极光激酶A通过促进巨噬细胞源性泡沫细胞形成参与动脉粥样硬化发生发展
The role of Aurora kinase A in atherosclerosis by promoting macrophage-derived foam cell formation
耿戈戈 1张峰华 2刘晓炎 1樊雯婷 1甘滔1
作者信息
- 1. 赣南医科大学基础医学院
- 2. 赣南医科大学康复学院,江西 赣州 341000
- 折叠
摘要
目的:明确极光激酶A(Aurora kinase A,AurkA)是否通过调控巨噬细胞源性泡沫细胞形成参与动脉粥样硬化发生发展.方法:从GEO数据库提取动脉粥样硬化小鼠主动脉基因表达谱数据(GSE 19286),选择特异性探针分析AurkA表达.选用8周龄普通饲料喂养的C57BL/6J健康雄性小鼠作为CC组(喂养普通饲料),将8周龄C57BL/6J背景的ApoE-/-(Apolipoprotein E KO)健康雄性小鼠随机分为AN组(喂养普通饲料)和AH组(喂养21%脂肪,0.5%胆固醇的高脂饲料),喂养16周.收集外周血及主动脉、肝脏、脾脏等组织样本,检测血清总胆固醇(Total cholesterol,TC)、甘油三酯(Triglycerides,TG)、低密度脂蛋白胆固醇(Low density lipoprotein cholesterol,LDL-C)水平和主动脉大体油红染色确认动脉粥样硬化小鼠模型建模成功后,利用qPCR方法检测各组织AurkA mRNA表达.选用8周龄C57BL/6J背景的ApoE-/-健康雄性小鼠,随机分为MC组(溶剂处理的高脂喂养的ApoE-/-小鼠)和M组(AurkA抑制剂MLN8237处理的高脂喂养的ApoE-/-小鼠),以同样方法高脂喂食16周构建动脉粥样硬化小鼠模型.在建模第12周开始M组给予小鼠20 mg·kg-1 MLN8237灌胃,每周2次,持续4周,MC组给予小鼠同等体积溶剂(10%2-羟丙基-β-环糊精和1%碳酸氢钠)处理.在建模16周后取外周血,检测血脂水平的同时,经流式细胞术检测外周血中髓性细胞占比;取主动脉经大体油红染色检测斑块总面积;取心脏制备主动脉根部切片经HE和油红染色后分析斑块面积以及斑块内脂质累积情况.基于Bloodspot数据库分析AurkA mRNA在各类血液细胞中的表达模式并构建细胞模型,使用8周龄C57BL/6J小鼠获取骨髓细胞悬液,加入10 ng·mL-1 M-CSF诱导成为骨髓源性巨噬细胞,重新铺板分为对照组(Con)、建模组(ox-LDL)以及AurkA抑制剂处理组(ox-LDL+TCS).在建模组(ox-LDL)和AurkA抑制剂处理组(ox-LDL+TCS)细胞中加入100 μg·mL-1氧化低密度脂蛋白(ox-LDL)构建泡沫细胞模型,同时给予ox-LDL+TCS组10 nM AurkA抑制剂TCS7010处理,给予ox-LDL组等量溶剂处理,48 h后利用油红染色检测泡沫细胞的脂质累积情况.结果:动脉粥样硬化小鼠主动脉基因表达谱数据显示,AurkA mRNA在病灶区的表达呈上升趋势.与CC组和AN组相比,AH组血清中的TC、TG以及LCL-C水平均升高(P＜0.05),同时主动脉中存在大量粥样斑块沉积,提示造模成功.相较AN组,AH组小鼠肝脏、脾脏以及主动脉中的AurkA mRNA水平均有上调(P＜0.05).在给予MLN8237处理后,与MC组相比,M组的体重、血脂水平以及主动脉斑块总面积均无显著改变(P＞0.05),但其主动脉根部的斑块面积以及斑块内的脂质累积均减少(P＜0.05).流式细胞术分析结果显示,M组小鼠外周血中的髓性细胞占比与MC组相当(P＞0.05),但单核细胞占比降低(P＜0.05).AurkA在血液细胞中的表达谱分析显示,其在髓性细胞尤其是巨噬细胞中特异性高表达.在细胞实验中,与ox-LDL组相比,ox-LDL+TCS组的油红阳性面积占比下降(P＜0.05),提示AurkA抑制剂能够下调巨噬细胞的脂质累积从而抑制泡沫细胞生成.结论:AurkA通过调控外周血中的单核细胞数量以及斑块中巨噬细胞源性泡沫细胞生成促进动脉粥样硬化发生发展,抑制AurkA活性可有效改善动脉粥样硬化进程.
