CN115252759A - 酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 - Google Patents
酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用 Download PDFInfo
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Abstract
本发明属于生物技术及生物制药技术领域,公开了一种酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用。本发明发掘了酸奶衍生多肽的医疗新用途,开拓了一个新的应用领域,有望成为延缓端粒缩短和抗衰老的有效药物,同事为开发和扩展酸奶衍生多肽的抗衰老提供了基础。
Description
技术领域
本发明属于生物技术及生物制药技术领域,涉及酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用。
背景技术
端粒是存在于真核细胞线状染色体末端的DNA-蛋白质复合体,它与端粒结合蛋白一起构成了染色体特殊的“帽子”结构,其主要功能是保持染色体的完整性使其免受损伤。随着细胞的分裂,端粒会进行性缩短,而当端粒缩短至一定的阈值时,细胞会发出损伤信号,并启动细胞衰老和凋亡程序,从而使得细胞走向不可逆性衰老,进而死亡,因此,端粒长度的维持对细胞衰老乃至个体衰老至关重要。经典的“端粒学说”表明,氧化应激能够加剧端粒磨损:一方面,富含鸟嘌呤的端粒DNA序列易成为急性氧化损伤的靶点;另一方面,活性氧等氧化应激产物可通过破坏端粒长度调节机制从而加速端粒磨损。因此,细胞内氧化损伤累积可能是端粒磨损的重要原因。此外,端粒酶逆转录酶活性是端粒长度的另一重要调节因子,通过利用自身RNA模板功能和逆转录酶活性合成新的端粒DNA重复序列,从而在细胞增殖的过程中参与端粒长度的维持。
酸奶由牛奶经微生物发酵而来,发酵过程为酸奶提供各类微生物活性代谢产物的同时也引入了多种具有预防和保健功能的活性肽,这些活性肽为心血管系统、神经系统、肠道健康和免疫防御等方面提供了多重的健康益处。此外,酸奶中的活性肽在胃肠消化阶段能够进一步释放出生物学活性更高的小分子衍生多肽。然而,酸奶中的活性肽能否通过抗氧化作用延缓端粒长度的磨损速度尚不明确。
发明内容
针对氧化应激加速端粒缩短进而导致细胞不可逆衰老的技术问题,本发明目的旨在提供酸奶衍生多肽在制备延缓端粒药物中的应用。
经研究发现,酸奶可通过增强抗氧化能力而延缓衰老模型小鼠端粒长度的磨损速度。通过与牛奶经消化后的多肽种类进行对比,筛选出酸奶中特有的具有潜在抗氧化生物活性的低分子量衍生肽,其序列分别为PLGTQ(脯氨酸-亮氨酸-甘氨酸-苏氨酸-谷氨酰胺)和VLNPW(缬氨酸-亮氨酸-天冬酰胺-脯氨酸-色氨酸),具体筛选过程参见(Peptideprofiling and the bioactivity character of yogurt in the simulatedgastrointestinal digestion,Journal of Proteomics,Volume 141,1June 2016,Pages24-46)。
编码PLGTQ和VLNPW的核酸序列如下所示:
PLGTQ:ccgctgggcacccag
VLNPW:gtgctgaacccgtgg
本发明提供了酸奶衍生多肽在制备延缓端粒缩短药物中的应用。所述酸奶衍生多肽为具有序列PLGTQ或VLNPW的多肽。本发明提供的酸奶衍生多肽在制备延缓端粒缩短药物中的应用,对药物剂型和制备方法没有特别限制,可采用本领域常规通用的制备方法制成片剂、胶囊、颗粒剂、缓释剂、注射剂等各种剂型。
鉴于上述酸奶衍生多肽能够有效延缓端粒缩短,进而延缓衰老和死亡,本发明进一步提供了上述酸奶衍生多肽在制备抗衰老药物中的应用。本发明提供的酸奶衍生多肽在制备抗衰老药物中的应用,对药物剂型和制备方法没有特别限制,可采用本领域常规通用的制备方法制成片剂、胶囊、颗粒剂、缓释剂、注射剂等各种剂型。
与现有技术相比,本发明提供的酸奶衍生多肽在制备延缓端粒缩短药物及抗衰老药物中的应用具有以下有益效果:
(1)本发明发掘了酸奶衍生多肽的医疗新用途,开拓了一个新的应用领域,有望成为延缓端粒缩短和抗衰老的有效药物,同事为开发和扩展酸奶衍生多肽的抗衰老提供了基础。
(2)酸奶衍生多肽(例如具有序列PLGTQ和VLNPW的多肽)能够通过调节c-Myc/TERT信号通路和氧化应激水平,进而延缓细胞端粒磨损速度。
