CN111748015B - 一种活性多肽os-ll11及其应用 - Google Patents
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Abstract
本发明公开了一种活性多肽OS‑LL11及其应用。所述的多肽OS‑LL11的氨基酸序列为LLPPWLCPRNK。本发明系统地研究了局部使用该肽对UVB诱导的昆明小鼠背部皮肤以及PAM 212细胞损伤的保护作用，通过检测相关的抗氧化酶以及组织病理学的分析评估来获得实验结果，来自该实验的结果可以为临床应用OS‑LL11作为一种治疗或美容产品提供了强有力的依据；本发明所述的活性多肽OS‑LL11可以减轻UVB辐射诱导的氧化应激并可以保护皮肤免受光损伤。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种活性多肽OS-LL11及其应用。
背景技术
暴露在阳光下会引起多种皮肤损伤，包括晒伤、光免疫抑制、光老化和光致癌，紫外线约占阳光的13%，分为长波紫外线（UVA:310-400nm)，中波紫外线(UVB:290-320nm)以及短波紫外线(UVC:200-290nm).UVC完全被大气吸收，只有部分UVB和UVA辐射到达地球表面，UVB主要作用于皮肤的表皮基底细胞层。它引起直接和间接的生物效应，包括嘧啶光产物的形成、DNA合成的刺激、皮肤自由基的产生、细胞周期的生长、光老化和光致癌。
UVB辐射引起的活性氧（ROS）可损伤生物大分子（如脂质、蛋白质和DNA）并诱发严重氧化应激，导致皮肤色素沉着增加、细胞凋亡和皮肤癌。如果DNA损伤不能及时修复，UVB损伤的细胞将被凋亡细胞清除，p53抑癌蛋白是凋亡细胞的关键调控因子。细胞凋亡级联反应主要通过两个通路触发：外在死亡受体介导的信号通路以及内在线粒体介导的信号通路。P53的非转录依赖促细胞凋亡的功能，主要通过线粒体途径发挥作用，其过程是：激活的p53快速转位到线粒体，与位于线粒体膜上的Bcl家族蛋白相互作用，从而改变线粒体外膜的通透性，使线粒体内的一些促凋亡蛋白（如细胞色素C、AIF、SMAC/Diablo、核酸内切酶G等）释放到胞浆，激活胞浆中的效应分子—胱天蛋白酶（caspases）,从而启动细胞凋亡级联反应、诱导细胞凋亡[9]。因此，开发具有高活性成分的防晒产品、抵抗紫外线辐射、保护皮肤免受光损伤、预防和延缓皮肤衰老，已成为目前医学研究的热点。而开发以天然动植物为有效成分的抗氧化剂是近年来抗皮肤光损伤研究的新方向。
发明内容
本发明的第一目的在于提供一种多肽OS-LL11；第二目的在于提供所述的活性多肽OS-LL11的应用。
本发明的第一目的是这样实现的，所述的活性多肽OS-LL11的氨基酸序列为LLPPWLCPRNK。
本发明所述的抗皮肤光损伤保护活性多肽RL-PL9的鉴定步骤如下：
A、采集活体花臭蛙皮肤（物料a）；
B、提取总RNA，并用按标准程序纯化mRNA，用cDNA文库构建试剂盒合成cDNA；
C、利用聚合酶链反应技术得到产物b，并将产物b用DNA凝胶提取试剂盒回收，克隆到大肠杆菌活性细胞中，构建特异性cDNA文库。
D、用多肽合成仪通过固相合成法合成目标物活性多肽OS-LL11。
本发明的第二目的是这样实现的，所述的活性多肽OS-LL11在制备美容产品中的应用。
本发明提供的抗痛风活性多肽OS-LL11，其制备方法可以通过生物化学分离纯化方法，由花臭蛙的皮肤分泌物中得到。上述方法制备的活性多肽OS-LL11，可通过分离纯化及活性鉴定。
本发明所述的活性多肽OS-LL11的制备也可通过本领域常规的方法，采用化学合成或基因表达的方式得到，氨基酸序列与本发明SEQ ID NO: 1相同。
本发明系统地研究了局部使用该肽对UVB诱导的昆明小鼠背部皮肤以及PAM 212细胞损伤的保护作用，通过检测相关的抗氧化酶以及组织病理学的分析评估来获得实验结果，来自该实验的结果可以为临床应用OS-LL11作为一种治疗或美容产品提供了强有力的依据；本发明所述的活性多肽OS-LL11可以减轻UVB辐射诱导的氧化应激并可以保护皮肤免受光损伤。
附图说明
图1为本发明OS-LL11呈剂量依赖性的ABTS+自由基清除活性示意图；
