补肾益精法对雷公藤多苷诱发大鼠精子线粒体超微结构与膜电位损伤的影响

Influences of therapy of tonifying kidney and replenishing essence on damage of sperm mitochondrial ultrastructure and membrane potential induced by tripterygium glycosides in rats

目的观察雷公藤多苷制备的少弱精症模型大鼠精子线粒体超微结构与膜电位水平的变化,探讨补肾益精法治疗少弱精症的干预机制。方法取体重为200～220 g雄性大鼠,随机分成正常组、模型组、黄精赞育胶囊组(3.24 g/kg)、补肾益精方小、中、大剂量组(分别为0.68、1.35、2.70 g/kg),除正常组外,其他各组动物连续每天灌服雷公藤多苷8周(30 mg/kg),制备少弱精症模型。造模结束后,各组动物按剂量灌胃给药,连续30 d,正常组、模型组给予等容量蒸馏水。末次给药后30 min,处理动物,采用JC-1荧光染色流式细胞技术测定各组动物线粒体膜电位正常精子百分率(JC-1+%),并通过透射电子显微镜观察精子线粒体超微结构的改变。结果雷公藤造模8周后,模型动物JC-1+%显著下降,橙红色荧光强度明显降低;精子尾部出现水肿、呈现空腔,线粒体肿胀、大小不一,轴丝出现断裂。模型大鼠连续30 d给予补肾益精方后,大鼠JC-1+%明显增加;精子外膜较完整、线粒体肿胀减轻、轴丝轻度变性。结论保护精子线粒体结构可能为拮抗雷公藤多苷生殖毒性的有效途径。补肾益精方可通过抑制精子线粒体膜电位下降和保护线粒体结构完整性达到治疗少弱精症的目的。

Objective To observe the changes of sperm mitochondrial uhrastructure and membrane potential in rat model of oligoasthenotspermia prepared with tripterygium glycosides （GTW）, and discuss the intervention mechanism of therapy of tonifying kidney and replenishing essence to oligoasthenotspermia. Methods Male rats （weighing from 200 g to 220 g） were randomly divided into normal group, model group, Huangjing Zanyu Capsules group （positive group, 3.24 g/kg）, and groups of low-dose, mid-dose and high-dose Bushen Yijing Fang （low-dose group, mid-dose group and high-dose group, 0. 68 g/kg, 1.35 g/kg and 2. 70 g/kg）. Except of normal group, other groups were orally given GTW（30 mg/kgc/d） every day continuously for 8 weeks for preparing the model of oligoasthenotspermia. After modeling, all medicated groups were given corresponding medicinal for 30 days, and normal group and model group were given the same amount of distilled water. The level of sperm mitochondrial membrane potential （MMP） was detected by applying JC-1 fluorescence staining flow cytometry, and changes of sperm mitochondrial ultrastructure were observed with transmission electron microscope （TEM）. Results After modeling by GTW for 8 weeks, the level of MMP and fluorescent intensity of salmon pink decreased significantly in model rats. The tail of sperm showed edema and cavity, and mitochondria appeared swelling and unevenness with fracture of axoneme. After administration of Bushen Yijing Fang for 30 days, the level of MMP increased significantly. The sperm membrane showed integrity, mitochondrial swelling was alleviated and degeneration of axoneme was mild. Conclusion The protection of sperm mitochondrial structure may be one of effective ways for antagonizing the reproductive toxicity of GTW. Bushen Yijing Fang can treat oligoasthenotspermia through inhibiting the decrease of sperm MMP and protecting the integrity of mitochondrial structure.