CN111053764B - 丹酚酸a在制备治疗耐甲氧西林金黄色葡萄球菌感染性肺炎药物中的应用 - Google Patents
丹酚酸a在制备治疗耐甲氧西林金黄色葡萄球菌感染性肺炎药物中的应用 Download PDFInfo
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Abstract
本发明公开了丹酚酸A在制备治疗金黄色葡萄球菌感染性疾病药物中的应用。具体的,本发明公开了丹酚酸A在单独或联合抗生素治疗金黄色葡萄球菌,尤其是耐甲氧西林金黄色葡萄球菌感染性疾病中的应用。本发明的目的在于将丹酚酸引入金黄色葡萄球菌感染的治疗中,通过使用丹酚酸A抑制重要毒力因子Sortase A,显著降低金黄色葡萄球菌的致病力,有助于机体免疫***对病菌的清除。本发明还明确了丹酚酸A抑制金黄色葡萄球菌Sortase A的分子机制,为丹酚酸A治疗金黄色葡萄球菌感染提供可靠依据,为研发新的治疗药物提供了重要的前体化合物,也为同类中药的开发奠定一定的基础。与用抗生素治疗相比较,使用丹酚酸A治疗具有无耐药性,治愈率高的特点。
Description
技术领域
本发明涉及医药技术领域,尤其涉及一种丹酚酸A在制备治疗耐甲氧西林金黄色葡萄球菌感染性肺炎药物中的应用。
背景技术
金黄色葡萄球菌性肺炎是由金黄色葡萄球菌感染所引起的疾病。近年来,由于抗菌药物的广泛使用,细菌出现多重耐药性,使得细菌性肺炎的治疗面临无药可治的局面。目前,临床上金黄色葡萄球菌尤其是耐甲氧西林金黄色葡萄球菌(MRSA)已成为医院内肺部感染的重要致病菌,其所导致的肺炎病情较为严重,在全球具有较高的发生率和病死率,是儿童、老年人和免疫缺陷患者死亡的重要原因。目前治疗金黄色葡萄球菌肺炎的药物通常使用苯挫西林和氯挫西林,严重的MRSA感染可以使用糖肽类抗生素万古霉素治疗,但由于抗万古霉素的MRSA菌的出现,常常会导致治疗失败。因此,临床仍急需疗效显著且安全性高的治疗药物,寻找有效抑制病菌致病力的化合物仍是当前重要的研究课题。
金黄色葡萄球菌的发病机制受到大量不同毒力蛋白的调节,这些蛋白质可介导与宿主细胞外基质的附着,抑制补体活性,抑制抗体功能,裂解宿主细胞,使其定殖于身体的不同组织并通过宿主天然防御***逃避免疫消除。因此,靶向毒力蛋白是一种有前景的替代抗生素治疗金黄色葡萄球菌的方法。金黄色葡萄球菌分选酶SrtA可以将金黄色葡萄球菌的细胞壁锚定(CWA)蛋白锚定到细胞壁上。缺乏CWA蛋白的金黄色葡萄球菌的SrtA突变株在各种小鼠感染模型中的毒力显著降低。因此,通过使用抑制剂阻断SrtA的活性来破坏CWA蛋白在菌表面的锚定可以有效地降低细菌毒力,从而促进宿主对细菌的清除。由于SrtA不是细菌生长和增殖的必要组分,抑制SrtA不会引起细菌耐药性。此外,SrtA是一种“管家”分选酶,其在各种临床金黄色葡萄球菌株中是保守的。因此,通过抑制金黄色葡萄球菌分选酶SrtA从而减弱毒力以阻碍金黄色葡萄球菌感染是一个有行之有效的策略。
发明内容
有鉴于此,本发明要解决的技术问题在于提供一种丹酚酸A在制备治疗耐甲氧西林金黄色葡萄球菌感染性肺炎药物中的应用,对金黄色葡萄球菌的 SrtA具有较高的抑制活性。
