CN108670964A - 甘油二酯在削减镉中毒方面的应用 - Google Patents
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Abstract
本发明公开了甘油二酯在削减镉中毒方面的新应用，通过构建动物模型，探究食品型甘油二酯在介导镉吸收及其毒性方面的应用，我们采用给小鼠灌胃的给药方式，模拟了人体通过口摄入镉到体内，在体内消化吸收的过程，利用激光共聚焦，以Leadmium Green AM dye为指示剂测定镉在细胞内分布和含量。本发明从源头上降低了“镉从口入”的风险，为构建降镉食谱提供了新思路，为从膳食层面构建消减镉中毒策略提供借鉴和科学依据。
Description
技术领域
本发明涉及甘油二酯的新用途，尤其是涉及甘油二酯在消减镉中毒方便的应用。
背景技术
镉(Cadmium,Cd)是中国土壤的首要重金属污染物，并且国际癌症研究机构IARC已确认镉及其化合物为人类致癌物质。镉污染食品的摄入是人体吸收镉的主要途径，其经小肠吸收进入血液循环，在肝、肾、心及其他组织中蓄积。镉离子与钙离子的半径几乎相同，故镉离子可与钙离子竞争钙泵上的结合位点，通过钙离子通道进入细胞内。有文献报道和我们的前期研究显示，抑制小肠上皮细胞不同类型钙离子通道一定程度上可以减少镉离子进入细胞。
甘油二酯(diacylglycerol,DAG)是一种天然的油脂，是由甘油和两分子脂肪酸酯化而成。甘油二酯在植物中的含量很少，是公认的食品安全成分，是人体代谢的重要中间产物。在治疗肥胖，高血压，高血脂方面有重要的作用。已有文章报道过甘油二酯在细胞水平上对TRPC钙离子通道的调节作用，低浓度的甘油二酯激活钙离子通道，高浓度甘油二酯抑制钙离子通道，然而并没有报道过甘油二酯介导镉吸收及其毒性。
发明内容
针对现有技术存在的上述问题，本发明申请提供了一种甘油二酯在削减镉中毒方面的应用。本发明通过构建动物模型，探究食品型甘油二酯在介导镉吸收及其毒性方面的应用，我们采用给小鼠灌胃的给药方式，模拟了人体通过口摄入镉到体内，在体内消化吸收的过程，利用激光共聚焦，以Leadmium Green AM dye为指示剂测定镉在细胞内分布和含量。
本发明的技术方案如下：
甘油二酯在削减镉中毒方面的应用。
所述甘油二酯在制备削减镉中毒药物中的应用。
所述甘油二酯在制备削减镉中毒保健品中的应用。
所述甘油二酯在制备削减镉中毒食品中的应用。
所述甘油二酯在削减人体或动物体镉中毒方面的应用。
所述甘油二酯为甘油二酯油，其主要成分为1,2-甘油二酯与1,3-甘油二酯的混合物。
所述甘油二酯为口服制剂。
本发明有益的技术效果在于：
本发明利用激光共聚焦，以Leadmium Green AM dye为指示剂测定镉在细胞内分布和含量，参考临床常用检测指标，通过测定小鼠血清谷丙转氨酶(ALT)活性，评价受试鼠肝脏受损程度；测定尿液尿肌酐及尿微量白蛋白含量，以评价受试鼠肾脏功能。
经研究发现与野生型小鼠，野生镉中毒小鼠相比，高剂量的甘油二酯饲喂的野生镉中毒小鼠可以有效抑制镉吸收，减少镉毒性。从源头上降低了“镉从口入”的风险，为构建降镉食谱提供了新思路，为从膳食层面构建消减镉中毒策略提供借鉴和科学依据。
附图说明
图1为七种剂量甘油二酯干预下急性镉中毒小鼠细胞内镉离子荧光强度变化图；
图2为七种剂量甘油二酯干预下急性镉中毒小鼠细胞内镉离子荧光强度变化统计图；
图3为七种剂量甘油二酯干预下急性镉中毒小鼠生存曲线图；
图4为七种剂量甘油二酯干预下急性镉中毒小鼠尿液中尿肌酐浓度统计图；
图5为七种剂量甘油二酯干预下急性镉中毒小鼠尿液中微量白蛋白浓度统计图；
图6为六种剂量甘油二酯干预下慢性镉中毒小鼠细胞内镉离子荧光强度变化图；
图7为六种剂量甘油二酯干预下慢性镉中毒小鼠细胞内镉离子荧光强度变化统计图；
图8为六种剂量甘油二酯干预下慢性镉中毒小鼠血清中谷丙转氨酶活性统计图；
