CN107951896A - 一类非甾体类抗炎药在抗寨卡病毒中的应用 - Google Patents
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Abstract
本发明属于已知化合物的新用途领域，具体公开了一类非甾体类抗炎药在制备抗寨卡病毒药物中的应用，所述非甾体类抗炎药为阿司匹林、布洛芬、对乙酰氨基酚、萘普生钠或氯诺昔康。本发明发现阿司匹林，布洛芬，对乙酰氨基酚，萘普生钠，氯诺昔康等非甾体类抗炎药能够有效地抑制寨卡病毒，在制备抑制寨卡病毒药物中具备重大的开发意义和应用前景。
Description
技术领域
本发明涉及已知化合物的新用途领域，具体地，涉及一类非甾体类抗炎药在抗寨卡病毒中的应用。
背景技术
寨卡病毒属黄病毒科，黄病毒属，单股正链RNA病毒，直径20nm，是一种通过蚊虫进行传播的虫媒病毒，主要在巴西各地区蔓延。寨卡病毒最早于1940年代在非洲被发现，此后也传播到东南亚、太平洋岛国和美洲地区。2015年巴西的寨卡暴发流行中发现了很多小头畸形的新生儿（出生的新生儿头围与匹配的相同性别和孕龄的孩子比，低于平均值超过了两个标准差），越来越多的证据表明寨卡病毒与小头症之间存有关联。寨卡病毒可能导致婴儿患上“小头症”，导致患者的脑神经失调，脑部发育不全，终身不能自理。由于传播该病毒的伊蚊在全世界都可以找到，病毒的爆发极有可能会传播到更多国家。然而，目前世界上仍缺乏有效的抗寨卡病毒的疫苗和药物。
非甾体类抗炎药(NSAIDs) 是一类具有解热、镇痛，多数还有抗炎、抗风湿作用的药物，对于一些风湿性疾病，如早期类风湿关节炎、老年性关节炎及早期强直性脊柱炎等是首选药物。现有技术中未公开非甾体类抗炎药还可以用于抗寨卡病毒。
发明内容
本发明为了克服现有技术的上述缺陷，提供一类非甾体类抗炎药在抗寨卡病毒中的应用。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
非甾体类抗炎药在制备抗寨卡病毒药物中的应用，所述非甾体类抗炎药为阿司匹林、布洛芬、对乙酰氨基酚、萘普生钠或氯诺昔康。
不同非甾体类抗炎药对寨卡病毒的抑制程度不同，优选地，所述非甾体类抗炎药为萘普生钠或氯诺昔康。
一种抗寨卡病毒药物，含有非甾体类抗炎药阿司匹林、布洛芬、对乙酰氨基酚、萘普生钠或氯诺昔康。
所述非甾体抗炎药通过抑制寨卡病毒入侵宿主细胞，从而实现抗寨卡病毒。
所述非甾体抗炎药通过抑制寨卡病毒入侵细胞时所结合的宿主细胞表面受体AXL蛋白的表达，实现抑制寨卡病毒入侵宿主细胞。
所述非甾体抗炎药通过泛素化通路降解宿主细胞的AXL，从而抑制了AXL蛋白的表达。
所述非甾体抗炎药通过PGE2/cAMP/PKA通路降解宿主细胞的AXL，从而抑制了AXL蛋白的表达。
所述非甾体抗炎药在NOD-SCID小鼠体内抑制AXL蛋白在颅内的表达，从而抑制小鼠颅内的寨卡病毒复制。
与现有技术相比，本发明具有如下有益效果：
本发明首次发现阿司匹林，布洛芬，对乙酰氨基酚，萘普生钠，氯诺昔康等非甾体类抗炎药能够有效地抑制寨卡病毒， IC50约为0.21～3.11uM。
更进一步地，本发明发现阿司匹林，布洛芬，对乙酰氨基酚，萘普生钠，氯诺昔康等非甾体类抗炎药通过抑制寨卡病毒入侵细胞时所结合的宿主细胞表面受体AXL蛋白的表达，抑制了寨卡病毒与宿主细胞的结合。而非甾体抗炎药可以通过泛素化通路降解宿主细胞的AXL，从而抑制了AXL蛋白的表达，或者，非甾体抗炎药可以通过PGE2/cAMP/PKA通路降解宿主细胞的AXL，从而抑制了AXL蛋白的表达，或者，所述非甾体抗炎药可以在NOD-SCID小鼠体内抑制AXL蛋白在颅内的蛋白表达，从而抑制小鼠颅内的寨卡病毒复制。
附图说明
图1为构建针对寨卡包膜E蛋白的体外高通量药物筛选模型以及不同非甾体类抗炎药对寨卡包膜蛋白的抑制效果。
