CN110483652A - 一种人乳寡糖及其在制备用于通过缓解肠道缺氧损伤而治疗或预防nec的药物中的用途 - Google Patents
一种人乳寡糖及其在制备用于通过缓解肠道缺氧损伤而治疗或预防nec的药物中的用途 Download PDFInfo
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- CN110483652A CN110483652A CN201910501997.1A CN201910501997A CN110483652A CN 110483652 A CN110483652 A CN 110483652A CN 201910501997 A CN201910501997 A CN 201910501997A CN 110483652 A CN110483652 A CN 110483652A
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Abstract
本发明公开了一种人乳寡糖及其在制备用于通过缓解肠道缺氧损伤而治疗或预防NEC的药物中的用途，人乳经过离心去除脂肪得到乳清，乳清经超滤、乙醇处理，得到粗寡糖，粗寡糖经过色谱、质谱去除含有乳糖的组分，冻干后即得到纯化后的人乳寡糖(HMOs)，该纯化后的HMOs与天然人乳中的HMOs组成成分相似。经大量的动物实验证实，得到的纯化后的HMOs能够有效缓解肠道缺氧损伤，表现为促进肠上皮细胞增殖，抵抗细胞凋亡，降低细胞炎性反应，从而治疗坏死性小肠结肠炎或降低其发病率。
Description
技术领域
本发明属于食品生物技术领域，特别涉及一种人乳寡糖及其在制备用于通过缓解肠道缺氧损伤而治疗或预防NEC的药物中的用途。
背景技术
坏死性小肠结肠炎(Necrotizing enterocolitis,NEC)是一种常见于早产儿的高致死率疾病，致死率在10％～50％之间。目前针对NEC的治疗手段十分有限，早期通常采取抗生素疗法进行缓解，晚期只能通过手术进行治疗，但术后常伴有神经系统及消化系统的并发症，严重影响婴儿的生长发育。因此，针对NEC可能造成的风险进行预防是比较理想的防治手段。
目前研究认为，NEC有两大发病机制。其一是过度的炎症反应造成的机体损伤。肠道微生物菌群异位定植是炎症反应的来源之一，一些肠道致病菌在回肠末端及结肠定植会刺激婴幼儿的肠屏障，导致肠屏障损伤和通透性上升。其释放的内毒素还会进入血液刺激身体其他器官造成系统性免疫反应。除此之外，新生儿由于呼吸系统发育不良，或其他原因造成的肠供血不足进而引起的缺氧，也会导致肠上皮细胞受损，进而导致炎性反应的发生。其二，是婴幼儿自身发育不良导致机体免疫调控和损伤修复功能不良造成防御力下降。除此之外，喂养方式的不同也会导致发病率的变化。大量研究已经证实，母乳喂养的早产婴儿罹患NEC的风险比非母乳喂养的婴儿低6-10倍。目前的婴幼儿配方奶粉还未证明能够有效降低NEC的患病率。因此，确定母乳中能够预防NEC的组分具有重要的研究价值和经济价值。
人乳寡糖(Human milkoligosaccharides,HMOs)，是人乳中第三大固形物，初乳中含量在15-25g/L，常乳含量在5-10g/L。远远高于牛乳及其他哺乳动物乳中含量(<1g/L)。HMOs由五种单糖单元组成：葡萄糖，半乳糖，岩藻糖，唾液酸及N-乙酰葡萄糖胺，已经发现的HMOs结构在150种以上。目前婴幼儿配方奶粉中只允许添加两种HMOs，其余则有低聚半乳糖和低聚果糖替代，无论在含量还是在结构复杂性都远远低于人乳中实际情况，但目前关于HMOs的生理功能还存在诸多争议，对其作用机制还不甚清楚。因此限制了其的广泛应用。
发明内容
本发明的目的是提供一种人乳寡糖及其在制备用于通过缓解肠道缺氧损伤而治疗或预防NEC的药物中的用途。
本发明的目的是通过以下技术方案实现的：
