NTE/LPC信号通路在TOCP诱导小鼠卵巢颗粒细胞自噬和凋亡中的作用及机制研究

Tri-ortho -cresyl phosphate (TOCP) has been widely used as plasticizers, plastic softeners, and flame-retardants in industry. It has been shown that inhibition of neuropathy target esterase (NTE) activity by TOCP leads to reproduction toxicity. Our preliminary results found that TOCP could decrease estradiol concentration, injure granulosa cells in the ovay tissue, and induce autophagy of mice granulose cells. Inasmuch as TOCP can inhibit NTE activity and increase LPC content, and LPC can induce apoptosis, and granulosa cells play an important role in synthesis and secretion of estradiol. These results indicate that TOCP might induce autophagy and apoptosis of mice granulosa cells via NTE/LPC signal. Therefore, we will test this hypothesis by the following approaches: Firstly, we will employ transmission electron microscopy (TEM), western blotting, and flow cytometry to investigate whether TOCP induces autophagy and apoptosis of mice granulosa cells; Secondly, phenyl valerate hydrolysis and thin-layer chromatography (TLC) will be used to assess the effect of TOCP on NTE activity and LPC content in these cells. Finally, we will determine whether TOCP induces autophagy and apoptosis of mice granulosa cells via NTE/LPC signal. The present study will provide a novel insight into the mechanism for TOCP- induced reprodcution toxicity in female mice.

三邻甲苯磷酸酯（TOCP）常作为增塑剂、软化剂及阻燃剂等，在工业中使用非常广泛。研究显示，TOCP可通过抑制神经病靶酯酶（NTE）活性而导致生殖毒性。我们的初步结果发现，TOCP可显著降低雌性小鼠体内雌二醇的含量，特异性地损伤小鼠卵巢中的颗粒细胞，并诱导颗粒细胞自噬。.鉴于TOCP可显著抑制NTE活性并增加溶血磷脂酰胆碱（LPC）含量，LPC可诱导细胞凋亡以及颗粒细胞在雌二醇合成和分泌中的重要作用，提示TOCP很可能通过NTE/LPC而诱导颗粒细胞自噬和凋亡。本课题通过分离的小鼠颗粒细胞，首先用透射电镜、免疫印迹和流式细胞仪等方法确定TOCP诱导颗粒细胞自噬和凋亡，然后用戊酸苯酯水解法及薄层层析等方法确定TOCP对细胞内NTE活性及LPC含量的影响，并最终确定TOCP可通过NTE/LPC而诱导细胞自噬和凋亡。为深入探讨TOCP的雌性生殖毒性的调控机理提供理论基础和实验依据。

三邻甲苯磷酸酯（TOCP）常作为增塑剂、软化剂及阻燃剂等，在工业中使用非常广泛。我们的研究发现，TOCP可导致雌性小鼠卵巢颗粒细胞的排列明显紊乱，且可降低血浆中的雌激素浓度，并可诱导小鼠卵巢组织自噬。细胞实验显示，TOCP可降低小鼠卵巢颗粒细胞活力，诱导小鼠卵巢颗粒细胞自噬并产生氧化应激，而对细胞周期和凋亡无显著影响。免疫组织化学结果显示，NTE在小鼠卵巢颗粒细胞中高表达，且TOCP可显著抑制小鼠颗粒细胞内NTE蛋白及 mRNA表达。鉴于TOCP可增加细胞内LPC含量，我们发现LPC可显著抑制小鼠卵巢颗粒细胞活性，诱导小鼠卵巢颗粒细胞自噬和氧化应激，但对细胞周期和凋亡无显著影响。用氧化应激抑制剂NAC抑制氧化应激，可缓解LPC对细胞活性的抑制和细胞自噬的诱导。本课题的研究为深入探讨TOCP的雌性生殖毒性的调控机理提供理论基础和实验依据。