丹酚酸A,结构式如式Ⅰ所示,化学名为(2R)-3-(3,4-二羟基苯基)-2-[(E)-3-[2-[(E)-2-(3,4-二羟基苯基)乙烯基]-3,4-二羟基苯基]丙-2-烯酰]氧丙酸,是一种从临床常用的唇形科植物丹参(Salvia miltiorrhiza Bunge)的根茎中分离得到的水溶性酚酸类化合物。药理学研究表明,丹酚酸A具有多种药理活性,如抗氧化、抗炎、抗动脉粥样硬化、抗血栓形成以及神经保护作用等。
本发明首次公开了丹酚酸A具有预防和/或治疗金黄色葡萄球菌感染的作用,可用于治疗由金黄色葡萄球菌引起的肺炎等疾病。通过使用丹酚酸A抑制金黄色葡萄球菌的重要毒力因子SrtA,显著降低金黄色葡萄球菌的致病力,有助于机体免疫***对病菌的清除。
具体的,本发明公开了丹酚酸A在制备治疗金黄色葡萄球菌感染性疾病药物中的应用。
进一步的,本发明公开了丹酚酸A在制备治疗金黄色葡萄球菌感染性肺炎药物中的应用。
更进一步的,本发明公开了丹酚酸A在制备治疗耐甲氧西林金黄色葡萄球菌感染性肺炎药物中的应用。
本发明公开了丹酚酸A在制备抑制金黄色葡萄球菌分选酶Sortase A药物中的应用。
本发明中,所述丹酚酸A可单独使用,或与其他药物多药联合使用。如与抗生素联合使用,对金黄色葡萄球菌感染性肺炎进行治疗。
本发明中,丹酚酸A可以和药学上可接受的辅料制备成药学上可接受的各种剂型,如胶囊剂、注射剂或片剂等本领域技术人员熟知的剂型,本发明对此并无限定。
本发明的有益效果:
1、本发明的目的在于将丹酚酸A引入到金黄色葡萄球菌感染性疾病的治疗中,公开了丹酚酸A在制备治疗金黄色葡萄球菌感染性疾病药物中的新用途,公开了丹酚酸A对金黄色葡萄球菌的毒力因子Sortase A具有抑制作用,从而降低金黄色葡萄球菌对Fg的粘附作用,降低金黄色葡萄球菌生物被膜的形成以及表面蛋白SpA的展示以及降低金黄色葡萄球菌对肺细胞A549的侵袭。进一步体内实验显示丹酚酸A可以显著提高小鼠的致死性MRSA菌感染性肺炎的生存率。
2、本发明公开了丹酚酸A在多药联合治疗金黄色葡萄球菌感染性肺炎中的应用,采用丹酚酸A和抗生素多药联合方式对金黄色葡萄球菌感染性肺炎进行治疗,引入的丹酚酸A在显著降低金黄色葡萄球菌的毒力的同时,还可以显著降低肺脏组织的炎症反应,起到增效作用,有助于提高抗生素临床治疗的有效性。
3、本发明还明确了丹酚酸A可有效减轻肺炎模型小鼠肺脏损伤严重程度,减少炎症细胞聚集,降低肺脏内病菌的载量,这些研究成果为丹酚酸A 治疗金黄色葡萄球菌感染性肺炎提供可靠依据,为研发新的治疗药物开辟新的途径,也为同类中药的开发奠定一定的基础。
4、由于丹酚酸A通过抑制金黄色葡萄球菌重要的毒力因子SrtA降低其致病力,对细菌的生存不施加压力,因而同抗生素治疗相比,不会引起耐药性的产生。
附图说明
图1为丹酚酸A对金黄色葡萄球菌USA300 SrtA的体外活性抑制作用柱形图;
图2为丹酚酸A对金黄色葡萄球菌USA300生长的影响曲线图;
图3为丹酚酸A抑制金黄色葡萄球菌对人肺上皮细胞的侵袭柱形图;
图4为丹酚酸A对金黄色葡萄球菌肺炎的肺组织保护切片图;
图5为丹酚酸A对小鼠金黄色葡萄球菌肺炎的保护率曲线图。
具体实施方式
为了进一步说明本发明,下面结合实施例对本发明提供的丹酚酸A在制备治疗耐甲氧西林金黄色葡萄球菌感染性肺炎药物中的应用进行详细描述。
