CN115887444A - 水飞蓟宾在制备硫酸酯酶成熟酶抑制剂中的抗菌医药用途 - Google Patents
水飞蓟宾在制备硫酸酯酶成熟酶抑制剂中的抗菌医药用途 Download PDFInfo
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Abstract
本发明提供了水飞蓟宾在制备硫酸酯酶成熟酶抑制剂中的抗菌医药用途，通过体外抑制产气荚膜梭菌分解粘蛋白实验、HPLC活性检测实验和雏鸡产气荚膜梭菌感染治疗学实验等，发现水飞蓟宾能抑制产气荚膜梭菌硫酸酯酶成熟酶介导的硫酸酯酶活性以及，水飞蓟宾对产气荚膜梭菌感染具有显著治疗作用。本发明用水飞蓟宾治疗产气荚膜梭菌感染，与传统抗生素治疗相比较，具有无耐药性，治疗效果显著的优点，水飞蓟宾可以用于产气荚膜梭菌感染的新药开发，并对药物靶标确认有着重要意义，本发明为产气荚膜梭菌引起的坏死性肠炎防治提供了一种安全、有效的新手段。
Description
技术领域
本发明公开了水飞蓟宾在制备硫酸酯酶成熟酶抑制剂中的抗菌医药用途，属于医学制药领域。
背景技术
水飞蓟宾（Silibinin）是从菊科植物水飞蓟干燥的果实和种子中提取得到的天然产物，是一种黄酮木脂素类化合物。
产气荚膜梭菌是全球公共卫生领域具有重要地位的人兽共患病原菌。产气荚膜梭菌感染引起的鸡坏死性肠炎一直是困扰养禽业可持续健康发展的重要问题，每年会造成全球养禽业巨大的经济损失，同时引发食物中毒威胁人类的生命健康。产气荚膜梭菌的致病力与其携带的各种毒力因子密切相关，其中产气荚膜梭菌产生的硫酸酯酶成熟酶能催化自身硫酸酯酶的形成并使其具有活性，产气荚膜梭菌的硫酸酯酶可以分解利用肠道内作为保护屏障的硫酸化粘蛋白，为其在宿主肠道的定植和引发坏死性肠炎创造有利条件。因此产气荚膜梭菌硫酸酯酶成熟酶是产气荚膜梭菌感染防治药物研发的潜在治疗靶点。过去人们通过在饲料中添加大量抗生素进行防治，进而导致了细菌多重耐药性的产生。近年来随着各个国家禁止在饲粮中使用抗生素以后，导致畜禽养殖过程中坏死性肠炎发病率显著升高，阻碍着养殖业的可持续健康发展。因此开发新的可持续发展的产气荚膜梭菌感染防治药物具有十分重要的意义。
发明内容
本发明公开了水飞蓟宾在制备硫酸酯酶成熟酶抑制剂中的抗菌医药用途，公开了水飞蓟宾新的医用用途，可以用于治疗产气荚膜梭菌感染的疾病。
本发明所述的水飞蓟宾可抑制产气荚膜梭菌硫酸酯酶成熟酶的活性，并在体内对产气荚膜梭菌感染具有良好的防治作用。
本发明涉及的水飞蓟宾的分子式：C25H22O10，分子量：482.436，分子结构如下：
水飞蓟宾在制备产气荚膜梭菌硫酸酯酶成熟酶抑制剂中的医用用途。
所述水飞蓟宾有效抑制产气荚膜梭菌硫酸酯酶成熟酶的修饰活性。
所述的产气荚膜梭菌硫酸酯酶成熟酶抑制剂，可有效抑制产气荚膜梭菌对粘蛋白的分解利用，以及有效防治由产气荚膜梭菌引起的感染。产气荚膜梭菌感染是指由产气荚膜梭菌ATCC 13124引起的雏鸡坏死性肠炎。
所述的水飞蓟宾药物制剂为药学上可接受的载体，如胶囊剂、片剂。
本发明通过体外抑制产气荚膜梭菌分解粘蛋白实验、HPLC活性检测实验和雏鸡产气荚膜梭菌感染治疗学实验等，发现水飞蓟宾能抑制产气荚膜梭菌硫酸酯酶成熟酶介导的硫酸酯酶活性以及，水飞蓟宾对产气荚膜梭菌感染具有显著治疗作用。
本发明的积极效果在于：
