miR-32-5p通过STAT3/PTEN通路对ApoE基因敲除小鼠动脉粥样硬化斑块的影响  

Effect of miR-32-5p on atherosclerotic plaque in ApoE gene knockout mice via the STAT3/PTEN pathway

作　　者：马香书[1] 苑可心[2] 李永辉[3] 赵培[1] 路永刚[1] 帖彦清[1] MA Xiangshu;YUAN Kexin;LI Yonghui;ZHAO Pei;LU Yonggang;TIE Yanqing(Department of Clinical Laboratory,Hebei General Hospital,Shijiazhuang,Hebei 050051,China;不详)

机构地区：[1]河北省人民医院检验科,河北石家庄050051 [2]河北省人民医院心内一科 [3]河北省疾病预防控制中心

出　　处：《医学动物防制》2023年第10期989-993,997,F0003,共7页Journal of Medical Pest Control

基　　金：河北省医学科学研究重点课题(20210058)。

摘　　要：目的研究微RNA(microRNA,miRNA)-32-5p(miR-32-5p)对载脂蛋白E(apolipoprotein E,ApoE)基因敲除(ApoE^(-/-))小鼠动脉粥样硬化的作用及其是否通过信号转导和转录活化因子3(signal transducer and activator of transcription 3,STAT3)/磷酸酶及张力蛋白同源物(phosphates and tensin homologue deleted on chromosome ten,PTEN)通路影响巨噬细胞极化与动脉粥样硬化之间的联系。方法将6~8周龄动脉粥样硬化雄性ApoE^(-/-)小鼠随机分为实验组和模型组,实验组通过给小鼠注射miR-32-5p慢病毒构建过表达模型,模型组注射同等剂量生理盐水。4周后解剖小鼠,留取主动脉组织常规石蜡切片,采用苏木精-伊红(hematoxylin and eosin staining,HE)染色、免疫组化、免疫荧光法分别检测主动脉斑块面积及斑块内巨噬细胞含量;通过Western blotting(WB)检测主动脉斑块和体外培养巨噬细胞中磷酸化的信号转导和转录活化因子3(phosphorylated signal transducer and activator of transcription 3,p-STAT3)、PTEN、CCAAT增强子结合蛋白β(CCAT/enhancer-binding protein beta,C/EBP-β)等相关蛋白表达量。结果与模型组相比,实验组小鼠主动脉斑块面积明显缩小(Z=-3.477,P<0.01)、斑块内巨噬细胞数减少(Z=-3.628,P<0.01)、巨噬细胞向M2表型转化(Z=-3.628,P<0.01)。WB提示实验组p-STAT3(t=-10.381,P<0.01)、C/EBPβ(t=-6.205,P<0.01)表达量升高,而PTEN(t=7.057,P<0.01)表达量降低;M2型巨噬细胞标记物甘露糖受体/CD206(mannose receptor/CD206)(t=-5.386,P<0.01)、精氨酸酶(arginase)(t=-4.486,P<0.01)、发现于炎症区域(found in inflammatory zone,FIZZ)(t=-5.701,P<0.01)的表达量升高;体外实验同样显示实验组CD206(t=-11.431,P<0.01)、p-STAT3(t=-12.401,P<0.01)、C/EBP-β(t=-4.755,P<0.01)表达升高,PTEN(t=21.729,P<0.01)表达降低。结论miR-32-5p过表达可通过激活STAT3通路抑制PTEN,促进巨噬细胞向M2表型转化,发挥保护动脉粥样硬化的作用。Objective To study the effect of microRNA-32-5p(miR-32-5p)on atherosclerosis in ApoE knockout(ApoE^(-/-))mice and whether it affects the relationship between macrophage polarization and atherosclerosis through STAT3/PTEN pathway.Methods The male ApoE^(-/-)mice aged 6-8 weeks were randomly divided into experimental group and model group.The overexpression model was established by injecting miR-32-5p Lentivirus into the experimental group mice,and the model group mice were injected with the same dose of normal saline.Four weeks later,the mice were dissected.Aortic tissue was taken for routine paraffin sections.HE staining,immunohistochemistry and immunofluorescence were used to detect the size of aortic plaques and the contents of macrophages in plaques.Western blotting was used to verify the expression level of phosphorylated-STAT3(p-STAT3),PTEN,C/EBP-βand other related proteins in aortic plaques and in vitro cultured macrophages.Results Compared with the model group,the aortic plaque area in the experimental group was significantly reduced(Z=-3.477,P<0.01),the number of macrophages in the plaque was decreased(Z=-3.628,P<0.01),and macrophages transformed into M2 phenotype(Z=-3.628,P<0.01).Western blotting showed that the expression of p-STAT3(t=-10.381,P<0.01),C/EBP-β(t=-6.205,P<0.01)increased,while the expression of PTEN(t=7.057,P<0.01)decreased in the experimental group,and the expression of M2 macrophage markers including CD206(t=-5.386,P<0.01),arginase(t=-4.486,P<0.01)and FIZZ(t=-5.701,P<0.01)increased.In vitro experiments also showed the same result,the expression of CD206(t=-11.431,P<0.01),p-STAT3(t=-12.401,P<0.01)C/EBP-β(t=-4.755,P<0.01)in the experimental group was increased,and the expression of PTEN(t=21.729,P<0.01)was decreased.Conclusion The overexpression of miR-32-5p can inhibit PTEN by activating STAT3 pathway,promotes the transformation of macrophages to M2 phenotype and plays a protective role in atherosclerosis.

关 键 词：miR-32-5p PTEN 动脉粥样硬化 巨噬细胞 APOE基因敲除小鼠 

分 类 号：R543.5[医药卫生—心血管疾病]