CN116459245A - 漆黄素在制备沙门氏菌ⅲ型分泌***抑制剂中的应用 - Google Patents
漆黄素在制备沙门氏菌ⅲ型分泌***抑制剂中的应用 Download PDFInfo
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Abstract
本发明涉及漆黄素在制备沙门氏菌Ⅲ型分泌***抑制剂及抗沙门氏菌感染药物中的应用，以上皮样细胞人***细胞系(HeLa)为感染载体，通过细胞损伤保护试验及小鼠沙门氏菌感染治疗试验证实漆黄素通过影响沙门氏菌Ⅲ型分泌***的功能对沙门氏菌感染具有显著的抑制作用。相比传统抗生素，中药活性化合物漆黄素可通过靶向抑制沙门氏菌Ⅲ型分泌***的功能来发挥抗感染的作用，给予细菌较小的生存压力，不易诱导耐药性的产生，且具有来源广泛、成本低廉、治愈率高的特点。
Description
技术领域
本发明属于医学制药技术领域，具体涉及漆黄素在抑制沙门氏菌III型分泌***(T3SS)及在制备治疗沙门氏菌感染药物中的应用。
背景技术
沙门氏菌病是指由各种类型沙门氏菌所引起的对人类、家畜以及野生禽兽不同形式疾病的总称，在公共卫生领域被认为是一种严重的人畜共患病。鼠伤寒沙门氏菌对环境适应性强，广泛的存在自然环境中。它具有极广泛的宿主谱，对畜禽和哺乳动物都有致病性，是一种重要的人畜共患病病原菌，被感染的畜禽通常表现为胃肠道炎症或败血病，大大降低动物的繁殖生产力。临床上沙门氏菌感染较难控制，治疗效果非常不理想，给畜禽养殖业造成了巨大的经济损失，并严重威胁公共健康和食品安全。目前，由于抗生素被广泛用于鼠伤寒沙门氏菌感染，极大增加了细菌耐药性的产生。同时随着抗生素耐药性的增强，对于细菌感染的治疗和预防变得十分困难，所以迫切需要一种新的抗感染疗法。
抗毒力策略是解决抗生素耐药性问题最有希望的解决方案之一。沙门氏菌毒力岛1编码的沙门氏菌Ⅲ型分泌***(T3SS-1)在鼠伤寒沙门氏菌感染过程中起着至关重要的作用，T3SS-1主要作用于细菌侵袭的初始阶段，是一种类似注射器的精密分子装置，促使细菌与宿主的非吞噬细胞之间接触，紧接着在真核细胞膜上形成一个孔隙，便于T3SS-1注射效应蛋白进入宿主细胞，将效应蛋白转运到真核宿主细胞中以诱导感染。T3SS靶向治疗策略可解除病原菌的武装，对细菌的生存产生较小的生存压力，从而降低诱导耐药性的风险。因此，针对沙门氏菌T3SS开发的创新药物，对于防控沙门氏菌感染具有极其重要的现实意义。
漆黄素(Fisetin)是一种黄酮类化合物，广泛存在于草莓、苹果、洋葱、黄瓜等水果和蔬菜中，来源广泛，价廉易得，具广泛的药理活性，如抗炎、抗氧化、抗凝血、抗血栓、解痉、治疗糖尿病肾损伤等。近年来研究发现，漆黄素可以通过抑制肿瘤细胞增殖和信号转导通路、诱导肿瘤细胞凋亡等途径发挥抗肿瘤作用，故其日益受到大家的关注。目前，国内外未见Fisetin通过抑制沙门氏菌Ⅲ型分泌***的功能来抵抗沙门氏菌感染的报道。
发明内容
本发明所述漆黄素CAS登录号为528-48-3，分子式为C15H10O6，分子量为286.236。
漆黄素的化学结构式如下：
本研究以沙门氏菌Ⅲ型分泌***效应蛋白为药物靶标筛选得到的天然化合物漆黄素在较低浓度下通过抑制T3SS关键效应蛋白SipA的转运进而抑制沙门氏菌T3SS的功能，进而阻断沙门氏菌的入侵，最终导致效应蛋白(SipA和SipB)的表达水平显著降低。体外试验表明，在有效浓度范围内(≤32μg/mL)漆黄素不具有细胞毒性，且不影响细菌生长，经漆黄素预处理沙门氏菌后，沙门氏菌侵入HeLa细胞的能力显著降低。体内试验中，经漆黄素治疗后沙门氏菌感染小鼠存活率具有一定的升高，靶器官(肝脏和脾脏)细菌定殖数显著降低。
