玉米赤霉烯酮通过PI3K/Akt/mTOR通路诱导大鼠睾丸支持细胞自噬及对细胞周期分布的影响

[目的]探究磷脂酰肌醇3激酶/蛋白激酶/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）信号通路在玉米赤霉烯酮（ZEA）诱导睾丸支持细胞（SC）自噬及影响SC细胞周期分布中的作用，揭示ZEA对雄性生殖毒性的机制。[方法]以Wistar大鼠原代睾丸支持细胞为材料，利用Western blot、流式细胞术和免疫荧光技术等方法检测PI3K/Akt/mTOR信号通路在ZEA对SC自噬及细胞周期分布的影响。[结果]随着ZEA浓度升高，PI3K/Akt/mTOR信号通路关键蛋白磷酸化水平显著或极显著下降（P<0.05或P<0.01）；加入PI3K抑制剂LY294002后，微管相关蛋白1轻链3（LC3）的荧光信号强度显著增强，荧光聚点更加明显（P<0.01）；同时，与ZEA单独处理组比，Akt、mTOR磷酸化水平显著或极显著下降（P<0.05或P<0.01），LC3Ⅱ/LC3Ⅰ表达量极显著上升（P<0.01）；G2/M期关键蛋白Cdc2、Cdc25B等表达水平随着ZEA浓度的升高显著或极显著下降（P<0.05或P<0.01），磷酸化组蛋白H3（p-histone H3）的荧光明显减少；加入PI3K抑制剂LY294002后，ZEA致睾丸支持细胞G2/M期阻滞的比例显著降低（P<0.05），且细胞周期关键激酶Cdc2的活性显著增加（P<0.05）。[结论]通过PI3K/Akt/mTOR信号通路，一定浓度范围的ZEA在诱导SC自噬中起负调控作用，而此通路的激活可以部分逆转ZEA对SC细胞G2/M期的阻滞，从而起到保护作用。
[Objectives]This study aimed to reveal the mechanism of the male reproductive toxicity of zearalenone(ZEA),this study explored the role of PI3K/Akt/mTOR signaling pathway in ZEA induced autophagy in sertoli cells(SC)and its influence on the distribution of cell cycle.[Methods]Using primary SC as test materials,the effects of PI3K/Akt/mTOR signaling pathway on autophagy and cell cycle distribution of SC were detected by Western blot technique,flow cytometry and immunofluorescence techniques.[Results]With the increasing concentration of ZEA,the phosphorylation level of key protein in PI3K/Akt/mTOR pathway decreased gradually(P<0.01 or P<0.05). After adding the PI3K inhibitor LY294002,the fluorescence signal intensity of LC3 significantly increased,which resulted in the more obvious fluorescence accumulation(P<0.01). At the same time,comparing the combined treatment group to the ZEA individual treatment group,the phosphorylation levels of Akt proteins and mTOR proteins significantly decreased(P<0.01 or P<0.05).The expression of LC3Ⅱ/LC3Ⅰ significantly increased(P<0.01). In addition,the expression of key proteins Cdc2,CDC25B in G2/M phase decreased gradually with the increase of ZEA concentrations(P<0.05 or P<0.01). Fluorescence reduction of p-histone H3 was detected by immunofluorescence. After adding the PI3K inhibitor LY294002,the proportion of ZEA induced SC G2/M arrest significantly declined(P<0.05). Moreover,the activity of the cyclic key protease Cdc2 significantly increased(P<0.05).[Conclusions]The results suggest that in a certain concentration range,ZEA can induce autophagy through the activation of the PI3K/Akt/mTOR signaling pathway which plays a negative regulatory role in autophagy process. ZEA can affect the cycle distribution of SC,induce the cell cycle arrest and inhibit proliferation. However,the activation of the