CN111617069B - 漆黄素在抗水产养殖动物嗜水气单胞菌感染中的应用 - Google Patents
漆黄素在抗水产养殖动物嗜水气单胞菌感染中的应用 Download PDFInfo
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Abstract
本发明公开了漆黄素在抗水产养殖动物嗜水气单胞菌感染中的应用，漆黄素能通过直接抑制气溶素的活性降低嗜水气单胞菌的体内外致病力，而漆黄素对嗜水气单胞菌的生长没有影响。进一步通过构建试验动物模型发现漆黄素对斑点叉尾鮰嗜水气单胞菌人工感染模型有显著的治疗作用，8天后的死亡率降至25%。漆黄素为植物黄栌的有效成分，同时还分布于洋葱、草莓等蔬菜和水果中，是一种安全性较高的黄酮类化合物，可以作为药物或饲料添加剂用于治疗和预防水产养殖动物的嗜水气单胞菌感染。
Description
技术领域
本发明属于水产养殖技术领域，具体涉及漆黄素在抗水产养殖动物嗜水气单胞菌感染中的应用。
背景技术
漆黄素又称为非瑟酮、紫铆素等，是一种黄酮类化合物。漆黄素主要分布于漆树科黄栌等植物中，在蔬菜和水果如洋葱、草莓、苹果和黄瓜中也有分布。黄栌具有清热解毒、散瘀止痛等功效，可用于治疗肝炎、丹毒、感冒等疾病。现代研究发现，漆黄素具有抗氧化、抗炎、神经保护、抗肿瘤、抗抑郁等药理学活性。
嗜水气单胞菌是一种广泛分布于自然界中的革兰氏阴性菌，是一种条件性致病菌，能导致鱼类、陆生动物和人的多种疾病。嗜水气单胞菌是淡水养殖鱼类的常见病原菌，能导致多种水产养殖动物的感染性疾病，严重威胁了淡水水产养殖的的健康发展。嗜水气单胞菌感染的治疗主要依赖于化学抗菌药物，但由于长期不合理使用导致耐药菌甚至多重耐药菌的产生。此外，由于化学抗菌药物的使用导致了其在水产品中的蓄积和残留，是威胁水产品质量安全和食品安全的重要因素之一。因此，研发具有全新机制的抗嗜水气单胞菌感染药物对于水产养殖业的健康发展具有重要意义。
气溶素是嗜水气单胞菌分泌的主要毒力因子，是嗜水气单胞菌导致各种疾病的物质基础，具有肠毒性、溶血性和细胞毒性。气溶素是一种成孔毒素，结合到易感细胞细胞膜上后可形成具有孔道的七聚体，导致细胞内容物外流而引起细胞死亡。大量研究表明，嗜水气单胞菌缺失气溶素后其致病力显著下降，气溶素是鉴别致病性嗜水气单胞菌的标志。因此，以气溶素成为研究抗嗜水气单胞菌感染药物的热门靶标，具有广阔的应用前景。
发明内容
本发明的目的在于提供了漆黄素在抗水产养殖动物嗜水气单胞菌感染中的应用，漆黄素对嗜水气单胞菌的生长几乎没有影响，但能直接抑制嗜水气单胞菌主要毒力因子气溶素的溶血活性和细胞毒性，降低斑点叉尾鮰嗜水气单胞菌感染模型的死亡率。
为了实现上述目的，本发明采用以下技术方案：
漆黄素在抗水产养殖动物嗜水气单胞菌感染中的应用：经试验验证，漆黄素对嗜水气单胞菌的最小抑菌浓度大于512μg/mL，在16μg/mL以下对嗜水气单胞菌的生长几乎没有影响，但漆黄素能剂量依赖性的抑制气溶素的溶血活性；进一步研究发现，当漆黄素浓度达到2μg/mL及以上后能显著抑制气溶素导致的A549细胞损伤；对感染嗜水气单胞菌的斑点叉尾鮰灌服50mg/kg剂量的漆黄素，每12h使用一次，连续3天，可使其死亡率降低到25％，而阳性对照组死亡率为100％。以上表明，漆黄素可以作为一种渔药用于抑制水产养殖动物嗜水气单胞菌感染。