附图说明
图1：漆黄素抑制沙门氏菌Ⅲ型分泌***的功能(蓝色表示效应蛋白SipA的转运功能正常；绿色表示效应蛋白SipA不能正常转运)
图2：漆黄素可显著抑制沙门氏菌效应蛋白基因的表达
图3：漆黄素治疗可一定程度上提高沙门氏菌感染小鼠保护率(横轴表示时间，纵轴表示存活率)
图4：漆黄素显著降低感染小鼠靶器官菌落定殖数(横轴表示不同处理组肝脏、脾脏组织，纵轴为菌落定殖数)
具体实施方式
通过以下实施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
漆黄素作为沙门氏菌T3SS调控蛋白的抑制剂及在制备治疗沙门氏菌感染药物中的作用，用于药学上可接受的任何载体。
实施例2
漆黄素作为沙门氏菌T3SS抑制剂用于制备治疗感染性疾病的药物。
实施例3
漆黄素作为沙门氏菌T3SS调控蛋白的抑制剂用于治疗细菌引起的感染性疾病，特别是由沙门氏菌引起的人畜感染等疾病。
1.沙门氏菌III型分泌***抑制剂的筛选
以沙门氏菌效应蛋白SipA与β-内酰胺酶(SipA-TEM)融合报告质粒***及TEM荧光底物CCF-4检测受试化合物对沙门氏菌Ⅲ型分泌***功能的影响。TEM荧光底物CCF-4可进入细胞，细胞内的CCF-4在无β-内酰胺酶活性的条件下，在409nm可激发完整CCF4分子，其完整的CCF-4中的香豆素会使荧光共振能量转移至荧光素，使荧光素发出518nm的绿色荧光信号。在存在β-内酰胺酶活性条件下，CCF4的切割能从空间上将两种染料分开并干扰荧光共振能量转移，此时如果用409nm的光激发香豆素，产生的将是447nm的蓝色荧光信号。这种蓝色信号在显微镜下易于观察，也可通过荧光酶标仪蓝色通道读数的增加进行检测。通过天然化合物与沙门氏菌提前共孵育后感染细胞，再以感染细胞后呈现不同颜色荧光信号进行抑制剂的筛选，若信号由绿变蓝受到抑制，则表明该化合物可能抑制SipA-TEM的向细胞内的转运，检测漆黄素对T3SS入侵相关效应蛋白表达的影响。
结果表明：SipA-TEM融合报告质粒***与免疫荧光图显示，与阳性对照组相比(T3SS效应蛋白SipA能够正常转运至细胞内，细胞呈蓝色荧光)，漆黄素在浓度为16μg/ml时可以显著抑制沙门氏菌T3SS效应蛋白SipA由胞外转运至胞内，失去正常功能(视野内绝大部分细胞呈绿色荧光)，与沙门氏菌invA基因敲除株感染组结果相似(invA基因编码的蛋白InvA是介导T3SS***效应蛋白转运的关键基因，敲除后失去正常的功能。因此，细胞呈绿色荧光)(见附图1)。
2.漆黄素对沙门氏菌生长的影响
沙门氏菌接种至LB液体培养基(0.3M NaCl)中过夜培养(37℃，200rpm)，次日1:100扩培至OD600nm为0.1左右，分装至锥形瓶(20ml/瓶)，分别设定不加药物组和不同浓度漆黄素处理组。37℃，200rpm继续培养，每隔30min测定并记录每组样品的OD600nm，直至细菌生长至平台期。
结论：在7h的培养时间内，漆黄素处理组(0～32μg/ml)与未加药物处理组相比，细菌生长至平台期OD600nm值无显著差异，表明漆黄素在有效浓度范围内不影响细菌正常生长(见附表1)。
附表1漆黄素对沙门氏菌(SL1344)生长的影响测定
3漆黄素对沙门氏菌入侵宿主细胞的影响
3.1漆黄素对宿主细胞无细胞毒性
