CN117959272A - 异阿魏酸在制备肺炎克雷伯菌荚膜抑制剂中的应用 - Google Patents
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Abstract
肺炎克雷伯菌可导致牛、猪、鹿、鸡等畜禽及人类呼吸系统感染、消化系统感染、菌血症及其他化脓性感染,是临床重要致病菌。荚膜作为肺炎克雷伯菌的基本毒力因子,可通过阻断补体结合和杀伤、阻碍免疫细胞吞噬等多种途径促进感染,是抗肺炎克雷伯菌感染的潜在靶点。本发明以K2型临床分离肺炎克雷伯菌K7菌株为研究模式菌株,通过糖醛酸试验筛选获得了可显著抑制荚膜合成的化合物异阿魏酸,并通过电镜观察、离心抵抗及小鼠肺炎克雷伯菌肺炎治疗实验等阐明异阿魏酸能够通过抑制荚膜合成有效保护小鼠。异阿魏酸作为荚膜抑制剂将为抗肺炎克雷伯菌感染提供新途径和先导化合物,在耐药水平居高不下的现况之下,具有极高的开发意义和应用前景。
Description
技术领域
本发明属于医学制药技术领域,涉及异阿魏酸在制备肺炎克雷伯菌荚膜抑制剂中的医药用途。
背景技术
肺炎克雷伯菌(Klebsiella pneumoniae,K.pneumoniae)是引起牛、猪、鹿、鸡等畜禽以及人类医院和社区获得性感染的重要人兽共患病原菌,可引起呼吸系统感染、尿路感染、菌血症及其他化脓性感染。近年来多重耐药肺炎克雷伯菌危害日益加重,给养殖业特别是奶牛养殖业造成了巨大经济损失。目前,抗生素依然是治疗细菌感染的首选,然而多重耐药肺炎克雷伯菌对一线抗生素几乎全部耐药,碳青霉烯类抗生素作为最后一道防线也被逐渐突破。流行病学调查结果显示我国已成为耐碳青霉烯肺炎克雷伯菌(CRKP)的高发地区,CRKP检出率从2005年的3%骤然上升至25%,临床亟需抗生素以外的治疗策略。
荚膜是主要由多糖和水分组成、包围在细菌表面的一层保护性结构,也被称为“K抗原”。大量研究表明肺炎克雷伯菌可利用荚膜在干燥、极酸及饥饿等不良条件下生存,并在突破机体皮肤、黏膜等天然屏障侵入宿主体内后利用荚膜通过以下机制促进感染:①阻止免疫细胞对细菌的吞噬作用和调理吞噬作用;②通过与外膜远端的分子结合阻碍防御素、乳铁蛋白等抗菌肽的杀菌作用;③阻断补体成分与细菌膜的互作,进而阻止补体的裂解和调理作用;④通过协助激活NOD依赖性途径和屏蔽LPS被免疫细胞受体识别,减少活性氧(ROS)、IL-8、IL-6和TNF-α,避免免疫反应的暴发性激活。荚膜作为最基本的毒力因子对肺炎克雷伯菌致病至关重要,以其为靶点进行抑制剂筛选将为抗肺炎克雷伯菌感染提供新的途径和先导化合物,在耐药水平居高不下甚至依然呈上升态势的背景下,对多肺炎克雷伯菌感染防治具有重要意义。
异阿魏酸是广泛存在于丹参及升麻等植物的一种酚酸类化合物,其化学结构式见附图1。现代药理学研究表明其有抗糖尿病、抗流感病毒和增强β-内啡肽分泌等药理活性。目前,国内外尚未见异阿魏酸在制备肺炎克雷伯菌荚膜抑制剂的报道。
发明内容
本发明的目的:提出了异阿魏酸在制备荚膜抑制剂中的医用用途,为防治肺炎克雷伯菌感染提供了一种不依赖抗生素效应的新型策略和前导化合物。
本发明以K2型临床分离肺炎克雷伯菌K7菌株为研究模式菌株,通过糖醛酸试验筛选获得了在不影响细菌生长的浓度下可显著抑制荚膜合成的化合物异阿魏酸,其可显著降低荚膜厚度,有效削弱荚膜介导的离心抵抗,小鼠肺炎克雷伯菌肺炎治疗实验确证异阿魏酸能够通过抑制荚膜合成有效保护小鼠。
附图说明
图1:异阿魏酸的化学结构式。
图2:异阿魏酸在一定浓度范围内不影响肺炎克雷伯菌的生长(横轴表示时间,纵轴表示OD600测定值)。
图3:异阿魏酸剂量依赖性地降低肺炎克雷伯菌表面荚膜多糖的含量(横轴表示异阿魏酸浓度,纵轴表示糖醛酸含量)
图4:异阿魏酸抑制肺炎克雷伯菌的荚膜厚度。
图5:异阿魏酸剂量依赖性地削弱荚膜介导的离心抵抗(横轴表示异阿魏酸浓度,纵轴表示离心后上清的OD600测定值与初始菌液OD600测定值的比值)。
图6:异阿魏酸在小鼠肺炎克雷伯菌肺炎模型中的保护作用。
具体实施方式
通过以下实施例进一步举例描述本发明,并不以任何方式限制本发明,在不背离本发明的技术解决方案的前提下,对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
异阿魏酸可作为肺炎克雷伯菌荚膜抑制剂用于药学上可接受的任何载体。