公开了水飞蓟宾新的医用用途；使用水飞蓟宾与用抗生素治疗相比较，治疗具有无耐药性、治疗效果良好的优势；本发明用水飞蓟宾治疗产气荚膜梭菌感染，与传统抗生素治疗相比较，具有无耐药性，治疗效果显著的优点，水飞蓟宾可以用于产气荚膜梭菌感染的新药开发，并对药物靶标确认有着重要意义，为产气荚膜梭菌引起的坏死性肠炎防治提供了一种安全、有效的新手段。
附图说明
图1为产气荚膜梭菌ATC13124在M9Y和M9YM培养基中的生长曲线；
图2为水飞蓟宾对产气荚膜梭菌ATCC 13124在M9YM和BHI培养基中的生长曲线；
图3为产气荚膜梭菌厌氧硫酸酯酶成熟酶（anSMEcpe）的重组表达蛋白；
图4为产气荚膜梭菌厌氧硫酸酯酶成熟酶（anSMEcpe）与水飞蓟宾的HPLC活性验证；
图5为水飞蓟宾对雏鸡产气荚膜梭菌感染治疗后回肠组织HE染色分析；
图6为水飞蓟宾对雏鸡产气荚膜梭菌感染治疗后回肠荷菌统计及病变评分；
图7为水飞蓟宾对雏鸡产气荚膜梭菌感染治疗后回肠粘蛋白的数量及表达情况；
图8为水飞蓟宾对雏鸡产气荚膜梭菌感染治疗后回肠组织各蛋白及炎性因子的表达情况。
具体实施方式
通过体外抑制产气荚膜梭菌对粘蛋白的分解利用实验、生长曲线、HPLC抑制活性验证实验以及对雏鸡产气荚膜梭菌感染的防治作用证实了水飞蓟宾可以抑制产气荚膜梭菌硫酸酯酶成熟酶的活性及在产气荚膜梭菌感染中的防治作用。本实验中所用产气荚膜梭菌菌株编号为ATCC 13124。
实施例1
体外抑制产气荚膜梭菌对粘蛋白的分解利用实验
配置M9Y培养基（Na2HPO4 6.78 g/L；KH2PO4 3 g/L；NaCl 0.5 g/L；NH4Cl 1 g/L；葡萄糖4 g/L；MgSO4 0.493 g/L；CaCl2 0.0111 g/L；胰蛋白胨5 g/L，添加至1000 mL蒸馏水中溶解，灭菌备用）；配置M9YM培养基（Na2HPO4 6.78 g/L；KH2PO43 g/L；NaCl 0.5 g/L；NH4Cl 1g/L；葡萄糖4 g/L；MgSO4 0.493 g/L；CaCl2 0.0111 g/L；胰蛋白胨5 g/L；粘蛋白（购买自sigma公司）2.5 g/L；添加至1000 mL蒸馏水中溶解，灭菌备用）；
将接种于BHI培养基中过夜培养的产气荚膜梭菌ATCC13124使用M9Y培养基洗涤三次，并将细菌悬液的OD600值调为1，分别吸取30 μL菌液接种于各自装有3 mL M9Y和M9YM培养基的培养板中，静置培养于37℃厌氧培养箱中，每隔1h测量细菌的OD600值，绘制产气荚膜梭菌在两种培养基中的生长曲线；
将使用DMSO溶解的水飞蓟宾化合物母液，按1%（v/v）加入M9YM和BHI培养基中至终浓度为1 mM，如上所述接种产气荚膜梭菌后，静置于37℃厌氧培养箱中，每隔1h测量细菌的OD600值，绘制产气荚膜梭菌的生长曲线；
结果如附图1所示，产气荚膜梭菌ATCC 13124在M9YM培养基中生长的更好，细菌生长的OD600值有明显差异，表明细菌能利用粘蛋白作为营养来源；
结果如附图2A所示，浓度为1mM的水飞蓟宾能在M9YM培养基中抑制产气荚膜梭菌对粘蛋白的分解利用；
如附图2B所示，同时在添加同等浓度水飞蓟宾的BHI培养基中，细菌生长的OD600值未表现出明显差异。
实施例2
HPLC抑制活性验证实验
