CN115813890B - 石斛酚及作为多粘菌素佐剂在制备治疗细菌感染性疾病药物中的应用 - Google Patents
石斛酚及作为多粘菌素佐剂在制备治疗细菌感染性疾病药物中的应用 Download PDFInfo
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Abstract
本发明公开了石斛酚及作为多粘菌素佐剂在制备治疗细菌感染性疾病药物中的应用。本发明提供了石斛酚作为抗菌药物的用途,恢复多粘菌素耐药菌的敏感性,可解决细菌耐药性问题。
Description
技术领域
本发明涉及医药用途,特别涉及石斛酚小分子作为多粘菌素佐剂在制备治疗多药耐药革兰氏阴性菌感染疾病药物中的应用。
背景技术
随着多重耐药细菌的出现和传播,细菌对抗生素的耐药性严重威胁着全球公共卫生安全。产碳青霉烯的肠杆菌是威胁人类及动物健康最重要的耐药细菌之一。粘菌素,一种阳离子环肽抗生素,是对抗产碳青霉烯类肠杆菌的最后手段。粘菌素通过与革兰氏阴性细菌外膜的脂多糖结合,介导细胞膜破裂,细胞内内容物渗漏,最终导致细菌溶解,从而发挥其抗菌作用。然而,编码磷乙醇胺转移酶的可转移粘菌素抗性基因(mcr-1)减少了脂类A的负电荷,并介导了获得性粘菌素抗性,随后,很快发现了其他mcr变体,包括mcr-2-10,由于该类基因可由质粒携带,并引起水平传播,使得流行率大大增强。在中国mcr-1及其变体的阳性率高达45%,这极大地挑战了粘菌素的有效性,导致产碳青霉烯类肠杆菌感染的治疗失败。因此,迫切需要克服MCR介导的粘菌素耐药性的手段。因为昂贵的成本、长的周期和低的盈利,新型抗生素的开发管道自二十世纪90年代末以来一直处于枯竭状态,新型抗菌药的研发速度已远远落后于耐药性的发展速度,使得人类进入后抗生素时代。相比之下,抗生素佐剂通过减缓耐药性的发展和提高抗生素的效力来克服抗生素耐药性,是对抗多药耐药(MDR)细菌的一种经济有效的策略。克拉维酸,一种β-内酰胺酶抑制剂,是一个最成功的例子,它在很大程度上挽救了青霉素和头孢菌素类抗生素的疗效。因此,开发粘菌素佐剂是对抗粘菌素耐药性的重要手段。
植物占据地球上最大的生物量,进化出了很多类似药物功能的次级代谢物以应对感染。据报道,从1981年到2010年,大约65%的批准药物要么属于天然化合物,要么属于它们的半合成衍生物。截止到2018年,FDA共收到800余份植物药物的研究申请或会前申请,并批准了两项植物新药申请(茶多酚和Fulyzaq)。表明植物源小分子是一种很有前途的抗菌先导化合物来源。且天然化合物确实的作用效果、结构的多样性,来源的丰富性、作用的安全性提示我们,从植物中寻找具有“协同增效”活性的小分子天然化合物,通过与现有重要抗菌药配伍使用从而提高其抗菌效果,使其恢复对多重耐药病原菌的敏感性,是目前解决细菌耐药性问题的重要策略之一。
石斛酚是从中药铁皮石斛中分离得到的一种双苄基多酚天然产物,具有抗炎、抗氧化、抗肿瘤、抗白内障等活性。尽管石斛酚的多种药理作用已被揭示,但其在治疗耐药菌感染中的应用潜力尚未确定。
发明内容
发明目的:本发明目的是提供石斛酚及作为多粘菌素佐剂在制备治疗细菌感染性疾病药物中的应用。
技术方案:本发明提供石斛酚在制备治疗细菌感染性疾病药物中的应用。
石斛酚小分子作为多粘菌素佐剂在制备治疗细菌感染性疾病药物中的应用。
进一步地,所述细菌包括E.coli B2(mcr-1)、沙门菌(Salmonella)15E343(mcr-3)、肺炎克雷伯菌(Klebsiella Pneumoniae)19-2-1(mcr-8)大肠杆菌DH5α(pUC19-mcr-1)。
所述的石斛酚与亚抑菌浓度的粘菌素联合破坏多重耐药大肠杆菌E.coli B2的细胞膜。
