刘小虎;赵志慧;周玥;王翠丽;韩艳军;周雯;
LIU Xiao-hu;ZHAO Zhi-hui;ZHOU Yue;WANG Cui-li;HAN Yan-jun;ZHOU Wen;Department of Histology and Embryology, School of Basic Medical Sciences, Dali University;

• 1:大理大学基础医学院组织学与胚胎学教研室

摘要(Abstract)：

研究磷脂酰肌醇3-激酶(posphoinositide 3-kinase,PI3K)、蛋白激酶B(protein kinase B,Akt)和哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)自噬信号通路在人参皂苷Rg_1(ginsenoside Rg_1,Rg_1)延缓D-半乳糖(D-galactose,D-gal)诱导的卵巢早衰小鼠模型卵巢早衰中的作用。将SPF级雌性BALB/c小鼠54只随机分成PBS组、D-gal组、Rg_1组。D-gal组给予颈背部皮下注射D-半乳糖200 mg·kg~(-1)·d~(-1),连续给药42 d;PBS组给予颈背部注射等时等量磷酸盐缓冲液(PBS),连续注射42 d;Rg_1组在D-gal组处理基础上,于第15天开始给予腹腔注射Rg_1 20 mg·kg~(-1)·d~(-1),连续给药28 d,同时D-gal组和PBS组也在第15天开始给予腹腔注射等时等量PBS,连续给药28 d。给药后,检测小鼠动情周期改变,卵巢SA-β-Gal苷酶染色检测卵巢衰老状态改变,Western blot法检测PI3K,Akt,mTOR,S6k,LC3-Ⅱ,P16~(INK4a)蛋白表达的改变。荧光定量PCR法检测衰老调控因子PI3K,Akt,mTOR,S6k,P16~(INK4a) mRNA表达的改变。研究结果显示,与PBS组比较,D-gal组第3周开始出现动情周期紊乱,卵巢SA-β-半乳糖苷酶染色阳性率升高,衰老标记物P16~(INK4a)表达量升高,自噬信号分子LC3-Ⅱ表达量降低;Rg_1作用后,Rg_1组卵巢SA-β-半乳糖苷酶染色阳性率降低,衰老标记物P16~(INK4a)表达量低于D-gal组,自噬信号分子LC3-Ⅱ表达量高于D-gal组。与PBS组比较,D-gal组PI3K,Akt,mTOR,S6k蛋白和mRNA表达上调,Rg_1组Akt,mTOR,S6k蛋白表达上调,PI3K,mTOR mRNA表达上调。Rg_1作用后,Rg_1组PI3K,Akt,mTOR,S6k蛋白表达量低于D-gal组,Akt,mTOR,S6k mRNA表达量低于D-gal组。研究结果提示,Rg_1具有延缓D-gal诱导的卵巢早衰小鼠模型中卵巢早衰的作用,PI3K/Akt/mTOR自噬信号通路在其中发挥重要功能。
The aim of this paper was to study the role of phosphoinositide 3-kinase(PI3 K), protein kinase B(Akt) and mamma-lian target of rapamycin(mTOR) in the inhibition of premature ovarian failure induced by D-galactose(D-gal) in mice model by ginsenoside Rg_1(Rg_1). Fifty-four female SPF BALB/c mice were randomly divided into PBS group, D-gal group, and Rg_1 group. In the D-gal group, D-galactose(200 mg·kg~(-1)·d~(-1)) was injected subcutaneously into the neck and back for 42 days. In the PBS group, an equal amount of phosphate buffered saline(PBS) was injected into the neck and back for 42 days. In addition to the therapy of D-gal group, Rg_1 group was given Rg_1(20 mg·kg~(-1)·d~(-1)) through intraperitoneal injection since the 15 th day for 28 days, at the same time, the D-gal group and the PBS group were also given an equal amount of PBS through intraperitoneal injection since the 15 th day for 28 days. After the treatment, the estrous cycle changes of the mice were detected, and the ovarian SA-β-Gal staining was used to detect the changes of ovarian aging. Western blot was used to detect the changes in protein expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). Fluorescence quantitative PCR was used to detect the changes in mRNA expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). According to the findings, compared with the PBS group, the D-gal group began to show estrous cycle disorder in the 3 rd week,the ovarian SA-β-Gal staining positive granulosa cells increased in the D-gal group, the expression of senescence marker P16~(INK4 a) increased, while the expression of autophagy signaling molecule LC3-Ⅱ decreased. After treatment with Rg_1, the positive rate of ovarian SA-β-Gal staining in Rg_1 group decreased, the expression level of autophagy signaling molecule LC3-Ⅱ in Rg_1 group was higher than that in D-gal group, while the expression level of senescence marker P16~(INK4 a) was lower than that in D-gal group. Compared with the PBS group, the protein and mRNA expressions of PI3 K, Akt, mTOR and S6 k in the D-gal group were up-regulated, the protein expressions of Akt, mTOR and S6 k in the Rg_1 group were up-regulated, and the mRNA expressions of PI3 K and mTOR were up-regulated. After treatment with Rg_1, the protein expressions of PI3 K, Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group, while the mRNA expressions of Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group. The finding ssuggested that Rg_1 has the effect in delaying ovarian premature failure in D-gal-induced mouse models, and PI3 K/Akt/mTOR autophagy signaling pathways play an important role.

