CN116726031A - 芦丁靶向f2/pld轴在制备银屑病药物中的应用 - Google Patents
芦丁靶向f2/pld轴在制备银屑病药物中的应用 Download PDFInfo
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Abstract
本发明涉及芦丁及F2/PLD轴在制备银屑病药物中的应用。本发明利用生物信息分析及体内外实验验证，探究芦丁防治银屑病的疗效和作用机制。研究发现外用芦丁干预咪喹莫特(Imiquimod，IMQ)诱导的银屑病样小鼠模型能显著减轻炎症反应，网络药理学分析显示F2/PLD是芦丁治疗银屑病的潜在核心靶标。在银屑病中高表达的F2可激活PLD通路促进炎症发展。IMQ诱导的银屑病样小鼠模型皮损处PLD1、F2表达升高，经芦丁干预后下调，表明芦丁可通过下调F2/PLD信号通路改善炎症浸润，减弱细胞过度增殖，改善银屑病样皮损。芦丁有望成为安全有效治疗银屑病和防治其复发的临床药物，而靶向F2/PLD轴可以作为一种新的防治方法，用于银屑病防治新策略。
Description
技术领域
本发明涉及医学科研技术领域，具体地说，是芦丁靶向F2/PLD轴在制备银屑病药物中的应用。
背景技术
银屑病是一种常见的慢性、复发性、免疫介导的皮肤疾病，影响全球约2-3％的人口[1]。银屑病有多种临床皮损表现，但最常见的表现为慢性、对称性、红斑性、鳞屑性丘疹和斑块，斑块是角质形成细胞过度增殖和分化紊乱引起的表皮增生作用而形成的[2]。银屑病在身心健康和经济方面都给患者和社会带来了十分严重的疾病负担[3][4]。
银屑病的免疫学特征是角质形成细胞的异常增殖和分化，以及淋巴细胞和中性粒细胞浸润，其中T细胞、树突状细胞和部分炎性细胞因子(包括TNF-α、IFN-γ、TGF-β和IL-17等)起主导作用[5]。因此，银屑病的机制研究主要集中在调控角质形成细胞增殖分化和减轻炎症反应等方面。现代医学已将TNF-α抑制剂、IL-17抑制剂、IL-23抑制剂等多种生物制剂用于临床并取得满意疗效，但禁忌症、潜在不良反应、复发率高等问题限制了它的广泛应用[6]。中医治疗银屑病具有适用性广、安全性高、复发率低等优势，是目前防治银屑病的重要方向。
中国专利文献CN202210349559.X，公开了“芦丁治疗银屑病的用途”，从抗氧化及免疫调节两方面探讨其治疗银屑病潜在的作用机制，为以芦丁为原料的纯中药作为银屑病临床治疗药物的研制提供理论依据。
决银方被认为是治疗银屑病的有效方剂，随机对照试验已证实决银方治疗寻常型银屑病的总有效率为72.5％[7]。为了进一步探究该方的核心成分及治疗银屑病的作用机制，申请人经LC-MS/MS和分子对接技术分析发现芦丁(rutin)在决银方中含量较高(50.14ug/ml)，是主要有效成分之一，并且与银屑病相关炎症靶标IL-17、TNF-α均具有较好的结合作用。前期研究显示外用芦丁干预IMQ诱导的银屑病样小鼠炎症模型，显著抑制了小鼠PASI积分、表皮增生、炎症细胞浸润等病理表型，但其分子机制有待阐释。
为进一步探索芦丁治疗银屑病的作用机制，进行网络药理学分析发现磷脂酶D(Phospholipase D，PLD)信号通路在调节皮肤炎症中发挥重要作用。PLD是一种酶，它切割磷脂酰胆碱产生磷脂酸(Phosphatidic acid，PA)，PA作为第二信使调节多种细胞过程[8]。已证实激活PLD能够诱导IL-6、IL-8、TNF-α等炎症因子的表达和分泌，它们刺激免疫细胞并促进炎症[9]，这与银屑病及其合并症关系密切。综上，表明PLD信号通路的激活可能是促进银屑病炎症发生、发展的重要因素。
KEGG富集分析结果表明，F2是PLD上游调控基因之一。F2指凝血因子II(coagulation factor II)，具有促炎、促进肿瘤增殖转移的作用[10]。在银屑病患者外周血中高表达[11]。新近研究表明，F2激活PLD后磷酸累积进而激活下游炎症通路促进炎症发展[9]，这可能是由细胞内Ca2+升高介导的[12]。
