CN114699411A - 中药化合物青藤碱作为topk激酶抑制剂在制备治疗银屑病产品中的应用 - Google Patents
中药化合物青藤碱作为topk激酶抑制剂在制备治疗银屑病产品中的应用 Download PDFInfo
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Abstract
本发明涉及中药化合物青藤碱作为TOPK激酶抑制剂在制备治疗银屑病产品中的应用。本发明从分子、细胞和动物等水平探讨青藤碱的抗银屑病活性及其具体的作用机制。研究表明青藤碱直接靶向TOPK，抑制TOPK活性,从而抑制银屑病样细胞模型病变，包括抑制银屑病特征性细胞因子IL‑17A的表达，抑制银屑病标志分子S100A7和p‑STAT3的表达，促进病变角质细胞凋亡；利用银屑病样动物模型发现青藤碱显著缓解IMQ诱导的银屑病样症状，表明青藤碱可作为TOPK抑制剂用于制备治疗银屑病的药物。
Description
技术领域：
本发明涉及抗银屑病的免疫调节药物技术领域，具体涉及中药化合物青藤碱及其类似物作为TOPK激酶抑制剂在制备治疗银屑病产品中的应用。
背景技术：
银屑病是常见的慢性炎症性皮肤病之一，主要的组织病理学特征为角质形成细胞的过度增殖和异常分化、免疫细胞浸润及血管扩张等，全球患病率为2％～3％，无性别差异性，且在任何一个年龄段都有可能发生，在20-40岁及55-70岁发病率最高。银屑病会干扰自尊、家庭和社会关系，从而负面影响生活质量，并且和心脏病、关节炎、抑郁症等共患疾病的风险增加。目前银屑病的治疗方案包括：局部治疗(如：皮质类固醇和维生素D类似物等)、光疗法(如：UVB和UVA等)、非生物系统治疗(如：环孢素、甲氨蝶呤和阿维A等)和生物治疗(如：TNF-α抑制剂、IL-23抑制剂和IL-17抑制剂等。在治疗时需要综合考虑合并症及个体化因素，如年龄、银屑病严重程度、银屑病的位置、银屑病的类型等选择特定的一种单独治疗或多种联合治疗。但现有疗法存在不同程度的毒副作用，比如：局部糖皮质激素存在皮肤萎缩，毛细血管扩张，纹胀和紫癜等。光疗法会造成皮肤红斑、灼伤、瘙痒和水泡，甚至会出现光老化或皮肤癌等。甲氨蝶呤的使用与不良的胃肠道副反应、疲劳、异常的肝功能及不太常见但更为严重的事件有关。
T-淋巴细胞来源的蛋白激酶(T-LAK-originated protein kinase，TOPK)是一种具有322个氨基酸的丝苏氨酸蛋白激酶，属于MEK蛋白家族成员，分子量大小为40kDa，在睾丸组织中大量表达，能够促进精子细胞增殖及脑神经干细胞增殖。TOPK参与有丝分裂过程，并在有丝分裂过程中表达升高，促进细胞增殖。TOPK磷酸化H2AX的Ser139位点，参与DNA损伤反应。TOPK在多种肿瘤中高表达，包括白血病等血液肿瘤、乳腺癌、结直肠癌等，促进肿瘤增殖、转化及转移，是潜在的肿瘤生物标志物。TOPK通过促进其底物(如JNK和PRPK等)的磷酸化促进皮肤癌，而其特异性抑制剂能够抑制SUV诱导的皮肤癌。除此之外，TOPK通过调控p38/JNK在LPS诱导的皮肤炎症中发挥重要的作用，是治疗皮肤炎症的有效靶标。但TOPK在银屑病中的作用及其机制未见报道，TOPK是否是银屑病独特的治疗靶标需要深入的研究。
