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目的:探讨五子衍宗方对环磷酰胺(Cyclophosphamide,CTX)致成年雄性小鼠睾丸生殖细胞DNA损伤及P53通路的影响,并探讨其可能的作用机制。方法:将雄性BALB/c小鼠随机分为正常对照组、模型组及五子衍宗方低、高剂量组,五子衍宗方低、高剂量组分别提前灌胃给予五子衍宗方140、280 mg/kg,其余各组灌胃给予生理盐水。从第3天开始,除正常对照组腹腔注射生理盐水外,其余各组注射CTX 50 mg/kg,隔2 d注射1次,连续10次。参照精液分析方法检测小鼠精子浓度、精子形态正常率、精子活率;单细胞凝胶电泳观察小鼠睾丸细胞DNA损伤的变化;免疫组化检测小鼠睾丸DNA损伤蛋白γ-H2AX的定位与表达变化;Western blot检测小鼠睾丸生殖细胞内DNA损伤相关蛋白γ-H2AX、Ku70及DNA损伤修复相关蛋白p-P53、P21核内表达水平。结果:与模型组比较,五子衍宗方低、高剂量均可显著升高模型组小鼠精子浓度、精子形态正常率及精子活率,显著降低彗星尾长、尾距、Olive尾矩和尾部DNA百分率及睾丸组织中γ-H2AX的阳性点数。Western blot结果表明,与模型组比较,五子衍宗方可显著降低睾丸生殖细胞核内DNA损伤相关蛋白γ-H2AX、Ku70及DNA损伤修复相关蛋白p-P53、P21的表达。结论:五子衍宗方能显著减轻CTX致小鼠睾丸生殖细胞DNA损伤,其机制可能与调控P53信号通路有关。
Objective: To investigate the effect of Wuzi Yanzong Recipe on DNA damage and P53 pathway of testicular germ cell in adult male mice induced by Cyclophosphamide (CTX) and to explore its possible mechanism. Methods: Male BALB / c mice were randomly divided into normal control group, Wuzi Yanzong Fang model group and Wuzi Yanzong Fang low and high dose group, Wuzi Yanzong Fang low and high dose group were given intragastric administration of Wuzi Yanzong Fang 140,280 mg / kg , The other groups were given intragastric saline. From the third day, except the normal control group, intraperitoneal injection of saline, the other groups were injected with CTX 50 mg / kg, injected once every 2 days for 10 times. The sperm concentration, sperm morphology normal rate and sperm motility of mice were detected by semen analysis. The changes of DNA damage in mouse testis were observed by single cell gel electrophoresis. The localization of DNA damage protein γ-H2AX in mouse testes Western blot was used to detect the expression of nuclear injury related proteins (γ-H2AX, Ku70) and DNA damage repair-related proteins (p-P53 and P21) in mouse testis germ cells. Results: Compared with the model group, Wu Zi Yan Zong Fang low and high doses could significantly increase the sperm concentration, sperm morphology and sperm motility in the model group, and significantly reduce tail length, tail distance, Olive tail moment and tail DNA Percentage and positive testicular gamma-H2AX points. Western blot results showed that Wuzi Yanzong decoction could significantly reduce the expression of DNA damage related proteins such as γ-H2AX, Ku70 and DNA damage repair-related proteins p-P53 and P21 in testis germ cells compared with model group. Conclusion: Wuzi Yanzong Decoction can significantly reduce DNA damage induced by CTX in testis germ cells of mice, which may be related to the regulation of P53 signaling pathway.