CN113768913A - 甜菜碱在制备改善卵巢功能的药物、食品、保健品的应用、药物、食品、保健品、动物模型 - Google Patents
甜菜碱在制备改善卵巢功能的药物、食品、保健品的应用、药物、食品、保健品、动物模型 Download PDFInfo
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Abstract
本发明公开了一种甜菜碱在制备改善卵巢功能的药物、食品、保健品的应用、药物、食品、保健品、动物模型，其公开了一种甜菜碱的新用途，即甜菜碱在制备抗卵巢衰老药物和抗卵巢衰老保健品或食品中的应用。本发明还建立了用于抗卵巢衰老化合物功效评价的动物模型，本发明通过借助卵巢病理损伤组(CTX)动物模型评估甜菜碱对OS引起的卵巢功能损伤的挽救作用，之后又借助更加典型的自然衰老动物模型评估甜菜碱对年龄因素导致的卵巢储备功能减退的挽救作用，全面的评估了甜菜碱对于病理及生理因素引起的卵巢功能损伤及卵巢储备功能减退的作用功效，揭示了甜菜碱用于开发临床改善卵巢储备减退与女性生殖功能损伤产品的潜在可能。
Description
技术领域
本发明涉及抗卵巢衰老药物技术领域，具体涉及一种甜菜碱在制备改善卵巢功能的药物、食品、保健品的应用、药物、食品、保健品、动物模型。
背景技术
研究显示，约有10％-15％的育龄夫妇患有不同程度的不孕症，其中多数由女性因素引起。吸烟、饮酒、肥胖、药物、年龄等多种因素都会损伤生殖系统(Turan V,OktayK.Sexual and fertility adverse effects associated with chemotherapy treatmentin women.Expert Opin Drug Saf.2014；13(6):775-83)，主要表现在生殖细胞质量和数量降低，难以形成优质胚胎。因此改善卵子和精子的质量和数量是治疗不孕不育的关键(LiJ,Eriksson M,Czene K,Hall P,Rodriguez-Wallberg KA.Common diseases asdeterminants of menopausal age.Hum Reprod.2016；31(12):2856-64；LoboRA.Hormone-replacement therapy:current thinking.Nat Rev Endocrinol.2017；13(4):220-31.)。
雌性生殖功能损伤涉及多种机制，其中氧化应激(OS)是影响卵母细胞质量与数量的重要原因之一。活性氧(ROS)参与调节卵泡发育及卵母细胞成熟(Ganesan S,KeatingAF.The ovarian DNA damage repair response is induced prior to phosphoramidemustard-induced follicle depletion,and ataxia telangiectasia mutatedinhibition prevents PM-induced follicle depletion.ToxicolApplPharmacol.2016；292:65-74；Chang EM,Lim E,Yoon S,Jeong K,Bae S,Lee DR,et al.Cisplatin InducesOveractivation of the Dormant Primordial Follicle through PTEN/AKT/FOXO3aPathway which Leads to Loss of Ovarian Reserve in Mice.PLoS One.2015；10(12):e0144245.)，同时，在诱导排卵以及促进黄体凋亡中起到重要作用(Delgado-Rosas F,Gaytán M,Morales C,Gómez R,Gaytán F.Superficial ovarian cortex vascularization isinversely related to the follicle reserve in normal cycling ovaries and isincreased in polycystic ovary syndrome.Hum Reprod.2009；24(5):1142-51；ErnstEH,
