CN107929724A - 神经氨酸酶及抑制剂在制备治疗肺纤维化和肾纤维化的药物中的应用 - Google Patents
神经氨酸酶及抑制剂在制备治疗肺纤维化和肾纤维化的药物中的应用 Download PDFInfo
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Abstract
本发明公开了神经氨酸酶及抑制剂在制备治疗肺纤维化和肾纤维化的药物中的应用。已知扎那米韦、磷酸奥司他韦为神经氨酸酶的有效抑制剂，黄连碱对神经氨酸酶的抑制作用也已在申请人先前的专利申请中公开，申请人又发现丹酚酸B在体外可以抑制神经氨酸酶的活性，为神经氨酸酶的抑制剂；试验证明，这几种神经氨酸酶抑制剂可以有效抑制肺和肾组织中神经氨酸酶活性，进而抑制肺纤维化和肾纤维化。由此可见，神经氨酸酶及其抑制剂可用于制备治疗肺纤维化和肾纤维化的药物。
Description
技术领域
本发明属于生物医药领域，涉及酶和酶抑制剂的应用，具体涉及神经氨酸酶及抑制剂在制备治疗肺纤维化和肾纤维化的药物中的应用。
背景技术
神经氨酸是广泛存在于生物体内的一类天然糖酸类化合物。现在确定神经氨酸有五十多种天然衍生物，N-乙酰神经氨酸、N-羟乙酰基神经氨酸是其中较为常见的衍生物。神经氨酸通常以短链残基的形式链接在糖蛋白、糖脂等糖缀合物的末端。神经氨酸是重要的生物信息传递分子，细胞表面糖蛋白和糖脂的神经氨酸化修饰在许多生物过程中发挥着至关重要的作用，包括细胞粘附、抗原识别和信号传导等。
在生物体内，神经氨酸酶是一种与神经氨酸相关的酶，在生物体内许多重要的生理过程中发挥着不可或缺的作用。神经氨酸酶可以断裂糖缀合物末端与神经氨酸残基相连接的糖苷键从而水解神经氨酸糖缀合物使得神经氨酸游离。
神经氨酸酶抑制剂是一类可以抑制神经氨酸酶活性的物质，如扎那米韦和磷酸奥司他韦。扎那米韦和磷酸奥司他韦是临床常用的治疗流感的药物。流感病毒通过其表面的红血球凝集素与宿主细胞表面的神经氨酸残基结合，继而侵入宿主细胞。随后，其基因在宿主细胞内复制和表达，形成新的流感病毒。在新病毒的释放阶段，神经氨酸酶催化水解成熟的流感病毒表面红血球凝集素与宿主细胞神经氨酸残基之间相连接的神经氨酸糖苷键，使得新病毒颗粒脱离被感染细胞，进一步在宿主体内扩散。扎那米韦和磷酸奥司他韦通过抑制神经氨酸酶的活性抑制新病毒颗粒脱离被感染细胞，抑制其在宿主体内扩散，达到治疗目的。
申请人于2016年3月22日提交了申请号为“201610166253.5”、发明名称为“神经氨酸酶及其抑制剂在心肌缺血及心肌梗死中的应用”的专利申请，该申请公开了神经氨酸酶及其抑制剂在心肌缺血及心肌梗死中的应用，提供了神经氨酸酶与心肌缺血损伤的关联性，证明通过抑制神经氨酸酶的活性可以缓解心肌缺血损伤，神经氨酸酶可以作为筛选预防、缓解和/或治疗心肌缺血损伤药物的靶标；该申请还证明了神经氨酸酶抑制剂对心肌缺血损伤的缓解作用，神经氨酸酶抑制剂通过降低神经氨酸酶的水平改善心肌缺血损伤。
申请人在随后的研究中还发现神经氨酸酶与多种疾病相关，经过检索，未发现现有技术公开过神经氨酸酶与这些疾病的相关性。
发明内容
本发明的目的在于提供神经氨酸酶及抑制剂在制备治疗肺和肾纤维化的药物中的应用。
本发明的上述目的是通过下面的技术方案得以实现的：
神经氨酸酶在制备治疗肺纤维化的药物中的应用。
神经氨酸酶在制备治疗肾纤维化的药物中的应用。
神经氨酸酶抑制剂在制备治疗肺纤维化的药物中的应用。
神经氨酸酶抑制剂在制备治疗肾纤维化的药物中的应用。
黄连碱或丹酚酸B及其可药用盐用于制备神经氨酸酶抑制剂的用途。
扎那米韦、磷酸奥司他韦、黄连碱或丹酚酸B及其可药用盐用于制备治疗肺纤维化和肾纤维化的药物的用途。