漆黄素是一种来源于植物中的黄酮类天然产物，对动物和人没有明显的毒副作用和残留，可广泛用于制备抗水产养殖动物嗜水气单胞菌感染的中药制剂和饲料添加剂。
与现有技术相比，本发明具有以下优点及有益效果：漆黄素不直接杀灭或抑制病原菌的生长，因此其不易诱导病原菌产生耐药性；对耐药性病原菌治疗效果较好；漆黄素为天然化合物，对鱼体、人及环境没有毒性作用，没有药物残留的风险。
附图说明
图1为漆黄素与嗜水气单胞菌168菌株共培养的生长曲线。
图2为漆黄素对气溶素溶血活性的影响。
图3为漆黄素对气溶素引起的A549细胞损伤的影响。
具体实施方式
实施例1：漆黄素对嗜水气单胞菌最小抑菌浓度测定
本发明所涉及的嗜水气单胞菌168菌株由长江水产研究所水产动物药理与药残控制技术实验室保存。采用CLSI推荐的肉汤微量稀释法测定了漆黄素对168菌株的最小抑菌浓度。步骤如下：
(1)将配置好的漆黄素(四川维克奇生物技术有限公司，纯度＞98％，CASNo.528-48-3)在96孔细胞培养板中倍比稀释，浓度分别为512μg/mL、256μg/mL、128μg/mL、64μg/mL、32μg/mL、16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mL；每孔药物体积100μL；
(2)分别无菌接种168菌株的单菌落至MH培养基中30℃培养过夜，离心取菌体用无菌生理盐水调整浓度为0.5麦氏比浊度，然后用培养基稀释至1×106CFU/mL，在96孔细胞培养板中加入菌液，使其最终浓度达到5×105CFU/mL，分别设置阴性和阳性对照组(阴性对照组不加药物不加菌液，阳性对照组只加菌液不加药物)，每个药物重复三次，在30℃生化培养箱中培养18-24h后观察结果，以没有细菌生长的最低药物浓度判定为该药物的最低抑菌浓度。结果发现，漆黄素对嗜水气单胞菌168菌株的最小抑菌浓度大于512μg/mL。
实施例2：漆黄素对嗜水气单胞菌生长的影响
本发明所涉及的嗜水气单胞菌168菌株由长江水产研究所水产动物药理与药残控制技术实验室保存。将168菌株在BHI液体培养基中培养至对数生长前期(OD600nm＝0.3，28℃)，将培养菌分别置于6个50mL的锥形瓶中，每瓶20mL，分别加入不同浓度(0，1，2，4，8，16μg/mL)的漆黄素，28℃条件下继续培养5h，每30min取菌液测定一次OD600nm的吸收值。附图1所示为不同浓度漆黄素与嗜水气单胞菌168菌株共培养的生长曲线，从图中可以看出漆黄素在浓度为16μg/mL及以下时对嗜水气单胞菌的生长没有影响。
实施例3：漆黄素对嗜水气单胞菌气溶素溶血活性的影响
本发明中所涉及的气溶素由长江水产研究所水产动物药理与药残控制技术实验室制备并保存。具体步骤如下：975μLPBS溶液加至6个1.5mL离心管中，分别加入2μL浓度为100μg/mL纯化气溶素，然后加入漆黄素使不同离心管中的浓度分别达到0，1，2，4，8,16μg/mL，混合均匀后放入37℃培养箱中；孵育15min后加入25μL脱纤维绵羊红细胞(上海源叶生物技术有限公司)，继续在37℃条件下孵育15min，高速离心后测量OD543nm吸收度，1％TritonX-100为阳性对照。如附图2所示，随着漆黄素浓度增加气溶素的溶血作用呈剂量依赖性的降低，当漆黄素浓度达到2μg/mL及以上时能显著抑制气溶素的活性。
实施例4：漆黄素对嗜水气单胞菌气溶素引起A549细胞损伤的保护作用