根据产气荚膜梭菌硫酸酯酶成熟酶(anSMEcpe)的核酸序列（NC_008261.1:744000-745112）和氨基酸序列（WP_011590280.1），构建了产气荚膜梭菌硫酸酯酶成熟酶的表达载体pET-28a-anSMEcpe，将表达载体转化至表达菌株BL21(DE3)中，后接种于LB培养基中培养至OD600值为0.6-0.8，添加异丙基-β-D-硫代半乳糖苷至终浓度为100 μmol/L进行诱导表达，收集菌体超声破碎后将上清与Ni -NTA Agarose混匀，按照纯化试剂盒说明书进行纯化，即得所述硫酸酯酶成熟酶anSMEcpe；通过超滤浓缩目的蛋白，使用12%的SDS-PAGE检测目的蛋白的纯度；
同时本发明通过比对多种细菌硫酸酯酶的氨基酸序列，参考Alhosna Benjdia等人（Benjdia A, Subramanian S, Leprince J, Vaudry H, Johnson MK, Berteau O.Anaerobic sulfatase-maturating enzyme--a mechanistic link with glycylradical-activating enzymes FEBS J. 2010 Apr;277(8):1906-20.）的试验结果，根据其保守序列合成多肽17C（所述序列为：Ac-TAVPSCIPSRASILTGM-NH2，上海波泰生物科技公司合成，纯度>95%）。将表征的1 mg厌氧硫酸酯酶成熟酶anSMEcpe与500 μM合成多肽在1 mL缓冲液（所述缓冲液配方为1 mmol/L AdoMet，6 mmol/L DTT，3 mmol/L 连二亚硫酸钠）中于厌氧环境下孵育，于0 h，4 h，6 h使用HPLC检测其在215 nm时修饰肽的吸光度。HPLC的C18柱使用溶剂A（所述溶剂A为0.1%三氟乙酸的纯水）平衡至90%，以1 mL/min的恒定流速应用10%-90%乙腈线性梯度进行洗脱，保存分析谱图；
同时将终浓度为1 mM的水飞蓟宾与表征纯化的1 mg厌氧硫酸酯酶成熟酶anSMEcpe、500 μM合成多肽在1 mL缓冲液（所述缓冲液配方为1 mmol/L AdoMet，6 mmol/LDTT，3 mmol/L 连二亚硫酸钠）中于厌氧环境下孵育，于0 h，4 h，6 h使用HPLC检测其在215nm时修饰肽的吸光度；HPLC的C18柱使用溶剂A（所述溶剂A为0.1%三氟乙酸的纯水）平衡至90%，以1 mL/min的恒定流速应用10%-90%乙腈线性梯度进行洗脱，保存分析谱图；
结果如附图3所示，通过表征纯化获得了大小约为44 kDa，纯度大于95%的硫酸酯酶成熟酶目的蛋白；
结果如附图4A所示，当表征的anSMEcpe目的蛋白与合成多肽17C一起在厌氧环境下孵育4 h后，产生了一个保留时间为8.6分钟的新肽，随着时间延长至6 h，产生的新肽进一步增加。结果表明表征的产气荚膜梭菌硫酸酯酶成熟酶anSMEcpe具有良好的硫酸酯酶修饰活性。如附图4B所示，当加入1mM水飞蓟宾共同孵育4 h和6 h后，于8.6分钟产生的新肽消失，表明水飞蓟宾能抑制硫酸酯酶成熟酶对硫酸酯酶17C的修饰作用。
实施例3
雏鸡产气荚膜梭菌感染的防治实验
