基于Fas/FasL途径的金匮肾气丸对肾阳虚模型睾丸细胞凋亡的影响

Effects of Jinguishenqi bolus on apoptosis of kidney-Yang deficiency model of testicular cells from the Fas/FasL pathway

目的以"劳倦过度、房室不节"肾阳虚小鼠为模型,从Fas/FasL途径探讨肾阳虚小鼠睾丸组织细胞凋亡发生机制及金匮肾气丸的治疗作用。方法以Colldege效应诱导雄性小鼠房室不节,用强迫小鼠游泳法造成劳倦过度,建立肾阳虚小鼠模型。4周后,分别用普通光学显微镜和电子显微镜进行睾丸组织的形态学观察,TUNEL法测定小鼠睾丸生殖细胞的凋亡率,利用RT-PCR检测小鼠睾丸组织Fas/FasL mRNA表达,用免疫组织化学分析Fas、FasL蛋白表达差异。结果1模型组小鼠睾丸组织曲细精管内支持细胞及各级生精细胞层次不清,结构破坏明显,金匮肾气丸组和睾酮组小鼠睾丸生殖细胞较模型组有一定程度改善。2在透射电镜下,模型组小鼠睾丸细胞中,可以观察到处于凋亡不同阶段的初级精母细胞,凋亡小体。3模型组TUNEL阳性细胞较正常组显著增加,主要为精原细胞和初级精母细胞,并可见精子细胞凋亡。金匮肾气丸治疗组睾丸细胞凋亡率明显低于模型组,但高于正常组,凋亡细胞类型以精原细胞和初级精母细胞为主。4模型组与正常组相比,Fas/FasL的mRNA表达量明显增高,金匮肾气丸组和睾酮组Fas、FasL mRNA表达较正常组稍高,但与模型组比较,明显降低。5模型组Fas在支持细胞和精原细胞及初级精母细胞高表达增多,精子细胞高表达增加明显;模型组FasL高表达细胞明显增多,在支持细胞、各级生精细胞及精子细胞、精子中均有分布,无明显定位特异性;金匮肾气丸组和甲基睾酮对肾阳虚模型小鼠的Fas/FasL均有明显的抑制作用。结论房劳-倦劳所致肾阳虚小鼠的睾丸内生殖细胞凋亡率上升,通过Fas、FasL的外源性死亡通路引起生殖细胞凋亡。金匮肾气丸可以通过调节Fas/FasL表达,降低小鼠睾丸生殖细胞凋亡。

Objective This experimental study focused on the germ cell apoptosis in Kidney – Yang deficiency syndrome mice model. Meanwhile,the effects of Jinguishenqi bolus were explored. Methods In this study,mice model was developed by overfatigue and sexual overstrain. The morphological changes of mice testis tissue were observed after HE staining. Ultramicrostructure were observed with transmission electron microscopy as well. In addition,the germ cell apoptosis ratios were detected by TUNEL.Results The structure of seminiferous tubule was destroyed obviously in model mice. The typical ultrastructural changes of apoptosis cell could be seen under ultramicroscope. These morphological changes in mice of the Jinguishenqi bolus group and testosterone group were much slighter when compared with model group. TUNEL positive cells in the model group were significantly increased,which mainly were the spermatogonia and primary spermatocytes. The testicular cell apoptosis rate of Jinguishenqi bolus group was significantly lower than that in the model group,but higher than the normal group. Conclusion There is a phenomenon of testicular cell apoptosis in the mice model developed by overfatigue and sexual overstrain,providing a ideal model which could be utilized to explore the theory of kidney Yang deficiency-related theory and traditional Chinese herb therapy.