HeLa细胞于96孔板内过夜培养(2.0×104个/孔)，次日分组，漆黄素处理组(加入不同浓度漆黄素)、DMEM处理组(不加漆黄素)和无细胞对照组(加入DMEM，不加漆黄素)，每组设定三个重复，提前将漆黄素和DMEM混合均匀，加入各自对应组别，并于37℃温箱培养8h后1000rpm离心10min弃去培养基，按照CCK-8试剂盒说明处理并测定450nm处吸光度，计算各组细胞存活率。公式如下：细胞存活率(％)＝(待测组-无细胞对照组)/(DMEM处理组-无细胞对照组)×100％
结论：与未加漆黄素处理组相比，在2～32μg/ml浓度范围内漆黄素对HeLa细胞几乎无毒性(见附表2)。
附表2漆黄素对HeLa细胞毒性测定
4漆黄素对T3SS效应蛋白基因表达的影响
沙门氏菌接种至LB液体培养基(0.3M NaCl)中过夜培养(37℃，200rpm)，次日1:100扩培至OD600nm为0.3左右，分别设定不加药物组和不同浓度漆黄素处理组。37℃，200rpm继续培养5h，取菌液，离心，取适量菌液，加入适量的SDS-loading buffer，煮沸10min，SDS-PAGE和蛋白免疫印迹(Western Blotting)分析T3SS效应蛋白基因的表达情况。
结论：在16-32μg/mL漆黄素预处理后，沙门氏菌T3SS入侵相关效应蛋白(SipA和SipB)表达水平均出现不同程度的下降，该结果表明漆黄素通过抑制沙门氏菌Ⅲ型分泌***效应蛋白SipA的转运和入侵相关效应蛋白(SipA和SipB)的表达而影响沙门氏菌T3SS的功能(见附图2)。
4漆黄素对小鼠沙门氏菌感染的保护作用
4.1沙门氏菌感染小鼠模型的建立
6-8周龄雌性Balb/C小鼠(18～20g)，适应性饲养3天，期间自由采食和饮水，随后在饮水中添加5g/L链霉素，预处理3天，在感染前禁食禁水12h，采用口服灌胃的方式灌服沙门氏菌(1×107CFU/只)建立小鼠感染沙门氏菌肠炎模型。
5.2保护率试验
链霉素预处理小鼠随机分为健康对照组，沙门氏菌感染+PBS处理组、沙门氏菌感染+漆黄素处理组，沙门氏菌感染+漆黄素处理组在细菌感染小鼠后灌服50mg/kg漆黄素，2次/天，连续治疗4天，其中沙门氏菌感染+生理盐水处理组在相同时间内灌服相应体积PBS，健康对照组不做任何处理，连续观察10天统计不同处理组小鼠的精神状态和死亡情况。
结论：健康对照组10d内均未出现死亡，存活率为100.00％；感染组第5天和第9的死亡率分别为60.00％和100.00％；感染治疗组第9天存活率为20.00％，可见漆黄素对于沙门氏菌肠炎模型的小鼠具有一定的保护作用(见附图3)。
5.3靶器官菌落定殖数检测
感染后第5天颈椎脱臼处死小鼠，取各组小鼠的肝脏和脾脏组织，称重研磨制备组织匀浆，PBS倍比稀释后涂于含有链霉素抗性的LB琼脂细菌培养平板，37℃培养12h后进行菌落计数。
结论：与感染组相比，漆黄素治疗后，小鼠靶器官(肝和脾)的细菌载量显著减少(见附图4)。
综上所述，本研究以沙门氏菌感染上皮样细胞人***细胞系(HeLa)，建立细胞水平的药物保护效果评价模型；以口服感染沙门氏菌的方式成功建立沙门氏菌感染小鼠模型，发现漆黄素可在一定程度上降低沙门氏菌感染小鼠的死亡率，并降低肝脏和脾脏的细菌载量，这些研究将为沙门氏菌感染新药研发提供思路和基础。
Claims (3)
1.漆黄素在制备沙门氏菌Ⅲ型分泌***及其抗沙门氏菌感染药物中的应用。
2.如权利要求1所述漆黄素在制备治疗沙门氏菌感染药物中的应用，其特征在于所述的药物漆黄素为药学上可接受的载体。
3.如权利要求1所述漆黄素在制备治疗沙门氏菌感染药物中的应用，其特征在于所述的感染是指由沙门氏菌引起的感染。
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