实施例1、丹酚酸A对金黄色葡萄球菌毒力因子SrtA的抑制作用
将含有pET28a-SrtA载体的表达菌大肠杆菌BL21,接种到LB培养基中, 37℃,200rpm震荡过夜。次日,以1:100的比例重新接种到加入卡纳抗性的 LB培养基中,37℃,200rpm培养至OD600达0.6-0.8。取1mL菌液作为诱导前对照。其余冰浴30min,加入终浓度为1mM的IPTG 16℃,180rpm诱导过夜。次日,取1mL菌液作为诱导后对照,离心收集菌体(4000rpm,30min)。用蛋白缓冲液将收集回的菌体重悬,使用超声破碎仪对重悬的菌体进行超声破碎,使用离心机分别收集破碎后的上清和沉淀(12000rpm,1h)。最后,通过SDS-PAGE电泳检测诱导前、诱导后、上清、沉淀中蛋白的表达并采用 Ni-NTA亲和层析技术,对带有His标签的SrtA蛋白进行分离纯化。为了确保纯化后的蛋白有活性,实验需在4℃下进行。
通过荧光共振能量转移(FRET)方法测定分选酶A的活性。该测定在 96孔黑色板中进行。合成的底物肽Abz-LPATG-Dap(Dnp)-NH2用作荧光内部淬灭的底物。孔中含有10μM纯化的SrtA蛋白,不同浓度的丹酚酸A以及反应缓冲液,终体积为300μl。反应在37℃下进行1小时。然后加入底物肽,并在37℃再孵育1小时,最后使用酶标仪读取荧光(激发波长350nm,发射波长495nm)。只加入缓冲液和底物肽作为对照。通过下式确定药物对SrtA活性的抑制率:100%×(C-T)/C,其中C是未处理组的荧光值,T是实验组的荧光值。
结果如附图1所示,丹酚酸A对SrtA的活性有明显的抑制作用,并且可以以剂量依赖性方式降低荧光信号。通过计算,丹酚酸A的半数最大抑制浓度(IC50)值为5.75μg/mL。
实施例2,丹酚酸A对金黄色葡萄球菌USA300生长的影响
为了进一步确定丹酚酸A对金黄色葡萄球菌USA300的生长没有影响,测定了在256μg/mL的丹酚酸A存在下金黄色葡萄球菌USA300的生长。将过夜培养的金黄色葡萄球菌USA300以1:100的比例加入含有或不含有256μg/mL中药单体的无菌BHI肉汤中培养,并在37℃下振荡生长24小时。以只加入DMSO组作为对照。使用紫外分光光度计在不同的时间间隔测量 600nm处的吸光度,并绘制生长曲线。结果如附图2所示。即使丹酚酸A的剂量是其IC50的40倍,金黄色葡萄球菌的生长速率仍与WT相似。结果表明,丹酚酸A可用作潜在的抗毒力因子而不影响金黄色葡萄球菌USA300的生长,应用丹酚酸A处理金黄色葡萄球菌不会诱导耐药性的产生。
实施例3、丹酚酸A抑制金黄色葡萄球菌对A549细胞的侵袭
将人肺上皮细胞A549悬浮于DMEM/高葡萄糖培养基(含10%胎牛血清) 中,然后以3×105个细胞接种到24孔细胞培养板中(含有12mm直径的盖玻片),置于细胞培养箱中,于37℃,5%CO2培养过夜。将过夜培养的金黄色葡萄球菌1:100接种到含有终浓度为64μg/mL和128μg/mL丹酚酸A的培养基中,继续培养至吸光度为OD600nm=1.0后备用。从各组取出1mL金黄色葡萄球菌悬液(吸光度为OD600nm=1.0),加到24孔细胞培养板内对应的细胞中,每组3个重复,在37℃,5%CO2的条件下孵育60分钟。每个孔用PBS冲洗 3次,再加入1mL补充有300μg/mL庆大霉素的DMEM/高葡萄糖培养基,孵育30分钟(37℃,5%CO2),清除未进入细胞的金黄色葡萄球菌。每个孔用 PBS冲洗3次,将盖玻片上的A549细胞放在无菌蒸馏水中裂解,然后在BHI 琼脂培养板上计数CFU。结果如附图3所示,表明丹酚酸A能够显著减少金黄色葡萄球菌对人肺上皮细胞侵袭,该作用呈现剂量依赖性。
