CN107050011A - 染料木素在制备抗自身免疫性肝炎药物中的应用 - Google Patents

染料木素在制备抗自身免疫性肝炎药物中的应用 Download PDF

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CN107050011A
CN107050011A CN201710135584.7A CN201710135584A CN107050011A CN 107050011 A CN107050011 A CN 107050011A CN 201710135584 A CN201710135584 A CN 201710135584A CN 107050011 A CN107050011 A CN 107050011A
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immunity hepatitis
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尤强
徐澈
张晨
潘金顺
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 

Abstract

本发明公开了染料木素在制备抗自身免疫性肝炎药物中的应用，染料木素本身无毒副作用，且可以有效抑制肝脏单核细胞的浸润而导致的肝脏组织水肿坏死，可作为免疫研究的活性药物以及作为药物靶点开发抗自身免疫性肝炎的药物，具有良好的基础及临床应用前景。

Description

染料木素在制备抗自身免疫性肝炎药物中的应用
技术领域
本发明属于抗自身免疫性肝炎药物研发领域，具体涉及染料木素在制备抗自身免疫性肝炎药物中的应用。
背景技术
肝脏位于右上腹，隐藏在右侧膈下和肋骨深面，大部分肝为肋弓所覆盖，仅在腹上区、右肋弓间露出并直接接触腹前壁，肝上面则与膈及腹前壁相接。肝脏是人体内脏里最大的器官，也是消化系统内最大的消化腺，其对来自体内和体外的许多非营养性物质如各种药物、毒物以及体内某些代谢产物，具有生物转化作用，通过新陈代谢将它们彻底分解或以原形排出体外，这种作用也被称作“解毒功能”。
早在1962年，就有研究者发现肝脏具有免疫耐受原性，是机体最重要的免疫器官。其一方面需要强大的免疫应答能力抗感染,另一方面需要强有力的调节机制避免不必要的免疫激活,而后者主要通过免疫耐受来实现对于移植正常肝脏中各类免疫细胞处于稳态中。当免疫平衡机制打破时，机体对肝细胞产生自身抗体及自身反应性肝T细胞，则会出现自身免疫性肝炎(AIH)。
AIH是由自身免疫反应介导的慢性进行性肝脏炎症性疾病，其临床特征为不同程度的血清转氨酶升高、高γ-球蛋白血症、自身抗体阳性，组织学特征为以淋巴细胞、浆细胞浸润为主的界面性肝炎，严重病例可快速进展为肝硬化和肝衰竭。该病在世界范围内均有发生，在欧美国家发病率相对较高，在我国其确切发病率和患病率尚不清楚，但国内文献报道的病例数呈明显上升趋势。
根据血清自身抗体可将AIH分为3型，Ⅰ型AIH最为常见，约占AIH的80％，相关抗体为ANA和(或)SMA，免疫抑制剂疗效较差，目前没有有效的治疗方案；Ⅱ型AIH的特征为抗-LKM1阳性，儿童多发，可快速发展为肝硬化，复发率高，糖皮质激素治疗效果较差；Ⅲ型A IH的特征为血清抗-SLA/LP阳性，女性多发，有学者认为可归入Ⅰ型。AIH的预后差异较大，未经治疗的患者可缓慢进展为肝硬化，或发展为急性、亚急性、暴发性肝病，最终以各种并发症而死亡。寻找恰当有效治疗方案是如今迫在眉睫的难题。
染料木素是一种存在于豆类作物和齿状植物中的天然异黄酮，其结构与雌激素相似，并具有弱雌激素效应。其药理作用有：抗氧化作用；具有雌激素及抗雌激素性质；抑制拓析异物酶II的活性；抑制酪氨酸蛋白激酶PTK的活性；具有诱发细胞程序性死亡、提高抗癌药效、抑制血管生成等作用。
发明内容
本发明所要解决的技术问题是提供染料木素在制备抗自身免疫性肝炎药物中的应用，以解决现有技术中，自身免疫性肝炎(AIH)治疗效果差、容易复发的缺陷。
染料木素在制备抗自身免疫性肝炎药物中的应用在本发明的保护范围之内。
染料木素可以通透细胞，抑制酪氨酸蛋白激酶(tyrosine protein kinase)，抑制许多蛋白的酪氨酸磷酸化。CD36受体在酪氨酸激酶的作用下促进其与TLR4-TLR6形成二聚体，染料木素通过抑制CD36-TLR4-TLR6复合物形成从而抑制NF-κB信号通路的激活，进而减缓肝损伤。
有益效果：
