ATG12介导的Sertoli细胞自噬在5-羟甲基-2-糠醛导致血睾屏障破坏中的作用及机制研究

吸烟与男性生殖损害已成共识，但香烟烟雾成分复杂，关键组分及生殖损害机制仍然不明。5-羟甲基-2-糠醛（5-HMF）是课题组前期从香烟烟雾中分离的含量丰富毒性组分，可激活Sertoli细胞ATG12自噬通路，降低紧密连接蛋白表达引发血睾屏障破坏，自噬抑制剂或atg12敲除可缓解5-HMF诱发的上述损伤。提示5-HMF可能是吸烟相关生殖危害的重要组分，紧密连接蛋白经ATG12自噬降解可能是5-HMF诱发血睾屏障破坏的关键环节，但未见报道。为此本项目拟构建Sertoli细胞条件敲除atg12小鼠和细胞染毒模型，明确ATG12自噬与血睾屏障破坏关系；通过荧光共定位检测技术，剖析ATG12介导自噬与紧密连接蛋白降解的作用方式；通过下拉靶蛋白质谱技术筛选关键作用分子，解析其作用机制。试图以全新视角审视5-HMF引起血睾屏障破坏的过程及分子机制，为预防控制吸烟所致男性生殖危害提供依据和和技术支撑。

The association between cigarette and male infertility is well studied, however, due to the diversity of components of cigarette smoke, the key testicular toxicant and its mechanism remain unknown. 5-hydroxymethyl-2-furaldehyde (5-HMF) is an abundant toxic component that was recently isolated and identified from cigarette smoke extract (CSE) by our research group. 5-HMF was associated with decreased tight junctions (TJs) protein sand disrupted blood testis barrier (BTB). 5-HMF was also proved to increase the expression of ATG12 and subsequently activate autophagy flux. In addition, haploinsufficiency of atg12 or pharmacological inhibition of autophagy could significantly alleviate 5-HMF induced dysfunction, suggesting a relevant role of ATG12 in 5-HMF related BTB disruption. However, so far there is no related report about it. For this purpose, the following experiments will be performed in the present project: First of all, a Sertoli atg12 conditional knockout (atg12ckoSertoli-Cre) mice will be conducted as Sertoli cell is our potential target. Then, the 5-HMF exposed atg12ckoSertoli-Cre mice or Sertoli cells will be analyzed to further confirm the role of ATG12 mediated autophagy in 5-HMF induced BTB disassembly. Moreover, the biotin labeled fluorescence will be performed to illustrate the mode of autophagy related TJs degradation. Finally, the molecular mechanism of ATG12-mediated autophagy pathway in this BTB damage will be further analyzed by using Co-immunoprecipitation coupled UPLC-MS/MS method, and the autophagy related cargo proteins will be preferentially analyzed. The present study attempts to take new insight into the process and mechanism of 5-HMF induced BTB disruption, and also to provide theoretical and practical basis for the prevention and control of cigarette induced male reproductive toxicicty.