一种人乳寡糖的制备方法，包括以下步骤：
A.将人乳离心去除脂肪，收集的乳清，并将乳清经超滤膜超滤去除蛋白质及残余脂肪，滤液经无水乙醇处理，过夜沉淀，离心去除沉淀后，最终收集到的上清液经冻干得到粗寡糖；
B.粗寡糖经Luna HILIC column分离去除乳糖，色谱条件：A液超纯水，B液色谱纯乙睛，0–40min,80％–50％B；40–45min,20％B；45–55min,80％B，每分钟回收一管滤液，滤液经HPLC-MS/MS-QTOF进行鉴定，色谱柱为LunaHILIC column，色谱条件同上，质谱条件为负离子模式，碰撞电压30V，扫描范围m/z 300-2000(MS)，m/z 50-2000(MS/MS)，鞘气温度350℃，鞘气流速8L/min，经鉴定后去除含有乳糖的组分，剩余滤液冻干，即为纯化后的人乳寡糖。
本发明的另一方面：
一种人乳寡糖在制备用于通过缓解肠道缺氧损伤而治疗或预防NEC的药物或食品中的用途，其中所述人乳寡糖为上述的人乳寡糖。
进一步的，所述药物和食品均为组合物。
进一步的，所述食品包括奶粉、酸奶制品，调制乳饮料。
进一步的，所述药物中人乳寡糖的浓度为10mg/mL～20mg/mL。
进一步的，所述功能性食品中人乳寡糖的浓度为10mg/mL～20mg/mL。
本发明相比现有技术的有益效果为：
1、本发明所述的人乳寡糖，其制备方法可最大限度的去除乳糖而保留不同结构的HMOs，所提取的HMOs与天然人乳中的HMOs组成成分相似；
2、本发明明确了人乳寡糖能够降低机体过度的炎症反应，HMOs能够缓解缺氧环境对肠上皮细胞的损伤，表现为促进细胞增殖，抵抗细胞凋亡，降低细胞炎性反应，说明HMOs能够通过缓解肠道缺氧损伤来降低坏死性小肠结肠炎(NEC)的发病率，或对NCE进行有效的治疗；
3、本发明所述的人乳寡糖，可用于制备通过缓解肠道缺氧损伤而治疗或预防NEC的药物或食品，尤其可以用于添加至婴幼儿奶粉中，从而预防NEC的发病率，本发明的研究为其添加到婴幼儿奶粉提供了理论基础。
附图说明
图1为HMOs的纯化与鉴定结果图：其中，A)HPLC纯化色谱图；B)定量标准曲线；C)HMOs各组分组成比例；
图2为HMOs添加降低NEC的发病率和致死率的结果示意图；A)示H&E染色；B)示肠道外观；C)示NEC致死率；D)示NEC评分结果；
图3为HMOs降低促炎因子分泌水平的结果示意图；A)示血清IL-8浓度；B)示回肠IL-6，IL-8水平；
图4为HMOs抑制TLR4-NFκB的激活的结果示意图；A)示回肠TLR4表达；B)示磷酸化IKBα和NFκB的水平；
图5为HMOs恢复肠屏障增殖活性的结果示意图；A)示Ki67阳性细胞数；B)示SOX9阳性细胞数；
图6为回肠HIF1α免疫荧光染色的结果示意图；
图7为CCK8增殖实验结果示意图；A)示Western Blot检测HIF1α表达结果；B)示CCK8检测0,0.5,1,5,10mg/mlHMOs，1％O2或200μmolCocl2处理24小时；C)示CCK8检测10mg/mlHMOs,GOS,FOS或IFOS，1％O2或200μmolCocl2.处理24小时；
图8为缺氧24小时后的细胞凋亡结果示意图；A)示Annexin V-FITC细胞凋亡检测化学缺氧24小时，10mg/mlHMOs,GOS,FOS,或IFOS对细胞凋亡的作用；B)示Annexin V-FITC细胞凋亡检测物理缺氧24小时，10mg/mlHMOs,GOS,FOS,或IFOS对细胞凋亡的作用；
图9为IL-8的分泌水平的结果示意图；A)示Western Blot检测结果；B)示IL-8的分泌水平。
具体实施方式
实施例1
本实施例提供了一种人乳寡糖，所述人乳寡糖的制备方法为：
