CN113499426B - 一种乳源多肽在制备防治酒精性脂肪肝药物、保健品或食品添加物中的应用 - Google Patents
一种乳源多肽在制备防治酒精性脂肪肝药物、保健品或食品添加物中的应用 Download PDFInfo
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Abstract
本发明公开了一种乳源多肽在制备防治酒精性脂肪肝药物、保健品或食品添加物中的应用，通过体外实验证实其可以有效抑制游离脂肪酸（FFAs）诱导的小鼠原代肝细胞内脂质堆积；在体研究发现其具有降低高脂饮食诱导的脂肪肝小鼠模型肝脏体重比、减少脂质积聚的功效。因此，该乳源多肽及其嵌合多肽可用于在制备防治脂肪肝的药物、保健品以及食品添加物。
Description
技术领域
本发明涉及多肽和代谢调控领域，具体地说涉及一种乳源多肽在制备防治酒精性脂肪肝药物、保健品或食品添加物中的应用。
背景技术
随着肥胖和代谢综合征的全球化流行，非酒精性脂肪肝的发病率逐年上升且呈低龄化趋势，已经成为我国第一大肝病。作为代谢综合征的重要组成，非酒精性脂肪肝不仅与心脑血管疾病密切相关，还可发展为肝硬化、肝癌，最终导致 30%-40%的患者死亡，严重威胁人类健康。非酒精性脂肪肝的治疗尚无理想的药物，目前以控制饮食、改变不良生活方式为主，但耗时长、见效慢且易反复。因此，积极探索防治非酒精性脂肪肝的新手段迫在眉睫。
脂质代谢紊乱是非酒精性脂肪肝发生的关键环节之一。肝脏作为能量代谢的中心器官，是脂质合成与分解的重要场所。在正常生理状态下，肝细胞内的脂质处于合成与分解的动态平衡之中，一旦这种平衡被打破，就会使肝细胞中的游离脂肪酸（Free FattyAcids, FFAs）大量累积，甘油三酯含量剧增进而发生脂肪变性，最终导致非酒精性脂肪肝。近年来多肽因其具有分子量低、单位活性好、易于合成制备的特点，成为各大药企研发的焦点。因此，从多肽角度寻找调控脂质代谢的新途径，或许能为非酒精性脂肪肝的防治提供新思路。
发明内容
发明目的：本发明的目的是提供一种乳源多肽或嵌合多肽在用于制备治疗非酒精性脂肪肝药物中的应用，以及在用于制备降低高脂诱导肝细胞脂质堆积的药物中的应用；并对该多肽在细胞水平和腹腔注射的有效浓度进行确定。
技术方案：为了实现上述发明目的，本发明所述乳源多肽包含SEQ ID NO.1所示的氨基酸序列，β-酪蛋白第113-120位氨基酸，本领域技术人员可通过生物工程技术扩增、表达获得，也可通过化学合成方法（例如固相/液相合成方法）获得。该乳源多肽作为活性氨基酸序列，可与一条穿膜氨基酸序列形成的嵌合多肽，所述穿膜氨基酸序列位于活性氨基酸序列的N端。
对于上述的乳源多肽或嵌合多肽，经研究发现了其可用于治疗非酒精性脂肪肝，降低高脂诱导肝细胞脂质堆积。因此可被进一步用于开发脂肪肝防治、养肝护肝的药物。
作为本发明的优选方案，所述穿膜氨基酸序列包括至少一段寡聚精氨酸序列，所述穿膜氨基酸序列中精氨酸数量的占比不低于40%。
更优选地，所述穿膜氨基酸序列含有活性氨基酸序列N端依次连接的谷氨酰胺、脯氨酸的寡聚氨基酸片段，这些寡聚氨基酸形成的穿膜氨基酸序列可有效连接活性序列，高效转运穿越细胞膜且不会引发免疫反应。所述寡聚氨基酸片段为至少2个相邻且相同的氨基酸组成的短肽。
进一步的，作为本发明的最优方案，所述嵌合多肽选自如下氨基酸序列：
SEQ ID NO.2: GRKKRRQRRRVKEAMAPK，
SEQ ID NO.3: GRKKRRQRRRPPQVKEAMAPK，
SEQ ID NO.4: GRKKRRQRRRPPQQVKEAMAPK。