已知扎那米韦、磷酸奥司他韦为神经氨酸酶的有效抑制剂，黄连碱对神经氨酸酶的抑制作用也已在申请人先前的专利申请中公开，申请人又发现丹酚酸B在体外可以抑制神经氨酸酶的活性，为神经氨酸酶的抑制剂；试验证明，这几种神经氨酸酶抑制剂可以有效抑制肺和肾组织中神经氨酸酶活性，进而抑制肺纤维化和肾纤维化。由此可见，神经氨酸酶及其抑制剂可用于制备治疗肺纤维化和肾纤维化的药物。
附图说明
图1为各组小鼠肺纤维化面积、N-乙酰神经氨酸含量和神经氨酸酶活性比较；
图2为各组大鼠肾纤维化面积和肾组织中神经氨酸酶活性比较。
具体实施方式
下面结合实施例具体介绍本发明实质性内容，但并不以此限定本发明的保护范围。
实施例1：神经氨酸酶及抑制剂与肺纤维化的关系
一、实验方法
分组及给药：SPF级雄性C57BL/6小鼠(体重20-24g)适应性饲养3-5d后，随机分为假手术组(对照组)、模型组和扎那米韦、磷酸奥司他韦、黄连碱、丹酚酸B给药组。除对照组经气管注射等容量灭菌生理盐水，其余各组均经气管注射BLM(5mg/kg)造模。2h后，给药组小鼠分别给予0.2mg/kg/d的扎那米韦(i.v.)、5mg/kg/d磷酸奥司他韦(p.o.)、40mg/kg/d黄连碱(p.o.)、40mg/kg/d丹酚酸B(p.o.)，模型组和对照组灌胃给予等体积溶媒0.5％CMC-Na，连续给药4周。末次给药2h后腹主动脉放血处死，收集支气管肺泡灌洗液(BALF，回收率≥80％)，取肺组织。肺左上叶于4％多聚甲醛溶液固定以病理检查，其余肺组织-80℃冻存。
肺纤维化造模方法：小鼠以戊巴比妥钠(30mg/kg)腹腔注射麻醉后施行手术。暴露气管，以微量进样器向气管内缓慢注入BLM(5mg/kg)，注射后立即将动物直立旋转1min，使药物在肺内分布均匀。BALF收集方法：小鼠处死后，分离气管。以7号头皮针沿向心方向穿入气管，缓慢推入灭菌生理盐水1mL，再缓慢回抽，得回收液约0.75-0.8mL，来回2次。回收液以1500r/min离心15min，取上清即BALF，-80℃保存待测。
肺组织病理学检查：肺左上叶于4％多聚甲醛中固定，石蜡包埋、切片(厚度5μm)，Masson's染色，观察肺组织病理形态学改变及胶原纤维含量。
BALF中N-乙酰神经氨酸含量测定：使用齐一生物提供的N-乙酰神经氨酸含量检测试剂盒测定，具体测定方法参照试剂盒说明书。
肺组织神经氨酸酶活性测定：将病理学检测取材后的其余肺组织置于冰上，用冰冷的生理盐水冲洗干净，用滤纸吸干表面水分，称重，按500g/L的比例加入含0.25mmol/L蔗糖和1mmol/L EDTA的匀浆液匀浆，充分匀浆后将匀浆液离心，2000g×10min，将所得上清液进一步以78000g离心90min，取上清液，使用ELISA法检测肺组织中神经氨酸酶水平，检测试剂盒为神经氨酸酶检测试剂盒(购自碧云天)，按照检测试剂盒的操作说明书进行测定。
二、实验结果
与对照组相比，模型组小鼠肺纤维化面积显著升高，证明造模成功；与模型组相比，各给药组小鼠肺纤维化面积显著降低。模型组小鼠BALF中N-乙酰神经氨酸比对照组显著升高，肺组织中神经氨酸酶活性显著升高；各给药组小鼠BALF中N-乙酰神经氨酸比模型组显著降低，肺组织中神经氨酸酶活性也显著降低。结果如表1和图1所示。
表1各组小鼠肺纤维化面积、N-乙酰神经氨酸含量和神经氨酸酶活性比较
该实验证明，抑制神经氨酸酶活性，N-乙酰神经氨酸含量下降，肺纤维化小鼠的肺纤维化得到显著改善，神经氨酸酶为抑制肺纤维化的靶点。
实施例2：神经氨酸酶及抑制剂与肾纤维化的关系
一、实验方法
分组及给药：雄性清洁级Wistar大鼠(体重160-180g)适应性饲养3-5d后，随机分为假手术组(对照组)、模型组和扎那米韦、磷酸奥司他韦、黄连碱、丹酚酸B给药组。除对照组外，其余各组大鼠均进行造模。造模方法如下：通过ip 10％水合氯醛(4ml/kg)将大鼠麻醉后，将大鼠右侧卧位固定于手术台上，常规备皮消毒，行左侧腹切口，逐层分离组织直至腹膜，暴露并游离左侧输尿管，分别用丝线结扎输尿管上1/3处以及中1/3处，再在两结扎点之间切断输尿管，最后逐层缝合。对照组大鼠仅打开腹腔和游离左侧输尿管，不结扎，不切断。造模后，可通过肾脏组织病理观察到炎症细胞浸润，胶原成分增加，结扎侧肾脏明显增大，肾小管上皮细胞变性、脱落甚至坏死，肾小管部分萎缩或者扩张，即表明造模成功。
