PI3K/Akt/mTOR信号通路在巨噬细胞自噬及动脉粥样硬化斑块不稳定中的作用

 目的：探讨磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在巨噬细胞自体吞噬以及动脉粥样硬化斑块不稳定中的作用。方法：利用Akt抑制剂康士得（20 μmol/L）、mTOR抑制剂雷帕霉素（10 nmol/L）及mTOR-siRNA（30 nmol/L）体外处理小鼠RAW 264.7 巨噬细胞株 48 h后，透射电镜观察巨噬细胞自噬体的变化，细胞免疫荧光法及Western blotting法检测微管相关蛋白LC3-II表达，实时荧光定量qRT-PCR和Western blotting法检测Akt、mTOR及自噬相关蛋白Beclin 1的表达，ELISA检测巨噬细胞分泌炎症因子水平。体内实验中， 24只雄性新西兰兔给予球囊损伤+ 1%胆固醇喂养8周，然后随机分为对照组、康士得（1.0 mg·kg-1·d-1）组和雷帕霉素（0.5 mg·kg-1·d-1）组，每组8只，干预4周。血管内超声（IVUS）检测斑块的影像学特征，透射电镜观察斑块中巨噬细胞超微结构的改变，免疫荧光法检测微管相关蛋白LC3-II表达，免疫组织化学法检测巨噬细胞Akt和mTOR的蛋白表达。 结果：与对照组比较，康士得、雷帕霉素及mTOR-siRNA干预巨噬细胞后，透射电镜下观察到自噬体明显增多，微管相关蛋白LC3-II和自噬相关蛋白Beclin 1的表达水平明显上调，而Akt及mTOR 的mRNA及蛋白表达水平明显减少，巨噬细胞分泌的IL-10明显降低，而IFN-γ的分泌显著增加。体内实验： IVUS显示，与对照组比较，康士得组及雷帕霉素组的外弹性膜面积（EEMA）、斑块面积（PA）及斑块负荷（PB）明显减少，透射电镜下观察到巨噬细胞中自噬体增加，组织免疫荧光法示LC3-II明显增加，HE染色显示斑块纤维帽的厚度明显增加，内、中膜厚度显著减低，组织免疫组化染色显示巨噬细胞RAM-11及p-mTOR染色显著减少。结论：选择性抑制PI3K/Akt/mTOR信号通路能诱导巨噬细胞自噬，减少斑块巨噬细胞的浸润, 抑制炎症反应进而稳定动脉粥样硬化易损斑块。

Roles of PI3K/Akt/mTOR signaling pathway in macrophage autophagy and atherosclerotic plaque instability

AIM：To investigate whether selective inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.METHODS：In in vitro study, casodex (20 μmol/L), rapamycin (10 nmol/L) or mTOR-siRNA (30 nmol/L) was used to treat mouse macrophage cell line RAW 264.7. Inflammation-related cytokines secreted by macrophages were measured by means of ELISA. Ultrastructural changes of the macrophages were examined by transmission electron microscopy. The mRNA and protein expression levels of Akt, mTOR and autophage-related protein Beclin 1 were assayed by real-time fluorescence quantitative RT-PCR and Western blotting. The expression of autophagy-related indicator LC3-II was detected by immunofluorescence and Western blotting. In in vivo study, 24 New Zealand white rabbits underwent balloon-induced abdominal aortic wall injury and were fed with a diet of 1% cholesterol for 8 weeks. The rabbits were randomly divided into control group (n=8), casodex group (1.0 mg·kg-1·d-1, n=8) and rapamycin group (0.5 mg·kg-1·d-1, n=8). Four weeks after drug administration, intravascular ultrasound (IVUS) was carried out to observe the plaque imaging. Ultrastructural changes of the macrophages and the protein expression of Akt, mTOR and LC3-II in the macrophages were also measured.RESULTS：In in vitro study, more typical autophagosomes were detected in casodex-, rapamycin- or mTOR-siRNA-treated cells. The expression level of LC3-II increased, but Beclin 1,p-Akt and p-mTOR significantly decreased in the 3 treatment groups. The concentration of IL-10 decreased while IFN-γ significantly increased in the treatment groups. In in vivo study, IVUS found that external elastic membrane area (EEMA),plaque area(PA) and plaque burden (PB) significantly decreased in casodex and rapamycin treatment groups. Expression of LC3-II increased significantly in the 2 treatment groups. The staining of RAM-11 and p-mTOR in the macrophages was significantly reduced as compared with control group.CONCLUSION：Selective inhibition of PI3K/Akt/mTOR signaling pathway reduces the infiltration of macrophages and stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.