(3)酸奶衍生多肽(例如具有序列PLGTQ和VLNPW的多肽)能够降低β-半乳糖苷酶活性,提高抗氧化能力,进而延缓端粒磨损速度,从而发挥抗衰老作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的方案,以下将对实施例或现有技术描述中所需要使用的附图作简单介绍。
图1为不同条件下衰老小鼠外周血白细胞端粒长度测试结果。
图2为不同条件下衰老小鼠肝脏β-gal活性测试结果。
图3为CCK8法检测酸奶衍生多肽对细胞衰老模型细胞活力的测试结果。
图4为不同条件下细胞衰老模型相对端粒长度的测试结果。
图5为不同条件下细胞衰老模型抗氧化酶(GSH-Px、CAT和SOD活性)和氧化应激代谢产物(ROS水平和MDA水平)的影响。
图6为不同条件下细胞衰老模型凋亡率的测试结果。
图7为不同条件下细胞衰老模型TERT和c-Myc蛋白表达的测试结果;其中β-actin(肌动蛋白)作为内参。
具体实施方式
以将结合附图对本发明各实施例的技术方案进行清楚、完整的描述,显然,所描述实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施例,都属于本发明。
具有序列PLGTQ和VLNPW的酸奶衍生多肽采用常规技术手段得到,参见(Peptideprofiling and the bioactivity character of yogurt in thesimulatedgastrointestinal digestion,Journal of Proteomics,Volume 141,1 June2016,Pages 24-46)。
实施例1—qPCR法检测酸奶衍生多肽对小鼠端粒长度的影响
实验材料:昆明小鼠(SPF级),雄性,3月龄,购于北京维通利华实验动物公司。
实施步骤:
(1)动物饲养及分组:
将80只5周龄雄性昆明小鼠随机分为空白对照组,衰老模型组、阳性对照组、PLGTQ和VLNPW。除空白对照组外,其余四组均进行建模处理。
建模处理:动物于温度20~25℃,相对湿度65~70%,明暗交替周期为12h,通风良好的环境中饲养。适应性喂养一周后,通过持续6周皮下注射80mg/kg D-gal制备衰老小鼠模型,第6周末,小鼠出现反应迟缓,活动量减少,毛发枯黄无光泽,精神萎靡等现象,表明衰老模型构建成功。
待模型构建成功后,给予阳性对照组(维生素E 100mg/kg/天)、PLGTQ和VLNPW(20mg/kg/天)持续8周每日灌胃相应剂量的药物,对照组灌胃无菌生理盐水(20mg/kg/天)。
(2)端粒长度检测:
按DNA提取试剂盒说明书分别提取各组小鼠外周血白细胞DNA后,以36B4为内参进行PCR扩增检测相对端粒长度,每个样本重复检测三次,循环条件为95℃预变性2min,95℃变性5s,60℃退火和延伸10s,进行40个循环,采用法计算基因相对表达量,引物序列见表1。
表1引物名称和序列
实验结果如图1所示,与模型组相比,阳性对照组、PLGTQ和VLNPW组端粒长度显著延长,且差异均具有统计学意义,表明两种多肽与抗衰老阳性对照维生素E一样可以显著缓解衰老模型小鼠外周血端粒长度的磨损速度。
实施例2—染色法检测小鼠肝组织β-半乳糖酶活性
实施步骤:按照实施例1设置动物分组,进行药物处理。剖取药物干预后的各组小鼠肝脏组织,利用多聚甲醛固定24h,以蔗糖浸泡过夜后制备冰冻切片,经孵育液显色过夜,中性红复染后于油镜下观察各组切片蓝色阳性信号颗粒数量。
实验结果如图2所示,与模型组相比,阳性对照组、PLGTQ和VLNPW组蓝色信号颗粒数量显著减少,表明β-gal活性减弱,提示两种多肽与维生素E具有相同的缓解模型小鼠衰老的作用。
实施例3—CCK8法检测酸奶衍生多肽对细胞衰老模型细胞活力的影响
实验材料:LO2细胞购于上海中科院典藏细胞库
实施步骤:将处于对数期的LO2细胞接种于96孔板中,细胞密度为5×103个/孔,每组设置6个重复孔,待细胞贴壁后按照不同实验分组要求给予相应的干预处理,空白组是不含任何细胞的DMEM完全培养基;对照组是不含多肽的细胞培养液(DMEM完全培养基作为培养液);实验组是分别加入0.01、0.05、0.1、0.5、1、5、10、50和100μg/ml多肽的细胞培养液。
分别干预24h后去除原有培养液,每孔按照CCK8与培养基1:9的体积比加入混合溶液,避光孵育2h后,采用酶标仪于450nm波长处测定各孔吸光度值,并按公式计算出细胞存活率。计算公式为:
