CN110478357A - 硝唑尼特及其体内代谢物在抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化中的应用 - Google Patents
硝唑尼特及其体内代谢物在抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化中的应用 Download PDFInfo
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Abstract
本发明公开硝唑尼特及其体内代谢物在抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化中的应用。本发明通过研究发现硝唑尼特(式I)具有抑制高脂饲料诱导的仓鼠体重增加和脂肪重量增加，抑制肝脏重量增加和脂肪肝，降低血清总胆固醇和总甘油三酯增加的作用。进一步的，本发明测定了替唑尼特对抑制脂肪酸合成和促进脂肪酸氧化的代谢酶‑乙酰辅酶A羧化酶活性的影响，发现替唑尼特具有与硝唑尼特相似的抑制乙酰辅酶A羧化酶活性的作用。因此，本发明提出了硝唑尼特及其体内代谢物在抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化中的应用。本发明的提出为抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化提供了新的有效的技术手段，具有广阔的应用前景。
Description
技术领域
本发明涉及硝唑尼特(Nitazoxanide)及其结构修饰物、硝唑尼特体内代谢物替唑尼特(Tizoxanide)及替唑尼特的结构修饰物在抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化中的应用。本发明属于生物医药领域。
背景技术
肥胖已成为当代流行的代谢性疾病，肥胖可导致高血脂、脂肪肝、动脉粥样硬化等疾病，造成沉重的社会经济负担，目前亟待开发新型的抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化药物。
发明内容
本发明的目的之一是提供硝唑尼特(Nitazoxanide，邻[N-(5-硝基噻唑-2-基)氨基甲酰]苯酚乙酸酯)(式I所示)在制备抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化药物中的应用；
本发明的目的之二是提供硝唑尼特药学上可接受的盐或以其为基础进行改造后的化合物在制备抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化药物中的应用；
本发明的目的之三是提供硝唑尼特的体内代谢物替唑尼特(Tizoxanide)(式II所示)在制备抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化药物中的应用；
本发明的目的之四是提供替唑尼特药学上可接受的盐或以其为基础进行改造后的化合物在制备抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化药物中的应用。
为了达到以上目的本发明采用了以下技术手段：
本发明通过建立高脂饲料给予仓鼠喂养方式，建立肥胖、高血脂和脂肪肝模型，同时口服途径给予硝唑尼特，发现硝唑尼特显著抑制高脂饲料诱导的仓鼠体重增加和脂肪重量增加，抑制高脂饲料诱导的仓鼠肝脏重量增加和脂肪肝，降低高脂饲料诱导的仓鼠血清总胆固醇和总甘油三酯增加。高胆固醇和高甘油三酯可直接导致动脉粥样斑块形成，因此硝唑尼特也同样会抑制动脉粥样硬化。
因此，本发明提出了硝唑尼特(Nitazoxanide，邻[N-(5-硝基噻唑-2-基)氨基甲酰]苯酚乙酸酯)及其药学上可接受的盐或以其为基础进行改造后的化合物在制备抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化药物中的应用；其中硝唑尼特的化学结构式如式I所示。
