玉米赤霉烯酮对湖羊睾丸间质细胞毒性及m^(6)A甲基化修饰相关基因表达的影响

Effects of zearalenone on cytotoxicity and m^(6)A methylationrelated gene expression in leydig cells of Hu sheep

[目的]本文旨在探究玉米赤霉烯酮(zearalenone, ZEA)对湖羊睾丸间质细胞(leydig cells, LC)的毒性作用。[方法]用200μmol·L^(-1)ZEA处理湖羊LC 8 h,通过EdU法检测细胞增殖情况,RT-qPCR和Western blot分别检测与细胞增殖和凋亡、氧化应激、m^(6)A甲基化修饰相关基因和蛋白的表达变化。[结果]与对照组相比,ZEA处理组LC活性和增殖数量极显著下降(P<0.01);细胞增殖相关基因pcna及其蛋白表达水平均极显著下降(P<0.01);促凋亡基因表达水平(bax、caspase3,P<0.01;tp53、caspase9,P<0.05)均显著升高,相对应蛋白表达水平(TP53、CASPASE9,P<0.01)极显著升高;抗凋亡基因bcl-2及蛋白表达水平显著降低(P<0.05);氧化应激相关基因相对表达水平(cat,P<0.05;gsh-px、sod2,P<0.01)显著或极显著上升,其相关蛋白表达水平(SOD2,P<0.01)极显著上升;m^(6)A相关基因alkbh5的相对表达水平显著降低(P<0.05),mettl3和ythdc1水平极显著升高(P<0.01),ythdf3水平显著升高(P<0.05)。[结论]ZEA可抑制湖羊LC增殖,促进湖羊LC凋亡,导致LC发生氧化损伤,产生毒性作用,且其毒性作用可能跟m^(6)A甲基化修饰相关。

[Objectives]The paper aimed to explore the toxic effect of zearalenone(ZEA)on leydig cells(LC)of Hu sheep.[Methods]Using Hu sheep LC as experimental materials,treating the LC with 200μmol·L^(-1) ZEA for 8 h,cell proliferation was measured by the EdU assay.RT-qPCR and Western blot were used to detect the expression changes of genes and proteins related to cell proliferation,apoptosis,oxidative stress and m^(6)A methylation modification,respectively.[Results]Compared with the control group,the activity and proliferation of LC cells in the ZEA-treated group decreased extremely significantly(P<0.01).The relative expression levels of cell proliferation related gene pcna and its related proteins decreased extremely significantly(P<0.01).The relative expression levels of pro-apoptotic genes increased(bax,caspase3,P<0.01;tp53,caspase9,P<0.05),and its related proteins increased(TP53,Caspase9,P<0.01).The relative expression levels of anti-apoptotic gene bcl-2 and its related proteins reduced significantly(P<0.05).The relative expression levels of oxidative stress-related genes increased(cat,P<0.05;gsh-px,sod2,P<0.01),and its related proteins increased(SOD2,P<0.01).The relative expression levels of m^(6)A related genes alkbh5 reduced significantly(P<0.05),mettl3 and ythdc1 extremely significantly increased(P<0.01),ythdf3 significantly increased(P<0.05).[Conclusions]ZEA could inhibit the proliferation and promote the apoptosis of LC in Hu sheep,leading to oxidative damage and toxic effect of LC,and its toxic effect may be related to the change of m^(6)A methylation modification.