ML,Grund S,Hardy K,Heuck A,Sunde L,et al.Dormancy and activationof human oocytes from primordial and primary follicles:molecular clues tooocyte regulation.Hum Reprod.2017；32(8):1684-700.)。当ROS的产生超过抗氧化系统的清除能力时，抗氧化系统稳态失衡，诱导颗粒细胞凋亡、影响细胞间信号作用传导，由此导致生殖细胞数量和功能异常(Nguyen QN,Zerafa N,Liew SH,Morgan FH,Strasser A,Scott CL,et al.Loss of PUMA protects the ovarian reserve during DNA-damagingchemotherapy and preserves fertility.Cell Death Dis.2018；9(6):618.)。
大量研究证实，过量ROS对生殖系统存在负面影响，因此清除过多的ROS对提高生殖细胞质量至关重要。为此，研究者通过借助药物诱导、基因敲除、自然衰老等动物模型(Delkhosh A,Delashoub M,Tehrani AA,Bahrami AM,Niazi V,Shoorei H,etal.Upregulation of FSHR and PCNA by administration of coenzyme Q10 oncyclophosphamide-induced premature ovarian failure in a mouse model.JBiochemMolToxicol.2019；33(11):e22398；Liu XM,Yan MQ,Ji SY,Sha QQ,Huang T,ZhaoH,et al.Loss of oocyte Rps26 in mice arrests oocyte growth and causespremature ovarian failure.Cell Death Dis.2018；9(12):1144；Liu X,Lin X,Zhang S,Guo C,Li J,Mi Y,et al.Lycopene ameliorates oxidative stress in the agingchicken ovary via activation of Nrf2/HO-1pathway.Aging(Albany NY).2018；10(8):2016-36.)，模拟生殖功能损伤的病理状态，借此研究OS损伤机制并寻求可能的挽救措施。其中，CTX是一种与闭经/卵巢功能不全和卵巢功能障碍相关的临床化疗药物。研究发现ROS是CTX引起卵巢毒性的重要潜在机制(Kalich-Philosoph L,Roness H,Carmely A,Fishel-Bartal M,Ligumsky H,Paglin S,et al.Cyclophosphamide triggers follicleactivation and"burnout"；AS101prevents follicle loss and preservesfertility.SciTransl Med.2013；5(185):185ra62.)