CN112574282A - 一种抗氧化损伤皮肤保护活性多肽rl-pp10及其应用 - Google Patents
一种抗氧化损伤皮肤保护活性多肽rl-pp10及其应用 Download PDFInfo
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Abstract
本发明公开了一种抗氧化损伤皮肤保护活性多肽RL‑PP10及其应用。所述的抗氧化损伤皮肤保护活性多肽RL‑PP10的氨基酸序列为PPRWPKEECP。应用为所述的抗氧化损伤皮肤保护活性多肽RL‑PP10在制备抗氧化化妆品、保健食品和药品中的应用。本发明从云南产泽蛙的皮肤分泌物中鉴定到一种具有抵抗皮肤光损伤和氧化应激活性的天然多肽RL‑PP10,该多肽具有高活性、高稳定性等特点,表明本发明抗氧化损伤皮肤保护活性多肽RL‑PP10有较大的医疗方面应用潜力。本发明所述的抗氧化损伤皮肤保护活性多肽RL‑PP10可作为抗紫外线药物候选物来源的有益特点。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种抗氧化损伤皮肤保护活性多肽RL-PP10及其应用。
背景技术
过度暴露于紫外线(UV)辐射是多种皮肤疾病如晒伤、光老化、皮肤癌等发生发展的主要原因之一,其中UVB(290-320 nm)辐射对皮肤的损伤最为主要。抵抗UVB辐射是目前保护皮肤光损伤的主要方法之一,故近年来,具有抗氧化功能的保护剂应运而生。就已存在的皮肤抗氧化剂来讲,大多存在活性低、合成成本高及见光易分解的特点,且数量不能满足人们日益增长的抵抗皮肤氧化损伤的需要。因此,新型天然抗氧化剂的开发至关重要。多肽类抗氧化剂由于其高活性、较少的毒性代谢产物和易于大规模合成等特点而广泛引起人们的关注。
两栖动物是多肽类药物来源的天然宝库,其原因为两栖动物生长环境之复杂,其裸露的皮肤极容易受到外界伤害。故它们的皮肤可以分泌多种生物活性肽,保护其生存。目前我们已从两栖动物皮肤分泌物中提取多种天然活性肽,包括抗病毒多肽、抗氧化损伤肽、抗菌活性肽等,但这些多肽的数量对两栖动物皮肤分泌物宝库来讲远远不够,还有更多的天然多肽有待开发。
发明内容
本发明的第一目的在于提供一种抗氧化损伤皮肤保护活性多肽RL-PP10;第二目的在于提供所述的抗氧化损伤皮肤保护活性多肽RL-PP10的应用。
本发明的第一目的是这样实现的,所述的抗氧化损伤皮肤保护活性多肽RL-PP10的氨基酸序列为PPRWPKEECP。
本发明所述的抗氧化损伤皮肤保护活性多肽RL-PP10的制备鉴定步骤如下:
A、获取泽蛙活体皮肤(物料a);
B、将物料a通过总RNA提取、mRNA纯化、cDNA文库构建、PCR产物测序、生物信息学分析得到目标物——抗氧化损伤皮肤保护活性多肽RL-PP10的氨基酸序列。
本发明的第二目的是这样实现的,所述的抗氧化损伤皮肤保护活性多肽RL-PP10在制备抗氧化化妆品、保健食品和药品中的应用。
我们从生长在高海拔强紫外辐射地区的泽蛙蛙皮肤中鉴定了一种新型活性肽RL-PP10,其稳定性和抗氧化活性较高,可以有效保护皮肤免受紫外线辐射所引起的光损伤及氧化应激。因此,天然抗氧化活性肽RL-PP10可作为抗紫外线药物及氧化应激的候选物之一。
本发明从云南臭蛙的皮肤分泌物中提取到一种具有抵抗皮肤光损伤和氧化应激活性的天然多肽RL-PP10,该多肽具有高活性、高稳定性等特点,表明本发明抗氧化损伤皮肤保护活性多肽RL-PP10有较大的医疗方面应用潜力。本发明所述的抗氧化损伤皮肤保护活性多肽RL-PP10可作为抗紫外线药物候选物来源的有益特点。
附图说明
图1为本发明抗氧化多肽RL-PP10的体外DPPH、ABTS+清除率图;
图2为本发明抗氧化多肽RL-PP10对HaCaT细胞活力影响图;
图3为本发明抗氧化多肽RL-PP10在细胞水平丙二醛,乳酸脱氢酶和过氧化氢酶表达图;
