王钦 司华新 伏蓉 孔彩华 刘克娜 隋世燕

【摘要】目的 研究喙尾琵琶甲乙醇提取物（BRFAE）对环磷酰胺诱导的卵巢早衰小鼠的保护作用，初步探索沉默信息调节因子1 /肿瘤相关蛋白53 （SIRT1/p53）信号通路在此过程中的作用。方法 将40只无特定病原体级雌性KM小鼠随机分为对照组、模型组和BRFAE低剂量和高剂量组。模型组和BRFAE低、高剂量组予腹腔注射环磷酰胺160 mg/kg，对照组则注射等体积的生理盐水，次日对照组和模型组予生理盐水灌胃，BRFAE低、高剂量组分别予BRFAE 100、200 mg/kg灌胃，连续14 d。干预结束后采集小鼠血液检测小鼠血浆丙二醛、一氧化氮和超氧化物歧化酶（SOD）水平，摘取卵巢稱重并制作HE切片进行卵泡计数，同时检测卵巢组织中SIRT1和p53 mRNA及蛋白表达。结果 低、高剂量的BRFAE均能改善环磷酰胺导致的卵巢增重和卵巢指数降低（P均<0.01），增加原始、初级卵泡和各级卵泡总数并减少闭锁卵泡的数量（P均< 0.05），其卵巢体积增大、黄体纤维化和血管减少，一氧化氮和丙二醛的水平降低，SOD活力升高（P均< 0.01），且高剂量BRFAE各项指标改善效果比低剂量BRFAE更为明显（P均<0.05）。免疫组织化学染色显示，与对照组相比，模型组SIRT1蛋白表达减弱（P < 0.01），而p53蛋白表达增强（P < 0.01），经BRFAE治疗后SIRT1蛋白表达增强（P < 0.01），而p53蛋白表达减弱（P < 0.01）。SIRT1和p53的qPCR检测结果与免疫组织化学染色结果基本一致。结论 BRFAE能够通过抗氧化作用抑制环磷酰胺所致的小鼠卵巢早衰，SIRT1/p53信号通路可能参与了其抗卵巢早衰作用。

【关键词】喙尾琵琶甲乙醇提取物;SIRT1/p53通路;KM小鼠;卵巢早衰

Protective effect of Blaps Rynchopetera Fairmaire alcohol extract on cyclophosphamide-induced premature ovarian failure in mice Wang Qin， Si Huaxin， Fu Rong， Kong Caihua， Liu Kena， Sui Shiyan. School of Public Health， Dali University， Dali 671000， China

Corresponding author， Sui Shiyan， E-mail： sysui569@163.com

【Abstract】Objective To evaluate the effect of Blaps Rynchopetera Fairmaire alcohol extract （BRFAE） on cyclophosphamide （CP）-induced premature ovarian failure in mouse models and investigate the role of SIRT1/p53 signaling pathway in this process. Methods Forty SPF female KM mice were randomly divided into the control， model， low- and high-dose BRFAE groups. Mice in the model， low- and high-dose BRFAE groups were given with CP at a dose of 160 mg/kg， and those in the control group were given with normal saline. On the next day， mice in the control and model groups were intragastrically administered with normal saline， and those in the low- and high-dose BRFAE groups were intragastrically administered with BRFAE （100 mg/kg and 200 mg/kg） for 14 consecutive d. After intragastric administration， mouse plasma samples were taken to detect the levels of malondialdehyde （MDA）， nitric oxide （NO） and superoxide dismutase （SOD）. The ovarian tissues were collected， weighed and prepared for HE staining. The quantity of follicles was counted. The expression levels of SIRT1 and p53 mRNA and proteins in the ovarian tissues were also determined. Results Administration of low- and high-dose BRFAE significantly improved the CP-induced increase in ovarian weight and the reduction of ovarian index （all P < 0.01）， significantly increased the total quantity of primitive， primary and all stages of follicles， and significantly reduced the quantity of atretic follicles （all P < 0.05）. In both low- and high-dose BRFAE groups， the ovarian volume was significantly increased， the corpus luteum fibrosis and blood vessels were significantly decreased， the NO and MDA levels were significantly declined， and the activity of SOD enzyme was significantly elevated （all P < 0.001）. The improvement effect of high-dose BRFAE was more pronounced compared with that of low-dose BRFAE （all P <

0.05）. The results of immunohistochemistry showed that compared with the control group， the expression level of SIRT1 protein in the model group was significantly down-regulated （P < 0.01）， whereas that of p53 protein was significantly up-regulated （P < 0.01）. After BRFAE treatment， the expression level of SIRT1 was significantly up-regulated （P < 0.01）， whereas that of p53 was significantly down-regulated （P < 0.01）. The expression levels of SIRT1 and p53 mRNA detected by q-PCR were basically consistent with the results of immunohistochemistry. Conclusion BRFAE inhibits CP-induced premature ovarian failure in mice through its antioxidant effect， and the SIRT1/p53 signaling pathway may be involved in its anti-premature ovarian failure effect.

【Key words】Blaps Rynchopetera Fairmaire alcohol extract; SIRT1/p53 signaling pathway;

KM mouse; Premature ovarian failure

卵巢早衰指婦女40岁之前由于某些原因引起的卵巢功能衰竭，其表现有闭经、不育、雌激素缺乏，以及促性腺激素水平升高[1]。……