抑制PI3K/Akt/mTOR信号通路对兔原代巨噬细胞自体吞噬的影响及机制

Roles of Selective Inhibition of PI3K /Akt /mTOR Signaling Pathway on Rabbit Primary Macrophage Autophagy and Possible Pechnism

目的探讨抑制PI3K/Akt/mTOR信号通路对兔原代巨噬细胞自体吞噬中的影响。方法分离培养纯种新西兰兔腹腔原代巨噬细胞并分为4组,加入磷脂酰肌醇3激酶(PI3K)抑制剂LY294002(10μmol/L)组、哺乳动物雷帕霉素靶蛋白(mTOR)抑制剂雷帕霉素(10 nmol/L)组、蛋白激酶B(Akt)抑制剂曲西立滨组(20μmol/L)以及空白对照组。共培养4 h、12 h后分别收集细胞,运用透射电镜观察巨噬细胞自噬体的变化,细胞免疫荧光法检测微管相关蛋白轻链3Ⅱ(LC3Ⅱ)分子的表达,Western blot检测Akt、mTOR、磷酸化Akt(p-Akt)、磷酸化mTOR(pmTOR)及自噬相关蛋白Beclin-1和自噬蛋白Atg5-Atg12连接体的表达,单丹酰尸胺(MDC)染色法观察自噬溶酶体的变化。结果与空白对照组相比,透射电镜下LY294002组自噬体、自噬空泡、髓磷脂图像等自噬标记物明显减少,雷帕霉素组、曲西立滨组明显增多;激光共聚焦显微镜下LY294002组LC3Ⅱ表达显著减少,雷帕霉素组、曲西立滨组表达显著增多;Western blot结果显示LY294002组Beclin-1及Atg5-Atg12蛋白表达水平显著下降,p-mTOR、p-Akt蛋白表达显著减少;雷帕霉素组、曲西立滨组Beclin-1及Atg5-Atg12蛋白表达水平明显上调,共培养4 h后pAkt表达增多,雷帕霉素组p-mTOR表达增多,曲西立滨组减少;共培养12 h后雷帕霉素组、曲西立滨组p-mTOR表达显著减少,雷帕霉素组p-Akt表达显著增多,曲西立滨组显著减少;MDC染色显示LY294002组自噬溶酶体明显减少,雷帕霉素组、曲西立滨组明显增多。结论抑制PI3K/Akt/mTOR信号通路能促进兔原代巨噬细胞自体吞噬,抑制PI3K能减少兔原代巨噬细胞自体吞噬,可能是不同类型的PI3K分子通过其他通路起作用。

Aim To investigate the role of selective inhibition of the PI3K/Akt/mTOR signaling pathway on prompting rabbit primary macrophage autophagy. Methods Primary macrophages were obtained intraperitoneally from the New Zealand rabbits and then were co-cultured with the phosphatidylinositol 3-kinase （PI3K） inhibitor LY294002 （10 μmol/L）, protein kinase B （Akt） inhibitor triciribine （20μmol/L）, mammalian target of rapamycin （mTOR） inhibitor rapamyein （ 10 μmol/L） and no drugs respectively for 12 hours. Ultrastructural changes of macrophages were examined by transmission electron microscopy. The expression level of microtubule-associated protein light chain 3 Ⅱ （ LC3 Ⅱ ） was assayed by immunofluorescence. Protein expression levels of Akt, roTOR, phosphorylation of protein kinase B （ p-Akt）, phosphorylation of mammalian target of rapamycin （p-mTOR） and autophage related protein Beclin-1 and autophagy protein 5 and 12 conjugated form （Atg5-Atgl2） were measured by Western blot. Mondansylcadaverine （MDC） staining was used to see autophagy lysosome changes. Results Compared with the control group, few autophagosomes and vacuoles were detected in group LY294002 while plenty of typical autophagosomes were detected in group rapamcin and triciribine. Theexpression levels of Beclin-1 and AtgS-Atgl2 decreased significantly in group rapamcin, while increased significantly in group rapamcin and triciribine. The fluorescence microscope showed few dots of LC3 1I in group LY294002 and many in group rapamcin and triciribine. The expression of p-Akt and p-mTOR increased obviously in group rapamcin while the former increased a lot and the latter decreased in group rapacin after co-culturing of 4 hours. The expression of p-mTOR decreased significantly in the treated groups, however, p-Akt decreased significantly in group rapamcin and triciribine but increased obviously in group rapamci after 12 hours＇ co-culture. There were no significant differences on the total AKT and roTOR levels among the treated groups. MDC staining showed decreased autophagic lysosomes in group LY294002 and increased autophagic lysosomes in group rapamcin and triciribine. Conclusion Selective inhibition of PI3K/Akt/ mTOR signaling pathway can promote rabbit primary macrophage autophagy while inhibition of PI3K suppress macrophage autophagy by other signaling pathway.