基于PI3K/Akt/mTOR信号通路调控巨噬细胞自噬探讨黄芪甲苷抗动脉粥样硬化的作用机制

Mechanism of anti-atherosclerosis of astragaloside Ⅳ based on regulation of macrophage autophagy by PI3K/Akt/mTOR signaling pathway

目的观察黄芪甲苷对磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的调控,研究黄芪甲苷抗动脉粥样硬化的作用机制。方法体外细胞实验中,将小鼠巨噬细胞RAW264.7随机分为5组,即对照组、黄芪甲苷组、康士得组、雷帕霉素组以及si RNA组。分别用黄芪甲苷含药血清、Akt抑制剂康士得、mTOR抑制剂雷帕霉素及mTOR-si RNA体外处理RAW264.7细胞48 h,透射电镜观察巨噬细胞自噬体的变化,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PCR(q RT-PCR)和Western blotting法检测Akt、mTOR及自噬相关蛋白Beclin 1的表达,ELISA检测RAW264.7细胞分泌炎症因子白细胞介素-4(IL-4)、IL-10、IL-2和γ-干扰素(IFN-γ)水平。体内实验采用HE染色检测各组小鼠主动脉横截面病理损伤程度;q RT-PCR和Western blotting法分别检测小鼠主动脉组织中Akt、mTOR的mRNA和蛋白表达。结果体外实验结果显示,与对照组比较,黄芪甲苷含药血清组和各抑制剂组细胞透射电镜下观察到自噬体明显增多(P<0.05),LC3-II和Beclin1蛋白的表达水平明显上调(P<0.05),而Akt及mTOR的mRNA及蛋白表达水平明显减少(P<0.05),巨噬细胞分泌的IL-10明显降低,而分泌的IFN-γ显著增加(P<0.05)。小鼠主动脉HE染色结果显示,与模型组比较,黄芪甲苷组小鼠血管各层结构正常,排列整齐,局部有小灶性的钙化颗粒物沉积,病变轻、斑块小,泡沫细胞和脂质减少,弹力板基本完整,病变程度明显较轻,并较各抑制剂组轻。黄芪甲苷组小鼠主动脉组织Akt、mTOR的mRNA和蛋白表达均较低(P<0.05)。结论黄芪甲苷抑制动脉粥样硬化斑块的形成机制与调控PI3K/Akt/mTOR信号通路、抑制炎症反应有关。

Objective To study the mechanism of anti-atherosclerosis of astragaloside IV by observing the regulation of PI3K/Akt/mTOR signaling pathway. Methods In the in vitro experiments, macrophages were randomly divided into control group, astragaloside IV group, Kangshide group, rapamycin group and si RNA group. The changes of autophagy of macrophages were observed by transmission electron microscopy（TEM） after 48 h of mouse RAW264.7 macrophages in vitro treated by astragaloside IV-containing serum, Akt inhibitor Kangshide, mTOR inhibitor rapamycin and mTOR-si RNA. The expression of Akt, mTOR and autophagy-associated protein Beclin 1 was detected by real-time quantitative RT-PCR and Western blotting. The expression of Beclin 1 was detected by immunofluorescence and Western blotting. IL-4, IL-10, IL-2, and IFN-γ in RAW264.7 cells were detected by ELISA. In the in vivo experiments, the pathological changes of aorta were detected by HE staining. The expression of Akt, mTOR mRNA and protein in aorta of mice was detected by q RT-PCR and Western blotting, respectively. Results In vitro experimental results showed that compared with control group, the autophagus of astragaloside IV-containing serum group and each inhibitor group was significantly increased under the TEM（P〈0.05）. The expression levels of LC3-II and Beclin1 were significantly up-regulated（P〈0.05）. The expression of Akt and mTOR mRNA and protein was significantly decreased（P〈0.05）. The secretion of IL-10 was significantly decreased and the secretion of IFN-γ was significantly increased（P〈0.05）. HE staining results showed that, compared with the model group, the blood vessels of astragalus membranaceus group were normal, arranged neatly, with small focal calcification of the granules. The lesions were mild, the patches were small, the foam cells and the lipids were reduced, the elastic plates were basically complete, The degree of lesion was significantly lighter and lighter than that of the inhibitor groups. The expression of Akt, mTOR mRNA and protein were significantly lower in the aorta of astragaloside IV group（P〈0.05）. Conclusion Astragaloside IV inhibition of atherosclerotic plaque formation mechanism and regulation of PI3K/Akt/mTOR signaling pathway, inhibition of inflammatory response.