对于上述SEQ ID NO.1 - SEQ ID NO.4所示的多肽序列，可进行必要的修饰，包括但不限于特定基团的保护/去保护，C端和/或N端和/或侧链的酰基化、烷基化、酰胺化、酯基化，以及其他特殊偶联或螯合修饰。通常地，可在N端添加羧基，C端添加羟基或氨基进行修饰。
对于前述的乳源多肽或嵌合多肽，均可用于制备治疗非酒精性脂肪肝的药物、用于制备防治非酒精性脂肪肝的保健品或食品添加物、以及用于制备降低高脂诱导肝细胞脂质堆积的药物。其中，所述乳源多肽或嵌合多肽在细胞水平发挥作用的浓度为50-200 μM，优选为80-150 μM；在腹腔注射动物试验中发挥生物活性的浓度为5-15mg/Kg，优选为5-10mg/Kg。
附图说明
图1为本发明实施例2采用CCK8法检测不同浓度嵌合多肽处理下原代肝细胞活力变化以评估多肽药物毒性的曲线；
图2为本发明实施例2采用油红O染色法检测不同浓度嵌合多肽处理下原代肝细胞脂质堆积变化；
图3为实施例2采用有尼罗红染色法检测不同浓度嵌合多肽处理下原代肝细胞脂质堆积变化；
图4为实施例3关于嵌合多肽对非酒精脂肪肝小鼠采食量的影响；
图5为实施例3关于嵌合多肽对非酒精脂肪肝小鼠肝脏/体重比的影响；
图6为实施例3关于H&E染色和油红O染色检测乳源多肽衍生干预下2个月对非酒精脂肪肝小鼠肝脏脂质积聚的影响。
具体实施方式
下面结合附图和具体实施方式对本发明进行进一步说明。
实施例1
本实施例提供了以下四条氨基酸序列，除特别说明外，本实施例及以下实施例中的肽链均委托自上海科肽生物有限公司通过化学合成获得，纯度>98%。
本实施例提供了如下多肽序列：
SEQ ID NO.1：VKEAMAPK；分子式C38H68N10O11S，平均分子量873.07 g/mol，理论等电点pH 9.88；
SEQ ID NO.2：GRKKRRQRRRVKEAMAPK；分子式C93H175N41O22S，平均分子量2251.7 g/mol，平均等电点pH 12.471。
SEQ ID NO.3：GRKKRRQRRRPPQVKEAMAPK；分子式C105H189N43O25S2，平均分子量2518.02 g/mol，平均等电点pH 11.911。
SEQ ID NO.4：GRKKRRQRRRPPQQVKEAMAPK；分子式C113H205N47O28S，平均分子量2702.19 g/mol，平均等电点pH 12.471。
以上序列SEQ ID NO.1- SEQ ID NO.4的任意一种或多种的组合作为活性成分，通过添加其他药学上可接受的载体或赋形剂，可制备成不同剂型、载体的药物，还可以在其基础上拓展为保肝养肝的保健品。
本领域技术人员在不脱离本发明保护范围的基础上，可进行必要的修饰，包括但不限于特定基团的保护/去保护，C端和/或N端和/或侧链的酰基化、烷基化、酰胺化、酯基化，以及其他特殊偶联或螯合修饰。优选在N端添加羧基，C端添加羟基或氨基进行修饰。
实施例2 嵌合多肽对肝细胞脂质堆积的影响
1. 实验方法
以原代分离的小鼠肝细胞为研究对象；按照50 μM和200 μM浓度梯度用SEQ IDNO.4嵌合多肽处理细胞，于12h、24h、36h、48h、60h和72h时间点采用CCK-8（Abcam,ab228554）实验检测细胞活性，评估嵌合多肽对小鼠肝细胞活性的影响；同时，以1mM FFAs（棕榈酸和油酸1:1配置）以及50 μM和200 μM浓度嵌合多肽处理原代小鼠肝细胞48h小鼠，通过油红O染色和尼罗红染色检测，二者可以通过与脂质结合呈现红色，进而评估其对FFAs引起的脂质堆积的影响。
