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目的:研究阿伐他汀对白血病细胞K562自噬的影响及相关信号机制。方法:取对数生长期的K562细胞,分为阴性对照组、阿伐他汀组、阿伐他汀+PI3K抑制剂(LY294002)组、阿伐他汀+mTOR抑制剂(雷帕霉素)组。共培养24h后,采用吖啶橙染色观察细胞自噬体的变化,Western Blot检测Ⅱ型自噬标志蛋白微管相关蛋白1轻链3(LC3-Ⅱ)和自噬血管基因Beclin-1的表达,RT-PCR和Western Blot检测磷酸化Akt(p-Akt)、磷酸化mTOR(p-mTOR)的表达。结果:与阴性对照组相比,阿伐他汀抑制白血病细胞K562自噬体的形成(P<0.05),下调LC3-Ⅱ和Beclin-1的表达(P<0.05)。与阿伐他汀组相比,阿伐他汀+PI3K抑制剂(LY294002)组LC3-Ⅱ和Beclin-1的表达增加(P<0.05);阿伐他汀+mTOR抑制剂(雷帕霉素)组LC3-Ⅱ、Beclin-1蛋白表达增加(P<0.05)。与阿伐他汀组相比,阿伐他汀+PI3K抑制剂(LY294002)组p-Akt和p-mTOR基因的mRNA和蛋白表达水平均下降(P<0.05);阿伐他汀+mTOR抑制剂(雷帕霉素)组p-Akt和p-mTOR基因的mRNA和蛋白表达水平均下降(P<0.05)。结论:阿伐他汀可以抑制白血病细胞K562自噬,推测其机制可能与激活PI3K/Akt/mTOR信号通路有关。
Objective: To study the effect of atorvastatin on autophagy of K562 leukemia cells and its related signaling mechanisms. Methods: K562 cells in logarithmic growth phase were divided into negative control group, atorvastatin group, atorvastatin + PI3K inhibitor (LY294002) group, atorvastatin + mTOR inhibitor (rapamycin) group. After co-cultured for 24 hours, the changes of autophagosomes were observed by acridine orange staining. The expression of type II autophagic marker protein microtubule-associated protein 1 light chain 3 (LC3-Ⅱ) and autophagy gene Beclin-1 was detected by Western Blot The expressions of phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR) were detected by RT-PCR and Western Blot. Results: Compared with the negative control group, atorvastatin inhibited the formation of autophagosome K562 leukemia cells (P <0.05) and decreased the expression of LC3-Ⅱ and Beclin-1 (P <0.05). Compared with atorvastatin group, the expression of LC3-Ⅱ and Beclin-1 in atorvastatin + PI3K inhibitor (LY294002) group increased (P <0.05); atorvastatin + mTOR inhibitor (rapamycin) -Ⅱ, Beclin-1 protein expression increased (P <0.05). Compared with atorvastatin group, the mRNA and protein expression of p-Akt and p-mTOR gene in atorvastatin + PI3K inhibitor (LY294002) group decreased (P <0.05); atorvastatin + mTOR inhibitor (P <0.05). The mRNA and protein expression levels of p-Akt and p-mTOR in both groups were decreased (P <0.05). Conclusion: Atorvastatin can inhibit the autophagy of K562 leukemia cells, suggesting that its mechanism may be related to the activation of PI3K / Akt / mTOR signaling pathway.