图9为六种剂量甘油二酯干预下慢性镉中毒小鼠尿液中尿肌酐浓度统计图；
图10为六种剂量甘油二酯干预下慢性镉中毒小鼠尿液中微量白蛋白浓度统计图。
具体实施方式
下面结合附图和实施例，对本发明进行具体描述。
实施例1
1材料和方法
1.1材料
1.1.1动物健康的野生型SPF级6周龄左右雄性小鼠100只购买自南京模式动物研究所公司，饲养在江南大学医学院动物中心的SPF级动物饲养屏障环境中，环境湿度保持在50％，温度维持在22-25℃，自由进食饮水，通过灯光控制系统使其生活在为12h:12h的昼夜节律中。
1.1.2材料氯化镉(CdCl2·2.5H2O)江南大学食品学院赠与；DMEM(Dulbecco'sModified Eagle Medium)培养基购自Gibco公司；胎牛血清(fetal bovine serum,FBS)购自四季青公司；青霉素-链霉素溶液(100×)、牛血清白蛋白(BSA)均购自碧云天生物技术有限公司；胰岛素购自万邦医药公司；表皮生长因子(epidermal growth factor，EGF)购自Peprotech公司；鼠尾胶原购自sigma公司；肌酐检测试剂盒(比色法)、小鼠白蛋白ELISA试剂盒购自Abcam公司；谷丙氨酸氨基转移酶(ALT或SGPT)活性比色/荧光检测试剂盒购自BioVision公司；铅离子/镉离子绿色荧光探针(Leadmium GreenAM dye)购自ThermoFisher Scientific公司；吐温-80购自国药集团；甘油二酯油(DAG)购自西安德朗生物科技有限公司，胶原酶Ⅺ购自sigma公司。
1.1.3仪器全功能微孔板检测仪(BioTek)；激光共聚焦显微镜(Leica)；冷冻切片机(Leica)；细胞培养箱(Thermo)；全自动正置荧光显微镜(Zeiss)；生物安全柜(Thermo)。
1.2实验方法
1.2.1动物模型构建
(1)预实验随机取SPF级野生雄性小鼠24只，平均分为8组，每组3只。实验组小鼠先以20mg/kg CdCl2溶液(生理盐水配制)加不同剂量甘油二酯油灌胃3天，然后以80mg/kgCdCl2溶液(生理盐水配制)加不同剂量甘油二酯油灌胃4天，2％吐温-80助溶。甘油二酯油(DAG)由低到高的灌胃剂量依次是0ml/kg DAG、0.5ml/kg DAG、1ml/kg DAG、2.5ml/kg DAG、5ml/kg DAG、10ml/kg DAG、20ml/kg DAG；对照组小鼠用生理盐水加2％吐温-80灌胃，连续灌胃7天。造模结束后检测十二指肠肠细胞内镉离子荧光强度。
(2)随机取SPF级野生雄性小鼠40只，平均分为8组，每组5只。实验组小鼠以80mg/kg CdCl2溶液(生理盐水配制)加不同剂量甘油二酯油灌胃，2％吐温-80助溶，DAG由低到高的灌胃剂量依次是0ml/kg DAG、0.5ml/kg DAG、1ml/kg DAG、2.5ml/kg DAG、5ml/kg DAG、10ml/kg DAG、20ml/kg DAG；对照组小鼠用生理盐水加2％吐温-80灌胃，连续灌胃5天。在灌胃第二天收取小鼠尿液检测尿肌酐、微量白蛋白含量，造模期间记录小鼠的死亡日期。
(3)随机取SPF级野生雄性小鼠35只，平均分为7组，每组5只。实验组小鼠以10mg/kg CdCl2溶液(生理盐水配制)加不同剂量甘油二酯油灌胃，2％吐温-80助溶，DAG由低到高的灌胃剂量依次是0ml/kg DAG、0.5ml/kg DAG、1ml/kg DAG、2.5ml/kg DAG、5ml/kg DAG、10ml/kg DAG；对照组小鼠用生理盐水加2％吐温-80灌胃，连续灌胃30天。造模结束后收取小鼠尿液检测尿肌酐、微量白蛋白，取小鼠血清检测谷丙转氨酶活性，检测小鼠十二指肠肠细胞内镉离子荧光强度。
1.2.2检测小鼠尿肌酐、微量白蛋白浓度取小鼠任意时间段尿液，按试剂盒说明书进行检测。
