CN114832000B - Lpe 16:0在制备抗呼吸道合胞病毒感染的药物中的应用 - Google Patents
Lpe 16:0在制备抗呼吸道合胞病毒感染的药物中的应用 Download PDFInfo
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Abstract
本发明公开了LPE 16:0在制备抗呼吸道合胞病毒感染的药物中的应用。本发明发现，LPE16:0具有明显的抗呼吸道合胞病毒感染的活性，且LPE 16:0为一种人体内的内源性物质，副作用低，药物安全性高。因此，LPE 16:0具备开发成抗呼吸道合胞病毒感染的药物的前景。
Description
技术领域
本发明属于医药领域，涉及已知化合物的新用途，具体涉及LPE 16:0在制备抗呼吸道合胞病毒感染的药物中的应用。
背景技术
呼吸道合胞病毒(respiratory syncytial virus，RSV)是一种副黏液病毒类型的RNA病毒，主要通过飞沫、接触等传播，它是引起全球5岁以下儿童急性下呼吸道感染(acutelower respiratory tract infection，ALRTI)最重要的病毒体，也是婴幼儿住院甚至死亡的重要因素。。
目前尚无LPE 16:0用于抗呼吸道合胞病毒感染的报道。
发明内容
本发明的目的在于提供LPE 16:0在制备抗呼吸道合胞病毒感染的药物中的应用。
本发明上述目的通过如下技术方案实现：
一种LPE 16:0在制备抗呼吸道合胞病毒感染的药物中的应用。
进一步地，所述药物以LPE 16:0为活性成分，还含有药学上可以接受的载体，制成药学上可以的剂型。
更进一步地，所述载体包括固体、液体和半固体载体。
更进一步地，所述剂型包括片剂、胶囊、注射剂和滴剂。
有益效果：
本发明发现，LPE 16:0具有明显的抗呼吸道合胞病毒感染的活性，且LPE 16:0为一种人体内的内源性物质，副作用低，药物安全性高。因此，LPE 16:0具备开发成抗呼吸道合胞病毒感染的药物的前景。
附图说明
图1为各组小鼠的肺组织病理(200×)；
图2为各组小鼠的肺组织病理评分；
图3为各组小鼠指标MCP-1检测情况。
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。
一、实验材料
1、仪器
ALLegra 64R高速冷冻离心机(美国Beckman公司)；Revco UXF超低温冰箱(美国Thermo Fisher公司)；CPA225D十万分之一电子天平(德国Sartorius公司)；Nikon Ti荧光显微镜(日本尼康公司)；RM2125 RTS石蜡切片机、HistoCore包埋机(德国Lecia公司)；DP45烘片机、DK45摊片机、LTF-K脱水机(中国派斯杰公司)；DHG-9071A恒温箱(中国精宏公司)；MM400混合球磨仪(德国RetSch公司)；蛋白核酸分析仪、PCR逆转录仪(德国Eppendorf公司)；Quantstudio 7Flex荧光定量PCR仪(美国Life technologies公司)。
2、试剂
磷酸盐缓冲液(PBS)购于上海源培生物科技股份有限公司；溶血磷脂酰乙醇胺(LPE 16:0)、溶血磷脂酰胆碱(LPC 16:0)、溶血磷脂酰甘油(LPG 16:0)均购于上海源叶生物科技有限公司；无水乙醇、二甲苯购于南京化学试剂股份有限公司；苏木素、伊红购于雷根生物技术有限公司；HiScript III RT SuperMix for qPCR(+gDNAwiper)、ChamQUniversal SYBR qPCR Master Mix、FastPure Cell/Tissue Total RNA Isolation KitV2均购于南京诺唯赞生物科技有限公司；SYBR Green染料购于美国Bio-Rad公司。
二、实验方法
1、动物培养、造模、分组和给药
SPF级BALB/c雌性健康小鼠108只，体重18-22g，6-8周龄，购于南京青龙山动物养殖场，动物合格证号：SCXK(沪)2018-0006。适应性饲养3天，开始进行动物实验，饲养条件：室温24-26℃，相对湿度55％，12小时光暗昼夜循环，分笼饲养，自由进食饮水。
适应性饲养3天后，将108只BALB/c雌性小鼠随机分为9组，分别为空白对照组(n＝12)、3天RSV模型组(n＝12)、7天RSV模型组(n＝12)、3天LPC 16:0给药组(n＝12)、7天LPC16:0给药组(n＝12)、3天LPE 16:0给药组(n＝12)、7天LPE 16:0给药组(n＝12)、3天LPG16:0给药组(n＝12)、7天LPG 16:0给药组(n＝12)。除空白对照组外，其余各组小鼠在乙醚浅麻醉后，以RSV病毒(1×106PFU)给小鼠滴鼻感染，每鼠100μL，空白对照组采用同法滴入等体积无菌生理盐水。各组小鼠于造模当天开始每日滴鼻给药，空白对照组、RSV模型组给予生理盐水，3天LPC 16:0给药组、3天LPE 16:0给药组、3天LPG 16:0给药组分别滴鼻给药LPC 16:0、LPE 16:0、LPG 16:0，连续给药3天，7天LPC 16:0给药组、7天LPE 16:0给药组、7天LPG 16:0给药组分别滴鼻给药LPC 16:0、LPE 16:0、LPG 16:0，连续给药7天，所有组给药剂量均为400μg/只/天。
