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

Effects of zearalenone on the autophagy and cell cycle of sertoli cells by PI3K/Akt/mTOR pathway

[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/m TOR)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/m TOR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/m TOR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、m TOR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/m TOR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/LC3Ⅰsignificantly increased(P<0.01).In addition,the expression of key proteins Cdc2,CDC25B in G 2/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 G 2/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 PI3K/Akt/mTOR signaling pathway may partially reverse cell G 2/M arrest,which plays a protective role.