银屑病尚无特效疗法，适当的对症治疗可控制症状，但存在易反复发作的特点，且长期治疗容易导致不良反应，因此亟需开发安全、有效、复发率地的银屑病防治药物。目前未见芦丁及其通过F2/PLD轴防治银屑病的报道。
发明内容
本发明的目的是针对现有技术中的不足，本发明的第一方面，提供芦丁或其上调剂在制备防治银屑病的药物中的应用。
优选地，所述的芦丁或其上调剂是通过下调F2/PLD信号通路来改善银屑病样皮损。
本发明的第二方面，提供一种F2或其抑制剂在制备防治银屑病的药物中的应用。
本发明的第三方面，提供一种PLD或其抑制剂在制备防治银屑病的药物中的应用。
本发明的第四方面，提供一种防治银屑病的药物组合物，所述的药物组合物以芦丁为活性成分和药学上可接受的载体组成。
本发明的第五方面，提供一种防治银屑病的药物组合物，所述的药物组合物以F2或其抑制剂为活性成分和药学上可接受的载体组成。
本发明的第六方面，提供一种防治银屑病的药物组合物，所述的药物组合物以PLD或其抑制剂为活性成分和药学上可接受的载体组成。
优选地，所述药物组合物的剂型为外用剂型或内用剂型。
优选地，所述药物组合物的剂型为贴剂、糊剂、软膏剂、微针剂、涂膜剂、巴布剂、喷雾剂、胶囊剂、颗粒剂、片剂、丸剂、口服液或注射液。
本发明优点在于：
1.本发明利用动物实验结合生物信息分析，验证芦丁防治银屑病的作用机制。发现芦丁作用于IMQ诱导的小鼠模型后显著抑制了小鼠PASI积分、表皮增生、炎症细胞浸润等病理表型。
2.进行网络药理学分析发现PLD信号通路在调节皮肤炎症中发挥重要作用。KEGG富集分析结果表明，F2是PLD上游调控基因之一，在银屑病患者外周血中高表达。IMQ诱导的银屑病样小鼠皮损部位PLD1、F2表达升高，经芦丁外用干预后显著下调。表明F2/PLD信号通路是调节皮肤炎症的重要通路，在银屑病中高表达的F2通过激活PLD促进炎症发展。芦丁通过下调F2/PLD信号通路，改善炎症浸润，减弱细胞过度增殖，改善银屑病样皮损。这代表了银屑病病理中的一个新的分子特征，也是防治银屑病的一个有前途的靶点。
需要说明的是：
在本发明中，所用的F2、PLD可以是天然存在的，比如其可以被分离或纯化自哺乳动物。此外，所述的F2、PLD也可以是人工制备的，比如可以根据常规的基因工程重组技术来生产重组F2、PLD。本发明优选采用重组F2、PLD。
任何适合的F2、PLD均可用于本发明。所述的F2、PLD包括全长的F2、PLD或其生物活性片段。经过一个或多个氨基酸残基的取代、缺失、或添加而成的F2、PLD的氨基酸序列也包括在本发明中。F2、PLD或其生物活性片段包括一部分保守氨基酸的替代序列，所述经氨基酸替换的序列并不影响其活性或保留了其部分的活性。适当替换氨基酸是本领域公知的技术，所述技术可以很容易地被实施并且确保不改变所得分子的生物学活性。这些技术使本领域技术人员认识到，通常在一种多肽的非必要区域改变单个氨基酸基本上不会改变生物活性见Watson，Molecular Biology of The Gene，第四版，1987，The Benjamin/CummingsPub.Co.P224。
任何一种F2、PLD的生物活性片段都可以应用到本发明中。在这里，F2、PLD的生物活性片段的含义是指作为一种多肽，仍然能保持全长的F2、PLD的全部或部分功能。所述优选的生物活性片段至少保持50％的全长F2、PLD活性。在更优选的条件下，所述活性片段能够保持全长F2、PLD的60％、70％、80％、90％、95％、99％、或100％活性。
本发明也可采用经修饰或改良的F2、PLD，比如，可采用为了促进其半衰期、有效性、代谢、和/或蛋白的效力而加以修饰或改良的F2、PLD。所述经过修饰或改良的F2、PLD可以是一种F2、PLD的共轭物，或其可包含被取代的或人工的氨基酸。所述经过修饰或改良的F2、PLD可以是与天然存在的F2、PLD具有较小的共同点，但也能缓解银屑病或缓解银屑病样炎症，且不会带来其它不良影响或毒性。也就是说，任何不影响F2、PLD的生物活性的变化形式都可用于本发明中。
根据F2、PLD的氨基酸序列可以方便地得出其相应的核苷酸编码序列。
附图说明