硝唑尼特口服吸收完全，且在体内完全代谢为代谢产物替唑尼特(Tizoxanide)，因此，硝唑尼特的抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化作用是来自于替唑尼特的作用，为验证此推测，本发明测定了替唑尼特对抑制脂肪酸合成和促进脂肪酸氧化的代谢酶-乙酰辅酶A羧化酶(acetyl CoA carboxylase,ACC)活性的影响，发现替唑尼特具有与硝唑尼特相似的抑制乙酰辅酶A羧化酶活性的作用，因此本发明进一步提出替唑尼特及其药学上可接受的盐或以其为基础进行改造后的化合物在制备抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化药物中的应用，其中替唑尼特的化学结构式如式II所示。
相较于现有技术，本发明的有益效果是：发现新型改善脂代谢的药物。
本发明为抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化提供了新的有效的技术手段。
附图说明
图1为硝唑尼特抑制高脂饲料诱导的仓鼠体重增加；
其中：左图为饲养七周后动物的代表照片；右图为7周过程中各组的动物体重变化；*P<0.05,**P<0.01vs HFD.ND，正常饲料；HFD,高脂饲料；Nit，硝唑尼特；
图2为硝唑尼特抑制高脂饲料诱导的仓鼠肝脏重量增加和脂肪肝，降低肝脏总胆固醇(TC)和低密度脂蛋白胆固醇(LDL-C)；
其中：A图为饲养七周后动物肝脏的代表照片；B图为肝脏重量的统计结果。C图为肝脏总胆固醇(TC)和低密度脂蛋白胆固醇(LDL-C)统计结果。**P<0.01vs ND.##P<0.01vsHFD.ND，正常饲料；HFD,高脂饲料；Nit，硝唑尼特；
图3为硝唑尼特抑制高脂饲料诱导的仓鼠双侧肾周白色脂肪(perirenal whiteadipose tissue，prWAT)脂肪增加；
*P<0.05vs ND.#P<0.05vs HFD.ND，正常饲料；HFD,高脂饲料；Nit，硝唑尼特；
图4为硝唑尼特抑制高脂饲料诱导的仓鼠血清甘油三酯和胆固醇增加；
血清甘油三酯和胆固醇的统计结果；**P<0.01vs ND.#P<0.05，##P<0.01vsHFD.ND，正常饲料；HFD,高脂饲料；Nit，硝唑尼特。
图5为替唑尼特抑制HepG2细胞乙酰辅酶A羧化酶(ACC)活性的结果；
其中：A为替唑尼特抑制HepG2细胞乙酰辅酶A羧化酶(ACC)活性的代表Westernblot结果，B为统计结果；C为硝唑尼特抑制HepG2细胞乙酰辅酶A羧化酶(ACC)活性的Westernblot结果；*P<0.05，**P<0.01vs CTL.Tizoxanide,替唑尼特；Nitazoxanide，硝唑尼特；p-ACC：磷酸化ACC；T-ACC：总ACC。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1硝唑尼特抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化的作用
1材料和方法
1.1实验动物：
叙利亚金黄地鼠，雄性，8周龄，125～145g，40只，购于辽宁长生生物技术股份有限公司。
1.2饲料：
ND：大小鼠维持饲料，购于北京科澳协力饲料有限公司。
HFD：猪油10％、蔗糖15％、蛋黄粉15％、酪蛋白5％、胆固醇1.2％、胆酸钠0.2％、碳酸氢钙0.6％、石粉0.4％、鼠维持饲料52.6％，于北京华阜康生物科技股份有限公司定制。
1.3试剂：
硝唑尼特(Nitazoxanide)：上海阿达玛斯试剂，CAS号：55981-09-4。
羧甲基纤维素钠(CMC-Na)：天津市福晨化学试剂厂。
1.4试剂盒：
液体样本甘油三酯(TG)酶法测定试剂盒(E1003-250)、血液总胆固醇(TC)酶法测定试剂盒(E1005-250)购于北京普利莱基因技术有限公司。
1.5实验方法:
1.5.1建立高脂动物模型
本实验采用高脂饲料喂饲法诱导叙利亚金黄地鼠建立高脂动物模型。40只叙利亚金黄地鼠适应性饲养1周后，随机分组4组，分为ND组，HFD组，HFD+Nit 50mg/kg组及HFD+Nit100mg/kg组，药物处理组每日1次灌胃给予相应剂量硝唑尼特混悬液(0.5％羧甲基纤维素钠悬助)，非药物处理组给予等剂量0.5％羧甲基纤维素钠。连续给药7周，并记录动物体重变化及摄食量。
1.5.2血清分离：
动物禁食12小时后，10％水合氯醛麻醉，采用心尖取血法收集血样，室温静置4小时后，5000g离心30分钟收集血清。
1.5.3脏器及脂肪重量：
精确分离心脏、肝脏、双侧肾脏、双侧附睾白色脂肪、双侧肾周白色脂肪及肩胛间棕色脂肪，称湿重。
2、结果
硝唑尼特抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化的作用