，CTX暴露可产生大量ROS，引起多种抗氧化酶水平异常(Meirow D,Biederman H,Anderson RA,Wallace WH.Toxicity ofchemotherapy and radiation on female reproduction.ClinObstet Gynecol.2010；53(4):727-39.)，继而诱导细胞凋亡、减少细胞增殖，严重影响卵母细胞质量与数量。同样，自然衰老动物模型也存在生殖系统ROS状态。深入研究发现，随着生殖系统的衰老，卵巢中原始卵泡数量减少、卵巢储备水平降低，年龄依赖性内分泌发生改变、生殖道异常，卵细胞和胚胎质量显著下降。其中卵子质量显著下降的主要原因是衰老的卵母细胞容易产生染色体分裂异常及线粒体功能障碍，导致抗氧化水平降低，氧化应激增加，ROS(活性氧)水平升高。
抗氧化剂可有效清除体内多余ROS，抗氧化剂应用简单、效果明显，逐渐成为近年来的研究热点。其在改善生殖系统ROS上发挥强抗氧化效应，逐渐成为新型生殖营养素(国作登字-2017-A-00496585)。甜菜碱为一碳代谢中重要的甲基供体参与同型半胱氨酸-蛋氨酸循环。甜菜碱可通过提高蛋氨酸、腺苷甲硫氨酸、半胱氨酸含量，促进GSH合成、增强机体抗氧化能力(Gonfloni S,Di Tella L,Caldarola S,Cannata SM,Klinger FG,DiBartolomeo C,et al.Inhibition of the c-Abl-TAp63 pathway protects mouseoocytes from chemotherapy-induced death.Nat Med.2009；15(10):1179-85.)，被视为一种新兴的重要抗氧化剂。
但是，迄今为止，尚未见关于甜菜碱在应对抗卵巢衰老和卵巢储备功能减退的临床应用的相关报道。
发明内容
本发明通过借助卵巢病理损伤组(CTX)动物模型评估甜菜碱对ROS引起的卵巢功能损伤的挽救作用，之后又借助更加典型的自然衰老动物模型评估甜菜碱对年龄因素导致的卵巢储备功能减退的挽救作用，全面的评估了甜菜碱对于病理及生理因素引起的卵巢功能损伤及卵巢储备功能减退的作用功效，揭示了甜菜碱用于开发临床改善卵巢储备功能减退与女性生殖功能损伤产品的潜在可能。
本发明提供了一种甜菜碱在制备改善卵巢功能的药物中的应用。本发明对甜菜碱在增加卵泡数量、降低卵母细胞ROS水平、提高成熟卵母细胞数量、改善IVF结局等方面的作用进行了研究，实验结果表明甜菜碱可作为改善卵巢功能储备、提高卵母细胞质量、改善ART结局的重要抗氧化组分。
本发明还提供了一种甜菜碱用于抗卵巢衰老药物的动物模型，其包括空白对照组、卵巢生理衰老组、阴性对照组、卵巢病理损伤组、病理损伤甜菜碱补充组和生理衰老挽救组。利用本发明的动物模型，能够将甜菜碱同临床所对应的病理或高龄导致的卵巢储备功能减退人群的情形相联系，并取得良好的模拟效果。
进一步的，其按照如下方法构建，所述空白对照组采用8周龄野生型ICR小鼠，利用生理盐水200μL灌胃28天；所述卵巢生理衰老组采用38周龄ICR小鼠，生理盐水200μL灌胃28天；所述阴性对照组采用8周龄野生型ICR小鼠，每日以等量生理盐水灌胃，灌胃28天，第22日腹腔注射等量生理盐水；所述卵巢病理损伤组采用8周龄野生型ICR小鼠，每日以等量生理盐水灌胃，灌胃28天，第22日腹腔注射CTX150mg/kg；所述病理损伤甜菜碱补充组采用8周龄野生型ICR小鼠，甜菜碱400mg/kg/d灌胃28天，第22日腹腔注射CTX150mg/kg；所述生理衰老挽救组采用38周龄ICR小鼠，甜菜碱400mg/kg/d灌胃28天。
本发明还提供了一种改善卵巢功能的药物，其包含甜菜碱。
本发明还提供了一种甜菜碱在制备改善卵巢功能的保健品或食品中的应用。
本发明还提供了一种改善卵巢功能的食品，其包含甜菜碱。
本发明还提供了一种甜菜碱在制备保护雌性动物生殖系统药物中的应用。通过对卵巢病理损伤组小鼠补充甜菜碱能显著提高其卵泡数量和促排取卵数量，并能使卵巢的DDX4蛋白水平恢复至接近正常的水平，由此说明甜菜碱对雌性动物生殖系统具有保护作用。