所述的石斛酚可以下调参与LPS修饰基因的表达,并且亚抑菌浓度的粘菌素可增强石斛酚对参与LPS修饰基因的表达的抑制效果。
有益效果:本发明提供了新型的粘菌素佐剂可以恢复mcr阳性耐药菌对粘菌素的敏感性并且减缓粘菌素耐药性的发展,提高了细菌耐药性。
附图说明
图1石斛酚与粘菌素的体外协同活性,图A显示了石斛酚联合粘菌素对E.coli DH5α(pUC19-mcr-1)的协同抗菌活性;图B显示了石斛酚联合粘菌素对E.coli B2的协同抗菌活性;图C显示了石斛酚联合粘菌素对其他mcr变体的协同抗菌活性;图D显示了石斛酚和粘菌素组合的时间杀菌曲线;
图2石斛酚的溶血活性以及对粘菌素耐药性发展的影响,图A显示了石斛酚表现出较低的溶血率;图B显示了石斛酚可减弱高浓度粘菌素的溶血性;图C为E.coli B2在亚抑菌浓度的粘菌素或粘菌素与石斛酚的组合的药物作用压力下连续传代后细菌对粘菌素敏感性的变化,结果表明石斛酚可以减缓细菌耐药性的发展;
图3石斛酚与粘菌素联合对动物感染模型的治疗效果,图A为实验的示意图,(1)大蜡螟幼虫被E.coli B2感染1小时后给予药物治疗,记录大蜡螟幼虫48小时内的存活率,(2)小鼠通过腹腔注射感染E.coli B2,1小时后给药治疗,计算72小时内的存活率,取肝、肾、脾研磨并稀释后滴在LB琼脂上,计算内脏载菌量,图B显示了石斛酚和粘菌素的组合可以显著提高E.coli B2感染大蜡螟幼虫的存活率,图C显示了石斛酚和粘菌素联合治疗可以显著提高E.coli B2感染小鼠的存活率,图D、E、F显示联合治疗显著降低小鼠肝、肾、脾的载菌量;
图4石斛酚可增强多粘菌素对细菌细胞内、外膜的损伤,图A表示石斛酚增强粘菌素对细菌外膜的损伤;图B显示石斛酚增强粘菌素对细菌细胞膜的损伤;图C说明石斛酚不会影响细菌ROS的产生;图D显示了石斛酚增强粘菌素对细菌膜电势的破坏;
图5石斛酚以浓度依赖的方式下调参与LPS修饰基因的表达,图A、B、C显示了石斛酚可以下调mcr-1、eptA、pagP的表达;图D、E、F显示了石斛酚与亚抑菌浓度的粘菌素组合更显著地抑制mcr-1、eptA、pagP的表达。
具体实施方式
实施例1石斛酚与多粘菌素的协同抗菌活性
采用棋盘稀释法测定石斛酚和多粘菌素联合使用对多重耐药菌E.coli B2(mcr-1)、沙门菌(Salmonella)15E343(mcr-3)、肺炎克雷伯菌(Klebsiella Pneumoniae)19-2-1(mcr-8)、大肠杆菌DH5α(pUC19-mcr-1)的协同抗菌活性。
棋盘稀释法具体步骤如下:
(1)用CAMHB肉汤培养基稀释待测菌株的菌液,使细菌悬液浓度为1×106CFU/mL。
(2)多粘菌素以CLSI准则推荐的溶剂溶解并用CAMHB肉汤培养基稀释,得到浓度为32μg/mL的多粘菌素溶液。
(3)石斛酚用DMSO溶解并用CAMHB肉汤培养基稀释,得到浓度为1024μg/mL的石斛酚溶液。
(4)取96孔平底板,每孔加入100μL MHB肉汤培养基,最后一行每孔加入100μL步骤(2)中制备的多粘菌素溶液,自第八行倍比稀释至第二行;第一列每孔加入步骤(3)制备的石斛酚溶液(每孔100μL),倍比稀释至第七列,之后每孔加入100μL步骤(1)制备的细菌悬液,37℃静置培养16h-20h,观察石斛酚和多粘菌素联合使用抑制细菌生长的最低浓度组合。
分级抑菌浓度(FICI)计算方法如下:
FIC=MIC(A药联合)/MIC(A药单用)+MIC(B药联合)/MIC(B药单用)
实验结果如图1所示,石斛酚可以恢复mcr阳性的粘菌素耐药菌对粘菌素的敏感性,并且石斛酚联合粘菌素可以在短时间将多药耐药菌大肠杆菌B2(mcr-1)的菌量降低至检测限(102CFUs/mL)以下,但对于对多粘菌素敏感的细菌,石斛酚并不会增强粘菌素的抗菌活性。