口服途径给予硝唑尼特抑制高脂饲料诱导的仓鼠体重增加(见图1)、抑制高脂饲料诱导的仓鼠肝脏重量增加和脂肪肝，降低肝脏总胆固醇(TC)和低密度脂蛋白胆固醇(LDL-C)(见图2)、抑制高脂饲料诱导的仓鼠肾周白色脂肪增加(见图3)、降低高脂饲料诱导的仓鼠血清总胆固醇和总甘油三酯增加(见图4)。
实施例2替唑尼特和硝唑尼特对HepG2细胞乙酰辅酶A羧化酶活性的影响
1材料和方法
1.1细胞
HepG2人肝癌细胞，购于上海中乔新舟生物科技有限公司，货号：ZQ0022
1.2试剂
替唑尼特(Tizoxanide)，购于MedChemExpress，货号：HY-12687/CS-3893
ACC抗体，购于Cell Signaling Technology,货号：3676
p-ACC(Ser79)抗体，购于Cell Signaling Technology,货号：11818
β-actin抗体，购于ABclonal，货号：AC026
1.3 Westernblot技术
细胞处理：用含有15％胎牛血清，1％青链霉素的DMEM培养液培养细胞至80％密度，换无血清DMEM培养液饥饿处理12小时，无血清条件下加入终浓度(μM)为0.5、1、5、10、25的替唑尼特和硝唑尼特，对照孔加入等体积DMSO,作用24小时。
细胞蛋白的提取：弃去培养液，PBS洗板3次后吸干。加入适量RIPA裂解液(含10％体积磷酸酶抑制剂，1％体积蛋白酶抑制剂)，冰浴裂解5分钟，刮取细胞裂解液置于1.5mL离心管中，细胞超声破碎仪破碎后，4℃，13500rpm/min条件离心15分钟，收集上清液。BCA法测定上清液中蛋白质浓度，按70μg蛋白上样量稀释各样品至相同浓度，加入五分之一体积的Loading Buffer(6×)缓冲液，涡旋混匀后100℃煮样5分钟备用。
PAGE凝胶电泳：8％丙烯酰胺凝胶，每个泳道加入算得上样体积的蛋白样品，于Tris-Glycine-SDS电泳缓冲液(25mM Tris,0.2M Glycine,0.1％SDS)中，恒压80V电泳至样品前沿进入分离胶层，转为恒压120V继续电泳至所需条带充分分离，停止电泳。具体步骤如下：
(1)转膜：切取所需位置凝胶，选用0.45μm孔径NC膜，于Tris-Glycine-Methanol转膜缓冲液(25mM Tris,0.2M Glycine,20％Methanol)中，冰浴条件下，300mA恒流转膜适宜时间(β-actin:60分钟，ACC：120分钟)。
(2)封闭：转膜完毕后，去离子水洗膜3次，每次5分钟，之后于5％脱脂牛奶中室温封闭1～2小时。
(3)一抗孵育：去离子水洗膜3次，每次5分钟，于适宜比例稀释的一抗溶液中4℃过夜。(ACC，p-ACC按1:500稀释，β-actin按1:20000稀释，稀释介质TBS)
(4)二抗孵育：一抗孵育完毕后，TBS-T洗膜4次,每次5分钟，之后于LI-COR 800CWGoat anti-Rabbit二抗(1:5000，TBS稀释)中室温孵育1小时。
(5)扫描成像：二抗孵育完毕后，TBS-T避光洗膜4次,每次5分钟，使用ODYSSEY CLx红外激光成像系统，800波长扫描成像，Image Studio软件统计数据。
2结果
替唑尼特(Tizoxanide)分别以0.5、1、5、10、25μM浓度处理HepG2人肝癌细胞24小时，可显著抑制乙酰辅酶A羧化酶(acetyl CoA carboxylase，ACC)活性(ACC磷酸化水平增高表示活性抑制，p-ACC)(见图5A、B)。同时，测定了硝唑尼特对HepG2人肝癌细胞ACC活性的影响，结果显示硝唑尼特抑制HepG2细胞乙酰辅酶A羧化酶(ACC)的活性(见图5C)。
Claims (2)
1.硝唑尼特(Nitazoxanide，邻[N-(5-硝基噻唑-2-基)氨基甲酰]苯酚乙酸酯)及其药学上可接受的盐或以其为基础进行改造后的化合物在制备抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化药物中的应用；其中硝唑尼特的化学结构式如式I所示：
2.替唑尼特(Tizoxanide)及其药学上可接受的盐或以其为基础进行改造后的化合物在制备抗肥胖、降血脂、抗脂肪肝及抗动脉粥样硬化药物中的应用；其中替唑尼特的化学结构式如式II所示：
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