五子衍宗方对自然衰老大鼠睾丸生殖细胞DNA损伤和凋亡的保护作用研究

Protective effect of Wuzi Yanzong recipe on testicular DNA damage and apoptosis in natural ageing rats

该文探讨五子衍宗方对自然衰老大鼠睾丸生殖细胞DNA损伤和凋亡的保护作用及其可能机制。将SPF级16月龄SD雄性大鼠随机分为3组,衰老模型组（Ageing）、五子衍宗方低剂量组（WZ,1 g·kg^-1）、五子衍宗方高剂量组（WZ,4 g·kg^-1）,2月龄SD大鼠作为青年对照组（Adult）,每组10只。普通饲料或含药饲料喂养4个月后,处死大鼠,取出睾丸组织,称取睾丸质量,计算睾丸指数。HE染色观察睾丸组织形态。免疫荧光检测DNA损伤相关蛋白ATR的表达和定位。免疫印迹检测睾丸组织中DNA损伤相关蛋白γ-H2AX,Chk1,p-p53及凋亡相关蛋白Bcl-2和Bax的表达。TUNEL检测大鼠睾丸生殖细胞凋亡变化。结果显示,五子衍宗方能显著提高睾丸质量及睾丸指数,明显改善睾丸组织形态。免疫印迹结果显示,与青年对照组相比,衰老模型组大鼠睾丸组织γ-H2AX,Chk1,p-p53,Bax蛋白表达水平显著升高,Bcl-2蛋白表达水平显著下降。五子衍宗方干预后能显著降低γ-H2AX,Chk1,p-p53,Bax蛋白表达水平,升高Bcl-2蛋白表达水平,且能升高Bcl-2/Bax的比值。免疫荧光结果显示,五子衍宗方能显著降低衰老所致睾丸组织ATR升高。TUNEL结果显示,五子衍宗方能显著减少睾丸组织生殖细胞凋亡。以上结果表明,五子衍宗方可保护自然衰老大鼠睾丸生殖细胞DNA损伤和凋亡,机制可能与调节p53信号通路有关。

To study the protective effect of Wuzi Yanzong recipe on testicular DNA damage and apoptosis in natural ageing rats,SPF grade 16-month-old SD male rats were randomly divided into three groups： ageing model group,low and high dose Wuzi Yanzong recipe groups（ WZ,1,4 g·kg^-1）. In addition,2-month-old SD rats were used as adult control group（ 10 rats in each group）. The ageing model group and the adult control group were fed with normal diet for 4 months. Wuzi Yanzong groups received medicated feed for 4 months. After fasting for 12 hours,the rats were sacrificed. Then testis tissues were taken and weighed to calculate the testis index. The change of testicular tissue morphology was observed by HE staining. Expression and localization of DNA damage-associated protein ATR were observed by immunofluorescence. The expressions of DNA damage-related proteins γ-H2 AX,Chk1,p-p53 and apoptosis-related proteins Bcl-2 and Bax in testes were detected by Western blot. The apoptosis of testis tissue in rats was detected by using TUNEL. The results showed that as compared with the youth control group,the protein expression levels of γ-H2 AX,Chk1,p-p53 and Bax were significantly increased while Bcl-2 protein expression level was significantly decreased intestis tissues of ageing model group. Wuzi Yanzong recipe significantly decreased protein expression levels of γ-H2 AX,Chk1,p-p53 and Bax and increased Bcl-2 protein expression level as well as Bcl-2/Bax ratio. Immunofluorescence results showed that Wuzi Yanzong recipe could significantly decrease the ageing-induced ATR,increase in testis tissues. TUNEL results showed that Wuzi Yanzong recipe could significantly attenuate the germ cell apoptosis in testicular tissues. All the above results suggest that Wuzi Yanzong recipe could protect the germ cell in testicular tissues of natural ageing rates from DNA damage and apoptosis,and the mechanism may be associated with regulating p53 signaling pathway.