10L人乳混合均匀，4℃，14000g离心30min去除脂肪。收集的乳清经10kd超滤膜超滤去除蛋白质及残余脂肪。滤液经2倍体积无水乙醇处理，4℃过夜沉淀。12000g离心去除沉淀后，最终收集到的上清液冻干得到粗寡糖。粗寡糖经Luna HILIC column(10×250mm,5μm,Phenomenex)分离去除乳糖，色谱条件：A液超纯水，B液色谱纯乙睛，0–40min,80％–50％B；40–45min,20％B；45–55min,80％B.每分钟回收一管滤液，滤液经HPLC-MS/MS-QTOF(Agilent 6545,美国)进行鉴定，色谱柱为Luna HILICcolumn(2.1mm×250mm,3μm,Phenomenex)，色谱条件同上，质谱条件为负离子模式，碰撞电压30V，扫描范围m/z 300-2000(MS)，m/z 50-2000(MS/MS)，鞘气温度350℃，鞘气流速8L/min。经鉴定后去除含有乳糖的组分，剩余滤液冻干，即为纯化后的HMOs。标准HMOs分析单品购买自Carbosynth。
纯化后的HMOs经LC-QTOF鉴定出含有的几种主要组成成分如图1所示，本实施例中所使用的纯化分离方法可最大限度的去除乳糖而保留不同结构的HMOs。所提取的HMOs与天然人乳中的HMOs组成成分相似。
其中，人乳样本来源于10位中国成年健康女性，哺乳期不超过1个月。C57BL/6小鼠购自维通利华(北京，中国)。所有人群实验及动物实验均已通过中国人民解放军空军总医院伦理审查委员会的批准(No.100)，本实验所使用的动物实验模型和饲养过程符合动物福利的相关要求。
利用上述制备得到的人乳寡糖进行NEC动物实验：
90只7日龄C57BL/6乳鼠随机分成6组，母乳喂养组(BF)，配方粉组(FF)，配方粉+LPS处理组(FF+LPS)，人乳寡糖组(FF+HMOs)，人乳寡糖+LPS处理组(FF+HMOs+LPS)，配方粉寡糖组(FF+IFOS)。根据HMOs在初乳中的含量设定为浓度为20g/L，配方粉寡糖由低聚半乳糖和低聚果糖按9:1比例配置，浓度为20g/L。NEC造模方法为冷刺激+缺氧造模，每次将乳鼠暴露在95％氮气+5％氧气环境下10min，之后给予4℃冷刺激10min，一天三次。LPS添加量为4mg/kg/d，以模拟致病菌侵袭效果。整个实验过程进行3天，三天后取1cm回肠做HE染色。NEC确诊根据病理切片评分进行判断。每张片子由两位专业医生单独进行判断，分为0-4分。0分：无损伤；1分：轻微绒毛损伤或与基质层分离；2分：中度绒毛受损；3分：绒毛严重受损甚至消失；4分：完全坏死，绒毛隐窝结构消失。当高于或等于2分时可判定为发生了NEC。
鼠代乳根据鼠乳营养成分进行设计，配方如下：雅培婴幼儿配方奶粉35g，4g汤臣倍健蛋白粉，溶于100ml无菌水中。总能量为8.2MJ/L.T。每8小时灌胃一次，第一天灌胃100μl/次，之后每天增加50μl/次。
本实施例中所用的检测方法如下所述。
炎症因子检测：
小鼠血清IL-8检测使用ELISA检测试剂盒进行(CXCL15 mouse ELISA kit,invitrogen)，新鲜血液室温放置2小时后，3000rpm离心15min，收集血清，-80℃保存，根据ELISA试剂盒说明书操作。小鼠肠道组织IL-8,IL-6使用ELISA试剂盒进行检测(IL-6mouseELISA Kit,invitrogen)。100mg小鼠回肠段组织经RIPA裂解液裂解，12000rpm离心后收集上清，-80℃保存。参照试剂盒说明书操作，最终血清中浓度按体积换算，肠道组织按蛋白浓度进行换算。
Western Blot：
提取后的蛋白质经5×上样缓冲液变性处理后，使用10％SDS-PAGE变性蛋白凝胶电泳进行分离，后将蛋白转移到0.45μmPVDF膜上，脱脂奶粉封闭1小时，4℃孵育一抗过夜，孵育二抗1小时后，经化学发光液显色拍照。
表1 Western Blot一抗使用信息表
免疫荧光染色：