附图1为芦丁网络药理学分析图。(A.芦丁与银屑病交集靶点的韦恩图，紫色表示疾病，蓝色表示成分芦丁。B.PPI网络互作图，圈中为芦丁与银屑病共有靶点F2。C.84个靶基因的KEGG富集分析，箭头所指为PLD信号通路)；
附图2为芦丁治疗IMQ诱导的小鼠皮肤皮损改善图。(A.第12天时各组典型耳背皮损外观。B.不同芦丁浓度下各组小鼠背部皮损PASI评分。C.不同芦丁浓度下各组小鼠耳朵厚度。D.各组耳部及背部皮损典型H&E照片(芦丁浓度：1.5％)，200X，比例尺＝150μM。E.各组耳部表皮厚度(芦丁浓度：1.5％)。F.各组背部表皮厚度(芦丁浓度：1.5％)。所有数据表示为平均值±标准差。
###p<0.001，与正常对照组(Control)比较，*p<0.05，**p<0.01，***p<0.001，与模型组(IMQ)比较)；
附图3为测定芦丁干预后PLD、F2表达图。(A.采用PLD活性比色测定试剂盒检测芦丁对PLD1、PLD2活性的影响。B.采用Westernblot法检测芦丁对F2表达的影响。***p<0.001与正常对照组(Control)比较，###p<0.001，与模型组(IMQ)比较)；
附图4为测定芦丁作用于HaCaT细胞的药物浓度图。(A.CCK-8测定在48小时检测芦丁对HaCaT细胞活力的影响，IC50＝10uM。B.采用qRT-PCR检测芦丁对不同浓度下炎性细胞因子IL-6和TNF-αmRNA表达的影响。###p<0.001，##p<0.01，与对照组相比，**p<0.01，*p<0.05，与M5组相比)。
具体实施方式
下面结合具体实施方式，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1动物实验
1.方法和材料
1.1网络药理学筛选芦丁治疗银屑病干预靶点
通过TCMSP、TCMIP、HIT2.0、Swiss Target Prediction平台筛选芦丁靶点，通过Disgenet、GeneCards、OMIM数据库筛选获得银屑病疾病靶点。利用Draw Venn Diagram在线工具将药物与疾病靶点取交集。将共有靶点通过Cytoscape3.6.0构建成分-疾病-靶点PPI网络图。将共有靶点映射到David数据库中进行KEGG通路富集分析。
1.2芦丁乳膏的制备
将油相(十六十八醇5％、鲸蜡硬脂醇2％、单双硬脂酸甘油酯3％、轻质液体石蜡5％、聚氧乙烯(21)硬脂醇醚2％、聚氧乙烯(2)硬脂醇醚2％、羟苯乙酯0.1％)水浴加热至80℃熔化，之后冷却至65℃，分别加入1％、1.5％、2％芦丁，搅拌混合均匀。水相(甘油8％、丙二醇2％、HEPES缓冲液70.9％)加热至80℃。在搅拌下将水相缓慢加入油相中，边加边搅拌，直至冷却至室温，即分别得到1％、1.5％、2％乳膏成品，应用于后续银屑病样小鼠模型疗效实验。
2.实验方法
2.1小鼠建模及干预
采用IMQ诱导的银屑病样小鼠模型进行体内研究。将雄性、6-8周龄、体重20g±2g的BABLc小鼠随机分为6组，每组4只小鼠，分组及治疗方案如下：(1)正常对照组(Control)组：用凡士林乳膏62.5mg每日涂抹小鼠背部与双耳廓内外侧；(2)模型组(IMQ)组：将5％IMQ乳膏62.5mg分别涂抹小鼠背部与双耳廓内外侧。6h后用凡士林乳膏62.5mg涂抹小鼠背部与双耳廓内外侧；(3)1％芦丁用药组(IMQ+1％Rutin)组：将5％IMQ乳膏62.5mg涂抹小鼠背部与双耳廓内外侧。6h后用1％芦丁乳膏60mg均匀涂抹小鼠背部与双耳廓内外侧；(4)1.5％芦丁用药组(IMQ+1.5％Rutin)组：将5％IMQ乳膏62.5mg涂抹小鼠背部与双耳廓内外侧。6h后用1.5％芦丁乳膏60mg均匀涂抹小鼠背部与双耳廓内外侧；(4)2％芦丁用药组(IMQ+2％Rutin)组：将5％IMQ乳膏62.5mg涂抹小鼠背部与双耳廓内外侧。6h后用2％芦丁乳膏60mg均匀涂抹小鼠背部与双耳廓内外侧。(5)阳性对照组(IMQ+Cal)：将5％IMQ乳膏62.5mg涂抹小鼠背部与双耳廓内外侧。6h后用0.005％卡泊三醇软膏60mg均匀涂抹小鼠背部与双耳廓内外侧。所有动物实验均经上海中医药大学伦理委员会批准(No.YYLAC-2021-107-11)。
