异鼠李素通过抑制泡沫细胞形成增加动脉粥样硬化斑块稳定性的机制研究

Mechanism of Isorhamnetin Increasing the Stability of Atherosclerotic Plaques by Inhibiting Foam Cell Formation

背景泡沫细胞的形成是动脉粥样硬化重要的病理基础,其是由巨噬细胞吞噬大量胆固醇和三酰甘油后转化而来。因此,如何促进巨噬细胞的脂质代谢、抑制其转化为泡沫细胞是延缓动脉粥样硬化病情进展的关键。目的分析异鼠李素通过抑制泡沫细胞形成增加动脉粥样硬化斑块稳定性的机制。方法本实验时间为2020年8月至2021年10月。动物实验:将10只雄性C57BL小鼠作为空白对照组(不进行干预);将20只ApoE^(-/-)小鼠随机分为动脉粥样硬化组(采用高脂饲料喂养16周以构建动脉粥样硬化小鼠模型)和动脉粥样硬化+异鼠李素组(采用高脂饲料喂养16周以构建动脉粥样硬化小鼠模型,于高脂饲料喂养第9周开始给予异鼠李素,连续给药8周),每组10只。检测三组小鼠动脉粥样硬化斑块中脂质核心面积百分比、泡沫细胞所占比例。细胞实验:取对数生长期的RAW 264.7巨噬细胞,将其分为空白对照组(不进行干预)、氧化低密度脂蛋白(ox-LDL)干预组(采用ox-LDL干预24 h以体外诱导泡沫细胞形成)、ox-LDL+异鼠李素干预组(采用异鼠李素孵育8 h,之后采用ox-LDL干预24 h以体外诱导泡沫细胞形成),检测各组巨噬细胞中SIRT6表达水平。取对数生长期的RAW 264.7巨噬细胞,将其分为空白对照组(不进行干预)、ox-LDL组(采用ox-LDL干预24 h以体外诱导泡沫细胞形成)、ox-LDL+异鼠李素组(采用异鼠李素孵育8 h,之后采用ox-LDL干预24 h以体外诱导泡沫细胞形成)、ox-LDL+异鼠李素+Ad-sh-SIRT6组〔采用异鼠李素孵育8 h,转染Ad-sh-SIRT6腺病毒以干扰SIRT6的表达,12 h后用ox-LDL干预24 h以体外模拟动脉粥样硬化状态〕,检测各组巨噬细胞中脂滴数目。结果动物实验:动脉粥样硬化+异鼠李素组小鼠动脉粥样硬化斑块中脂质核心面积百分比低于动脉粥样硬化组(P<0.05)。动脉粥样硬化组小鼠动脉粥样硬化斑块中泡沫细胞所占比例高于动脉粥样硬化+异鼠李素组(P<0.05)。细胞实验:ox-LDL干预组、ox-LDL+异鼠李素干预组巨噬细胞中SIRT6表达水平低于空白对照组(P<0.05);ox-LDL+异鼠李素干预组巨噬细胞中SIRT6表达水平高于ox-LDL干预组(P<0.05)。ox-LDL组、ox-LDL+异鼠李素组、ox-LDL+异鼠李素+Ad-sh-SIRT6组巨噬细胞中脂滴数目多于空白对照组(P<0.05);ox-LDL+异鼠李素组巨噬细胞中脂滴数目少于ox-LDL组(P<0.05);ox-LDL+异鼠李素+Ad-sh-SIRT6组巨噬细胞中脂滴数目多于ox-LDL组、ox-LDL+异鼠李素组(P<0.05)。结论异鼠李素通过上调SIRT6表达水平,减少巨噬细胞中脂滴数目,抑制动脉粥样硬化斑块中泡沫细胞形成,缩小动脉粥样硬化斑块中脂质核心面积,进而增加动脉粥样硬化斑块稳定性。

Background The formation of foam cells is an important pathological basis of atherosclerosis,which is transformed by macrophages after phagocytosis of large amounts of cholesterol and triacylglycerols.Therefore,how to promote the lipid metabolism of macrophages and inhibit their transformation into foam cells is the key to delay the progression of atherosclerosis.Objective To analyze the mechanism of isorhamnetin increasing the stability of atherosclerotic plaques by inhibiting foam cell formation.Methods The experiment time was from August 2020 to October 2021.Animal experiment:10 male C57BL mice were used as blank control group(no intervention);20 ApoE^(-/-)mice were randomly divided into atherosclerosis group(fed with high-fat diet for 16 weeks to construct atherosclerosis mouse model)and atherosclerosis+isorhamnetin group(fed with high-fat diet for 16 weeks to construct atherosclerosis mouse model,and given isorhamnetin from the ninth week of high-fat diet for 8 consecutive weeks),10 mice in each group.The percentage of lipid core area and the proportion of foam cells in the atherosclerotic plaques of the three groups of mice were detected.Cell experiment:RAW 264.7 macrophages in logarithmic growth phase were taken and divided into blank control group(no intervention),oxidized low-density lipoprotein(ox-LDL)intervention group(with ox-LDL intervention for 24 h to induce foam cell formation in vitro),and ox-LDL+isorhamnetin intervention group(incubated with isorhamnetin for 8 h,and then intervened with ox-LDL for 24 h).The expression levels of SIRT6 in macrophages in each group were detected.RAW 264.7 macrophages in the logarithmic growth phase were taken and divided into blank control group(no intervention),ox-LDL group(with ox-LDL intervention for 24 h to induce foam cell formation in vitro),ox-LDL+isorhamnetin group(incubated with isorhamnetin for 8 h,then intervened with ox-LDL for 24 h to induce foam cell formation in vitro),ox-LDL+isorhamnetin+Ad-sh-SIRT6 group[incubated with isorhamnetin for 8 h,transfected with Ad-sh-SIRT6 adenovirus to interfere with the expression of SIRT6,and 12 h later intervented with ox-LDL for 24 h to simulate atherosclerosis in vitro].The number of lipid droplets in macrophages in each group was detected.Results Animal experiment:the percentage of lipid core in atherosclerotic plaques in the atherosclerosis+isorhamnetin group was lower than that in the atherosclerosis group(P<0.05).The proportion of foam cells in atherosclerotic plaques in the atherosclerosis group was higher than that in the atherosclerosis+isorhamnetin group(P<0.05).Cell experiment:the expression level of SIRT6 in macrophages in the ox-LDL intervention group and ox-LDL+isorhamnetin intervention group was lower than that in the blank control group(P<0.05);the expression level of SIRT6 in macrophages in the ox-LDL+isorhamnetin intervention group was higher than that in the ox-LDL intervention group(P<0.05).The number of lipid droplets in macrophages in ox-LDL group,ox-LDL+isorhamnetin group,ox-LDL+isorhamnetin+Ad-sh-SIRT6 group was more than that in blank control group(P<0.05);the number of lipid droplets in macrophages in the ox-LDL+isorhamnetin group was less than that in the ox-LDL group(P<0.05);the number of lipid droplets in macrophages in ox-LDL+isorhamnetin+Ad-sh-SIRT6 group was more than that in ox-LDL group and ox-LDL+isorhamnetin group(P<0.05).Conclusion Isorhamnetin reduces the number of lipid droplets in macrophages by up-regulating the expression level of SIRT6,inhibits the formation of foam cells in atherosclerotic plaques,and reduces the lipid core area in atherosclerotic plaques,thereby increasing atherosclerotic plaques stability.