免疫负调节分子MKP-1在睾丸炎中的保护作用及其机制研究

Orchitis may lead to male infertility. When Orchitis occurs, immunogenic germ cells are exposed to the immune antigens and attacked by their own immune systems, which usually results in irreversible testicular dysfunction. MAP phosphatase 1 (MKP)-1 has been regarded as a crucial negative regulator of innate immune response. MKP-1 could relieve the impaired progression of tight junction protein (occludin) in Sertoli cells by LPS stimulus. However, it remains unknown about the roles of MKP-1 in controlling the seminiferous tubules when Orchitis occurs. In the present study, we will use the Mkp-1-/- mouse as model to compare the differences of the proteotome between WT and KO Sertoli cells, compare the structure and function of blood-testis barrier of WT and KO mouse testis, explore the effects of Mkp-1 knockdown on the expression /distribution and endocytosis / degradation function of tight junction protein. Additionally, the mechanisms whether MKP-1 affects the structure and function of BTB by regulating the cytokines expression and phosphorylation of MAPK signaling pathway need to be clarified. Meanwhile, this project will detect the expression levels of MKP-1 and related tight junction protein in the testis samples of the Orchitis patients to assess the protective effect of MKP-1 in Orchitis, and it may lay scientific basis and offer new targets for the diagnosis and treatment of male infertility caused by Orchitis.

睾丸炎症造成血-睾屏障结构与功能的破坏，可诱发机体对自身精子产生免疫反应，造成不可逆的生育障碍。MAPK磷酸酶-1（MKP-1）是重要的炎症负调节分子。MKP-1能够体外抑制炎症刺激下Sertoli细胞紧密连接蛋白occludin表达的下降。然而， MKP-1在睾丸炎中是否发挥作用？目前未见报道。本项目以Mkp-1-/-小鼠为模型，通过比较正常与敲除小鼠Sertoli细胞的蛋白组学差异，血-睾屏障的结构功能改变，分析Mkp-1敲除对睾丸紧密连接结构和Sertoli细胞蛋白內吞/降解功能的影响，并通过观察细胞因子和相关信号分子对紧密连接蛋白表达和定位的影响，阐述MKP-1影响血-睾屏障的关键分子和作用机理。通过检测睾丸炎患者睾丸组织中MKP-1及相关连接蛋白的表达水平，进一步明确MKP-1在维持睾丸免疫自稳及睾丸炎中的保护作用，为炎症及免疫相关男性不育患者的诊治提供新的理论和实验依据。

睾丸属于免疫豁免器官，拥有特定的免疫微环境。血-睾屏障可以隔离精子抗原的识别，并能有效阻止外来有害物质的损害，对建立和维持免疫豁免微环境至关重要。研究表明，血-睾屏障蛋白的功能受到丝裂原蛋白激酶（MAPK）的调节，并参与维持血-睾屏障的正常结构与功能。MAPK磷酸酶-1（MKP-1）是炎症反应中重要的负调节分子，能够通过调节MAPK信号分子的磷酸化水平，维持免疫应答处于合适的强度。我们前期研究发现，MKP-1能够体外抑制炎症刺激下紧密连接蛋白occludin表达的下降，在睾丸炎症中发挥保护作用。然而，MKP-1在睾丸免疫微环境中的作用，目前仍不清楚。本研究发现基因敲除Mkp-1后，小鼠睾丸紧密连接蛋白（如occludin）的表达下降、定位改变，血-睾屏障结构改变、通透性增加，睾丸免疫微环境发生改变，造成精子发生异常，生育力下降。与此同时，MKP-1可以通过抑制clathrin介导的蛋白內吞活动以及V-ATPase依赖的囊泡酸化，影响Sertoli细胞蛋白內吞/降解功能，从而减少occludin表达的下降。进一步研究发现，基因敲除Mkp-1后，小鼠睾丸及sertoli细胞内信号分子p38的磷酸化水平亦显著增强；RNA干扰抑制MKP-1表达后，可造成sertoli细胞内p38介导occludin表达的进一步下降，提示MKP-1可通过抑制p38的磷酸化，在体影响血-睾屏障分子的表达。综上所述，本研究表明免疫负调节分子MKP-1可以通过抑制信号分子p38的磷酸化，减少Sertoli细胞内occludin蛋白的內吞/降解，影响occludin的表达和定位，保持血-睾屏障的结构与功能，进而维持睾丸免疫豁免微环境，为炎症及免疫相关男性不育患者的诊治提供新的理论和实验依据。