进一步的，所述药物的使用期间为雌性动物的妊娠期和哺乳期之外。
本发明还提供了一种用于保护雌性动物生殖系统的药物，其包含甜菜碱，所述药物的使用期间为雌性动物的妊娠期和哺乳期之外。
附图说明
图1(a)-(c)为本发明的卵巢石蜡切片HE染色对比图,其中，Control为空白对照组，CTX为卵巢病理损伤组，Betaine为病理损伤甜菜碱补充组；
图2(a)-(d)为各级卵泡计数分析对比，其中，Primordial为原始卵泡，Primary为初级卵泡，Secondary为次级卵泡，Antral为窦卵泡；
图3(a)-(c)为卵母细胞数量镜检对比图，其中，标尺为500μm，Control为空白对照组，Aging为卵巢生理衰老组，Aging-res为生理衰老挽救组；
图4(a)为MII卵母细胞计数对比；
图4(b)为DDX4蛋白的WesternBlot结果分析；
图4(c)为灰度统计结果对比，其中，Control为空白对照组，Aging为卵巢生理衰老组，Aging-res为生理衰老挽救组；
图5为补充甜菜碱对Aging组小鼠的卵母细胞的ROS免疫荧光染色图；
图6为卵母细胞H2DCFDA阳性率统计；
图7为补充甜菜碱对卵巢病理损伤组(CTX)小鼠的卵母细胞的ROS免疫荧光染色图；
图8为卵母细胞H2DCFDA荧光强度统计；
图9(a)-(c)为补充甜菜碱对卵巢病理损伤组(CTX)小鼠的2-cell胚胎的镜检对比图；
图9(d)-(f)为补充甜菜碱对卵巢病理损伤组(CTX)小鼠的4-cell胚胎的镜检对比图；
图9(g)-(i)为补充甜菜碱对卵巢病理损伤组(CTX)小鼠的囊胚的镜检对比图；
图9(j)为补充甜菜碱对卵巢病理损伤组(CTX)小鼠的2-cell胚胎的数量的影响对比图；
图9(k)为补充甜菜碱对卵巢病理损伤组(CTX)小鼠的囊胚的数量的影响对比图；
图10(a)-(c)为补充甜菜碱对卵巢生理衰老组小鼠的受精卵的镜检对比图；
图10(d)-(f)为补充甜菜碱对卵巢生理衰老组小鼠的2-cell胚胎的镜检对比图；
图10(g)-(i)为补充甜菜碱对卵巢生理衰老组小鼠的囊胚的镜检对比图；
图10(j)为补充甜菜碱对卵巢生理衰老组小鼠的2-cell胚胎的数量的影响对比图；
图10(k)为补充甜菜碱对卵巢生理衰老组小鼠的囊胚的数量的影响对比图。
具体实施方式
本发明各个实施例所用的小鼠均为雌鼠，购自维通利华，为SPF级别ICR小鼠；小鼠严格按照标准进行饲养，4-5只/标准笼，环境温度控制在22-24℃，湿度为40-70％，12h光照-12h黑暗循环。食物和水充足任取。实验动物的使用依照《中华人民共和国实验动物管理条例》严格执行。
每周测量所有实验小鼠的体重、观察生存状态，直至实验结束。实验结束时对所有小鼠进行脱颈椎处死。
实施例1：本发明的动物实验模型如下：
空白对照组(Control)：8周龄野生型ICR小鼠，生理盐水200μL灌胃28天；
卵巢生理衰老组(Aging)：38周龄ICR小鼠，生理盐水200μL灌胃28天；
阴性对照组(Negative control)：8周龄，野生型，ICR小鼠，每日以等量生理盐水灌胃，灌胃28天，第22日腹腔注射等量生理盐水；
卵巢病理损伤组(CTX)：8周龄，野生型，ICR小鼠，每日以等量生理盐水灌胃，灌胃28天，第22日腹腔注射CTX150mg/kg；
病理损伤甜菜碱补充组：8周龄，野生型，ICR小鼠，甜菜碱400mg/kg/d灌胃28天，第22日腹腔注射CTX150mg/kg；
生理衰老挽救组(Aging-res)：38周龄ICR小鼠，甜菜碱400mg/kg/d灌胃28天。
本发明所使用的甜菜碱采购自Sigma，规格为B2629O，在使用时将甜菜碱溶解于无菌的小鼠饮水中(ddH2O)，备用。
本发明分别利用卵巢生理衰老组(Aging)小鼠和卵巢病理损伤组(CTX)小鼠作为损伤模型，并对损伤模型使用甜菜碱予以挽救，并与正常模型小鼠进行卵巢储备功能、卵巢发育状态和生育结局等结果的对比。
