玉米赤霉烯酮对山羊睾丸间质细胞的毒性机制研究

Toxicity mechanism of zearalenone on goat testicular interstitial cells

【目的】探究玉米赤霉烯酮(ZEA)对山羊睾丸间质细胞活力、形态及细胞凋亡相关基因表达的影响,明确其对山羊睾丸间质细胞的毒性作用,为后续揭示ZEA对雄性山羊生殖系统的毒性机制提供理论依据。【方法】以山羊睾丸间质细胞为试验材料,采用不同浓度ZEA处理细胞24 h,以CCK-8法检测细胞增殖情况并筛选出最适浓度;然后通过光学显微镜、透射电子显微镜、细胞划痕试验、实时荧光定量PCR、JC-1试剂盒及ROS试剂盒分别检测ZEA处理山羊睾丸间质细胞的形态特征、超微结构变化、损伤愈合能力、细胞凋亡及氧化应激相关基因表达、线粒体膜电位及胞内活性氧(ROS)水平的变化。【结果】随着ZEA浓度的升高,山羊睾丸间质细胞活力呈剂量依赖效应极显著下降(P<0.01,下同),ZEA的半数抑制浓度(IC_(50))在20μmol/L附近,故选取20μmol/L作为后续试验的处理浓度。经20μmol/L ZEA处理后,山羊睾丸间质细胞大量死亡,细胞皱缩呈圆珠状,细胞间连接松散;核膜出现破损,内质网断裂、结构模糊,线粒体破裂,形成自噬囊泡,细胞核皱缩、深染;细胞愈合能力降低,划痕培养18 h后的细胞迁移率约80%;细胞中的Bcl-2、caspase-3和SOD2基因相对表达量显著上升(P<0.05),Bax、HO-1、Nrf2、GPX1和CAT基因相对表达量极显著上升;JC-1检测发现细胞中的红色荧光数量减少,荧光强度较弱,而绿色荧光数量明显增多且荧光强度较强;ROS检测显示细胞ROS水平极显著升高,即细胞氧化应激程度加剧。【结论】ZEA通过诱导山羊睾丸间质细胞形变及线粒体、内质网等细胞超微结构损伤,降低其损伤修复能力,促使线粒体膜电位水平降低和加剧氧化应激程度,而发挥其毒性作用。

【Objective】To explore the effects of zearalenone(ZEA)on the viability,morphology and apoptosis-related gene expression of goat testicular interstitial cells,and clarify the mechanism of its toxicity to goat testicular inter‐stitial cells,providing theoretical basis for the subsequent discovery of the toxic mechanism of ZEA on the reproductive system of male goats.【Method】Goat testicular interstitial cells were treated with different concentrations of ZEA for 24 h.The cell proliferation was detected by CCK-8 method and the optimal concentration was selected.Then,the morphologi‐cal characteristics,ultrastructural changes,damage healing ability,apoptosis and oxidative stress-related genes expres‐sion,mitochondrial membrane potential and intracellular reactive oxygen species(ROS)levels of testicular interstitial cells treated with ZEA were detected by optical microscopy,transmission electron microscopy,cell scratch test,real-time fluorescence quantitative PCR,JC-1 kit and ROS kit.【Result】With the increase of ZEA concentration,the viability of goat testicular interstitial cells decreased extremely significantly in a dose-dependent effect(P<0.01,the same below),and the half-inhibitory concentration of(IC_(50))ZEA was around 20μmol/L,so 20μmol/L was selected as the treatment concentration for subsequent tests.After being treated with 20μmol/L ZEA,a large number of goat testicular interstitial cells died,the cells were wrinkled and bead-like,and the intercellular connections were loose,nuclear membrane was damaged,the endoplasmic reticulum was broken,the structure was blurred,the mitochondria was broken,autophagy vesicles were formed,and the nucleus was wrinkled and deeply stained.The cell healing ability was decreased,and the cell mobility was about 80%after 18 h of scratch culture.The relative expressions of Bcl-2,caspase-3 and SOD2 genes were significantly increased(P<0.05),and the relative expressions of Bax,HO-1,Nrf2,GPX1 and CAT genes were ex‐tremely significantly increased.JC-1 detection showed that the amount of red fluorescence decreased and the fluorescence intensity was weak,while the amount of green fluorescence increased and the fluorescence intensity was strengthened.ROS detection showed that the level of ROS in cells was significantly increased,that was,the degree of cellular oxidative stress was intensified.【Conclusion】ZEA exerts its toxic effect by inducing the deformation,mitochondria,endoplasmic reticulum and other cell ultrastructural damage of goat testicular interstitial cells,reducing their damage repair ability,the reduction of mitochondrial membrane potential and aggravating oxidative stress.