基于IL-1α调节支持细胞紧密连接通透性研究五子衍宗丸生精机理

The timely restructuring of tight junction (TJ)is known to play an important role in spermatogenesis. Interleukin(IL)-1α,which is the cytokine secreted by sertoli cell, is shown to "open" TJ permeability barrier. Wuziyanzong pill is a classical formula in the treatment of male infertility. The mechanism of Wuziyanzong pill is under further study. The preliminary studies showed that IL-1αlevel increased significantly in Cox7a2 recombint plasmid group. Moreover, the claudin-11 expression in kidney essence deficiency rat testis decreased greatly following Wuziyanzong pill treatment. Regarding the TJ permeability barrier as the breakthrough point and relationship of "Cox7a2-IL-1α- TJ permeability -spermatogenesis", it is hypothesized that the improvement of spermatogenesis by Wuziyanzong pill be mediated via the action of IL-1αon TJ permeability. In this study, Wuziyanzong pill and its medicated serum is administered to kidney deficiency syndrome rats and primary purified rat sertoli cell respectively. With the method of cell biology, molecular biology and serum pharmacology, the IL-1αlevel, TJ permeability and spermatogenesis are to be observed both in vivo and in vitro. The present study is of great significance in elucidating the spermatogenetic mechanisms by Wuziyanzong pill via TJ permeability.

支持细胞紧密连接通透性的有序调节在精子形成中发挥重要作用，白细胞介素（IL）-1α可增加紧密连接通透性。五子衍宗丸是治疗男性不育经典方，该方是否可调控紧密连接仍待研究。前期实验表明Cox7a2重组质粒转染支持细胞后IL-1α水平升高，五子衍宗丸干预肾虚精亏证大鼠后紧密连接蛋白claudin-11表达下降。本课题以"IL-1α调节紧密连接通透性"为切入点，依据"Cox7a2-IL-1α-紧密连接-生精功能" 密切关联为理论基础，提出"五子衍宗丸通过IL-1α途径调节紧密连接通透性，进而改善生精功能"假说。本研究采用五子衍宗丸对肾精亏虚证生精障碍大鼠、原代培养大鼠支持细胞进行干预，通过细胞、分子生物学和血清药理学等方法观察IL-1α、紧密连接通透性及生精功能变化，对从支持细胞"紧密连接通透性调控"途径揭示五子衍宗丸改善生精功能机理具有重要意义。

五子衍宗丸是中医治疗男性不育的代表方。该法如何通过调控紧密连接通透性进而改善生精功能还所知甚少。我们通过离体和在体实验观察了五子衍宗丸干预后支持细胞分泌的炎症因子水平、紧密连接通透性及超微结构、组成蛋白表达及生精功能变化。研究显示：（1）离体实验：大鼠原代支持细胞：和正常细胞相比，转染cox7a2重组质粒后支持细胞紧密连接组成蛋白（JAM-A、ZO-1）表达下降，紧密连接结构松散，而五子衍宗丸含药血清干预后紧密连接组成蛋白（JAM-A、ZO-1）表达升高，紧密连接结构恢复正常。小鼠原代支持细胞：与正常细胞相比，采用Cox7a2过表达和Cox7a2-siRNA敲降对支持细胞炎症因子无明显影响。Cox7a2-siRNA转染支持细胞后紧密连接组成蛋白（JAM-A、ZO-1）表达升高。TNFa可降低紧密连接组成蛋白（Claudin-5、Claudin-11、Occludin）表达。五子衍宗丸含药血清干预后紧密连接组成蛋白（JAM-A、Occludin）表达有升高趋势。（2）在体实验：通过构建肾精亏虚证生精障碍小鼠发现：睾丸内各炎症因子水平(IL-1α、IL-6、IL-17和TNFα)均显著升高，紧密连接完整性和超微结构受损，组成蛋白表达无显著变化。五子衍宗丸干预后，睾丸内炎症因子水平显著下降，损伤的紧密连接完整性及超微结构得以恢复。本研究揭示了五子衍宗丸改善生精功能机理与其调控睾丸内炎症因子水平及紧密连接通透性有关。