青藤作为我国传统中草药，被应用治疗类风湿性关节炎已有2000多年的历史。青藤碱提取自青藤的根茎，属于生物碱，具有许多药理作用，如免疫抑制、细胞保护、镇痛、抗血管生成、抗心律失常及神经保护作用。青藤碱通过促进肿瘤细胞凋亡、抑制肿瘤细胞增殖发挥抗肿瘤，如胶质母细胞瘤、乳腺癌、肺癌的效应，还可抑制肿瘤细胞侵袭及迁移能力。此外，青藤碱通过抑制NF-κB信号通路发挥其抗炎作用，且已用于治疗人类的类风湿病。青藤碱通过抑制炎症介质(如PGE2等)的产生及血管的生成发挥抗类风湿的作用。青风藤通过调控JNK信号通路抑制LPS诱导的皮肤炎症。但青藤碱是否可以缓解银屑病症状尚不清楚，且其潜在机制有待阐明。为了探索治疗银屑病的药物，我们通过计算机辅助药物筛选技术及预实验，发现青藤碱可能具有抗银屑病作用。为了研究青藤碱治疗银屑病的作用，我们利用银屑病样细胞模型和动物模型，发现青藤碱能够抑制M5和IMQ诱导的银屑病样皮肤病变。进一步探索青藤碱抑制银屑病的作用机制，我们发现青藤碱结合并抑制TOPK激酶活性，可能是其抑制银屑病的作用靶标。本发明阐明了青藤碱靶向TOPK抑制银屑病的分子机制，为临床干预提供分子靶标及新的治疗策略。
发明内容：
(一)解决的技术问题
针对上述背景，本发明利用银屑病样细胞模型和动物模型，发现青藤碱能够抑制M5和IMQ诱导的银屑病样皮肤病变，能结合并抑制TOPK激酶活性，阐明了青藤碱靶向TOPK抑制银屑病的分子机制，提出了中药化合物青藤碱作为TOPK激酶抑制剂在制备治疗银屑病产品中的应用，为临床干预提供分子靶标及治疗银屑病提供新的策略。
(二)技术方案
为解决上述技术问题，本发明采用如下技术方案：中药化合物青藤碱作为TOPK激酶抑制剂在制备治疗银屑病产品中的应用，所述青藤碱的分子式为C19H23NO4，所述青藤碱具有以下结构式：
所述产品为药物、保健品、护肤品或化妆品中的一种或多种。
进一步地，所述青藤碱用于制备抑制TOPK激酶的活性的组合物，或用于制备TOPK激酶活性抑制剂。
进一步地，所述产品的活性成分为青藤碱或其二聚体的水合物或药学上可接受的盐。
进一步地，所述药物单独使用或与其他组合物一同使用。
进一步地，所述药物或所述组合物还用于抑制TOPK激酶磷酸化。
进一步地，所述组合物包括药物组合物、保健品组合物、食品组合物、护肤品或化妆品组合物。
进一步地，所述药物或所述组合物配置成气雾剂、乳膏剂、凝胶剂、透皮贴剂、可注射流体、丸剂、胶囊剂、糖浆剂或赋形剂。
进一步地，所述药物或所述组合物还用于治疗或预防与TOPK激酶活性相关的疾病，如日光性皮炎、湿疹、肿瘤等。
进一步地，所述药物或组合物用于治疗和预防银屑病。
进一步地，所述药物或组合物用于抑制选自下组的炎症因子mRNA的表达：IL-17(IL-17A和IL-17F)、IL-23、IL-6。
进一步地，所述药物或组合物用于抑制选自下组的趋化因子mRNA的表达：CXCL2、CCL20、BD2(β-defensin 2)、S100A8。
进一步地，所述药物或组合物用于抑制抗菌蛋白S100A7的蛋白表达水平和银屑病标志分子p-STAT3的磷酸化水平。
进一步地，所述药物或组合物用于缓解银屑病特征性标志：表皮增厚、角化过度或角化不全、棘皮症、表皮或真皮中性粒细胞增多积聚形成微囊肿。
本发明还提出了一种化合物青藤碱应用于治疗银屑病的方法，所述方法包括：口服、经皮肤表面局部或全身经皮用药、皮下注射、肌肉注射、静脉注射。
本发明还提出了化合物青藤碱应用于缓解银屑病症状和病理改变，抑制银屑病特征性细胞因子IL-17A的表达，抑制银屑病标志分子S100A7和p-STAT3的表达，促进病变角质细胞凋亡。通过qRT-PCR检测细胞因子的表达，WB检测蛋白分子的表达。
本发明还提出了一种TOPK激酶抑制剂，抑制TOPK激酶活性，所述TOPK激酶抑制剂以青藤碱或药学上可接受的盐作为有效成分。