1.2.3检测小鼠血清谷丙转氨酶活性小鼠CO2麻醉，心脏取血，放入1.5ml离心管中，室温放置2h，1500g，4℃离心20min取上清，然后按照试剂盒说明书进行检测。
1.2.4细胞培养小鼠断颈处死，取十二指肠2-3cm，立刻放入含有冰冷的2％PBS(PBS中加入2％青霉素-链霉素溶液(100×))的dish中，用移液管冲洗肠内容物，重复多次，移到新的含冰冷的2％PBS的dish中，利用剪刀纵切肠道，切碎约至2mm，用2％PBS湿润的移液管将组织转移至10ml离心管(含5ml冰冷的2％PBS)，轻轻摇动管子，清洗组织，然后放架子上使其沉淀，弃去PBS，再加新鲜的2％PBS清洗多遍，直至上清澄清，转移组织至含有3ml消化液(0.3mg/ml胶原酶Ⅺ，DMEM配制)的10ml离心管中，37℃震荡消化5min，轻轻上下摇晃管子约3s，静置，吸取上清丢弃，此操作重复3-4次，以丢弃消化下来的细胞碎片和单细胞，在剩下的未消化的组织中加入3ml消化液，37℃震荡消化10min，上下用力摇晃管子，静置，转移上清至新的10ml离心管，标记为消化液1，未消化的组织继续加入3ml新鲜的消化液，37℃震荡消化10min，上步得到的消化液1进行室温100g离心3min，弃上清，用完全培养基(含10％FBS，1％青霉素-链霉素溶液(100×)，1mg/ml EGF，0.25U/ml胰岛素的DMEM)重悬，此操作重复3-4次，直到大部分组织被消化，收集合并所有的消化液，100g室温离心3min，弃上清，完全培养基重悬，置于37℃、5％CO2及饱和湿度的培养箱中培养，48h之后换液，继续培养。
1.2.5检测细胞内镉离子荧光强度变化铅离子/镉离子绿色荧光探针(LeadmiumGreen AM dye)工作液配制：50μg的铅离子/镉离子绿色荧光探针先平衡到室温，然后加入50μl二甲基亚砜(DMSO)充分混匀制备成储存液。将分离的十二指肠细胞直接铺于鼠尾胶原包被的共聚焦小皿，48h之后换液，继续培养3-4天，吸去培养基，0.85％NaCl溶液清洗小皿3次，加入5ng/μl铅离子/镉离子绿色荧光探针(0.85％NaCl溶液配制)，37℃培养箱内避光孵育30min，之后0.85％NaCl溶液清洗小皿3次，最后加入500μl含有1％BSA的0.85％NaCl溶液，激光共聚焦63倍油镜拍照。
2结果
2.2甘油二酯对镉中毒的作用
2.2.1甘油二酯对急性镉中毒的作用利用Leadmium Green AM dye(ThermoFisher Scientific,USA)为镉离子的指示剂，通过观察细胞内镉离子荧光强度的变化来判断十二指肠细胞内镉离子的含量变化，图1为Leadmium Green AM dye检测未服用镉的野生型小鼠、急性镉中毒且同时灌胃不同剂量甘油二酯的野生型小鼠十二指肠细胞中镉含量，绿色：Leadmium Green，图中标尺为25μm。图1可以看出在预实验造模期间80mg/kg CdCl2+0ml/kg DAG小鼠全部死亡，剩下的其它实验组与野生对照小鼠相比，随着甘油二酯灌胃剂量的增加，细胞内镉离子的荧光强度先增强后减弱，提示细胞内镉离子的含量是一个增加又减少的过程。
图2为细胞内镉离子荧光强度变化的统计图，每个处理组n＝3，当甘油二酯灌胃剂量≥10ml/kg时可以有效抑制小鼠对镉离子的吸收。
图3显示了在急性镉中毒造模期间小鼠的生存曲线，在灌胃处理第二天，各组小鼠都出现了死亡现象，20ml/kg DAG处理的镉中毒小鼠死亡4只，解剖后发现小鼠胃部肿大，胃内有气泡，并且有大量的甘油二酯油，故猜测小鼠是由于甘油二酯油剂量太大，导致小鼠消化不良，撑胀而死。在灌胃处理第5天，80mg/kg CdCl2+10ml/kg DAG处理的小鼠生存率为80％，其他的实验组和对照组的生存率为0％。由此得出10ml/kg DAG处理的小鼠死亡率降低，大大减少了镉的吸收。