关键词(KeyWords)： 人参皂苷Rg_1;卵巢早衰;PI3K;Akt;mTOR;自噬;D-gal
ginsenoside Rg_1;premature ovarian failure;PI3K;Akt;mTOR;autophagy;D-gal

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81860038,81660731,81873103);; 云南省教育厅科学研究基金项目(2020Y0541)

作者(Author): 刘小虎;赵志慧;周玥;王翠丽;韩艳军;周雯;
LIU Xiao-hu;ZHAO Zhi-hui;ZHOU Yue;WANG Cui-li;HAN Yan-jun;ZHOU Wen;Department of Histology and Embryology, School of Basic Medical Sciences, Dali University;

Email:

DOI: 10.19540/j.cnki.cjcmm.20200901.405

参考文献(References)：

• [1] 林慧文,吴哲,梁莹,等.雷公藤多苷片诱导卵巢早衰模型小鼠的自身修复性研究[J].广州医药,2019,50(5):1.
• [2] 周翔.人工周期疗法联合小剂量左甲状腺素钠片治疗卵巢早衰的疗效观察[J].临床合理用药杂志,2017,10(10):82.
• [3] 陈醒,周应芳,白文佩.绝经期激素替代治疗的相关肿瘤风险研究进展[J].国际妇产科学杂志,2016,43(5):489.
• [4] CHU S F,ZHANG J T.New achievements in ginseng research and its future prospects[J].Chin J Integr Med,2009,15(6):403.
• [5] 黎阳,张铁军,刘素香,等.人参化学成分和药理研究进展[J].中草药,2009,40(1):164.
• [6] CHEN X,ZHANG J,FANG Y M,et al.Ginsenoside Rg1 delays tert-butyl hydroperoxide-induced premature sensecence in human WI-38 diploid fibroblast cells[J].J Gerontol A Biol Sci Med Sci,2008,63(3):253.
• [7] 徐静,贾力,赵余庆,等.人参的化学成分与人参产品的质量评价[J].药物评价研究,2011,34(3):199.
• [8] ZHOU W,LI J,LI X,et al.Development and validation of a reversed-phase HPLC method for quantitative determination of ginsenosides Rb1,Rd,F2,and compound K during the process of biotransformation of ginsenoside Rb1[J].J Sep Sci,2008,31(6/7):921.
• [9] 李琳,闫论,闫慧慧,等.早发性卵巢功能不全的遗传学最新研究进展[J].中华生殖与避孕杂志,2018,38(5):416.
• [10] 包秀芳,孙萍.不同浓度D-半乳糖致大鼠卵巢早衰的实验研究[J].实用妇科内分泌杂志:电子版,2017,4(15):73.
• [11] 郁琦.早发性卵巢功能不全诊治进展[J].中国实用妇科与产科杂志,2018,34(3):241.
• [12] 王宝娟,宫政,宋佳怡,等.SIRT1通过抑制卵母细胞衰老改善卵巢储备功能的研究进展[J].国际生殖健康/计划生育杂志,2020,39(1):45.
• [13] DE LOS SANTOS M J,MERCADER A,GALAN A,et al.Implantation rates after two,three or five day of embryo culture[J].Placenta,2003,24(suppl B):13.
• [14] 何连利.Rg1延缓D-gal POF小鼠模型生殖系统病理损害、改善生殖功能及其机制的研究[D].重庆:重庆医科大学,2017.