2、样本采集
滴鼻给药第三天，将空白对照组、3天模型组、3天LPC 16:0给药组、3天LPE 16:0给药组、3天LPG 16:0给药组的小鼠摘眼球采集血液样本，脱颈处死，保留肺组织样本。其中每组选6只采集小鼠右肺中叶用10％中性甲醛固定，剩余肺组织放入-80℃保存。
滴鼻给药第七天，将7天LPC 16:0给药组、7天LPE 16:0给药组、7天LPG 16:0给药组的小鼠摘眼球采集血液样本，脱颈处死，保留肺组织样本。其中每组选6只采集小鼠右肺中叶用10％中性甲醛固定，剩余肺组织放入-80℃保存。
3、指标检测
3.1肺组织病理切片处理
HE染色：取用多聚甲醛固定的肺组织，乙醇梯度脱水，2次二甲苯透明，石蜡包埋，切片，苏木精-伊红染色，通过光镜观察肺部形态，拍照。
组织病理学评分方法：参照苏宁、姚全胜等主编的《新药毒理实验动物组织病理学图谱》，盲法观察，以0-3的等级对肺组织的血管、间质、免疫细胞浸润情况等进行评价。无病变：0；轻度病变：1；中度病变：2；重度病变：3。
3.2实时荧光定量PCR实验
取第3天各组小鼠右肺组织15mg置于2ml的圆底EP管中，据FastPure Cell/TissueTotal RNA Isolation Kit V2说明书，提取肺组织中的总RNA，蛋白核酸分析仪对RNA进行浓度定量分析，按照qPCR试剂盒说明书将总RNA反转录成cDNA，SYBR Green II实时荧光定量PCR法检测肺组织中MCP-1的RNA转录水平。数据采用2-ΔΔCt法进行计算分析，以GAPDH作为内部对照对每个基因的表达水平进行标准化。表1为引物序列，实时荧光定量PCR反应条件(采用两步法)：95℃预变性15s，95℃变性5s，60℃退火34s，设置40个循环。
表1引物序列
3、数据分析
数据处理及作图采用统计软件Graphpad Prism 8，数据采用Mean±SD表示，多组数据采用非参数检验(Kruskal–Wallis test)分析，P<0.05表示差异有统计学意义。
三、实验结果
1、各组小鼠肺组织病理学观察
各组小鼠的肺组织HE染色结果见图1，评分见图2(注：#P<0.01，##P<0.01，###P<0.001，与空白对照组相比较；*P<0.05，**P<0.01，***P<0.01与模型组相比较)。结果显示，空白对照组小鼠的肺呈现正常形态，结构完整，肺泡壁很薄，支气管腔内无渗出物，黏膜上皮无变性坏死脱落，管壁及周围组织无淋巴细胞浸润。3天RSV模型组和7天RSV模型组小鼠肺形态出现异常，大量肺泡壁增厚，支气管上皮细胞出现病变，病变处结构不清晰，可见脱落的上皮细胞。3天LPE 16:0给药组、7天LPE 16:0给药组的小鼠肺组织结构均较模型组清晰，病理损伤明显改善。3天LPC 16:0给药组、7天LPC 16:0给药组小鼠肺形态出现异常，组织局部实质化，结构紊乱，肺泡壁结构不清晰，支气管管腔内可见脱落的上皮细胞，局部血管周围可见少量淋巴细胞浸润，并可见血管损伤，平滑肌细胞胞核固缩深染。3天LPG 16:0给药组、7天LPG 16:0给药组小鼠组织局部肺泡腔狭窄，肺泡壁结构较不清晰，肺泡腔内可见巨噬细胞，肺泡壁上有淋巴细胞与中性粒细胞浸润，局部可见较多肺泡腔内有嗜酸性物质渗出，局部支气管内可见脱落的上皮细胞团。提示，LPE 16:0对RSV感染导致的肺损伤改善作用显著，LPC 16:0、LPG 16:0对RSV感染导致的肺损伤未见明显改善作用。
2、实时荧光定量PCR实验
RSV感染机体后，机体中的巨噬细胞等会加强分泌趋化因子MCP-1以抵抗RSV感染。如图3，与空白对照组相比，模型组MCP-1表达水平显著升高，说明RSV感染模型造模成功；与模型组相比，LPE 16:0给药组MCP-1表达水平显著降低，说明RSV感染得到明显缓解。
上述实验表明，LPE 16:0具有明显的抗呼吸道合胞病毒感染的活性，另外2种测试内源性物质无明显抗呼吸道合胞病毒感染的活性。同时，LPE 16:0为一种人体内的内源性物质，副作用低，药物安全性高。因此，LPE 16:0具备开发成抗呼吸道合胞病毒感染的药物的前景。
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 (4)
1.一种LPE 16:0在制备抗呼吸道合胞病毒感染的药物中的应用，所述药物以LPE 16:0为活性成分。
2.根据权利要求1所述的应用，所述药物以LPE 16:0为活性成分，还含有药学上可以接受的载体，制成药学上可以的剂型。
3.根据权利要求2所述的应用，所述载体包括固体、液体和半固体载体。
4.根据权利要求2所述的应用，所述剂型包括片剂、胶囊、注射剂和滴剂。
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