图4为本发明抗氧化多肽RL-PP10对动物UVB损伤模型的保护作用;
图5为本发明抗氧化多肽RL-PP10对动物模型真皮与表皮的保护作用;
图6为本发明抗氧化多肽RL-PP10在组织中超氧化物歧化酶与还原性谷胱甘肽水平表达图;
图7为本发明抗氧化多肽RL-PP10在组织中对MAPK信号通路磷酸化抑制情况表达图。
具体实施方式
下面结合实施例和附图对本发明作进一步的说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或替换,均属于本发明的保护范围。
本发明所述的抗氧化损伤皮肤保护活性多肽RL-PP10的氨基酸序列为PPRWPKEECP。
本发明所述的抗氧化损伤皮肤保护活性多肽RL-PP10的制备鉴定步骤如下:
A、获取泽蛙活体皮肤(物料a);
B、将物料a通过总RNA提取、mRNA纯化、cDNA文库构建、PCR产物测序、生物信息学分析得到目标物——抗氧化损伤皮肤保护活性多肽RL-PP10的氨基酸序列。
本发明的第二目的是这样实现的,所述的抗氧化损伤皮肤保护活性多肽RL-PP10在制备抗氧化化妆品、保健食品和药品中的应用。
下面以具体实施案例对本发明做进一步说明:
实施例1
1. 样品收集和动物护理
泽蛙被分笼安置于容器中,并有足够面包虫可供其自由饮食。容器被放置于实验室中12/12小时明暗循环空调室中。实验前用去离子水清洗泽蛙皮肤表面后,通过快速捣毁延髓处死泽蛙、剥离其皮肤,快速浸入到预先准备好的液氮中保存,待下一步实验。
2. 纯化程序
将剥离的皮肤置于液氮中研磨至粉末,并用RNAiso(TaKaRa,大连,中国)提取总RNA。并用mRNA纯化试剂盒(Stratagene,加拿大)按标准程序纯化mRNA。用cDNA文库构建试剂盒(Clontech,大连,中国)合成cDNA文库。通过聚合酶链式反应(PCR技术)在94°C下进行2分钟,然后在92°C下进行30个10秒的循环,在50°C下进行30秒的循环,在72°C下进行40秒的循环,所得产物用DNA凝胶提取试剂盒(Bioteke,北京,中国)回收,然后连接到pMD19-T载体(TaKaRa,大连,中国)中。最后,利用42℃热刺激法将PCR产物克隆到大肠杆菌DH5a活性细胞中,构建了特异的cDNA文库。从每个特定的cDNA文库中随机选择插入量大于300 bp的30个克隆,在ABI 3730 XL DNA测序仪(美国加利福尼亚应用生物系统公司)上进行DNA测序。
3. 肽一级结构测定
DNA测序结果返回后,通过生物信息学手段,在NCBI数据里进行数据比对,确定其成熟肽序列。
4. RL-PP10对紫外线照射和过氧化氢处理下的HaCaT细胞的活性影响
将已贴壁的生长状态良好的HaCaT细胞用磷酸盐缓冲液(PBS)洗涤并分为四组(空白组、样品组、UVB辐照模型组和过氧化氢处理组),接种于96孔板(4000个细胞/孔)。在暴露于30 mJ/cm2 UVB辐射或200 μM过氧化氢下,样品组中的细胞用不同浓度的RL-PP10(1、5和10μM)或10 μM VC于37度下预处理2 h。而后将20 μL MTS加入至细胞培养基中2-4小时,于490 nm的测试波长下检测RL-PP10对HaCaT细胞活力的影响。
5. RL-PP10对过氧化氢处理或UVB照射下HaCaT细胞中丙二醛、乳酸脱氢酶和过氧化氢酶水平的影响
用PBS洗涤HaCaT细胞后用0.25%胰蛋白酶(Bi,Israel)消化3分钟,4°C 1000 g离心10分钟后用0.5 ml冷PBS悬浮细胞,使用超声波细胞粉碎机粉碎。然后再在4°C条件下,以12000 g离心15分钟并收集上清液,使用试剂盒测定丙二醛和过氧化氢酶的水平。乳酸脱氢酶水平为使用试剂盒用HaCaT细胞培养基上清液测定。
6. RL-PP10对皮肤光损伤的保护活性