2. 实验结果
图1展示了CCK8检测显示无论在高浓度200 μM下还是低浓度50 μM下，与溶剂组（Vehicle）相比原代肝细胞活力没有显著降低，说明本发明的嵌合多肽在50-200μM浓度范围内无显著药物毒性。从图2油红O染色结果可见，嵌合多肽可以剂量依赖性降低FFAs诱导的肝细胞脂质堆积；图3尼罗红染色同样表明嵌合多肽作用下原代肝细胞脂质含量降低。
实施例3 嵌合多肽在缓解非酒精性脂肪肝小鼠肝脏脂质积聚中的作用
1. 实验方法
选取C57BL/6小鼠为研究对象，给予6个月高脂饮食构建非酒精性脂肪肝小鼠模型；然后将非酒精性脂肪肝小鼠随机分为“对照组”和“多肽干预组”，通过腹腔注射嵌合多肽（10mg/Kg）干预8周，每周注射两次；取小鼠肝脏组织称量湿重，计算肝脏/体重比值，H&E和油红O染色观察肝脏形态及脂质堆积程度，评估嵌合多肽对肝脏脂质积聚的影响。
2. 实验结果
由图4和图5可以看出，给予嵌合多肽干预可以显著降低非酒精性脂肪肝小鼠肝脏/体重比值，提示小鼠肝脏脂质积聚降低，而小鼠摄食行为无显著变化。由图6中 H&E染色结果可见多肽干预组小鼠肝脏组织空泡状结构现在减少，说明细胞内含有较少的脂滴。图6油红O染色显示肝脏红色脂滴数目和大小均显著降低，同样说明嵌合多肽可有效降低非酒精性脂肪肝小鼠肝脏脂质的积聚。
序列表
<110> 南京市妇幼保健院
<120> 一种乳源多肽在制备防治酒精性脂肪肝药物、保健品或食品添加物中的应用
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<213> 人工序列(Artificial Sequence)
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Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gln Val Lys
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Claims (7)
1. 一种乳源多肽在用于制备治疗非酒精性脂肪肝药物中的应用，所述乳源多肽为SEQID NO.1所示的活性氨基酸序列与一条穿膜氨基酸序列形成的嵌合多肽，所述穿膜氨基酸序列位于活性氨基酸序列的N端。
2. 一种乳源多肽在用于制备降低高脂诱导肝细胞脂质堆积的药物中的应用，所述乳源多肽为SEQ ID NO.1所示的活性氨基酸序列与一条穿膜氨基酸序列形成的嵌合多肽，所述穿膜氨基酸序列位于活性氨基酸序列的N端。
3.根据权利要求1或2所述的应用，其特征在于：所述嵌合多肽的穿膜氨基酸序列包括至少一段寡聚精氨酸序列，所述穿膜氨基酸序列中精氨酸数量的占比不低于40%。
4.根据权利要求3所述的应用，其特征在于：所述穿膜氨基酸序列含有活性氨基酸序列N端依次连接的谷氨酰胺、脯氨酸的寡聚氨基酸片段；所述寡聚氨基酸片段为至少2个相邻且相同的氨基酸组成的短肽。
5. 根据权利要求4所述的应用，其特征在于，所述嵌合多肽选自SEQ ID NO.2 - 4所示的氨基酸序列。
6. 根据权利要求5所述的应用，其特征在于，所述嵌合多肽选自SEQ ID NO.4所示的氨基酸序列。
7. 根据权利要求4-6任意一项所述的应用，其特征在于：所述乳源多肽或嵌合多肽在细胞水平发挥作用的浓度为50-200 μM；在腹腔注射动物试验中发挥生物活性的浓度为5-15mg/Kg。
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