Sertoli细胞TLR信号通路在微波辐射损伤精子发生中作用的研究

Microwave radiation may impair spermatogenesis and male fertility. Mechanisms underlying impairment of spermatogenesis by microvave radiation have not been completely understood. We recently found that microwave radiation could increase the expression of pro-inflammatory cytokines in Sertoli cells, which may impair spermatogenesis. However, the mechanisms that microvave radiation induces the cytokine expression in Sertoli cells remain to be clarified. This proposal will investigate the effects of microvave radiation on the expression and function of Toll-like receptors (TLRs) in rat Sertoli cells, thus inducing pro-inflammatory cytokines that may damage germ cells. Three major aims will be focused in this proposal: (1) Effects of microvave radiation on the expression and function of TLRs in Sertoli cells. We will focus on the upregulation and activation of TLRs in Sertoli cells by microvave radiation. (2) Endogenous TLR agoanists released by damaged germ cells trigger TLR signaling in Sertoli cells. We will examine TLR signaling pathways that induce the cytokine expression in Sertoli cells, such as the activation of NF-kB and MAPKs (p38, JNK, ERK1/2). (3) Mechanisms that TLR-mediated pro-inflammatory cytokines damage germ cells.This proposal is important since it aims to answer an open questions in the field regarding the effects of working environments on reproductive health. Outcomes of this study will further understand the mechanisms underlying microvave radiation-induced impairment of spermatogenesis and male fertility, which may provide novel clues into the preventive and therapeutic strategies for the impairment of spermatogenesis and male fertility by microvave radiation.

微波辐射可致生精细胞凋亡/坏死、精子发生破坏，其机制尚未完全明确。我们近来发现微波辐射可诱导Sertoli细胞分泌多种炎症因子，影响精子发生，其调节机制有待深入研究。本项目利用大鼠为模型，采用整体与离体实验相结合，拟研究微波辐射通过上调与激活Sertoli细胞中的Toll样受体（TLR），诱导炎症因子分泌及精子发生损伤的分子机制。主要内容包括：（1）微波辐射对Sertoli细胞中TLR的表达与功能的影响，关注微波辐射通过TLR信号通路诱导炎症因子的表达；（2）微波辐射损伤生精细胞释放的内源性TLR配体对Sertoli细胞中TLR的激活，鉴定核转录因子（NF-kB）及丝分裂原激酶（P38，JNK与ERK1/2）的活化与炎症因子表达的关系；（3）TLR信号通路诱导的炎症因子对精子发生的影响。本项目旨在深入揭示微波辐射损害精子发生的机制，认识关键调节环节，为预防和治疗微波辐射致男性生育损伤提供新的线索。

微波辐射可致生精细胞凋亡/坏死、精子发生破坏，其机制尚未完全明确。我们以往研究发现微波辐射可诱导Sertoli细胞分泌多种炎症因子，影响精子发生，其调节机制有待深入研究。本项目利用大鼠为模型，采用整体与离体实验相结合，研究了微波辐射通过上调与激活Sertoli细胞中的Toll样受体（TLR），诱导炎症因子分泌及精子发生损伤的分子机制。主要结果如下：（1）建立了微波辐射损伤动物模型和Sertoli细胞模型，微波辐射致生精细胞变性、凋亡/坏死，有随时间延长损伤加重的趋势；100mw/cm2损伤重于30mw/cm2。体内、外的研究表明TLR2、TLR3、TLR4和TLR5，IL-1β、IL-6和TNF-α的mRNA 和蛋白水平均增加，p-P38、p-JNK、p-ERK1/2和p-NF-κB p65的蛋白水平均见不同程度的增加，30 mw/cm2与100 mw/cm2相比无明显规律；TLR信号通路分析显示MAPK抑制剂作用组可不同程度的降低IL-1β、IL-6和TNF-α的浓度，以30 mW/cm2组降低显著，而NF-κB抑制剂的作用无明显差别。提示S-HPM辐射活化TLR表达，上调炎症因子的表达主要通过MAPK通路所介导；（2）建立微波辐射Sertoli细胞与生精细胞共孵育模型，TLR内源性配体研究显示共孵育培养基中加入 DNase组IL-6和TNF-α的浓度较对照组降低，提示辐射损伤的生精细胞中可能存在类似于小分子DNA的内源性配体。（3）Sertoli细胞中TLR介导产生的炎症因子可致生精细胞的凋亡，采用TNFα、IL-1β和IL-6的中和抗体实验进一步证明微波辐射后Sertoli细胞的TNFα、IL-1β和IL-6致GC的早期凋亡率降低。本研究项目提示，微波辐射可能通过活化MAPK通路，激活Sertoli细胞的TLR2-5，诱导TNFα、IL-1β和IL-6的表达，进而诱导生精细胞的凋亡，而小分子的DNA类似物可做为TLR的内源性配体，加重此损伤过程。本项目为深入揭示微波损害精子发生的机制，为预防和治疗微波辐射致男性生育损伤提供新的线索。