图2为不同浓度的非甾体类抗炎药对野生型寨卡病毒的抑制效果。
图3为不同浓度的非甾体类抗炎药对寨卡病毒受体的抑制效果。
图4为不同浓度的非甾体类抗炎药通过PGE2/cAMP/PKA通路对寨卡病毒的抑制效果。
图5为不同的非甾体类抗炎药在小鼠体内对寨卡病毒的抑制效果。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1
构建针对寨卡包膜E蛋白的体外高通量药物筛选模型以及不同非甾体类抗炎药对寨卡包膜蛋白的抑制效果。
（1）包病毒：将4.5 μg pHIV-luciferase、4.5 μg pCMV-ΔR8.2和10 μg Zika-prM/E质粒转染至293T细胞（10 cm dish），48小时后，收集病毒上清，测p24。
（2）针对寨卡病毒包膜蛋白的化合物筛选：将p24-标准化的 Zika-prM/E 假病毒（含 8 μg/ml 促感染试剂Polybrene）感染96孔板中的A549细胞或Vero细胞，同时加入不同的非甾体类抗炎药。
（3）换液：感染12小时后，更换新鲜的DMEM培养基。
（4）检测荧光素酶活性：感染48小时后，每孔用PBS洗一次，然后加入100 μl 裂解缓冲液，震荡30 min，取10 μl裂解液检测荧光素酶活性。
根据实施例1的结果说明，阿司匹林，布洛芬，对乙酰氨基酚，萘普生钠，氯诺昔康等非甾类抗炎药可以特异性的作用于寨卡病毒包膜蛋白E，抑制寨卡病毒的进入；此外，非甾类抗炎药对登革病毒和水疱性口炎病毒的包膜蛋白并没有任何的抑制性作用（见图1）。
实施例2
不同浓度的非甾体类抗炎药对野生型寨卡病毒的抑制效果。
（1）包病毒：将4.5 μg pHIV-luciferase、4.5 μg pCMV-ΔR8.2和10 μg Zika-prM/E质粒转染至293T细胞（10 cm dish），48小时后，收集病毒上清，测p24。
（2）针对寨卡病毒包膜蛋白的化合物筛选：将p24-标准化的 MERS-CoV-S假病毒（含 8 μg/ml 促感染试剂Polybrene）感染96孔板中的293T细胞，同时加入不同浓度的非甾体类抗炎药。
（3）换液：感染12小时后，更换新鲜的DMEM培养基。
（4）检测荧光素酶活性：感染48小时后，每孔用PBS洗一次，然后加入100 μl lysisbufer，震荡30 min，取10 μl裂解液检测荧光素酶活性。
根据实施例2的结果说明，该类非甾体类抗炎药具有良好的抗病毒作用，IC50分别为阿司匹林1.63uM，布洛芬767nM，对乙酰氨基酚3.11uM，萘普生钠482nM，氯诺昔康210nM（见图2）
实施例3
一、不同浓度的非甾体类抗炎药对寨卡病毒受体蛋白AXL的抑制效果：
（1）铺板：将A549细胞传代铺板于6孔细胞培养板中，细胞密度为2x105个/每孔。
（2）药物处理：铺板12小时后，将不同浓度的非甾体类抗炎药加入到各个细胞培养板中。
（3）收样：药物处理48小时后，收取A549细胞，用RIPA裂解液进行细胞裂解，随后用western blot方法，检测细胞中AXL蛋白的表达情况
根据图3a的结果说明，该类非甾体类抗炎药可以抑制寨卡病毒受体AXL的蛋白表达。
二、非甾体类抗炎药对寨卡病毒受体蛋白AXL的降解作用：
（1）铺板：将A549细胞传代铺板于6孔细胞培养板中，细胞密度为2x105个/每孔。
（2）药物处理：铺板12小时后，将不同浓度的非甾体类抗炎药加入到各个细胞培养板中。
（3）收样：药物处理36小时后，加入2uM的MG132处理细胞，12小时后，收取A549细胞，用RIPA裂解液进行细胞裂解，随后用western blot方法，检测细胞中AXL蛋白的表达情况
根据图3b的结果说明，该类非甾体类抗炎药可以通过蛋白酶体泛素化通路降解寨卡病毒受体AXL蛋白。