ddH2O表示阴性对照，VC表示阳性对照。*P<0.05，**P<0.01，**P<0.001和**P<0.0001表示与阴性对照组（t检验）有显著性差异；数据是三个独立实验的平均值，一式三份；
图2为本发明OS-LL11呈剂量依赖性的DPPH自由基清除活性示意图；
ddH2O表示阴性对照，VC表示阳性对照。*P<0.05，**P<0.01，**P<0.001和**P<0.0001表示与阴性对照组（t检验）有显著性差异；数据是三个独立实验的平均值，一式三份；
图3 为本发明OS-LL11对PAM 212细胞活力的影响示意图；
用不同浓度的OS-LL11（0.1-1μM）在37℃下预处理细胞24小时，然后加入MTS（Promega，Madison，WI，USA）试剂检测OS-LL11对细胞活力的影响。所有数据均为三个独立实验的平均值（±SD），一式三份；
图4为本发明OS-LL11对UVB或H2O2诱导下PAM 212细胞CAT、LDH和MDA活性的影响；
其中：（A.B）CAT活性；（C.D）LDH和MDA活性；PAM 212细胞在UVB照射（A）和H2O2刺激（B，C，D）前用OS-LL11（1μM）或VC（10μM，阳性对照）预处理2小时；所有数据均为三个独立实验的平均值（±SD），一式三份；*p<0.05；**p<0.01；**p<0.001；**p<0.0001；一个对照组被认为是100%，数据相应地被标准化；
图5为OS-LL11对UVB照射小鼠背部皮肤的保护作用示意图；
小鼠分为5组（每组6只）。用UVB（150mJ/cm2）照射小鼠7天，后两周隔日照射UVB强度为300mJ/cm2；A. 每天拍摄小鼠背部皮肤损伤的照片，选取第1,3,5,7,9,11,13和15天的照片（A）；第15天小鼠背部皮肤放大的图片（B），显示了UVB引起的红斑、结痂和水肿等宏观形态学的变化；
图6为OS-LL11对UVB照射小鼠皮肤表皮和真皮厚度的影响。A. H&E染色；B.Masson三色染色；C.H&E染色表皮厚度矩形图；D.Masson三色染色真皮厚度矩形图；在最后一次UVB照射后，收集背部皮肤，切片6μm厚进行H&E和Masson三色染色；**p<0.01，***p<0.001，***p<0.0001，差异有显著性；误差条代表每个实验三个不同部分的平均值±标准差。一个对照组被认为是100%，数据相应地被标准化；
图7为OS-LL11对UVB照射小鼠背部皮肤SOD活性和GSH水平的影响；
其中，（A） UVB照射后SOD活性；(B) UVB照射后GSH水平；取皮肤组织（0.1g）于1ml预冷的生理盐水中匀浆，于4℃，10000 转离心15分钟，取上清液测定SOD活性和GSH水平。**p<0.01，***p<0.001，***p<0.0001，差异有显著性；误差条代表每个实验三个不同部分的平均值±标准差；一个对照组被认为是100%，数据相应地被标准化。
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。
本发明所述的活性多肽OS-LL11的氨基酸序列为LLPPWLCPRNK。
本发明所述的活性多肽OS-LL11的应用为所述的活性多肽OS-LL11在制备美容产品中的应用。
本发明所述的应用为所述的活性多肽OS-LL11在制备保护皮肤免受光损伤产品中的应用。
实施例1
1、自由基清除能力检测
本实施例进行了2,2'-联氮-二（3-乙基苯并噻唑啉-6-磺酸） (ABTS) 清除能力测试，ABTS自由基储备液(Sigma-Aldrich, USA)的制备是通过孵育2.8 mM过硫酸钾(Sigma-Aldrich, USA)和 7 mM溶于蒸馏水的ABTS避光保存至少6小时而得到的，之后立即使用。将储备液用蒸馏水稀释50倍，之后与溶于蒸馏水的样品溶液混合，同等体积的蒸馏水作为阴性对照组，溶于蒸馏水的VC作为阳性对照组。之后避光反应10分钟。415nm处吸光度的降低表明样品具有抗氧化活性。自由基清除率(%)的计算公式是：(A blank–A sample)×100/A blank。