实施例2
异阿魏酸可作为荚膜抑制剂用于制备治疗肺炎克雷伯菌感染的药物。
试验例1
异阿魏酸对肺炎克雷伯菌最小抑菌浓度(Minimal inhibition concentration,MIC)值测定
根据美国临床和实验室标准协会公布的最小抑菌浓度值测定试验标准稀释法测定异阿魏酸对肺炎克雷伯菌的MIC值。用LB培养基将过夜培养的K7菌液OD600调至0.1,待用。在1.5ml的离心管中用LB培养基倍比稀释异阿魏酸,使得其终浓度为4μg/ml,8μg/ml,16μg/ml,32μg/ml,64μg/ml,128μg/ml,256μg/ml和512μg/ml。将200μl含不同浓度异阿魏酸的培养基加至96孔板,再在每孔加入10μl调整好的细菌悬液(使细菌接种量为5×105CFU/ml)。将96孔板置于振板器上振荡混匀之后置于37℃,5% CO2温箱内培养。次日在适宜的光线下观察96孔板上的细菌生长情况。肉眼可见抑制细菌生长的化合物浓度判定为该化合物对肺炎克雷伯菌的MIC值。
结论:异阿魏酸对肺炎克雷伯菌的MIC值大于512μg/ml。
试验例2
生长曲线测定
将过夜培养的K7菌液按1:100扩培至新鲜LB培养基中,待OD600为0.3左右时量取菌液平均分装至五个50ml锥形瓶中(每瓶约20ml),分别加入不同浓度(0、4、8、16、32、64μg/ml)的异阿魏酸。在37℃,180rpm条件下继续培养,每隔1h测定每组样品的OD600,直至生长至平台期。
结论:异阿魏酸在受试范围内不影响肺炎克雷伯菌的生长,见附图2。
试验例3
糖醛酸分析实验
过夜培养的K7菌液按1:50扩培至2ml含不同浓度异阿魏酸的新鲜LB培养基中继续培养,4h后取500μl菌液与100μl 1%zwitergent(w/v,100mM柠檬酸)充分混合,在50℃下孵育20分钟;12000×g离心5min,取300μl上清与1.2ml 100%乙醇混合均匀,于4℃沉淀20min。再次离心并弃上清,待沉淀干燥后溶解于蒸馏水中,加入四硼酸钠-浓硫酸进行涡旋混合,煮沸5分钟后在冰上冷却,最后加入0.15%3-苯基苯酚(w/v,0.5% NaOH)检测520nm处的吸光值,根据半乳糖苷制作的标准曲线计算糖醛酸含量。
结论:异阿魏酸剂量依赖性地降低肺炎克雷伯菌表面荚膜多糖的含量,在16μg/ml时实现统计学差异,见附图2。
试验例4
透射电镜观察肺炎克雷伯菌荚膜结构
将肺炎克雷伯菌与32μg/ml异阿魏酸共培养至OD600=0.6左右,取1ml离心收集细菌团,用无菌PBS洗涤2遍后加入1ml 4%戊二醛溶液于4℃固定过夜;次日,将样品包埋于LR白色树脂中,制成超薄切片后用2%醋酸铀酰水溶液负染,利用透射电子显微镜检测荚膜厚度。
结论:32μg/ml异阿魏酸处理可以显著降低肺炎克雷伯菌表面荚膜结构的厚度,见附图4。
试验例5
肺炎克雷伯菌离心抵抗试验
将肺炎克雷伯菌与不同浓度(0、4、8、16、32、32μg/ml)的异阿魏酸共培养4h,取1ml培养物以1000×g离心5min,吸取上清,吹匀后用紫外分光光度计测OD600,统计离心后上清的OD600与菌液初始OD600的比值。
结论:溶剂对照组肺炎克雷伯菌由于较高的荚膜含量难以离心沉淀,而异阿魏酸处理剂量依赖性地削弱了由荚膜介导的离心抵抗,且在4μg/ml时即达到了显著差异水平,见附图5。
试验例6
建立小鼠肺炎克雷伯菌肺炎模型评估异阿魏酸的体内治疗效果
1×107CFU K7肺炎克雷伯菌以滴鼻的方式感染6-8周龄的雌性C57BL/6小鼠,感染1h后经皮下注射给予异阿魏酸(50mg/kg)进行治疗,每隔8h给一次药,连续治疗3天,在每隔时间点观察并记录各组的存活数量,记录至5天后绘制存活曲线。
结论:1×107K7感染导致溶剂对照组小鼠在72h内全部死亡,而50mg/kg异阿魏酸治疗可将肺炎克雷伯菌肺炎小鼠的存活率提高至50.0%,见附图6。
Claims (3)
1.异阿魏酸在制备肺炎克雷伯菌荚膜抑制剂中的应用。
2.如权利要求1所述的应用,其特征在于,所述的异阿魏酸对肺炎克雷伯菌荚膜合成具有显著的抑制作用。
3.如权利要求1所述的应用,其特征在于,异阿魏酸可作为荚膜抑制剂用于制备治疗肺炎克雷伯菌感染的药物。
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