三、非甾体类抗炎药对寨卡病毒受体蛋白AXL的泛素化作用：
（1）铺板：将A549细胞传代铺板于6孔细胞培养板中，细胞密度为1x106个/每板。
（2）药物处理：铺板12小时后，将不同的非甾体类抗炎药加入到各个细胞培养板中。
（3）收样：药物处理36小时后，加入2uM的MG132处理细胞，12小时后，收取A549细胞，用RIPA裂解液进行细胞裂解，随后用免疫共沉淀的方法，检测细胞中AXL蛋白的泛素化情况
根据图3c的结果说明，该类非甾体类抗炎药可以诱导寨卡病毒受体AXL蛋白的泛素化。
实施例4
不同浓度的非甾体类抗炎药通过PGE2/cAMP/PKA通路对寨卡病毒的抑制效果。
（1）铺板：将A549细胞传代铺板于6孔细胞培养板中，细胞密度为2x105个/每孔。
（2）药物处理：铺板12小时后，将不同浓度的非甾体类抗炎药加入到各个细胞培养板中。与此同时，分别加入PGE2，毛喉素(Forskolin)，H89三种不同化合物。
（3）收样：药物处理48小时后，收取A549细胞，用RIPA裂解液进行细胞裂解，随后用western blot方法，检测细胞中AXL蛋白的表达情况
根据实施例4的结果说明，该类非甾体类抗炎药可以通过PGE2/cAMP/PKA的信号通路来调控寨卡病毒受体AXL的蛋白表达（见图4）。
实施例5
不同的非甾体类抗炎药在小鼠体内对寨卡病毒的抑制效果。
（1）感染：将1x106PFU的寨卡病毒通过腹腔注射的方式，注射到NOD-SCID的小鼠体内，同时，将非甾体类抗炎药以50mg/kg的剂量通过腹腔注射到小鼠体内
（2）药物处理：腹腔注射药物，每隔一天注射一次，剂量50mg/kg
（3）收样：小鼠感染后第10天，处死小鼠，取脑组织，研磨，用空斑法滴定小鼠脑内的病毒含量。
根据实施例5的结果说明，该类非甾体类抗炎药可以在NOD-SCID小鼠体内抑制寨卡病毒的复制（见图5）。
Claims (8)
1.非甾体类抗炎药在制备抗寨卡病毒药物中的应用，所述非甾体类抗炎药为阿司匹林、布洛芬、对乙酰氨基酚、萘普生钠或氯诺昔康。
2.根据权利要求1所述的应用，其特征在于，所述非甾体类抗炎药为萘普生钠或氯诺昔康。
3.一种抗寨卡病毒药物，其特征在于，含有非甾体类抗炎药阿司匹林、布洛芬、对乙酰氨基酚、萘普生钠或氯诺昔康。
4.根据权利要求1或2所述的应用，其特征在于，所述非甾体抗炎药通过抑制寨卡病毒入侵宿主细胞，从而实现抗寨卡病毒。
5.根据权利要求4所述的应用，其特征在于，所述非甾体抗炎药通过抑制寨卡病毒入侵细胞时所结合的宿主细胞表面受体AXL蛋白的表达，实现抑制寨卡病毒入侵宿主细胞。
6.根据权利要求5所述的应用，其特征在于，所述非甾体抗炎药通过泛素化通路降解宿主细胞的AXL，从而抑制了AXL蛋白的表达。
7.根据权利要求5所述的应用，其特征在于，所述非甾体抗炎药通过PGE2/cAMP/PKA通路降解宿主细胞的AXL，从而抑制了AXL蛋白的表达。
8.根据权利要求5所述的应用，其特征在于，所述非甾体抗炎药在NOD-SCID小鼠体内抑制AXL蛋白在颅内的表达，从而抑制小鼠颅内的寨卡病毒复制。
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| CN111939155A (zh) * | 2020-08-03 | 2020-11-17 | 中山大学 | 吲哚生物碱在制备抗寨卡病毒和/或抗登革病毒药物中的应用 |
| CN111939155B (zh) * | 2020-08-03 | 2022-02-08 | 中山大学 | 吲哚生物碱在制备抗寨卡病毒和/或抗登革病毒药物中的应用 |
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