实施例2石斛酚与多粘菌素联用的时间杀菌曲线
多药耐药菌大肠杆菌B2(mcr-1)在CAMHB肉汤培养基中37℃培养至指数期,用CAMHB肉汤稀释菌液至106~107CFUs/mL的理想浓度。然后分别用石斛酚(64μg/mL)和多粘菌素(2μg/mL)单独或联合处理细菌,分别在0、4、8、12、24h取50μL菌液,10倍顺序稀释后滴在LB琼脂平板上,在37℃培养24h后计算菌落形成单位(CFUs/mL)。所有实验都至少进行了3次生物重复。
结果表明,石斛酚联合粘菌素可以在短时间将多药耐药菌大肠杆菌B2(mcr-1)的菌量降低至检测限(102CFU/mL)以下,说明石斛酚可增强多粘菌素的杀菌效果(图1)。
实施例3耐药性发展趋势预测传代试验
把过夜培养的E.coli B2在LB肉汤中以1∶1000稀释至含药的LB肉汤(亚抑菌浓度的粘菌素或粘菌素与石斛酚的组合)。在37℃,200r/min摇床中培养12小时后,将细菌培养物以1∶1000稀释,加入到新的含药培养基中,继续下一代培养。每隔四代,测定培养物的MIC。根据测得的MIC,增大多粘菌素和石斛酚的浓度。连续传代培养48代。
结果显示,石斛酚减缓了传代菌对多粘菌素的耐药性增长速度。如图2C所示,在亚抑菌浓度的抗生素压力下,多粘菌素对耐药菌的MIC增大了32倍,而加入石斛酚后,MIC仅增长2倍。这表明石斛酚可以抑制多粘菌素耐药基因的进化。
实施例4石斛酚的溶血性分析
为评价石斛酚的安全性,评估了石斛酚的溶血活性。在96孔平板中从1孔至10孔将石斛酚用磷酸盐缓冲液(PBS)倍比稀释,11孔以灭菌的PBS为阴性对照,12孔以双蒸水(ddH2O)为阳性对照。新鲜绵羊红细胞(RBC)用PBS洗涤两次后用PBS重悬,制得8%红细胞悬浮液,将红细胞悬浮液与不同浓度的石斛酚或石斛酚与多粘菌素共同混合,在37℃下孵育1h。之后,吸取120μL上清液3000rmp/min离心10min,吸取100μL测量释放的血红蛋白在576nm(OD576)处的吸光值,计算相应的溶血率。溶血率的计算如下所示:溶血率(%)=[(OD576样品-OD576阴性对照)/(OD576阳性对照-OD576阴性对照)]×100%。实验结果如图2所示,石斛酚对绵羊红细胞具有很低的溶血性,并且在与粘菌素联用时可以缓解高浓度粘菌素导致的溶血。
实施例5石斛酚与多粘菌素联合使用治疗大蜡螟幼虫细菌感染
(1)石斛酚用DMSO溶解配成2560mg/L的储备液,用PBS稀释成10、100mg/L的工作液。
(2)大肠杆菌B2接种于LB肉汤培养基,置于37℃培养到对数生长期。用PBS缓冲液重悬大肠杆菌B2使菌液浓度均为106CFU/mL。
(3)取50只体重300mg左右的大蜡螟幼虫随机分成4组,分别在左下最后1个腹足注射10μL步骤(2)中的菌液,感染1h后分别在右下最后1个腹足注射浓度10μL300mg/L(10mg/kg)的多粘菌素或多粘菌素加600mg/L(20mg/kg)乙酰紫草素,对照组给予10μL的PBS。
(4)在12、24、36、48h统计大蜡螟幼虫的存活率。
实验结果见图3。石斛酚联合粘菌素对抗耐药菌感染,在大蜡螟幼虫感染模型中,与粘菌素单药治疗相比,石斛酚与粘菌素联合治疗可以显著提高大蜡螟幼虫的存活率。
实施例6石斛酚与多粘菌素联用治疗小鼠腹膜炎
(1)小鼠分组处理
取24只体重20g左右的ICR小鼠,随机分成四组,分别为vehicle组、石斛酚50mg/kg、多粘菌素1.25mg/kg,石斛酚与多粘菌素联合组50mg/kg+1.25mg/kg(每组6只)。所有小鼠腹腔内注射0.5mL大肠杆菌B2菌悬液(7×108CFUs)。感染一小时后,分别给予PBS,石斛酚50mg/kg、多粘菌素1.25mg/kg,石斛酚与多粘菌素联合组50mg/kg+1.25mg/kg进行治疗。
