基于Rictor/mTORC2-PKCα通路调节支持细胞骨架肌动蛋白研究五子衍宗丸改善生精功能机理

The cytoskeletal actin microfilament networks of sertoli cell play an important role in maintaining the sertoli cell polarity, blood-testis barrier (BTB) integrity and spermatogenesis. Rictor/mTORC2-PKCαis the upstream signal pathway that regulate actin. Wuziyanzong pill is a classical formula in the treatment of male infertility. However, the mechanism of Wuziyanzong pill on spermatogenesis and actin microfilament networks is under further study. Our preliminary studies showed that the expression of Rictor and actin decreased in the testis of kidney essence deficiency syndrome animal with spermatogenic disorder induced by Tripterygium wilfordii. Meanwhile, the expression of Rictor and actin increased after the intervention of Wuziyanzong pills. Regarding Rictor/mTORC2-PKCαpathway modulating actin as the breakthrough point and relationship of “mTORC2-actin-spermatogenesis”, it is hypothesized that Wuziyanzong pill exerts its effects on actin expression and spermatogenesis through Rictor/mTORC2-PKCαpathway. In this study, Wuziyanzong pill is administered to kidney deficiency syndrome mice and primary purified mice sertoli cell respectively. With the method of cell biology, molecular biology and serum pharmacology, the expression of Rictor/ mTORC2、PKCα、actin microfilament expression and reorganization, actin bundling and polymerization, sertoli cell polarity, BTB integrity and spermatogenesis are to be observed both in vivo and in vitro. The present study is of great significance in elucidating the spermatogenetic mechanisms of Wuziyanzong pill.

支持细胞骨架肌动蛋白在维持支持细胞极性、血睾屏障完整性和生精功能中发挥重要作用。Rictor/mTORC2-PKCα是调节肌动蛋白的上游信号通路。五子衍宗丸是治疗男性不育经典方。前期实验表明肾精亏虚证生精障碍动物睾丸Rictor和肌动蛋白表达降低，五子衍宗丸干预后二者表达升高。本课题以“Rictor/mTORC2-PKCα调节肌动蛋白”为切入点，依据“mTORC2-肌动蛋白-生精功能”密切关联为理论基础，提出“五子衍宗丸通过Rictor/mTORC2-PKCα途径，调节肌动蛋白表达，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小鼠、原代培养小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Rictor/mTORC2、PKCα表达、肌动蛋白表达及重构、成束及聚合能力、支持细胞极性、血睾屏障完整性及生精功能变化，对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代表方，其如何通过调节肌动蛋白表达，进而改善生精功能还所知甚少。我们通过体外和体内实验观察五子衍宗丸干预后支持细胞Rictor/mTORC2-PKC通路、肌动蛋白、支持细胞极性、血睾屏障完整性及生精功能变化。研究显示：（1）体外实验：Rictor si-RNA转染后支持细胞Rictor mRAN表达明显下降，五子衍宗丸含药血清可以提高Rictor si-RNA转染后支持细胞Rictor/mTORC2-PKC通路中p-PKC/PKC的表达，并使支持细胞肌动蛋白F-actin与极性蛋白β-tubulin排列更为紧实、密集、荧光强度更强。同时，五子衍宗丸含药血清可以显著提高紧密连接相关蛋白（Occludin）及特化外质膜相关蛋白（N-cadherin）表达。（2）体内实验：通过构建肾精亏虚证生精障碍小鼠发现：五子衍宗丸可以拮抗GTW引起的生精小管损伤并修复睾丸超微结构与血睾屏障结构。同时，五子衍宗丸可以提高Rictor/mTORC2-PKC通路中PKC、肌动蛋白相关复合物（ARPC2）蛋白以及紧密连接相关蛋白ZO-1的表达。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支持细胞肌动蛋白以及Rictor/mTORC2-PKC通路有关。