将购买的60只三黄鸡分为3组，分别为感染组（CP）、治疗组（CP+SB）和健康组（NC）。感染组和治疗组每只三黄鸡（15d）每天经口服灌胃500 μL (108CFU/mL)产气荚膜梭菌ATCC13124菌悬液，治疗组2 h后每只三黄鸡灌服200 mg/kg（体积约为100 μL）的水飞蓟宾，感染组灌服同等剂量的溶剂。连续感染和治疗4天。模型对照组给予同等剂量的生理盐水，每组20只小鸡，至第19天时收集雏鸡的回肠、空肠和粪便样品进行检测和评价；
各组雏鸡回肠的HE染色结果如附图5所示，感染组（CP）雏鸡回肠绒毛形态被严重破坏且结构紊乱；水飞蓟宾治疗组（CP+SB）肠绒毛形态完整且结构清晰，与对照组（NC）的肠道无明显差异；
回肠的产气荚膜梭菌菌量如附图6A所示，水飞蓟宾治疗组（CP+SB）的产气荚膜梭菌菌载量显著低于感染组（CP）；回肠病变评分和病理评分如附图6B和6C所示，水飞蓟宾治疗组的评分显著低于感染组，表明水飞蓟宾可以有效的防治产气荚膜梭菌引起的雏鸡坏死性肠炎；
回肠的AB-PAS染色如附图7A和7B所示，水飞蓟宾治疗组（CP+SB）的粘蛋白数量明显多于感染组（CP），多个视野的统计学上有明显差异；回肠组织荧光定量结果如附图7C所示，水飞蓟宾治疗组（CP+SB）的Mucin-2表达量明显高于感染组（CP）和健康对照组（NC），表明水飞蓟宾可以减少产气荚膜梭菌对雏鸡肠道粘蛋白的分解利用，并提高组织Mucin-2的表达量，为防治产气荚膜梭菌感染起到了关键作用；
回肠组织的荧光定量结果如附图8A至8F所示，水飞蓟宾治疗组（CP+SB）的Occludin、Zonula occludens-1和Claudin-1三种紧密连接蛋白的表达量明显高于感染组（CP）和对照组（NC），并且水飞蓟宾降低了IL-6、TNF-α和IL-1β三种炎性因子的表达量，从而起到了防治产气荚膜梭菌感染的作用。
总结
水飞蓟宾可以抑制产气荚膜梭菌硫酸酯酶成熟酶对硫酸酯酶的活性修饰，导致产气荚膜梭菌硫酸酯酶活性丧失，使其不能分解利用肠道硫酸化的粘蛋白屏障，从而可以防治产气荚膜梭菌引起的雏鸡坏死性肠炎，特别是产气荚膜梭菌ATCC 13124引起的雏鸡坏死性肠炎。并进一步提高肠道相关连接蛋白的表达量，有利于维护雏鸡肠道环境的健康平衡。
Claims (5)
1.水飞蓟宾在制备产气荚膜梭菌硫酸酯酶成熟酶抑制剂中的医用用途。
2.根据权利要求1所述的水飞蓟宾在制备产气荚膜梭菌硫酸酯酶成熟酶抑制剂中的医用用途，其特征在于所述水飞蓟宾可有效抑制产气荚膜梭菌硫酸酯酶成熟酶的修饰活性。
3.根据权利要求1所述的水飞蓟宾在制备产气荚膜梭菌硫酸酯酶成熟酶抑制剂中的医用用途，其特征在于所述的产气荚膜梭菌硫酸酯酶成熟酶抑制剂，可有效抑制产气荚膜梭菌对粘蛋白的分解利用，以及有效防治由产气荚膜梭菌引起的感染。
4.根据权利要求1所述的水飞蓟宾在制备产气荚膜梭菌硫酸酯酶成熟酶抑制剂中的医用用途，其特征在于所述的水飞蓟宾药物为药学上可接受的载体，如胶囊剂、片剂。
5.根据权利要求3所述的产气荚膜梭菌引起的感染是指由产气荚膜梭菌ATCC 13124引起的雏鸡坏死性肠炎。
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| CN115896138B (zh) * | 2022-10-12 | 2023-08-08 | 吉林大学 | 一种厌氧硫酸酯酶成熟酶基因、厌氧硫酸酯酶成熟酶及其制备方法和应用 |
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