图4、图5为检测野生对照组、服镉小鼠、服镉和甘油二酯小鼠的尿肌酐、微量白蛋白浓度，指标升高提示着肾脏损伤。每个处理组n＝3。**P<0.01vs80mg/kg CdCl2；***P<0.001vs 80mg/kg CdCl2。
尿肌酐为血液中的肌酐经过肾滤过排出的肌酐含量，单独测定尿肌酐的浓度对肾功能评价很少有帮助。尿微量白蛋白是指尿液中出现微量白蛋白，白蛋白是血液中一种正常的蛋白质，生理条件下尿液中仅出现极少量的白蛋白。当肾脏组织受到损伤，或者发生病变，尿液中的微量白蛋白就会增多。结果显示与野生未服镉小鼠、野生服镉小鼠相比，按10ml/kg DAG灌胃处理镉中毒小鼠可以有效削减镉中毒。
2.2.2甘油二酯对慢性镉中毒的作用通过Leadmium Green AM dye(ThermoFisher Scientific,USA)检测十二指肠肠细胞内镉离子含量。图6为Leadmium Green AMdye检测未服用镉的野生型小鼠、慢性镉中毒野生型小鼠、慢性镉中毒且同时灌胃不同剂量甘油二酯的野生型小鼠十二指肠细胞中镉含量，绿色：Leadmium Green。图中标尺为25μm。由图6可以看出细胞内镉离子荧光强度先变亮再变暗，提示甘油二酯对镉离子的吸收随甘油二酯剂量增加，是一个先促进后抑制的过程。当甘油二酯剂量为10ml/kg时，细胞荧光强度与野生未服镉离子的细胞荧光强度差不多，与服用10mg/kg CdCl2+0ml/kg DAG的野生小鼠相比，荧光强度减弱，由此可知当甘油二酯剂量为10ml/kg时，可以有效的缓解小鼠镉中毒。
图7为镉离子荧光强度变化统计图，每个处理组n＝3。**P<0.01vs 10mg/kgCdCl2。由图可以看出在0-10ml/kg甘油二酯的剂量范围内，对小鼠镉中毒的影响先是促进后是抑制。
图8检测血清中谷丙转氨酶活性。收集野生型小鼠、慢性镉中毒野生型小鼠、慢性镉中毒同时饲喂不同剂量的甘油二酯的野生型小鼠的血清，用以检测血清谷丙转氨酶活性(ALT)。指标升高提示着肝脏损伤。每个处理组n＝3。谷丙转氨酶在肝脏中含量最多，通常血清中谷丙转氨酶活性很低。当肝脏组织发生病变或者受到损伤，细胞内的谷丙转氨酶就会释放到血液中，从而使血清中谷丙转氨酶活性增高。故检测的指标升高，提示着小鼠肝脏受损，发生病变。结果显示10ml/kg的甘油二酯处理的小鼠相比慢性镉中毒小鼠谷丙转氨酶活性降低，并且与野生未服镉小鼠相比活性有所下降。
图9、图10为检测野生对照组、服镉小鼠、服镉和甘油二酯小鼠的尿肌酐、微量白蛋白浓度。指标升高提示着肾脏损伤。每个处理组n＝5。*P<0.05vs 10mg/kg CdCl2。
由图可以看出，在0ml/kg-10ml/kg甘油二酯的处理下，尿肌酐和微量白蛋白的含量先增加随后降低，在10ml/kg甘油二酯的处理下，与野生型未服用镉小鼠的含量差不多。进一步证明了当甘油二酯灌胃剂量为10ml/kg时，可以有效的缓解小鼠镉中毒。综上所述，甘油二酯对镉离子的吸收为剂量依赖性，并且呈钟型曲线，当甘油二酯剂量≥10ml/kg时，可以有效的抑制镉吸收及其毒性。
Claims (7)
1.甘油二酯在削减镉中毒方面的应用。
2.根据权利要求1所述的应用，其特征在于，所述甘油二酯在制备削减镉中毒药物中的应用。
3.根据权利要求1所述的应用，其特征在于，所述甘油二酯在制备削减镉中毒保健品中的应用。
4.根据权利要求1所述的应用，其特征在于，所述甘油二酯在制备削减镉中毒食品中的应用。
5.根据权利要求1所述的应用，其特征在于，所述甘油二酯在削减人体或动物体镉中毒方面的应用。
6.根据权利要求1所述的应用，其特征在于，所述甘油二酯为甘油二酯油，其主要成分为1,2-甘油二酯与1,3-甘油二酯的混合物。
7.根据权利要求1所述的应用，其特征在于，所述甘油二酯为口服制剂。
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