细胞存活率=(A实验组-A空白组)/(A对照组-A空白组)×100%,A为检测波长为450nm出的吸光度。
实验结果如图3所示,10ng/ml~100μg/ml的两种多肽处理24h后,LO2细胞存活率呈现先增加后下降的趋势,表明衍生多肽与LO2细胞存在低浓度促进细胞存活率,高浓度抑制细胞存活率的剂量反应关系。
实施例4—qPCR法检测衍生多肽对LO2细胞端粒长度的影响
实施步骤:将对数生长期细胞随机分为对照组、模型组、PLGTQ和VLNPW组,空白对照组给予完全DMEM完全培养基作为培养液,模型组给予含40μmol/L t-BHP的培养液培养24h,PLGTQ和VLNPW组先分别给予含1μg/mlPLGTQ和0.1μg/mlVLNPW的多肽培养24h,再更换含40μmol/L t-BHP的培养液继续培养24h。分别对各组细胞干预后使用DNA提取试剂盒提取各组细胞总DNA,测定其浓度后以36B4为内参进行PCR扩增检测相对端粒长度,具体方法参照实施例1。
实验结果如图4所示,与模型组相比,PLGTQ和VLNPW组端粒长度显著增加,表明两种多肽可以有效缓解LO2细胞端粒长度的缩短速度。
实施例5—酸奶衍生多肽对细胞衰老模型抗氧化酶和氧化应激产物的影响
实施步骤:按照实施例4设置实验分组和药物处理。细胞处理结束后,按照试剂盒说明书操作,使用酶标仪检测并计算LO2细胞抗氧化酶GSH-Px、CAT和SOD活性以及氧化应激代谢产物ROS水平和MDA水平。
实验结果如图5所示,与模型组相比,PLGTQ组和VLNPW组抗氧化酶GSH-Px、CAT和SOD的活性显著增加,氧化应激产物ROS和MDA水平显著降低,表明两种多肽可以显著抑制LO2细胞内氧化应激产物的聚集并增强细胞抗氧化能力。
实施例6—酸奶衍生多肽对细胞衰老模型凋亡率的影响
实施步骤:按照实施例4设置实验分组和药物处理。细胞处理结束后,用胰酶消化并收集经干预处理后的各组LO2细胞,按照Annexin FITC/PI细胞凋亡检测试剂盒中说明书进行操作,流式细胞仪检测并计算细胞凋亡率,每个样本重复3次。
实验结果如图6所示,对照组细胞凋亡率为2.1%,模型组细胞凋亡率为15.5%,与对照组相比显著增加,PLGTQ和VLNPW组细胞凋亡率分别为5.6%和6.8%,与模型组相比显著降低,表明两种多肽可以有效缓解t-BHP引起的细胞凋亡。
实施例7—酸奶衍生多肽对细胞衰老模型TERT和c-Myc蛋白表达的影响
实施步骤:按照实施例4设置实验分组和药物处理。细胞处理结束后,提取各组细胞总蛋白,使用Bradford法对蛋白进行定量,Western blot法检测TERT和c-Myc蛋白表达水平。
实验结果如图7所示,与模型组相比,PLGTQ和VLNPW组c-Myc蛋白表达水平显著上调,同时TERT蛋白表达显著升高,表明两种多肽可以显著增加端粒酶活性。
综上所述,本发明首次证实了酸奶衍生多肽PLGTQ和VLNPW能够通过调节c-Myc/TERT信号通路和氧化应激水平延缓衰老模型细胞端粒磨损速度。同时,本发明证实了酸奶衍生多肽PLGTQ和VLNPW能够降低衰老模型小鼠脑内β-半乳糖苷酶活性,提高抗氧化能力,延缓端粒磨损速度,从而发挥抗衰老作用。因此,本发明提供了将酸奶衍生多肽PLGTQ和VLNPW在延缓端粒磨损及抗衰老中的应用,为开发和扩大酸奶衍生多肽的抗衰老提供了基础。
本领域的普通技术人员将会意识到,这里所述的实施例是为了帮助读者理解本发明的原理,应被理解为本发明的保护范围并不局限于这样的特别陈述和实施例。本领域的普通技术人员可以根据本发明公开的这些技术启示做出各种不脱离本发明实质的其它各种具体变形和组合,这些变形和组合仍然在本发明的保护范围内。
Claims (6)
1.酸奶衍生多肽在制备延缓端粒缩短药物中的应用。
2.根据权利要求1所述的酸奶衍生多肽在制备延缓端粒缩短药物中的应用,其特征在于,所述酸奶衍生多肽为具有序列PLGTQ或VLNPW的多肽。
3.根据权利要求1或2所述的酸奶衍生多肽在制备延缓端粒缩短药物中的应用,其特征在于,所述药物剂型为片剂、胶囊、颗粒剂、缓释剂或注射剂。
4.酸奶衍生多肽在制备抗衰老药物中的应用。
5.根据权利要求4所述的酸奶衍生多肽在制备抗衰老药物中的应用,其特征在于,所述酸奶衍生多肽为具有序列PLGTQ或VLNPW的多肽。
6.根据权利要求4或5所述的酸奶衍生多肽在制备抗衰老药物中的应用,其特征在于,所述药物剂型为片剂、胶囊、颗粒剂、缓释剂或注射剂。
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