2,2-二苯基-1-苦基肼(DPPH)清除测试实验，用于检测的混合液包含了190 μL5× 10-5 M 溶于甲醇DPPH溶液以及10 μL的样品溶液。混合液孵育在96孔板中置于室温下避光反应30分钟。在酶标仪517nm处读取吸光度值。DPPH清除率(%)的计算公式是(A blank–Asample)×100/A blank.。
2、细胞培养
PAM 212细胞是鼠的角质细胞。细胞培养在达尔伯克（氏）改良伊格尔（氏）培养基中(DMEM/F12, BI, Israel)。培养基中补充1%的抗生素（100单位/毫升青霉素，100单位/毫升链霉素）和10%胎牛血清(FBS, BI, Israel)置于37 °C含5% CO2孵育箱中。
3、MTS法测试细胞活力
当细胞达到80~90%的融合率时，用PBS清洗3次以去掉死细胞和杂质。然后种于96孔板，每孔3.0×103个细胞。用不同浓度OS-LL11 (0.1,0.25,0.5,1 μM)预处理细胞后置于37 °C孵育箱孵育24小时，之后加入 20 μL的MTS (Promega, Madison, WI, USA) 试剂再孵育2~4小时。在酶标仪490nm处读取吸光度值以得到该肽对细胞活力作用的数据结果。
4、检测LDH，MDA和CAT的水平
用PBS清洗PAM212细胞后，将细胞接种到6孔板并分为3组：对照组，样品组，空白组。用OS-LL11和VC预处理细胞2小时，之后用过氧化氢刺激或UVB辐射，置于孵育箱孵育24小时。孵育结束后培养皿内的培养基用来检测MDA(MDA分析试剂盒, 南京建成生物工程研究所, 南京)的水平。之后将培养皿置于冰上用裂解液（RIPA和PMSF，美伦生物科技，大连，中国；磷酸酶抑制剂，罗氏，上海，中国）裂解30分钟，用刮板刮取培养皿中裂解的细胞装于离心管中离心，12000转，5分钟。离心后取上清液用于检测LDH和CAT(LDH和CAT分析试剂盒，南京建成生物工程研究所, 南京)。实验操作按照制造商提供的标准方法。
5、动物实验
昆明小鼠（18-22g，雌性）取自昆明医科大学实验动物系。所有动物实验、护理和处理程序均按照昆明医科大学伦理委员会（kmmu 20180012）的要求进行。将小鼠置于特定的环境中（22 ± 2℃，明暗循环12小时，标准实验室饮食和水）。
6、UVB辐射
小鼠随机的分成5组（每组6只）：空白对照组，模型对照组（UVB辐射），溶剂组（UVB辐射+ddH2O）,样品组（UVB辐射+ddH2O）和阳性对照组（UVB辐射+VC）。实验开始前，小鼠静脉注射1%戊巴比妥钠（0.1ml/20g体重）麻醉，之后剔除背部皮肤毛发。前7天暴露小鼠背部皮肤在150mJ/cm2的紫外线（紫外线灯，TL20W/12，飞利浦，荷兰），之后两周隔天暴露于300mJ/cm2紫外灯下，用紫外辐射计（TM-213，temars，中国台湾）监测UVB的辐射强度。老鼠被放置在特制的辐射箱中，辐射高度为20厘米。每次照射结束后小鼠背部辐射皮肤涂抹OS-LL11（1μM）和VC（10μM）。在照射的最后一天，处死小鼠，取其背部皮肤进行后续实验。
7、苏木精伊红（H&E）和Masson三色染色
皮肤组织在石蜡中固定，在5-6μm处切片并放置在玻片上。根据标准方案，用H&E和Masson的三色染色试剂盒（Solarbio，中国北京）对切片进行染色。利用Image J软件对表皮和真皮厚度进行了评估。
8、皮肤组织超氧化物歧化酶和谷胱甘肽水平的测定
取小鼠皮肤组织，用预冷的生理盐水冲洗去除残余物，以1:9的体积比在冷生理盐水中匀浆，然后在10000 转4℃下离心15分钟。收集上清液测定SOD和GSH（SOD,GSH检测试剂盒，南京建城生物工程研究所，中国）的水平。所有试验程序均符合制造商提供的标准程序。
9、数据分析
所有结果均以平均标准差表示。统计分析使用Graph Pad Prism 6.0（Graph Pad软件，加利福尼亚州拉霍拉市，美国）。采用单因素方差分析进行两两比较。p值<0.05被认为具有统计学意义。
10、结果
10.1 自由基清除能力