本发明通过将所述药物或所述组合物应用于动物水平缓解银屑病症状和病理改变，通过实验得到：小鼠背部皮肤变薄，红斑及白色皮屑明显减少，背部皮肤的角质层变薄，角质层中中性粒细胞减少，颗粒层增厚，棘层变薄，真皮层血管变窄，银屑病面积严重指数(PASI)明显降低，银屑病特征性细胞因子(IL-17A等)和趋化因子(CXCL2等)的表达降低的方法。通过H&E染色观察皮肤病理改变、炎症细胞的浸润，qRT-PCR检测细胞因子和趋化因子的表达。
本发明从分子、细胞和动物水平等多个层次探讨青藤碱缓解银屑病的作用及其具体机制。研究机制表明，青藤碱通过靶向TOPK抑制银屑病的作用。分子实验结果显示，青藤碱可以与TOPK相互作用，同时呈浓度依赖性抑制TOPK激酶活性；细胞实验结果显示，青藤碱呈浓度及时间依赖性抑制TOPK激酶活性，抑制银屑病样细胞模型病变，包括抑制银屑病特征性细胞因子IL-17A的表达，抑制银屑病标志分子S100A7和p-STAT3的表达，促进病变角质细胞凋亡；最后，利用银屑病样动物模型发现，青藤碱显著缓解IMQ诱导的银屑病样症状，包括的小鼠背部皮肤变薄，红斑及白色皮屑明显减少，背部皮肤的角质层变薄，角质层中中性粒细胞减少，颗粒层增厚，棘层变薄，真皮层血管变窄，银屑病面积严重指数(PASI)明显降低，银屑病特征性细胞因子(IL-17A等)和趋化因子(CXCL2等)的表达降低。上述结果表明，青藤碱可作为TOPK抑制剂用于制备治疗银屑病的药物。
(三)有益效果
本发明产生的有益效果是：本发明通过研究发现青藤碱通过靶向抑制TOPK激酶活性缓解银屑病症状的作用，阐明了青藤碱通过抑制TOPK激酶活性缓解银屑病的分子机制，体外实验显示青藤碱可以抑制角质细胞增殖、促进角质细胞凋亡，提出了中药化合物青藤碱作为TOPK激酶抑制剂在制备治疗银屑病产品中的应用，为临床治疗银屑病提供新的策略。
附图说明：
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1为实施例一青藤碱在体外结合并呈浓度依赖性抑制TOPK激酶活性实验图；
其中图A为体外pull-down实验检测青藤碱与原核表达的TOPK蛋白(上)或HaCaT细胞中的TOPK蛋白(下)相互作用；
图B为体外激酶实验检测青藤碱呈浓度依赖性抑制TOPK激酶活性；
图2为实施例二青藤碱在细胞水平呈浓度及时间依赖性抑制TOPK激酶活性实验图；
图A和图B为青藤碱与M5共处理HaCaT细胞，并设置浓度梯度或时间梯度，通过WB检测TOPK及p-TOPK(Y74)；
图3为实施例三青藤碱抑制M5诱导的银屑病样皮肤病变实验图；
其中图A为青藤碱与M5共处理HaCaT细胞并设置浓度梯度，通过qRT-PCR检测银屑病特征性细胞因子IL-17A，MTX作为药物的阳性对照；
图B和图C为青藤碱与M5共处理HaCaT细胞，并设置浓度梯度或时间梯度，通过WB检测银屑病标志蛋白S100A7及p-STAT3；
图D和图E为青藤碱与M5共处理HaCaT细胞，并设置浓度梯度或时间梯度，通过WB检测凋亡标志分子Cleaved-PARP；
图4为实施例四青藤碱抑制IMQ诱导的银屑病样皮肤病变实验图；
其中图A为动物造模模式图；图B为小鼠背部形态学图片；图C为用PASI(PsoriasisArea Severity Index)指数对银屑病严重程度进行评估；图D为小鼠背部皮肤H&E染色图片；图E为小鼠背部皮肤H&E染色图片中表皮厚度、网脊和真皮层血管数量；
图F为小鼠背部皮肤匀浆后，qRT-PCR检测银屑病特征性细胞因子(IL-17A等)和趋化因子(CXCL2等)的表达。
具体实施方式：
