1, 4苯醌对K562细胞自噬的影响及机制
Effects and mechanism of autophagy induced by 1, 4-benzoquinone in K562 cells
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摘要:
目的 研究1，4苯醌（1，4-BQ）对人慢性髓系白血病K562细胞自噬的影响及其机制。
方法 选择人慢性髓系白血病K562细胞，采用1，4-BQ、自噬早期抑制剂LY294002和自噬晚期抑制剂氯喹（CQ）进行染毒处理，分为对照组、1，4-BQ染毒组（5、10、20 μmol/L）、LY294002组（10 μmol/L）、CQ组（10 μmol/L）、1，4-BQ（20 μmol/L）+LY294002（10 μmol/L）组和1，4-BQ（20 μmol/L）+CQ（10 μmol/L）组。应用MTT法检测细胞增殖率；采用CYTO-ID荧光探针并运用荧光染色法和流式细胞术法对细胞自噬水平进行定性、定量检测；采用丫啶橙染色法检测细胞溶酶体pH值；运用实时定量PCR法和Western blot法检测LC3-Ⅱ和P62自噬相关基因和蛋白的表达水平。
结果 10、20 μmol/L的1，4-BQ分别作用K562细胞24、48、72 h后，细胞相对增殖率明显降低（P < 0.05）。CYTO-ID自噬荧光染料特异性聚集在K562细胞胞质内，随着染毒浓度增加荧光强度明显增强。流式细胞术结果显示，与对照组相比，5、10、20 μmol/L 1，4-BQ组自噬水平均明显增加，差异具有统计学意义（P < 0.05）。丫啶橙染色结果显示，不同浓度1，4-BQ组红色荧光强度均高于对照组，即1，4-BQ组溶酶体pH明显降低。Western blot结果显示，当1，4-BQ浓度为10、20μmol/L时，LC3-Ⅱ和P62蛋白表达量明显高于对照组（P < 0.05），且在染毒24 h时增幅最大。干预自噬后，与1，4-BQ组相比，1，4-BQ+LY294002组的自噬水平和LC3-Ⅱ蛋白表达量均降低（P < 0.05）。与1，4-BQ组相比，1，4-BQ+CQ组自噬水平增加（P < 0.05），而与CQ组的差异无统计学意义。此外，与对照组相比，CQ组LC3-Ⅱ蛋白表达明显增加（P < 0.05）。与1，4-BQ组相比，1，4-BQ+CQ组LC3-Ⅱ蛋白表达水平差异无统计学意义（P>0.05）。
结论 1，4-BQ可引起K562细胞LC3-Ⅱ、P62蛋白表达增加，可能是通过增加自噬体的合成和减少自噬体的降解从而增加细胞自噬。
Abstract:Objective To investigate the effects and mechanism of 1, 4-benzoquinone (1, 4-BQ) on autophagy activity in K562 cells.
Methods Human chronic myelogenous leukemia K562 cells were divided into control group, l, 4-BQ (5, 10, and 20 μmol/L) groups, early-stage autophagy inhibitor LY294002 (10 μmol/L) group, late-stage autophage inhibitor chloroquine (CQ) (10 μmol/L) group, 1, 4-BQ (20μmol/L)+LY294002 (10μmol/L) group, and 1, 4-BQ (20μmol/L)+CQ (10μmol/L) group. The proliferation of K562 cells was evaluated by MTT assay, qualitative and quantitative autophagy level by fluorescent staining and flow cytometry assay using CYTO-ID fluorescence probe, lysosomal pH value by acridine orange staining method, and the gene and protein expressions of LC3-Ⅱ and P62 by real-time quantitative PCR and Western blot, respectively.
Results The relative proliferation rates of the K562 cells treated with 10 and 20 μmol/L 1, 4-BQ remarkably decreased at 24, 48, and 72 h (P < 0.05). CYTO-ID dye was typically accumulated in the cytoplasm of K562 cells, and the fluorescent signals raised with increasing 1, 4-BQ concentration. The flow cytometry results revealed that, compared with the control group, the autophagy level was significantly increased in the cells treated with 5, 10, and 20 μmol/L 1, 4-BQ (P < 0.05). The acridine orange staining results showed stronger red fluorescence of the 1, 4-BQ groups than that of the control group, indicating decreased lysosomal pH values of the 1, 4-BQ groups. The results of Western blot showed that the LC3-Ⅱ and P62 protein expression levels were increased in the 10 and 20μmol/L 1, 4-BQ groups compared with the control group (P < 0.05), and peaked at 24 h. After the designed intervention, the autophagy level and LC3-Ⅱprotein expression level were lower in the 1, 4-BQ+LY294002 group than that of the 1, 4-BQ group (P < 0.05). The autophagy level in the 1, 4-BQ+CQ group was higher than that in the 1, 4-BQ group (P < 0.05), but not different from that in the CQ group. Additionally, the protein expression level of LC3-Ⅱ in the CQ group was higher than that in the control group (P < 0.05). There was no difference in the protein expression level of LC3-Ⅱ between the 1, 4-BQ group and the 1, 4-BQ+CQ group (P>0.05).
Conclusion 1, 4-BQ can increase LC3-Ⅱ and P62 protein expression levels, indicating elevated autophagy level, with a potential pathway of increasing autophagic synthesis and decreasing autophagic degradation.
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