如图1所示，OS-LL11在0.1,0.25,0.5和1μM浓度下具有ABTS+自由基清除能力并具有剂量依赖的性。在最大浓度1μM时，OS-LL11可清除约26.6%的ABTS+，而VC清除率为23.7%。同时，我们还测试了其清除DPPH的活性，在图2中，浓度为1μM时，OS-LL11的清除率为8.88%，VC为6.87%。结果表明，OS-LL11具有抗氧化活性，并且其抗氧化能力强于VC。
10.2 OS-LL11对PAM 212细胞活力的影响
为了研究OS-LL11对PAM 212细胞活性的影响，本实施例用OS-LL11（0.1-1μM）处理PAM212细胞，并用MTT法进行分析，OS-LL1与未治疗组相比无明显差异（图3），表明OS-LL11在0.1-1 μM时对PAM 212细胞无毒性。
10.3 OS-LL11对经UVB辐射或H2O2处理的PAM 212细胞的抗氧化作用
通过检测抗氧化酶的水平，研究OS-LL11对PAM 212细胞的抗氧化作用。CAT、LDH和MDA指标可反映细胞氧化损伤程度，UVB照射或H2O2处理对PAM 212细胞有损伤作用，CAT水平显著降低，LDH和MDA水平升高，UVB照射和H2O2处理后CAT活性分别下降87.58±16.84%和65.57±10.37%，而AOP-P3（1μM）预处理使CAT水平分别升高181.8±13.57%和128.8±15.64%（图4A.B）。同样，H2O2刺激PAM 212细胞后，LDH和MDA分别升高58.56±5.778%和147.0±22.54%，OS-LL11预处理后LDH和MDA分别降低14.70±4.734%和130.1±23.70%。
10.4 OS-LL11对UVB诱导下小鼠背部皮肤光损伤的作用长期暴露在UVB辐射下可能导致慢性光损伤的症状包括红斑、水肿、脱皮。用UVB诱导的小鼠皮肤光损伤模型评估OS-LL11的光保护作用。选取辐射第15天的小鼠背部皮肤照片，可见模型组和溶剂组小鼠皮肤出现红斑、水肿、结痂和溃疡，说明光损伤小鼠模型构建成功。然而，用OS-LL11或VC治疗可明显减轻这些症状。皮肤变得更光滑，更少的疤痕和溃疡。UVB照射后表皮厚度较正常皮肤增加了107.5 ± 4.194 μm，而OS-LL11使表皮厚度减少87.81 ± 3.422 μm（图6中的A.C）。UVB照射后，皮肤厚度增加302.8 ± 6.354 μm，而OS-LL11使皮肤厚度降低了306.1 ±6.803 μm（图6中的B.D）。Masson三色染色显示UVB照射降低了胶原纤维的含量，而OS-LL11和VC处理则逆转了这一现象。说明OS-LL11能有效降低皮肤光损伤程度，其效果相当于VC。
10.5 OS-LL11可以降低UVB辐射引起的小鼠背部皮肤抗氧化酶的水平
UVB辐射可引起SOD、GSH等抗氧化酶的降低，进一步损伤细胞和组织。如图7所示，在UVB照射小鼠背部皮肤后，SOD和GSH水平显著降低了44.74±1.621%和88.40±3.473%，而OS-LL11作用后内源性的抗氧化酶的水平分别升高38.56 ± 1.261%和70.41 ± 1.877%（图7中的A, B），说明OS-LL11能增强内源性抗氧化酶活性以及对光损伤皮肤的保护作用，在本实验中其作用强于VC。
SEQUENCE LISTING
<110> 昆明医科大学
<120> 一种活性多肽OS-LL11及其应用
<130> 2020
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 11
<212> PRT
<213> 活性多肽（OS-LL11）的氨基酸序列
<400> 1
Leu Leu Pro Pro Trp Leu Cys Pro Arg Asn Lys
1 5 10
Claims (3)
1.一种活性多肽OS-LL11，其特征在于所述的活性多肽OS-LL11的氨基酸序列为LLPPWLCPRNK。
2.一种权利要求1所述的活性多肽OS-LL11的应用，其特征在于所述的活性多肽OS-LL11在制备美容产品中的应用。
3.根据权利要求2所述的活性多肽OS-LL11的应用，其特征在于所述的活性多肽OS-LL11在制备保护皮肤免受光损伤产品中的应用。
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