心理应激诱导β-内啡肽导致雄鼠生殖损伤的分子网络机制

Psychological stress can induce disorders in male reproductive and endocrine system, and could lead to male infertility. Our previous study found that psychological stress induced reproductive damage of male rats, and released β-endorphin(β-EP), while stressed rat injected with naloxone (the antagonists of β-EP receptor) could reverse this damage. which suggested that psychological stress induced beta-EP and caused male reproductive damage. In the current study, we first aimed to verify the reproductive damage of male rat induced by psychological stress, by constructing a series rat models, which including the control group, the psychological stress group, β-EP intervention group, naloxone intervention stressed group, the stressed β-EP knockout and β-EP group, and also by analysis of the behavior, reproductive organs morphology, sperm quality, germ cell apoptosis, reproductive endocrine hormone level. And then to confirm that the reproductive damage of stressed male rat could induced by β-EP. The molecular network mechanism of male reproductive damage was based on proteomic study. The proteomic profiling of the leydig cells of all rat group were screened by label free technical, and comparison analysis of proteomic profiling between any two groups, the different expressed proteins were selected, their function, pathways and interaction were analyzed by bioinformatics methods, and the verification of their expression level could be performed by immunohistochemistry, Western blot and qRT- PCR analysis. Based on all above analysis, the molecular network mechanism of male reproductive damage caused by β-endorphin, which induced by psychological stress should be elucidated, and this research could provide a new theory for male infertility caused by psychological stress.

心理应激可引起生殖内分泌系统紊乱、导致男性不育。课题组前期发现心理应激可导致雄鼠生殖损伤并引起β-内啡肽(β-EP)的释放，而纳洛酮（β-EP受体的拮抗剂）干预可逆转应激鼠的生殖损伤，提示心理应激可诱导β-EP导致雄鼠生殖损伤。本研究拟通过建立空白对照组、心理应激组、β-EP干预组、纳洛酮干预应激组、β-EP基因敲除应激组及对照组大鼠模型，并对其行为、生殖器官形态、精子质量、生殖细胞凋亡、生殖内分泌激素等多水平检测分析，明确β-EP导致应激鼠生殖损伤；采用lable free蛋白组技术分析各组雄鼠睾丸间质细胞的蛋白质表达全谱，通过组间多重比对分析、筛选差异表达蛋白，生物信息学方法分析其功能、通路及相互作用，采用免疫组化、Western blot及qRT-PCR技术多重验证筛选的关键蛋白，综合分析结果，阐明β-EP导致应激鼠生殖损伤的分子网络机制，为心理应激引起的男性不育理论机制提供新内容。

本项目建立了单纯的心理应激动物模型，发现慢性心理应激导致成年雄鼠糖水摄取快感缺乏，饮水量显著减少，应激组雄鼠体重增长明显低于对照组，且睾丸与附睾指数显著降低；精子活力与密度显著降低，HE染色显示应激组睾丸生精小管生精细胞数量显著减少；同时伴随神经内分泌激素CRH、ACTH和GC分泌显著增多，肾上腺CORT亢奋，以及生殖内分泌激素GnRH、LH、FSH和T显著降低；实验显示雄鼠睾丸ABP与BRDT表达下调。2. 发现慢性心理应激诱导雄鼠血清与睾丸组织中的β-EP 表达水平上调，且研究显示β-EP受体在睾丸组织中均有表达。应激组MOR显著升高，且阳性细胞区域主要分布在睾丸LCs与生精细胞中；应激组DOR与KOR表达水平也呈上调趋势，组化阳性细胞区域主要位于生精小管管腔以及少量的睾丸LCs当中。提示心理应激诱导β-EP释放及其受体水平升高可能参与在心理应激诱导的雄性生殖功能损伤。3.β-EP注射与慢性心理应激均可诱导雄鼠生殖功能受损，而Nal注射后的应激组可缓解心理应激与β-EP对雄鼠体重、睾丸和附睾指数的影响，同时逆转精子质量，且平衡神经内分泌激素（ACTH、CRH、GC和CORT）与生殖内分泌激素（GnRH、FSH、LH、T）与睾丸LCs与生精细胞凋亡，提示应激诱导β-EP释放可致雄鼠生殖损伤。4.研究发现在MAPK家族中，应激组p38 MAPK及其磷酸化水平显著上升，而ERK1/2与JNK及其磷酸化水平无明显变化，提示p38 MAPK参与生殖细胞凋亡。研究显示心理应激与β-EP干预促进LCs中p38 MAPK，Bax，caspase 3表达水平上升，Bcl2降低，而Nal作用后凋亡相关蛋白表达水平趋于对照组。提示MOR与p38 MAPK可能在应激诱导β-EP致LCs凋亡信号通路中起关键作用。进一步研究发现β-EP通过p38 MAPK途径促进TM3细胞凋亡：β-EP干预TM3细胞促进细胞凋亡， β-EP作用后细胞凋亡显著增多，且细胞增殖也受到抑综上：慢性心理应激可致雄性生殖功能受损。慢性心理应激诱导β-EP分泌。心理应激诱导β-EP参与雄鼠生殖功能损伤。 β-EP可作为凋亡信号与MOR结合激活细胞内p38 MAPK 凋亡信号通路，诱导下游分子促进LCs凋亡。本课题揭示了慢性心理应激对雄性生殖损伤的分子机制，为由于精神压力导致的不育的临床治疗提供新思路。