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例详细说明本发明的技术方案。
实施例一青藤碱在体外结合并呈浓度依赖性抑制TOPK激酶活性
1、体外pull-down实验检测青藤碱与TOPK的相互作用
(1)原核表达及纯化TOPK蛋白：将pET46-His-TOPK(BL21)菌种于37℃，200rpm恒温摇床复苏(8-12h)后，加入到无菌的500ml锥形瓶中，37℃，200rpm振摇约1.5h(OD600：0.6～0.8)，加入1M IPTG(终浓度1mM)，诱导4h(OD600：1.2～1.4)，菌液3000rpm离心10分钟，弃掉上清，加入1×PBS重悬细菌，3000rpm离心10分钟，重复三次。将细菌沉淀在-80℃放置10min，而后转至37℃水浴10min，反复冻融3次，充分裂解。加入10ml 1×PBS(提前预冷)，冰上超声20分钟后转至1.5ml EP管中，12000rpm，离心10分钟。转上清于15ml离心管中，加入500μl Ni-NTA-His-binding beads，4℃颠倒过夜。500rpm离心10分钟，beads分别用冰冷的PNI 20、PNI 40washing buffer各漂洗3次，用PNI400 elution buffer 500μl悬浮beads，考马斯亮蓝G250染色法及WB检测TOPK的表达。
(2)用青藤碱预先与CNBr-activated Sepharose 4B在4℃共孵育后，与通过原核表达系统得到TOPK蛋白，或者与HaCaT细胞裂解液，在4℃孵育过夜，通过WB检测青藤碱与TOPK是否相互作用。
2、体外激酶实验检测青藤碱抑制TOPK激酶活性
(1)原核表达及纯化Histone-H3蛋白：将GST-Histone-H3(BL21)菌种于37℃，200rpm恒温摇床复苏(8-12h)后，加入到无菌的500ml锥形瓶中，37℃，200rpm振摇约1.5h(OD600：0.6～0.8)，加入1M IPTG(终浓度1mM)，诱导4h(OD600：1.2～1.4)，菌液3000rpm离心10分钟，弃掉上清，加入1×PBS重悬细菌，3000rpm离心10分钟，重复三次。将细菌沉淀在-80℃放置10min，而后转至37℃水浴10min，反复冻融3次，充分裂解。加入10ml 1×PBS(提前预冷)，冰上超声20分钟后转至1.5ml EP管中，12000rpm，离心10分钟。转上清于15ml离心管中，加入500μlNi-NTA-His-binding beads，4℃颠倒过夜。500rpm离心10分钟，beads分别用冰冷的PNI 20、PNI 40washing buffer各漂洗3次，用PNI 400elution buffer 500μl悬浮beads，考马斯亮蓝G250染色法及WB检测GST-Histone-H3的表达。
(2)用不同浓度梯度的青藤碱与TOPK激酶在37℃共孵育后，再与原核表达的Histone-H3(TOPK的底物)，在ATP的存在进行体外激酶实验(in vitro kinase assay)，通过WB检测Histone-H3的磷酸化水平，验证青藤碱是否可以在体外抑制TOPK的激酶活性。
实施例二青藤碱在细胞水平呈浓度及时间依赖性抑制TOPK激酶活性
本实施例中所用的细胞为人角质形成细胞HaCaT，该细胞培养时需加入含10％FBS的DMEM培养基，于37℃，5％CO2培养箱中培养。
(1)建立银屑病样细胞模型：用10ng/ml的M5(TNF-α、IL-17A、IL-22、IL-1α和Oncostatin-M)刺激人角质形成细胞HaCaT，成功建立银屑病样细胞模型。