实施例2：甜菜碱对小鼠的卵巢形态影响
本发明采用HE染色方法对小鼠的卵巢形态进行评估，具体按如下方法进行：卵巢在4％多聚甲醛溶液(BL539A，白鲨)中固定过夜。然后用梯度浓度增加的乙醇(70％、80％、90％、100％)使卵巢脱水，在二甲苯中透明后，用石蜡包埋并切成5μm厚的切片。将切片脱蜡、再水化、用苏木精和伊红(C0105S,碧云天)染色，对卵巢切片在光学显微镜下计数各级卵泡，为防止重复计数，每张切片只选择带有细胞核者纳入计数范围。
利用上述的分析方法分别对空白对照组、卵巢病理损伤组和病理损伤甜菜碱补充组的小鼠的卵巢进行卵巢形态学评估，分别对卵巢的Primordial(原始卵泡)，Primary(初级卵泡)，Secondary(次级卵泡)和Antral(窦卵泡)的数量进行评估，结果参见附图1和2所示，结果表明：相较于空白对照组，卵巢病理(CTX)损伤组小鼠的原始卵泡、初级卵泡数量均有显著下降，而次级卵泡及窦卵泡数量并没有显著变化。
在甜菜碱(碧云天)补充后,虽然向小鼠体内注射了CTX，但是小鼠的原始卵泡，初级卵泡和次级卵泡的数量与卵巢病理损伤组相比，均有大幅度提高，同时，甜菜碱的补充对次级卵泡以及窦卵泡数量无显著影响。
实施例3：甜菜碱对小鼠的获卵数量的影响
本实施例利用卵巢生理衰老组(Aging)小鼠模型，并对比了同等促排条件下，是否补充甜菜碱对小鼠获卵数量的影响，并利用空白对照组做对比实验。
结果如图3(a)-(c)所示，结果表明显示Aging组小鼠的MII卵母细胞数量显著减少，甜菜碱补充(Aging-res)能够显著提高Aging小鼠的MII卵母细胞数量。但是，与年轻组小鼠(Control)相比，仍存在差距。
实施例4：甜菜碱对小鼠的卵巢中蛋白水平分析
本发明利用WesternBlot方法对小鼠卵巢中的蛋白含量进行分析，具体包括如下步骤：用含有1mM PMSF的8M尿素裂解缓冲液(8M尿素、75μMNaCl、50μMTris-Cl pH8.2)提取蛋白质，并使用蛋白质测定试剂盒(碧云天)进行蛋白浓度定量。蛋白质样品在7％SDS-PAGE凝胶中分离并转移到PVDF膜(Millipore)。在5％脱脂牛奶中封闭后，在对应的一抗中4℃下孵育过夜。抗体如下：抗DDX4(A7352，爱博泰克生物；1：1000)，抗Tublin(60004，武汉三鹰；1：2000)，之后在TBST中洗涤3次后，二抗(Invitrogen，31800；35560，1:3000)在室温下孵育2小时，ECL(180-506，天能)显色后记录结果。
参见图4(a)-(c)所示，利用上述的对小鼠卵巢中蛋白水平分析的方法对生殖细胞特异性蛋白Maker-DDX4表达水平进行分析，本实施例分别对空白对照组、卵巢病理损伤组和病理损伤甜菜碱补充组的小鼠卵巢中的DDX4蛋白水平进行分析，结果表明卵巢病理损伤组的小鼠卵巢中的DDX4蛋白水平显著降低，但是，补充甜菜碱后能显著升高DDX4水平，并能使其达到接近正常水平。
实施例5：甜菜碱对卵母细胞ROS水平的影响
取新鲜MII卵母细胞，置于H2DCFDA染色液内37℃培养箱中避光孵育30min，PBS-PVA洗涤液清洗3次，Hochest 33258(C1022，碧云天)37℃培养箱中避光染核5min，PBS-PVA洗涤3次，移至特殊皿中，激光共聚焦显微镜拍摄观察，计算荧光强度。
参见图5-8所示，本发明同时评估了卵巢病理损伤组(CTX)及卵巢生理衰老组(Aging)损伤两种模型的ROS水平，结果显示：同对照组相比，自然衰老组及病理损伤组的卵母细胞中ROS水平均显著升高，同时Aging组存在大量形态异常的卵母细胞，两种处理对应的病理损伤甜菜碱补充组中卵母细胞ROS水平均显著降低，其中针对于CTX病理损伤引起的ROS升高，补充甜菜碱能够将ROS水平纠正到同对照组几乎一致的水平。
实施例6：补充甜菜碱对小鼠生育结局的影响
