05) ; the apoptosis rate of macrophagesin ox-LDL+Ad-SIRT6 group and ox-LDL+Ad-SIRT6+Ad-CD36 group was lower than that in ox-LDL group (P <0.05) ; theapoptosis rate of macrophages in ox-LDL+Ad-SIRT6+Ad-CD36 group was higher than that in ox-LDL+Ad-SIRT6 group (P<0.05) .Conclusion SIRT6 can reduce the area of atherosclerotic plaques in ApoE-/- mice, increase the collagen content inatherosclerotic plaques, reduce the infiltration of macrophages in atherosclerotic plaques, and inhibit macrophages apoptosis,thereby increasing the stability of atherosclerotic plaques, and this effect is achieved by down-regulating the expression levelof CD36.

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