血管软化丸调控PI3K/Akt/mTOR通路影响细胞自噬及抗动脉粥样硬化的作用机制研究

Mechanism of Vascular Softening Pills Anti-Atherosclerosis Based on Regulation of Macrophage Autophagy by PI3K/Akt/mTOR Signaling Pathway

目的通过观察血管软化丸对磷脂酰肌醇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,研究血管软化丸抗动脉粥样硬化的作用机制。方法动物实验,观察各组小鼠主动脉横截面病理损伤程度,观察血管软化丸对小鼠主动脉组织Akt、mTOR mRNA和蛋白质表达。体外实验,观察血管软化丸含药血清、Akt抑制剂康士得、mTOR抑制剂雷帕霉素及mTOR-siRNA对小鼠RAW264.7巨噬细胞的影响,观察巨噬细胞自噬体的变化,观察含药血清对巨噬细胞微管相关蛋白LC3-II表达的影响,观察含药血清对巨噬细胞Akt、mTOR、微管相关蛋白LC3-II及自噬相关蛋白Beclin 1的表达的影响,观察含药血清对巨噬细胞分泌炎症因子水平的影响。结果动物实验显示:血管软化丸组动脉粥样硬化病变程度明显较轻。与对照组比较,血管软化丸组小鼠主动脉组织Akt和mTOR表达水平明显较低(均P<0.05)。体外实验发现,与对照组相比,血管软化丸含药血清组和各抑制剂组透射电镜下观察到自噬体数量明显较大(均P<0.05),巨噬细胞微管相关蛋白LC3-II和Beclin1表达明显升高(均P<0.05),而Akt及mTOR的mRNA及蛋白表达水平明显较低(均P<0.05),巨噬细胞分泌的炎症因子IL-10水平明显高低(P<0.05),而炎症因子IFN-γ水平明显较高(P<0.05)。结论血管软化丸通过抑制炎症反应治疗动脉粥样硬化的分子机制可能与调控PI3K/Akt/mTOR信号通路,调节巨噬细胞自噬,减少斑块巨噬细胞浸润有关。

Objective To study the mechanism of Vascular Softening Pills against atherosclerosis by observing the regulation of phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt)/mammalian rapamycin target protein(mTOR)signaling pathway.Methods The animal experiments were performed to observe the degree of pathological damage of aorta in each group.The expressions of Akt and mTOR mRNA and protein in aortic tissue of Vascular Softening Pills were observed.In vitro experiments were performed to observe the effects of Vascular Softening Pills-containing serum,Akt inhibitor Kangshide,mTOR inhibitor rapamycin and mTOR-siRNA on RAW264.7 macrophages and observe the changes of macrophage autophagosomes.We observed the effect of drug-containing serum on the expression of microtubule-associated protein LC3-II,Akt,mTOR,microtubule-associated protein LC3-II and autophagy-related protein Beclin 1 in macrophages.The effect of drug-containing serum on the levels of inflammatory factors secreted by macrophages was observed.Result The animal experiments showed that the degree of atherosclerotic lesions in the Vascular Softening Pills group was significantly lighter.Compared with the control group,the expression levels of Akt and mTOR in the aortic tissue of the Vascular Softening Pills group were significantly lower(P<0.05).In vitro experiments showed that compared with the control group,the autophagosomes were significantly increased under the transmission electron microscope(P<0.05),and the expression levels of LC3-II and Beclin1 were significantly higher(P<0.05),while the mRNA and protein expression levels of Akt and mTOR were significantly lower(P<0.05).The IL-10 levels secreted by macrophages were significantly lower(P<0.05)while IFN-γsecretion level was significantly higher(P<0.05).Conclusion The molecular mechanism of Vascular Softening Pills treating atherosclerosis by inhibiting inflammatory response may be related to the regulation of PI3 K/Akt/mTOR signaling pathway,regulation of macrophage autophagy and reduction of plaque macrophage infiltration.