本发明通过对小鼠的体外受精(IVF)过程中，甜菜碱对于小鼠生育结局的影响进行分析。本发明的IVF按照如下步骤完成:利用注射用孕马血清促性腺素PMSG(宁波三生；5IU)对小鼠注射48小时后，再次注射绒毛膜促性腺激素HCG(宁波三生；5IU)，13小时后从超排卵雌性小鼠中分离出卵丘-卵母细胞复合物(COC)。HTF培养基(易核)中的COC与获能的附睾精子混合并在37℃下在5％CO2下孵育，然后置于KSOM培养基(M1450，易核)中，不受干扰地培养4天，显微镜下观察发育中胚胎。
参见附图9(a)-(k)所示，与对照组相比，CTX模型组的小鼠的2-cell胚胎、4-cell胚胎以及囊胚率均显著降低，在补充了甜菜碱后，各级胚胎率均显著升高。
参见附图10(a)-(k)所示，与对照组相比，Aging模型组的小鼠的受精率、2-cell胚胎以及囊胚率均显著降低，在补充了甜菜碱后均显著升高。由此说明，甜菜碱能显著提高CTX病理损伤以及Aging自然衰老引起的小鼠的胚胎发育异常，对小鼠的生育结局具有良好的干预效果。
上述结果表明，甜菜碱对病理因素引起的卵巢功能异常或年龄因素导致的卵巢储备功能减退均有良好的干预效果。无论是病理(CTX)因素所导致的卵巢功能损伤还是生理(Aging)因素所导致的卵巢储备减退，均能够通过甜菜碱的补充得到显著改善，补充甜菜碱均能够有效增加卵泡数量、降低卵母细胞ROS水平、提高成熟卵母细胞数量、改善IVF结局。
由此可见，甜菜碱可作为改善卵巢功能储备、提高卵母细胞的数量和质量，改善ART(人类辅助生殖技术)结局的重要药物组分。本发明的各个实施例中是通过将甜菜碱直接向小鼠灌胃强饲，其效果等同于小鼠口服甜菜碱。很显然，甜菜碱的使用剂型和方式也可采用其他任意药物学可以接受的形式。抗卵巢衰老药物的剂型可以为口服制剂或注射制剂。口服制剂可以为片剂、丸剂、颗粒剂、口服液、胶囊剂、乳剂，注射制剂可以为注射液或注射粉针剂。
以上实施方式只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明精神实质所做的等效变化或修饰，都应涵盖在本发明的保护范围内。
Claims (10)
1.一种甜菜碱在制备改善卵巢功能的药物中的应用。
2.一种甜菜碱用于改善卵巢功能的药物的制备的动物模型，其特征在于，包括空白对照组、卵巢生理衰老组、阴性对照组、卵巢病理损伤组、病理损伤甜菜碱补充组和生理衰老挽救组。
3.根据权利要求2所述的动物模型，其特征在于，其按照如下方法构建，
所述空白对照组采用8周龄野生型ICR小鼠，利用生理盐水200μL灌胃28天；
所述卵巢生理衰老组采用38周龄ICR小鼠，生理盐水200μL灌胃28天；
所述阴性对照组采用8周龄野生型ICR小鼠，每日以等量生理盐水灌胃，灌胃28天，第22日腹腔注射等量生理盐水；
所述卵巢病理损伤组采用8周龄野生型ICR小鼠，每日以等量生理盐水灌胃，灌胃28天，第22日腹腔注射CTX150mg/kg；
所述病理损伤甜菜碱补充组采用8周龄野生型ICR小鼠，甜菜碱400mg/kg/d灌胃28天，第22日腹腔注射CTX150mg/kg；
所述生理衰老挽救组采用38周龄ICR小鼠，甜菜碱400mg/kg/d灌胃28天。
4.一种改善卵巢功能的药物，其特征在于，包含甜菜碱。
5.一种甜菜碱在制备改善卵巢功能的保健品或食品中的应用。
6.一种改善卵巢功能的保健品，其特征在于，包含甜菜碱。
7.一种改善卵巢功能的食品，其特征在于，包含甜菜碱。
8.一种甜菜碱在制备保护雌性动物生殖系统药物中的应用。
9.根据权利要求8所述的应用，其特征在于，所述药物的使用期间为在雌性动物的妊娠期和哺乳期之外。
10.一种用于保护雌性动物生殖系统的药物，其特征在于，包含甜菜碱，所述药物的使用期间为在雌性动物的妊娠期和哺乳期之外。
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