五子衍宗丸对衰老睾丸生殖细胞DNA损伤的保护作用与机制研究

随着有生育需求的中老年男性不断增多，因衰老而导致的生殖细胞DNA损伤与遗传缺陷，已成为影响人类生殖健康的主要因素之一，但目前仍缺乏有效的防治措施。五子衍宗丸是补肾填精代表方，具有改善男性生殖功能与延缓衰老作用；我们前期研究显示，五子衍宗丸可保护环磷酰胺所致小鼠生精细胞DNA损伤，其作用机制与抗氧化损伤等相关；而抗氧化能力下降与自由基产生增多是衰老导致睾丸生殖细胞DNA损伤的主要机制，提示五子衍宗丸亦可能对衰老导致的生殖细胞DNA损伤有保护作用。基于此，本项目以自然衰老大鼠为模型，采用TUNEL、comet assay等方法检测细胞凋亡与DNA损伤，Western blot和免疫荧光法检测Ku70、53BP1、RAD9A、Nrf2、HO-1等表达，从抗氧化损伤和促进DNA损伤修复的角度，研究五子衍宗丸对衰老睾丸生殖细胞DNA损伤的保护作用及机制，为临床应用五子衍宗丸防治生殖衰老提供依据。

With growing number of medium-elderly men of child-bearing, germ cells DNA damage and genetic disease resulted from testis aging have become a major concerned issue for reproductive health. However, the effective measures for preventing germ cells DNA damage with aging are also distressed. Wuziyanzong pills, a representative tonifying kidney recipe, significantly enhanced male reproductive function and delayed aging. Our previous study demonstrated that wuziyanzong pills protected germ cells from testicular DNA damage induced by cyclophosphamide of mice, which is related with its anti-oxidative effect. It is reported that increased free radical damage and decreased repair capacity were the main mechanism of testicular cells DNA damage of aging. These data suggested that wuziyanzong pills could protect germ cells from DNA damage with aging. Here, we first investigate germ cells apoptosis and DNA damage using TUNEL and comet assay in a natural aging rat model. Then, the protein expressions of Ku70, 53BP1, RAD9A, Nrf2 and HO-1 will be evaluated by western blot and immunofluorescenc analysis. Based on this, we will determine the protective effects of wuziyanzong pills on DNA damage of aging testicular germ cells through regulation of anti-oxidative damage and DNA damage repair, which will provide theoretical basis of wuziyanzong pills for prophylaxis and therapy of testis aging.