目的 基于ATR/Chk1/P53信号通路探讨三七总皂苷（PNS）对自然衰老大鼠睾丸生殖细胞DNA损伤的保护作用。方法 将SPF级SD大鼠随机分为青年对照组（3月龄），衰老模型组（18月龄），PNS低、中、高剂量（10、30、60 mg/kg）组（18月龄），每组10只。PNS低、中、高剂量组大鼠分别ig给予PNS 10、30、60 mg/kg，每周6 d，给药6个月。结束时称体质量后处死大鼠，迅速取出睾丸组织，称取睾丸质量，计算睾丸指数。HE染色法观察睾丸组织形态学变化，免疫组化法检测DNA损伤相关蛋白γ-H2AX、ATR的表达和定位，Western blotting检测睾丸组织中DNA损伤相关蛋白γ-H2AX、Chk1、p-P53、P21的表达水平。结果 与青年对照组比较，衰老模型组大鼠睾丸质量、睾丸指数显著降低，PNS能不同程度地升高睾丸质量、睾丸指数；HE结果表明，PNS能显著改善自然衰老所致的睾丸组织形态学变化；与青年对照组相比，衰老模型组大鼠睾丸组织DNA损伤标志蛋白γ-H2AX表达水平和阳性灶点数明显升高，ATR的阳性灶点数及其下游Chk1、p-P53、P21的蛋白表达明显升高，而PNS干预后可降低上述DNA损伤相关反应蛋白的表达。结论 PNS能够减轻自然衰老大鼠睾丸组织DNA损伤，其机制可能与调控ATR/Chk1/P53信号通路有关。

Objective To explore the protective effects of Panax notoginseng saponins (PNS) on testicular DNA damage in natural aging rats based on ATR/Chk1/P53 signal pathway. Methods SPF SD rats were randomly divided into five groups, with 10 rats in each group:young control group, aging model group, low, medium, and high dose of PNS-treated group. From 18 months, rats of PNS low, medium, and high dose groups were received PNS (10, 30, and 60 mg/kg)by stomach lavaging for 6 times per week for 6 months, respectively. The rats were weighed and euthanized by exsanguination under diethyl ether anesthesia, and testis were immediately removed, weighed, and the index of testis was calculated. The testicular tissue morphology was observed by HE staining, the expression and locations of γ-H2AX and ATR were detected by immunohistochemical analysis, and the relative expression levels of γ-H2AX, Chk1, p-P53, and P21 in testicular tissue were detected by Western blotting. Results Compared with young control group, the obvious changes of seminiferous tubule were observed in aging model group, accompanied with a reduction in weight and index of testis. However, PNS treatment in some extent improved seminiferous tubule structure, weight, and index of testis. In addition, the protein expression levels of γ-H2AX and the numbers of γ-H2AX-positive cells in testis were significantly upregulated in aging model group relative to young control group. Furthermore, the numbers of ATR-positive cells and the protein expression levels of Chk1, p-P53, and P21 were significantly upregulated in testis of rats in aging model group when compared with young control group. Conversely, PNS significantly restored these changes of aging induced DNA damage response related proteins. Conclusion PNS attenuates testicular DNA damage of natural aging rats, which may be associated with ATR/Chk1/P53 signaling.

国家自然科学基金资助项目（81573931，81373881，81503334）；三峡大学人才科研启动基金（KJ2014B067）