Abstract
Objective:To determine whether Aurora kinase A(AurkA)participates in the development of atherosclerosis(AS)by regulating the formation of macrophage-derived foam cells.Methods:Gene expression profile of atherosclerotic lesions from AS mice was downloaded from GEO data(GSE 19286)and analyzed for AurkA expression changes using specific probes.Eight-week-old C57BL/6J and ApoE-/-male mice were divided into normal-diet-fed C57BL/6J control group(CC group),normal-diet-fed ApoE-/-control group(AN group)and high-fat-diet-fed ApoE-/-AS group(AH group).The high-fat-diet contained 21%fat and 0.5%cholesterol.Peripheral blood,aorta,liver and spleen samples were collected at 16-week timepoint,and blood lipid(TC,TG,LDL-C)levels and en face aorta plaque buildup were analyzed to ensure the successful establishment of AS model.Total RNA was extracted from aorta,liver and spleen to assess AurkA mRNA levels using qPCR method.After that,8-week-old ApoE-/-male mice were randomly divided into vehicle-treated control group(MC group)and AurkA inhibitor MLN8237-treated group(M group),and fed with high-fat diet for 16 weeks.Starting at 12-week timepoint,M group mice were orally administrated with MLN8327(20 mg·kg-1,twice per week)for 4 weeks and MC group mice were treated with vehicle(10%2-hydroxypropyl-β-cyclodextrin和1%sodium bicarbonate)following the same routine.The mice of both groups were weighed weekly and tissues including peripheral blood,heart,aorta were excised at week 16.Peripheral blood samples were used to assess blood lipid levels and determine the proportion of myeloid cells using flowcytometry.Aorta samples were fixed for en face oil red staining and heart samples were used to prepare aortic root sections which were then stained with HE and oil red to evaluate the degree of plaque accumulation and lipid deposition.Lastly,the expression pattern of AurkA within different hematopoietic cell lineages were analyzed using bloodspot database and cellular model was constructed to study the role of AurkA during foam cell formation.Bone marrow cells were collected from 8-week-old C57BL/6J mice and stimulated with 10 ng·mL-1 M-CSF to induce bone marrow-derived macrophage cells.The induced cells were replated into control group(Con),ox-LDL-stimulated group(ox-LDL)and AurkA inhibitor-treated group(ox-LDL+TCS),and the latter two groups were stimulated with 100 μg·mL-1 ox-LDL to induce foam cell formation.Meanwhile,ox-LDL+TCS group received 10 nM AurkA inhibitor TCS7010 treatment and ox-LDL group received same amount of vehicle treatment.After 48-hour incubation,oil red staining was performed to assess the lipid accumulation within foam cells.Results:Gene expression profile data showed that AurkA was upregulated in the atherosclerotic lesion area.Compared to either CC group or AN group,the serum levels of TC,TG and LDL-C within AH group were increased(P＜0.05),and there was a substantial accumulation of atherosclerotic plaques in the aorta from AH group mice,indicating the successful construction of AS model.In the AH group,the AurkA mRNA levels were higher in liver,spleen and aorta than that in the AN group(P＜0.05).With the treatment of MLN8237,the body weight,serum lipid levels and aortic total plaque area were not different between MC group and M group(P＞0.05),but the plaque accumulation and its lipid content within the aortic root were decreased for M group compared to MC group(P＜0.05).Flow cytometry data revealed that the myeloid cell percentages in the peripheral blood were comparable between two groups(P＞0.05),but the monocyte proportion was lower in M group than that in MC group(P＜0.05).Expression pattern analysis showed that AurkA is highly expressed in myeloid cells,especially in macrophages.In the cellular experiments,the oil red-positive area was reduced for ox-LDL+TCS group compared to ox-LDL group(P＜0.05),suggesting that AurkA inhibitor decreased lipid accumulation in macrophage and thereby constrained the formation of foam cells.Conclusion:AurkA was overexpressed during the development of atherosclerosis,and it promoted the plaque formation by regulating the number of monocytes in peripheral blood and the generation of macrophage-derived foam cells.Overall,inhibition of AurkA activity effectively limited the progression of atherosclerosis.
关键词
极光激酶A/动脉粥样硬化/泡沫细胞形成/斑块生成/髓系细胞生成Key words
Aurora kinase A/Atherosclerosis/Foam cell formation/Plague formation/Myelogenesis引用本文复制引用
基金项目
国家自然科学基金项目(31960201)
江西省教育厅科学技术研究项目(GJJ170861)
赣南医学院校级科技创新团队课题(TD201903)
赣南医学院校级重点课题(ZD201702)
研究生创新创业基金项目(YC2022-X010)
出版年
2024
国家科技期刊平台
NSTL
万方数据