(2)青藤碱与M5共处理HaCaT细胞，并设置浓度梯度及时间梯度，通过WB检测p-TOPK(Y74)(可以代表细胞中TOPK的活性)，验证青藤碱呈浓度及时间依赖性对TOPK激酶活性的影响。
实施例三青藤碱(SIN)抑制M5诱导的银屑病样皮肤病变
1、qRT-PCR检测青藤碱对银屑病特征性细胞因子IL-17A的影响青藤碱与M5共处理HaCaT细胞，并设置浓度梯度及时间梯度，甲氨蝶呤(Methotrexate，MTX)是一种可用于治疗银屑病的药物，此处用作药物的阳性对照。通过qRT-PCR检测青藤碱呈浓度及时间依赖性对银屑病特征性细胞因子IL-17A的表达：使用RNA提取试剂盒(Yeasen，上海，CN)分离提取总RNA。使用具有1μg总RNA(Yeasen,Shanghai,CN)的随机引物进行cDNA合成。通过ABI Q3系统基于SYBR Green的定量PCR(Yeasen,Shanghai,CN)使用相应的引物进行实时PCR，分析相应靶基因mRNA的丰度并根据β-actin进行标准化。结果显示青藤碱抑制银屑病特征性细胞因子IL-17A的表达。
2、WB检测青藤碱对凋亡分子的影响
青藤碱与M5共处理HaCaT细胞，并设置浓度梯度及时间梯度，WB检测青藤碱凋亡标志分子Cleaved-PARP的表达。青藤碱促进凋亡分子Cleaved-PARP的表达。
3、WB检测青藤碱对银屑病标志性分子S100A7及p-STAT3的影响
处理条件同上，并通过WB检测银屑病标志性分子S100A7的表达，结果显示，青藤碱抑制银屑病标志性分子S100A7及p-STAT3的表达。
实施例四青藤碱(SIN)抑制IMQ诱导的银屑病样皮肤病变
1、动物实验模型构建：本发明中所用的小鼠为Balb/c雄鼠(8周龄)在小鼠背部侧均匀涂抹咪喹莫特(5％)建立银屑病样动物模型，利用银屑病样动物模型，研究青藤碱对银屑病样症状的作用。将Balb/c雄鼠(8周龄)随机分成五组：阴性处理组(Control，CON)、咪喹莫特组(IMQ)、青藤碱低剂量组(SIN(low))、青藤碱高剂量组(SIN(high))和丁酸氢化可的松(HYD)组，丁酸氢化可的松是一种可用于治疗银屑病的糖皮质激素药物，此处用作药物的阳性对照。每组6只小鼠。小鼠背部剃毛24h后，在青藤碱低剂量组、青藤碱高剂量组和丁酸氢化可的松组的小鼠背部均匀涂抹各含药凝胶或乳膏(30mg)，阴性处理组和咪喹莫特组用相同量的空白凝胶涂抹。药物处理5h后，在咪喹莫特组、青藤碱低剂量组、青藤碱高剂量组和丁酸氢化可的松组的小鼠背部均匀涂抹咪喹莫特(5％)乳膏(62.5mg)，阴性处理组用相同量的凡士林处理。以上处理均连续涂抹5-7天后处死小鼠，期间每天观察并拍照记录小鼠背部的银屑病发病情况。
2、通过PASI score评估青藤碱对银屑病皮损严重程度的影响
使用http://pasi.corti.li/评分网站对银屑病受损部位进行评分，根据银屑病病变区域占比、病变区域发红(0-4)、增厚(0-4)、鳞屑症状严重程度(0-4)来综合计算银屑病评分。PASI score评估显示青藤碱能够缓解银屑病皮损严重程度。
3、将小鼠背部皮肤进行H&E染色，观察青藤碱对银屑病标志性特征的影响，包括表皮棘皮症；角化过度和角化不全；表皮网脊明显拉长；真皮层血管扩张并到达尖端真皮乳头；真皮和表皮存在炎症浸润；中性粒细胞积聚在表皮形成角膜下微脓肿等。
(1)将小鼠背部皮肤置于10％多聚甲醛中固定24h，流水冲洗30min，酒精(由低到高)梯度脱水，二甲苯中透明，石蜡包埋，切成5-8μm的薄片，热水烫平，贴于载玻片上，烘干。