造模2h后，给药组大鼠分别给予0.2mg/kg/d的扎那米韦(i.v.)、5mg/kg/d磷酸奥司他韦(p.o.)、40mg/kg/d黄连碱(p.o.)、40mg/kg/d丹酚酸B(p.o.)，模型组和对照组灌胃给予等体积溶媒0.5％CMC-Na，连续给药4周。末次给药2h后腹主动脉放血处死，迅速取肾脏组织，一部分用4％多聚甲醛固定，用于病理检查；剩余-80℃条件下保存，用于指标检测。
肾组织病理学检查：肾脏组织于4％多聚甲醛中固定，石蜡包埋、切片(厚度5μm)，Masson's染色，观察肾组织病理形态学改变及胶原纤维含量。
肾组织神经氨酸酶活性测定：将病理学检测取材后的其余肾组织置于冰上，用冰冷的生理盐水冲洗干净，用滤纸吸干表面水分，称重，按500g/L的比例加入含0.25mmol/L蔗糖和1mmol/L EDTA的匀浆液匀浆，充分匀浆后将匀浆液离心，2000g×10min，将所得上清液进一步以78000g离心90min，取上清液，使用ELISA法检测肾组织中神经氨酸酶水平，检测试剂盒为神经氨酸酶检测试剂盒(购自碧云天)，按照检测试剂盒的操作说明书进行测定。
二、实验结果
与对照组相比，模型组大鼠肾纤维化面积显著升高，证明造模成功；与模型组相比，各给药组大鼠肾纤维化面积显著降低。模型组大鼠肾组织中神经氨酸酶活性显著升高，各给药组大鼠肾组织中神经氨酸酶活性也显著降低。结果如表2和图2所示。
表2各组大鼠肾纤维化面积和肾组织中神经氨酸酶活性比较
该实验证明，抑制神经氨酸酶活性，肾纤维化大鼠的肾纤维化得到显著改善，神经氨酸酶为抑制肾纤维化的靶点。
实施例3：
采用市售的神经氨酸酶抑制剂筛选试剂盒P0309(碧云天，Beyotime)对丹酚酸B进行体外抑制活性测试，阳性对照药为磷酸奥司他韦。在96孔板上每孔加入70μL缓冲液和10μL神经氨酸酶溶液，再加入10μL不同浓度的待测液，振动混匀，在37℃孵育5min，加入10μL含荧光底物的溶液，振动混匀，在37℃孵育30min，进行荧光测定，其中激发波长为322nm，发射波长为450nm。根据荧光读数计算出不同待测液的抑制率，并进一步获得阳性对照药磷酸奥司他韦和丹酚酸B的IC50值。结果如表3所示。
表3阳性对照药磷酸奥司他韦和丹酚酸B的IC50值
| 磷酸奥司他韦 | 丹酚酸B | |
| IC50值(nmol/L) | 13.5 | 195.5 |
上述实施例证明，丹酚酸B在体外可以抑制神经氨酸酶的活性，表现出中等强度的神经氨酸酶抑制活性，为神经氨酸酶的抑制剂。
已知扎那米韦、磷酸奥司他韦为神经氨酸酶的有效抑制剂，黄连碱对神经氨酸酶的抑制作用也已在申请人先前的专利申请中公开，申请人又发现丹酚酸B在体外可以抑制神经氨酸酶的活性，为神经氨酸酶的抑制剂；试验证明，这几种神经氨酸酶抑制剂可以有效抑制肺和肾组织中神经氨酸酶活性，进而抑制肺纤维化和肾纤维化。由此可见，神经氨酸酶及其抑制剂可用于制备治疗肺纤维化和肾纤维化的药物。
上述实施例的作用在于具体介绍本发明的实质性内容，但本领域技术人员应当知道，不应将本发明的保护范围局限于该具体实施例。
Claims (6)
1.神经氨酸酶在制备治疗肺纤维化的药物中的应用。
2.神经氨酸酶在制备治疗肾纤维化的药物中的应用。
3.神经氨酸酶抑制剂在制备治疗肺纤维化的药物中的应用。
4.神经氨酸酶抑制剂在制备治疗肾纤维化的药物中的应用。
5.黄连碱或丹酚酸B及其可药用盐用于制备神经氨酸酶抑制剂的用途。
6.扎那米韦、磷酸奥司他韦、黄连碱或丹酚酸B及其可药用盐用于制备治疗肺纤维化和肾纤维化的药物的用途。
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