本发明提供了一种成本低、简便易行的减缓免疫性肝损伤的方法，本发明所述的染料木素本身无毒副作用，且可以有效抑制肝脏单核细胞的浸润而导致的肝脏组织水肿坏死，可作为免疫研究的活性药物以及作为药物靶点开发抗自身免疫性肝炎的药物，具有良好的基础及临床应用前景。
附图说明
图1染料木素降低肝脏中谷丙转氨酶的水平，以及减轻肝脏水肿坏死。
图2染料木素减少肝脏中Con A诱导的单核细胞浸润。
图3染料木素抑制凋亡信号通路激活。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
本发明通过刀豆蛋白A(Con A)诱导野生型(WT)小鼠自身免疫性肝炎来研究染料木素自身免疫性肝炎对的影响。
实施例1：体内实验：
选取在同样饲养环境下性别、体重及周龄相同的WT小鼠18只，随机分为3组：阴性对照组，诱导组，处理组。阴性对照组腹腔注射0.5ml/kg的二甲基亚砜(DMSO)；诱导组小鼠尾静脉注射12mg/kg的Con A(溶于PBS，浓度为2.4mg/ml)后，同时腹腔注射0.5ml/kg的DMSO；处理组小鼠在尾静脉注射12mg/kg的Con A(溶解于PBS，浓度为2.4mg/ml)后，同时腹腔注射10mg/kg的染料木素(溶解于DMSO，浓度为20mg/ml)。
(1)8h后小鼠眼眶后静脉丛收集血液标本；二氧化碳窒息法处死小鼠后，取肝脏边缘3-5mm组织包埋，浸泡于10％多聚甲醛，剩余肝脏组织包入锡纸中冻于液氮，-80℃保存。血标本5,000×g,15min离心后，收集上清，检测谷丙转氨酶(ALT)水平；组织石蜡包埋后切片H&E染色，100倍倒置光学显微镜下观察不同处理条件下小鼠肝脏组织的坏死情况；
(2)提前将配制好的Hank’s缓冲盐溶液(HBSS)和0.2％Ⅳ型胶原酶液在水浴中预温敷至37℃。8h后，二氧化碳窒息法处死小鼠，仰卧固定，暴露小鼠胸腔与腹腔，门静脉插入导管，动脉夹固定，剪断下腔静脉腹腔段，经门静脉插管以匀速灌注预温孵的HBSS缓冲液以去除肝内血液及钙离子，当肝脏变苍白、下腔静脉流出液体变清时开始灌注预温的胶原酶液直至肝脏颜色变成土黄色、包膜下呈龟背状裂隙、压之凹陷不易恢复时停止灌注，完整摘除肝脏。在超净台内将肝脏细胞悬液通过70μm筛网后，700×g离心5min，35％percoll重悬后，500×g，15min离心后1.5ml红细胞裂解液重悬，500×g，3min离心后，100μl含有2％胎牛血清的HBSS溶液重悬，以上为肝脏单核细胞(MNCs)收集过程。
首先用抗小鼠CD16/32抗体孵育收集的MNCs10min，避免非特异性结合。接着用不同的流式抗体染色，包括：抗小鼠CD3、CD4、CD8、NK1.1、CD11b、Ly6G、Ly6C、F4/80及CD45抗体，流式细胞仪上机检测。
实验结果显示，Con A能够通过诱导小鼠ALT升高，肝脏组织水肿坏死以及增加CD3+、CD4+、CD8+、CD3-NK1.1+、CD11b+Ly6C+及CD11b+Ly6G+炎性细胞浸润介导肝脏损伤，而处理组ALT显著降低，肝脏坏死及细胞浸润也明显减轻，表明染料木素可以减轻小鼠免疫性肝损伤(图1，图2)。
实施例2：体外实验：
选取在同样饲养环境下性别、体重及周龄相同的WT小鼠3只，在体灌注肝脏获得MNCs，方法同前所述。台盼蓝染色观察细胞活力、计数后用含有5％灭活胎牛血清的William’s E培养基重悬，6孔板1ml/well，37℃培养箱中培养。培养过夜后用含有0.2％胎牛血清的William’s E培养基37℃培养箱中饥饿细胞4h，将细胞随机给予不同的处理。如下：
表1不同处理方法对照表
分别在5min、20min、60min、4h及8h时收集4种不同处理的细胞，提取蛋白后，蛋白印迹法检测目的蛋白在各处理细胞中的表达。
实验结果显示，CXCL10处理肝脏原代细胞后，从20min开始蛋白激酶B(Akt)和c-Jun氨基末端激酶(JNK)明显激活，激活时间可持续到8h；而同时给予CXCL10和染料木素的细胞，Akt和JNK的表达明显降低，表示染料木素可抑制肝脏炎症凋亡信号通路激活，从而减轻肝损伤(图3)。

Claims (1)

1.染料木素在制备抗自身免疫性肝炎药物中的应用。
CN201710135584.7A 2017-04-28 2017-04-28 染料木素在制备抗自身免疫性肝炎药物中的应用 Pending CN107050011A (zh)

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Application publication date: 20170818