2.2小鼠皮损程度评价
在第0、2、4、6、8、10、12天对小鼠背部和耳朵的外观拍照，并根据银屑病皮损面积及严重程度指数(psoriasis area and severity index，PASI)评分标准对小鼠背部与耳部皮肤炎症的严重程度进行评分，包括红斑、厚度和鳞屑。分值表示严重程度：0(无)，1(轻度)，2(中度)，3(重度)及4(极重度)。同时，在上述天数用游标卡尺对各组小鼠耳部中部进行耳厚测量，每只测量3次，取平均数记录。
2.3组织病理学
第12天记录上述数据后，对小鼠实施安乐死，取各组小鼠双耳及背部1×1cm皮肤组织，用4％福尔马林溶液固定48h后石蜡包埋，切片后用苏木精-伊红(hematoxylin-eosin，H&E)染色。用Image J软件在每只小鼠在5个不同的位置上测量表皮厚度取平均数作为表皮厚度。
2.4PLD活性检测试验
根据制造商的说明，使用PLD活性比色测定试剂盒(BioVision)测量PLD活性。简而言之，0.1g耳部组织加入缓冲液，4°匀浆后，10000g离心5min，取上清4°、100000g离心30min后弃上清，取沉淀加入反应混合物。使用分光光度计在500nm处测量吸光度，根据公式以计算样品的PLD活性。
2.5Westernblot分析
小鼠耳部皮肤组织用皮肤组织用Pro-read蛋白提取缓冲液(intRON，Sungnam)裂解，并使用Bradford试剂(Bio-Rad，Hercules，CA，USA)测定蛋白总量浓度。在电泳、转膜后与相关的抗体一起孵育，检测各组耳背组中的F2、PLD1、PLD2蛋白表达。使用的特异性抗体包括：抗F2抗体(1:1000，GTX101270，GeneTex)，抗PLD1抗体(1:1000，#3832，CST)和抗PLD2抗体(1:1000，PA5-104015，Thermo Fisher)。
3.统计分析
实验数据用GraphPad Prism 9进行分析，所有数据均表示为平均值(Mean)±标准差(SD)。用student’s t检验或方差分析比较组间差异。p＜0.05被认为有统计学差异。
4.结果
4.1通过网络药理学筛选芦丁治疗银屑病干预靶点
为了进一步筛选芦丁治疗银屑病干预靶点，通过TCMSP、TCMIP、HIT2.0、SwissTarget Prediction平台筛选获得芦丁靶点148个，通过Disgenet、GeneCards、OMIM数据库筛选获得银屑病疾病靶点4594个，利用Draw Venn Diagram在线工具将药物与疾病靶点取交集，得到包括F2在内的84个共有靶点，绘制韦恩图(图1A)。将共有靶点通过Cytoscape3.6.0构建成分-疾病-靶点网络(图1B)。将共有靶点映射到David数据库中进行KEGG通路富集分析，共得到包括PLD在内的信号通路164条(图1C)。
4.2芦丁显著改善IMQ诱导的小鼠银屑病样皮损
研究发现芦丁可以显著改善IMQ诱导的小鼠银屑病样皮损。IMQ诱导的病变表现出典型的红斑、鳞屑和增厚，在使用芦丁治疗后，小鼠的皮肤症状显著减轻(图2A)，银屑病小鼠耳部、背部皮损PASI评分和耳朵厚度较正常对照组明显增加，而各浓度芦丁乳膏干预后均显著下降(图2B-C)，其中以1.5％芦丁乳膏效果最为显著。
4.3芦丁显著改善IMQ诱导的银屑病样小鼠模型的组织病理
H&E染色显示Control组小鼠背部与耳部皮肤表皮层较薄，未出现炎性细胞浸润。与Control组相比，IMQ组小鼠背部与耳部皮肤表现为角化过度伴角化不全，角化不全区可见中性粒细胞微脓肿，颗粒层明显减少，基层增厚表皮突向下延伸。IMQ+Rutin组(1.5％芦丁乳膏)小鼠背部与耳部皮肤炎性细胞浸润和表皮增生显著减少(p<0.001)(图2D-F)。
4.4芦丁可降低IMQ诱导的银屑病样小鼠耳部PLD1、F2的表达