• [15] ZHANG T,HE W H,FENG L L.Effect of doxorubicin-induced ovarian toxicity on mouse ovarian granulosa cells[J].Regul Toxicol Pharmacol,2017,17(86):1.
• [16] 刘小虎,赵志慧,周玥,等.SIRT1在人参皂苷Rg1延缓D-gal诱导的卵巢早衰小鼠模型卵巢早衰中的作用[J].中国中药杂志,2020,45(19):4699.
• [17] 程霄.人参皂苷Rg1调控延缓脑衰老的机制研究[D].重庆:重庆医科大学,2019.
• [18] 刘不悔,何伟明,万毅刚,等.虫草菌丝抑制自噬相关AMPK/ULK1信号活性改善肾小管上皮细胞衰老的分子机制[J].中国中药杂志,2019,44(6):1258.
• [19] HANSEN M,CHANDRA A,MITIC L L.A role for aurophagy in the extension of lifespan by dietary restriction in C.elegans[J].PLoS Genet,2008,4(2):e24.
• [20] WOHLGEMUTH S E,SEO A Y,MARZETTI E,et al.Skeletal muscle autophagy and apoptosis during aging:effects of calorie restriction and life-long exercise[J].Exp Gerontol,2010,45(2):138.
• [21] OJHA R,BHATTACHARYYA S,SINGH S K.Autophagy in cancer stem cells:a potential link between chemoresistance,recurrence,and metastasis[J].Biores Open Access,2015,4(1):97.
• [22] 王鹤儒,于洋,陈丽艳.PI3K/mTOR抑制剂NVP-BEZ235抑制人乳腺癌细胞增殖及促进细胞自噬[J].基础医学与临床,2016,36(8):1139.
• [23] 白俊,吴也可,吴克明,等.雷公藤甲素通过PI3K/AKT/mTOR通路诱导卵巢颗粒细胞自噬的实验研究[J].中国中药杂志,2019,44(16):3429.
• [24] 潘斌才,梁颖雯,王春华,等.PTEN和mTOR蛋白在IDH1基因分型胶质母细胞瘤及PI3K/Akt/mTOR信号通路中的表达及其相关性[J].右江民族医学院学报,2018,40(2):108.
• [25] LIM S,KALDIS P.Cdks,cyclins and CKIS:roles beyond cell cycle regulation[J].Development,2013,140(15):3079.
• [26] YUAN H X,XIONG Y,GUAN K L.Nutrient sensing,metabolism,and cell growth control[J].Mol Cell,2013,49(3):379.
• [27] UM S H,FRIGERIO F,WATANABE M,et al.Absence of S6K1 protects against age-and diet-induced obesity while enhancing insulin sensitivity[J].Nature,2004,431(7007):200.
• [28] FENTONA T R,GOUTB I T.Functions and regulation of the 70 kDa ribosomal S6 kinase[J].Int J Biochem Cell Biol,2011,43(1):47.
• [29] 王冲,尹茂山,刘春霞.索马鲁肽对早期2型糖尿病心肌病大鼠mTOR信号通路的干预作用[J].中国现代应用药学,2019,36(14):1761.
• [30] SELMAN C,TULLET J M,WIESER D,et al.Ribosomal protein S6 kinase 1 signaling regulates mammalian life span[J].Science,2009,326(5949):140.