本发明所涉及的气溶素和A549细胞由长江水产研究所保存。A549细胞培养在37℃含有5％二氧化碳的细胞培养箱中，培养基为DMEM，添加10％胎牛血清，青链霉素双抗。试验时将细胞以1.5×105个/孔的浓度平铺于96孔细胞培养板上，在37℃二氧化碳培养箱中培养过夜。次日，向培养板中加入2μL浓度为100μg/mL纯化气溶素，随后分别加入漆黄素，使其终浓度达到0，0.5，1，2，4，8μg/mL；细胞继续置于二氧化碳培养箱中培养2h后取上清采用乳酸脱氢酶(LDH)释放试剂盒测定不同处理组LDH释放量。结果如附图3所示，当漆黄素浓度在2μg/mL及以上时能显著抑制A549细胞LDH的释放。结果表明，漆黄素能抑制气溶素引起的A549细胞损伤。
实施例5：漆黄素对斑点叉尾鮰嗜水气单胞菌感染模型的治疗作用
本发明所涉及的嗜水气单胞菌168菌株由长江水产研究所水产动物药理与药残控制技术实验室保存。试验在100cm×50cm×60cm的水族箱中进行，每天换水并保持水温在28±2℃左右。100g左右的健康斑点叉尾鮰经麻醉后通过腹腔注射嗜水气单胞菌168菌株建立人工感染模型。感染后2h开始对感染斑点叉尾鮰灌服50mg/kg剂量的漆黄素，每12h给药一次，连续给药3d，阳性对照组感染后灌服PBS作为对照。每个实验组使用斑点叉尾鮰20尾，感染后即开始记录死亡情况，结果见表1。该结果表明，斑点叉尾鮰人工感染嗜水气单胞菌后在8d内全部死亡；给予50mg/kg漆黄素治疗的斑点叉尾鮰发生了25％的死亡；其存活率远高于阳性对照组。通过以上研究充分表明，当斑点叉尾鮰感染嗜水气单胞菌后可通过给予50mg/kg剂量的漆黄素进行治疗，且该药物能取得较好的治疗效果。
表1漆黄素治疗斑点叉尾鮰嗜水气单胞菌感染模型后的死亡率
大量研究表明，嗜水气单胞菌的致病力与其自身携带的毒力因子的种类和数量密切相关，其中气溶素是主要的毒力因子之一，是鉴别致病性嗜水气单胞菌的标志。由于近年来水产源耐药菌逐渐增多，因此药物研发的方向从单纯的抑菌或杀菌转变为干预细菌感染的过程。由于气溶素在嗜水气单胞菌致病过程中不可或缺的地位，以气溶素为靶标的药物研发备受关注。通过实施例1发现，漆黄素对嗜水气单胞菌几乎没有抑菌作用，进一步通过实施例2表明在试验浓度下漆黄素对嗜水气单胞菌的生长没有任何影响。通过实施例3发现，漆黄素能显著降低气溶素所诱导的绵羊红细胞溶血，且呈剂量依赖性，表明漆黄素能直接抑制嗜水气单胞菌气溶素的活性。进一步通过细胞试验研究了漆黄素对气溶素诱导的A549细胞损伤的抑制作用，以上研究提示，漆黄素对嗜水气单胞菌引起的斑点叉尾鮰感染有潜在的治疗作用。因此，通过注射嗜水气单胞菌构建了斑点叉尾鮰嗜水气单胞菌感染模型，通过口灌50mg/kg剂量漆黄素研究其治疗作用。结果表明，漆黄素治疗后动物模型的死亡率可降低至25％，而模型动物组死亡率为100％，显著提升了动物模型的存活率。该结果表明，漆黄素能治疗嗜水气单胞菌引起的斑点叉尾鮰感染，是一种潜在的抗嗜水气单胞菌药物。
嗜水气单胞菌广泛存在于淡水养殖环境中，对多种淡水养殖水生动物有致病力，而气溶素是广泛分布于具有致病力的嗜水气单胞菌中，具有普遍性。由此可推断漆黄素对水产养殖动物嗜水气单胞菌感染均具有治疗作用。
Claims (1)
1.漆黄素在制备预防或治疗水产养殖动物嗜水气单胞菌感染的药物中的应用。
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