摘要
目的探讨月见草油(EPO)对高脂饮食联合来曲唑所致多囊卵巢综合征(PCOS)模型大鼠激素水平、代谢水平、氧化水平以及卵巢组织中NAD-依赖性去乙酰化酶Sirtuin-1(SIRT1)/转录因子叉头框蛋白O1(FoxO1)信号通路表达的影响。方法72只大鼠共分为6组,12只为正常对照,其余60只大鼠喂食高脂饲料(40%脂肪)8周,并在喂食方案的最后3周,联合浓度为1 mg/(kg·d)的来曲唑灌胃21 d,制备PCOS大鼠模型。模型制备结束后经尾部血清激素水平分析,每组剔除偏差较大的模型大鼠2只,并把正常对照组和模型制备成功的大鼠分为6组,每组10只,分别为正常对照组、PCOS模型组、二甲双胍组、EPO小剂量组、EPO中剂量组、EPO大剂量组。ELISA方法和生化酶法检测各组血清空腹血糖(FPG)、空腹胰岛素(FINS)、睾酮(T)、促卵泡生成激素(FSH)、促黄体生成激素(LH)、超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)和丙二醛(MDA),苏木精-伊红(HE)染色观察各组卵巢形态,免疫组织化学和蛋白质免疫印迹法(Western blotting)检测卵巢组织SIRT1/FoxO1通路和相关抗氧化因子SOD1和谷胱甘肽转移酶(GST)以及衰老因子SIRT1和FoxO1的定位与相对表达水平。结果与正常对照组相比,PCOS模型组体质量、FPG、FINS、T、LH和FSH水平均升高(P<0.001);SOD和T-AOC水平下降(P<0.001),MDA水平升高(P<0.001)。ELISA结果显示,EPO和二甲双胍治疗后,体质量、FPG、FINS水平较PCOS模型组均呈下降趋势且有统计学意义(P<0.001),各组SOD水平与PCOS模型组相比升高(P<0.001),MDA水平下降(P<0.05),二甲双胍组、EPO中剂量组和大剂量组T-AOC水平较PCOS模型组升高(P<0.001);二甲双胍组和EPO中剂量组、大剂量组T和LH水平较PCOS模型组下降(P<0.001);二甲双胍组和EPO治疗大剂量组FSH水平较PCOS模型组下降(P<0.001)。免疫组化结果显示,SOD1、GST和SIRT1蛋白在颗粒细胞、间质细胞、原始卵泡和黄体均有阳性表达;Western blotting结果显示,与正常对照组相比,PCOS模型组卵巢组织SIRT1(P<0.001)、FoxO1、P-FoxO1、P-FoxO1/FoxO1(P=0.003)、SOD1(P=0.003)和GST蛋白表达量均增加(P<0.001);EPO大剂量组SOD1(P=0.003)、GST(P=0.007)和P-FoxO1蛋白表达较PCOS模型组下降(P=0.003),EPO中剂量组SIRT1(P=0.011)和FoxO1(P=0.353)蛋白表达下降。结论EPO改善PCOS模型大鼠血清中氧化因子和激素水平,且抑制SIRT1/FoxO1信号通路因子表达,提高卵巢组织的抗氧化能力并改善其氧化应激。
Objective To investigate the effects of evening primrose oil(EPO)on the hormonal,metabolic and oxidation levels in rats with polycystic ovary syndrome(PCOS)induced by high-fat diet combined with letrozole,and to explore the expression of NAD-dependent deacetylase sirtuin-1(SIRT1)/Forkhead box O1(FoxO1)signaling pathway in the ovarian tissue.Methods A total of 72 rats were divided into 6 groups,with 12 as the normal control group,and the other 60 rats were fed high-fat diet(40%)for 8 weeks.In the last 3 weeks of the feeding program,letrozole at a concentration of 1 mg/kg/d was added to prepare PCOS models.After the models were established,the serum hormonal levels in the tail of rats were analyzed,and 2 rats with large deviation were removed from each group.Plus the normal control group,the PCOS models were divided into 5 groups,with 10 rats in each group:PCOS model group,metformin group,EPO low-dose group,EPO medium-dose group,and EPO high-dose group.The serum fasting plasma glucose(FPG),serum insulin(FINS),testosterone(T),luteinizing hormone(LH),follicle stimulating hormone(FSH),superoxide Dismutase(SOD),total antioxidant capacity(T-AOC)and malondialdehyde(MDA)of each group were detected with ELISA and biochemical method.The ovarian morphology was observed with hematoxylin and eosin(HE)staining.The locations and expressions of SIRT1/FoxO1 pathway,SOD1 and glutathione S-transferase(GST),SIRT1 and FoxO1 were detected with immunohistochemistry and Western blotting.Results Compared with the blank control group,the PCOS model group had significantly increased weight,FPG,FINS,T,LH,FSH(P<0.05),significantly decreased SOD and T-AOC(P<0.05),and significantly increased MDA(P<0.01).ELISA results showed that after EPO and metformin treatment,the weight,FPG,FINS and MDA decreased(P<0.05),but SOD increased(P<0.05)compared with the PCOS model group.Compared with PCOS model group,the metformin group,medium-dose and high-dose groups had significantly increased T-AOC(P<0.05),but significantly decreased T and LH(P<0.05).Compared with the PCOS model group,the metformin group and high-dose group had significantly decreased FSH level(P<0.05).Immunohistochemical results showed that SOD1,GST and SIRT1 were positively expressed in granulosa cells,mesenchymal cells,primordial follicles and corpus luteum.Western blotting results showed that compared with the blank control group,the PCOS model group had significantly increased expressions of SIRT1,FoxO1,P-FoxO1,P-FoxO1/FoxO1,SOD1 and GST(P<0.05).Compared with the PCOS model group,the high-dose group had significantly decreased levels of SOD1,GST and P-FoxO1(P<0.05);the medium-dose group had significantly decreased levels of SIRT1 and FoxO1(P<0.05).Conclusion EPO affects the levels of oxidative factors and hormones in the serum of PCOS model rats,inhibits the expression of SIRT1/FoxO1 signaling pathway,enhances the antioxidant capacity of ovarian tissue and improve the oxidative stress.
作者
虎娜
孙苗
邢莎莎
许丹霞
海小明
马玲
杨丽
勉昱琛
何瑞
陈冬梅
马会明
HU Na;SUN Miao;XING Shasha;XU Danxia;HAI Xiaoming;MA Ling;YANG Li;MIAN Yuchen;HE Rui;CHEN Dongmei;MA Huiming(Key Laboratory of Fertility Preservation and Maintenance(Ministry of Education),Ningxia Medical University,Yinchuan 750004,Ningxia,China;Department of Human Anatomy and Tissue Embryology,School of Basic Medical Science,Ningxia Medical University,Yinchuan 750004,Ningxia,China;Ningxia Key Laboratory of Stem Cell and Regenerative Medicine,The General Hospital of Ningxia Medical University,Yinchuan 750004,Ningxia,China;Experimental Animal Center of Ningxia Medical University,Yinchuan 750004,Ningxia,China)
出处
《山东大学学报（医学版）》
CAS
北大核心
2022年第5期22-30,共9页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金(81660813)
宁夏科技领军人才项目(2020GKLRLX11)
宁夏药物研发创新团队项目(NXKJT2019012)
宁夏高校大学生创新创业训练计划项目(S202110752060)