3.5μm回肠组织切片，经脱蜡，抗原修复，透膜后，使用免疫荧光封闭液封闭1小时。4℃，孵育一抗过夜，避光室温孵育二抗1小时，DAPI染色5分钟，抗荧光猝灭剂封片，荧光显微镜观察拍照(Leica DM4/6B,Leica)。
表2 免疫荧光一抗使用信息表
| 抗体名称 | 稀释比例 | 品牌 |
| Rabbit anti-TLR4 | 1:100 | Peprotech |
| Rabbit anti-pNFκB | 1:100 | Immunoway |
| Rabbit anti-Ki67 | 1:100 | abcam |
| Rabbit anti-SOX9 | 1:100 | abcam |
| Rabbit anti-Hif1a | 1:100 | Immunoway |
细胞缺氧实验：
Caco2细胞接种于6孔板中，培养条件为37℃，DMEM+10％FBS。正常对照组培养在5％CO2细胞培养箱中，缺氧组培养在95％N2+5％CO2的密封箱中。添加125mmol二氯化钴处理组做为化学缺氧阳性对照组培养于正常培养箱中。
CCK8细胞增殖活性检测：
Caco2细胞接种于96孔板中，每孔接种1000个细胞。HMOs浓度梯度为0,1,5,10,15mg/ml，GOS,FOS,IFOS浓度为10mg/ml，每个浓度设5个平行，缺氧24小时后，加入CCK8(碧云天，北京)10μl每孔。37℃孵育1小时后，450nm测定吸光值。计算细胞增殖活性。
细胞凋亡检测：
Caco2细胞接种于12孔细胞培养板中，接种密度为5×104每孔。HMOs浓度为10mg/ml的，GOS,FOS,IFOS浓度为10mg/ml，缺氧24小时后，参照细胞凋亡Annexin V-FITC试剂盒(碧云天，北京)的操作步骤操作，处理好的细胞用流式细胞仪进行检测。
数据处理：
数据用SPSS 18.0进行分析，分析方法为单因素方差分析(ANOVA)，参数检验为LSD检验，所有结果表述为平均值±标准差，当p<0.05时认为有显著性差异。所有图由GraphPad Prism7.0做图。
检测结果如下：
FF+HMOs组与FF+LPS+HMOs NEC致死率和发病率均显著下降，如图2所示，肠道病理学切片也显示，肠道损伤有所缓解，肠道胀气现象消失，而FF+IFOS组并无明显改善效果。
图2中，A)H&E染色结果表明HMOs能够缓解回肠段肠绒毛损伤。B)FF，FF+LPS，FF+IFOS组肠道外观有明显胀气C)FF+HMOs，FF+LPS+HMOs组NEC致死率显著降低.D)NEC评分结果显示，HMOs显著降低NEC的发病率。*表示与FF或FF+LPS组比较p<0.05。
血清中IL-8水平表明，HMOs添加显著降低了机体的炎症水平。回肠段IL-6和IL-8浓度进一步表明HMOs可以显著改善NEC的肠道炎症反应，IFOS添加对机体整体炎症水平无影响，对回肠IL-8水平亦无显著抑制效果(图2)。
如图3所示的HMOs降低促炎因子分泌水平。A)血清IL-8浓度表明HMOs能够降低血清中IL-8的水平。B)FF+HMOs和FF+LPS+HMOs组回肠IL-6，IL-8水平显著降低。*表示与FF或FF+LPS组有显著差异p<0.05。
TLR4是肠上皮细胞表面致病菌和内毒素的表面受体，在NEC中通常被激活后通过NFκB通路释放促炎因子进而引起炎症。已有研究表明抑制TLR4的激活可以显著降低NEC的发病率。免疫荧光结果表明添加HMOs组可以显著降低TLR4在回肠段的表达，进一步通过western blot结果显示，FF+HMOs组及FF+LPS+HMOs组核内pNFκB水平显著低于未添加组，这证明HMOs显著抑制了回肠段TLR4-NFκB信号通路的激活(图4)。IFOS并没有显著的抑制效果。
图4中，A)IHC-F表示FF+HMOs，FF+LPS+HMOs回肠TLR4表达显著降低B)HMOs显著降低磷酸化IKBα和NFκB的水平。*表示与FF和FF+LPS组有显著性差异p<0.05。