(2)二甲苯脱蜡，梯度酒精(由高到低)，最后入蒸馏水，苏木精染色，置酸水及氨水中分色，流水冲洗1小时，置70％和90％酒精中脱水，伊红染色液染色2-3分钟，纯酒精脱水，二甲苯透明，滴上树胶，盖上盖玻片封固，镜下拍照。结果显示，青藤碱缓解银屑病标志性特征，包括表皮棘皮症减轻；角化过度和角化不全缓解；表皮网脊缩短；真皮层血管收缩；真皮和表皮炎症浸润减少；中性粒细胞积聚在表皮形成的角膜下微脓肿消失等。
4、取小鼠背部皮肤进行qRT-PCR，检测青藤碱对银屑病特征性细胞因子(IL-17A等)和趋化因子(CXCL2等)表达的影响
使用RNA提取试剂盒(Yeasen，上海，CN)分离提取总RNA。使用具有0.2μg总RNA(Yeasen,Shanghai,CN)的随机引物进行cDNA合成。通过ABI Q3系统基于SYBR Green的定量PCR(Yeasen,Shanghai,CN)使用相应的引物进行实时PCR，分析相应靶基因mRNA的丰度并根据β-actin进行标准化。结果显示，青藤碱抑制银屑病特征性细胞因子(IL-17A等)和趋化因子(CXCL2等)的表达。
综上所述，本发明利用银屑病样细胞模型和动物模型，发现青藤碱能够抑制M5和IMQ诱导的银屑病样皮肤病变，能结合并抑制TOPK激酶活性，阐明了青藤碱靶向TOPK抑制银屑病的分子机制，提出了中药化合物青藤碱作为TOPK激酶抑制剂在制备治疗银屑病产品中的应用，为临床干预提供分子靶标及治疗银屑病提供新的策略。
最后需要说明的是，以上实施例仅用于说明本发明而非限制本发明的保护范围。另外，在阅读了本发明的技术内容之后，本领域技术人员可以对本发明作各种改动、修改或变型，所有的这些等价形式同样属于本申请所要求限定的保护范围之内。
Claims (10)
1.中药化合物青藤碱作为TOPK激酶抑制剂在制备治疗银屑病产品中的应用，其特征在于，所述青藤碱的分子式为C19H23NO4，所述产品为药物、保健品、护肤品或化妆品中的一种或多种。
2.如权利要求1所述的应用，其特征在于，所述产品的活性成分为青藤碱或其二聚体的水合物或药学上可接受的盐。
3.如权利要求1所述的应用，其特征在于，所述药物单独使用或与其他组合物一同使用。
4.如权利要求1或3所述的应用，其特征在于，所述药物或组合物配置成气雾剂、乳膏剂、凝胶剂、透皮贴剂、可注射流体、丸剂、胶囊剂、糖浆剂或赋形剂。
5.如权利要求1或3所述的应用，其特征在于，所述药物或组合物还用于治疗或预防与TOPK激酶活性相关的疾病，如日光性皮炎、湿疹、肿瘤。
6.如权利要求1或3所述的应用，其特征在于，所述药物或组合物用于抑制选自下组的炎症因子mRNA的表达：IL-17、IL-23、IL-6。
7.如权利要求1或3所述的应用，其特征在于，所述药物或组合物用于抑制选自下组的趋化因子mRNA的表达：CXCL2、CCL20、BD2、S100A8。
8.如权利要求1或3所述的应用，其特征在于，所述药物或组合物用于抑制抗菌蛋白S100A7的蛋白表达水平和银屑病标志分子p-STAT3的磷酸化水平。
9.如权利要求1或3所述的应用，其特征在于，所述药物或组合物用于缓解银屑病特征性标志：表皮增厚、角化过度或角化不全、棘皮症、表皮或真皮中性粒细胞增多积聚形成微囊肿。
10.一种TOPK激酶抑制剂，其特征在于，其活性成分为青藤碱或其二聚体的水合物或药学上可接受的盐。
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