选取6-8周龄雌性昆明小鼠(18-22g)进行试验。实验前驯养一周。随机分为4组(每组6只)即模型组(UVB)、对照组、样品组(UVB+RL-PP10)和对照组(UVB+VC)。
对小鼠背部皮肤局部脱毛,暴露于UVB辐射(UVB灯,TL20 W/12,飞利浦,荷兰)下,最初7天每天150 mJ/cm2进行辐射,而后接下来的2周内每隔一天辐射300 mJ/cm2。抗氧化保护肽RL-PP10(10 μM)或VC(10 μM)(均为液体)在小鼠背部皮肤的辐射区域局部施用一次/天。最后一次紫外辐射后,麻醉处死小鼠并迅速取下背部无毛皮肤以进行以下步骤(最后一天拍摄紫外线损伤小鼠皮肤的图像):
将皮肤组织分为两部分,一部分用4%多聚甲醛固定48小时,再由70%到100%的无水乙醇梯度脱水。最后二甲苯透明在石蜡中包埋。将组织样本制成6 μm厚的切片,并用H&E和Masson染色液染色。正置显微镜观察结果。
另一部分皮肤组织进行冰浴匀浆(0.1g皮肤组织在1ml冷PBS中匀浆3min),4°C条件下10000 g离心15min,测定匀浆蛋白浓度并应用试剂盒及抗体检测超氧化物歧化酶、谷胱甘肽水平和MAPK信号通路磷酸化情况。
结果如图1~图7,具体说明如下:如图1所示:RL-PP10于体外有显著的抗氧化活性(DPPH与ABTS+清除率高),当浓度达到10 μM时,ABTS+清除率达90%以上(图1中的A);DPPH清除率达60%以上(图1中的B)。图2中的A结果表明:RL-PP10(1、5、10 μM)对HaCaT的细胞活力无明显影响。但当暴露于UVB照射或用过氧化氢处理后,HaCaT的活性显著降低 (图2中的B),而RL-PP10的预处理可以显著地逆转这种降低(图2中的C),表明RL-PP10对细胞活力有显著的抗UVB辐射与抗氧化应激功能。如图3所示,UVB辐射和过氧化氢刺激可致HaCaT细胞内丙二醛水平(图3中的A、B)增加,过氧化氢酶活性(图3中的E、F)显著降低并释放大量乳酸脱氢酶(图3中C、D)。而RL-PP10(10 μM)的预处理能降低丙二醛水平和乳酸脱氢酶活性(图3中的A,B,C,D),增加过氧化氢酶的活性(图3中的E,F)。在历时21天的紫外线辐射后,小鼠背部皮肤发生一下病理性改变:损伤发生、表皮增厚、出现红斑。而抗氧化剂RL-PP10能有效抑制上述病理性改变(这点已于图4展示)。并且与正常皮肤相比,紫外线辐射后的小鼠表皮与真皮厚度(图5)显著增加, RL-PP10可以减少这种病理性真皮与表皮增厚。Masson三色染色显示,RL-PP10能抑制紫外线照射引起的胶原纤维的含量降低(图5中的B)。在UVB照射的小鼠皮肤中,超氧化物歧化酶活性(图6中的A)和谷胱甘肽水平(图6中的B)均显著降低,MAPK信号通路磷酸化水平增加,使用抗氧化保护肽RL-PP10可提高二者的表达水平并抑制MAPK信号通路磷酸化水平(图7),以此进行抗皮肤光损伤和抗氧化应激的作用。
SEQUENCE LISTING
<110> 昆明医科大学
<120> 一种抗氧化损伤皮肤保护活性多肽RL-PP10及其应用
<130> 2020
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> PRT
<213> 抗氧化损伤皮肤保护活性多肽RL-PP10的氨基酸序列
<400> 1
Pro Pro Arg Trp Pro Lys Glu Glu Cys Pro
1 5 10
Claims (2)
1.一种抗氧化损伤皮肤保护活性多肽RL-PP10,其特征在于所述的抗氧化损伤皮肤保护活性多肽RL-PP10的氨基酸序列为PPRWPKEECP。
2.一种权利要求1所述的抗氧化损伤皮肤保护活性多肽RL-PP10的的应用,其特征在于所述的抗氧化损伤皮肤保护活性多肽RL-PP10在制备抗氧化化妆品、保健食品和药品中的应用。
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