实施例4、小鼠金黄色葡萄球菌肺炎的实验治疗学研究
4.1建立小鼠金黄色葡萄球菌肺炎模型
挑取金黄色葡萄球菌单菌落于BHI培养基中,37℃,200rpm培养过夜。次日,以1:100的比例传代到新鲜的BHI培养基,继续摇至OD600nm=1.0,将菌液5000rpm离心10分钟收集菌体,用PBS洗涤3次,之后调OD600nm=1.0,备用。将雄性,体重18-22克的C57BL/6J小鼠使用***后,抓住小鼠背部使其直立,通过滴鼻接种制备好的菌液30μl,让菌液自然吸入,吸入后继续使小鼠保持直立30秒,再将小鼠放平,使其自然苏醒。对于致死率研究,滴鼻给金黄色葡萄球菌重悬液30μl(2×109CFU);对于病理组织学考察组,滴鼻接种金黄色葡萄球菌悬液30μl(1×109CFU)。
4.2丹酚酸A对小鼠肺脏组织病理学特征
肺部病理学组织切片结果表明(如附图4所示),接种金黄色葡萄球菌的小鼠注射生理盐水组病理切片显示肺组织显著充血,并且肺泡沟中有大量炎性细胞积聚,并且观察到明显的空泡化。用丹酚酸A处理后,肺组织充血较轻,肺泡腔的炎症细胞浸润明显减少,肺泡结构较完整,表明肺部炎症得到缓解。
4.3丹酚酸A对小鼠金黄色葡萄球菌肺炎的保护率实验
用2×109CFU的金黄色葡萄球菌USA300感染小鼠。将感染后用生理盐水处理的小鼠用作阴性对照。如图5所示,WT+生理盐水组在12h、24h、36h、 48h、60h、72h、84h、96h的死亡率分别为10%、40%、50%、70%、80%、 80%、80%、80%。在注射100mg/kg的丹酚酸A后,WT+丹酚酸A组在12h、 24h、36h、48h、60h、72h、84h、96h的死亡率分别为10%、10%、20%、20%、 20%、20%、20%、20%。通过生存分析显示WT+丹酚酸A组显示出显著的生存优势,特别是在感染后的早期时间点。这些结果表明,丹酚酸A可以使感染小鼠的存活时间增长,并且能保护小鼠不在感染初期死亡。
附图5表明,丹酚酸A处理,可以显著降低小鼠金黄色葡萄球菌肺炎的死亡率(P<0.05)。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求的保护范围内。
Claims (5)
1.丹酚酸A作为唯一活性成分在制备治疗金黄色葡萄球菌感染性疾病药物中的应用。
2.丹酚酸A作为唯一活性成分在制备治疗金黄色葡萄球菌感染性肺炎药物中的应用。
3.丹酚酸A作为唯一活性成分在制备治疗耐甲氧西林金黄色葡萄球菌感染性肺炎药物中的应用。
4.丹酚酸A作为唯一活性成分在制备抑制金黄色葡萄球菌分选酶Sortase A药物中的应用。
5.根据权利要求1~4任一项所述的应用,其特征在于,所述药物为胶囊剂、注射剂或片剂。
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