(2)统计存活率、测量载菌量
实验结果见图3,联合治疗不仅显著提高了小鼠的存活率,并且与单药治疗组相比,联合治疗组的小鼠腹腔脏器(肝、肾、脾)的载菌量均显著降低。这些结果表明石斛酚与粘菌素具有很好的体内协同效果。
实施例7石斛酚恢复mcr-1阳性细菌对多粘菌素敏感性的机制探究
外膜通透性测定:过夜培养的大肠杆菌B2用0.01mol/L PBS(pH 7.4)洗涤、悬浮。将OD600 nm为0.5的细菌悬液与1-N-苯基萘胺(NPN,10μm)在37℃下孵育30min。随后,在96孔板中,将190μL的探针细胞与10μL石斛酚(最终浓度为0~64μg/mL)单独或与粘菌素(最终浓度为2μg/mL)混合。37℃避光孵育1h后,用激发波长为350nm,发射波长为420nm的Infinite M200微板阅读器(TECAN)测定荧光值。
内膜通透性测定:过夜培养的大肠杆菌B2用0.01mol/L PBS(pH 7.4)洗涤、悬浮。,将OD600nm为0.5的细菌悬液与与碘化丙啶(PI)(10μm)在37℃下孵育30min。在37℃下孵育30min。随后,在96孔板中,将190μL的探针细胞与10μL石斛酚(最终浓度为0~64μg/mL)单独或与粘菌素(最终浓度为2μg/mL)混合。37℃避光孵育1h后,用激发波长为535nm,发射波长为615nm的Infinite M200微型平板阅读器(TECAN)测定荧光值。
用3,3-二丙基硫代羰花青碘化物(DiSC3(5),0.5μM)探测大肠埃希菌B2膜电位的动态曲线。简言之,用0.01mol L-1PBS(pH 7.4)清洗过夜培养的大肠杆菌B2,并将细菌混悬液调节至约0.5的OD600nm。然后,将细菌悬液与DiSC3(5)在37℃下避光孵育15min。随后,在96孔板中将190μL探针细胞与10μL石斛酚(最终浓度为0-64μg/mL)和粘菌素(最终浓度为2μg/mL)混合。以激发波长622nm,发射波长670nm,间隔五分钟测定荧光值。
RT-PCR测定石斛酚对mcr等基因表达的影响:在37℃的条件下,将大肠杆菌B2加入1mLLB肉汤中培养过夜。过夜培养物用1/100LB肉汤稀释,37℃扩大培养5h。用0.01m ol/LPBS(pH 7.4)洗涤、悬浮,使菌液的OD600nm约为0.5,分别与石斛酚(16~64μg/mL)、粘菌素(10μg/mL)或它们的组合在37℃下孵育4h,提取总RNA,用反转录试剂盒反转录成cDNA。使用7500快速实时荧光定量PCR系统,通过SYBR Green I嵌合荧光法进行RT-PCR分析。通过2-ΔΔCt法测定联合用药后mRNA表达量的变化。设置三个生物学重复。
结果发现:粘菌素和石斛酚联合作用增加了外膜和全膜的通透性(图4)。结果提示,石斛酚可通过恢复粘菌素的膜损伤能力来增强粘菌素的活性。此外,无论是否有粘菌素存在,石斛酚均以剂量依赖的方式下调mcr-1、eptA和pagP等LPS修饰基因的表达水平。说明石斛酚是通过抑制耐药基因的表达来与多粘菌素发挥协同作用(图5)。
Claims (1)
1.石斛酚小分子作为多粘菌素佐剂在制备治疗细菌感染性疾病药物中的应用,所述细菌为E.coli B2(mcr-1)、沙门菌(Salmonella)15E343(mcr-3)或肺炎克雷伯菌(KlebsiellaPneumoniae)19-2-1(mcr-8)。
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