采用IMQ诱导的银屑病样小鼠模型作为研究对象探究芦丁治疗银屑病的分子机制。PLD活性实验显示IMQ诱导的银屑病样小鼠模型皮损处PLD1活性升高，经1.5％芦丁乳膏干预后显著下调，而PLD2的表达差异无统计学意义(图3A)；Western-blot检测显示小鼠耳部皮损处F2表达升高，1.5％芦丁乳膏干预后显著下降(图3B)。提示芦丁可以下调银屑病引起的PLD1、F2表达升高。
实施例2细胞实验
1.材料与方法
1.1芦丁原液制备
芦丁(CAS：153-18-4)从上海雅吉生物科技有限公司购买(纯度≥98％)，称取20mg芦丁结晶，经超声后溶于DMSO，根据DMSO最小溶解度配置呈终浓度为81.90mM的芦丁母液，4℃冰箱贮存，用于后续实验。
1.2细胞培养和处理
HaCaT细胞系(300493)取自Cell Lines Service公司(Eppelheim,Germany)，在含10％胎牛血清、100U/ml青霉素和100μg/ml链霉素的DMEM中生长。上述细胞系于37℃培养箱中，在5％CO2的培养箱中。以0、1、2、5、10、20、50、100μM为终浓度梯度将芦丁母液稀释于DMEM，使用M5(IL-1α，IL-17A，IL-22，抑瘤素M和肿瘤坏死因子-α，2.5ng/ml)刺激HaCaT细胞模拟银屑病的炎症特征。
1.3HaCaT细胞活性检测
使用CCK8试剂盒(碧云天，上海，中国)进行CCK8检测。简而言之，在细胞铺板孵育48小时后，吸走培养液，并向每个孔中加入10μL CCK8和100μL培养基，在含有5％CO2的加湿培养箱中37℃孵育3小时后，使用分光光度计在450nm处测定细胞的增殖能力。
1.4RT-qPCR
通过Trizol法(碧云天，上海，中国)提取HaCaT细胞的总RNA，并且使用PrimeScriptTMRT MasterMix(TakaRa，Japan)逆转录合成cDNA。使用TB Green Premix ExTaq(TakaRa，Japan)进行qRT-PCR。所得到的目标基因表达的数值被归一化为β-肌动蛋白，并相对于对照样本中的表达进行定量。引物序列见表1。
表1引物序列
2.结果
2.1芦丁可抑制M5诱导的HaCaT细胞增殖，并降低IL-6、TNF-αmRNA表达
采用CCK-8检测芦丁在不同浓度下对M5刺激的HaCaT细胞增殖活性影响筛选芦丁体外有效浓度。我们发现芦丁半抑制浓度为10μM，即IC50＝10μM(图4A)。因此，后续分别对芦丁在5μM、10μM、20μM浓度刺激的HaCaT细胞进行RT-qPCR，检测其对炎症因子IL-6、TNF-αmRNA的影响。结果显示，M5诱导的HaCaT细胞中炎症因子IL-6、TNF-αmRNA表达在5μM、10μM、20μM浓度下均有下降，其中以10μM最为显著(TNF-α，p<0.001；IL-6，p<0.01)(图4B)。
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以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (9)
1.芦丁或其上调剂在制备防治银屑病药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述的芦丁或其上调剂是通过下调F2/PLD信号通路来改善银屑病样皮损。
3.F2或其抑制剂在制备防治银屑病药物中的应用。
4.PLD或其抑制剂在制备防治银屑病药物中的应用。
5.一种防治银屑病的药物组合物，其特征在于，所述的药物组合物以芦丁为活性成分和药学上可接受的载体组成。
6.一种防治银屑病的药物组合物，其特征在于，所述的药物组合物以F2或其抑制剂为活性成分和药学上可接受的载体组成。
7.一种防治银屑病的药物组合物，其特征在于，所述的药物组合物以PLD或其抑制剂为活性成分和药学上可接受的载体组成。
8.根据权利要求5-7任一所述的药物组合物，其特征在于，所述药物组合物的剂型为外用剂型或内用剂型。
9.根据权利要求8所述的药物组合物，其特征在于，所述药物组合物的剂型为贴剂、糊剂、软膏剂、微针剂、涂膜剂、巴布剂、喷雾剂、胶囊剂、颗粒剂、片剂、丸剂、口服液或注射液。
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