肠屏障因为NEC会受到损伤，表现为细胞增殖活性减弱，干细胞增殖速度降低。Ki67免疫荧光染色表明，FF+HMOs，FF+LPS+HMOs组回肠细胞增殖活性较FF,FF+LPS组有明显回收，但较BF组仍有一定差距。SOX9免疫荧光结果表明，回肠干细胞增殖活性受HMOs干预有显著回升，以上结果表明，HMOs干预对肠屏障损伤修复功能具有保护作用(图5)。
图5中，A)FF+HMOs组和FF+LPS+HMOs组Ki67阳性细胞数显著高于FF组和FF+LPS组。B)FF+HMOs组和FF+LPS+HMOs组SOX9阳性细胞数显著高于FF组和FF+LPS组。
综上所述，动物实验表明，HMOs添加可以显著改善NEC的症状，降低NEC的发病率，抑制TLR4-NFκB的激活，促进肠屏障的修复。同时我们发现针对LPS添加的NEC模型，HMOs作用效果同样显著。
HIF1a是细胞缺氧的指示蛋白，其表达量随缺氧程度的增加而上升。回肠段HIF1a免疫荧光染色结果表明，在发生NEC过程中回肠细胞缺氧程度明显上升，HMOs干预对缺氧程度有显著缓解，IFOS组并无此作用(图6)。
图6结果表明FF+HMOs和FF+LPS+HMOs组表达水平低于FF组和FF+LPS组。
如图7所示的CCK8增殖实验结果表明，缺氧24小时后细胞增值活力显著下降至60％，此时HMOs添加对细胞增殖活性有明显缓解作用，并呈现剂量关系，10mg/ml时细胞增殖活性恢复至正常组。GOS,FOS,IFOS组并无显著改善。Western Blot结果也表明，细胞缺氧程度随HMOs的添加而有所缓解。
图7表明，HMOs阻止缺氧造成的细胞增殖减缓。A)Western Blot检测HIF1α表达，结果显示高浓度HMOs可显著缓解细胞缺氧。B)CCK8检测0,0.5,1,5,10mg/mlHMOs，1％O2或200μmolCocl2处理24小时。C)CCK8检测10mg/mlHMOs,GOS,FOS或IFOS，1％O2或200μmolCocl2.处理24小时。*表示与Control组比有显著性差异(P≤0.05)。
如图8所示，缺氧24小时后凋亡细胞比例显著增加(右上+右下象限)，而HMOs添加后化学缺氧凋亡细胞比例由35.51％下调至19.21％，物理缺氧凋亡细胞比例由95.98％下调至77.73％，均有显著改善作用。GOS,FOS,IFOS组并无明显效果。
图8表明，HMOs阻止缺氧导致的细胞凋亡增加。其中，A)Annexin V-FITC细胞凋亡检测化学缺氧24小时，10mg/mlHMOs,GOS,FOS,或IFOS对细胞凋亡的作用.B)Annexin V-FITC细胞凋亡检测物理缺氧24小时，10mg/ml HMOs,GOS,FOS,或IFOS对细胞凋亡的作用。
细胞缺氧并不能显著促进IL-1β的分泌，但可显著促进IL-8的分泌，如图9所示。HMOs，GOS，FOS，IFOS均可显著抑制IL-8的分泌水平，但HMOs效果更为显著。Westernblot结果表明，随着HMOs添加浓度的上升。NFκB的激活受到抑制，这表明细胞炎症水平有所缓解。
如图9所示，HMOs抑制炎症因子IL1β，IL-8的分泌。抑制NFκB的激活。
结论：HMOs能够缓解缺氧环境对肠上皮细胞的损伤，表现为促进细胞增殖，抵抗细胞凋亡，降低细胞炎性反应。这表明HMOs能够通过缓解肠道缺氧损伤来降低NEC的发病率。
实施例2
本实施例提供了一种添加人乳寡糖的婴幼儿配方奶粉，其配方为：蛋白质10g/100g,脂肪28g/100g,碳水化合物52g/100g,牛磺酸34mg/100g,左旋肉碱7.5mg/100g,肌醇32mg/100g,β-胡萝卜素61mg/100g,叶黄素100μg/100g,钙380mg/100g，磷224mg/100g,镁40mg/100g,钠144mg/100g，钾625mg/100g,氯350mg/100g,锌4.5mg/100g,铁5.4mg/100g，铜400μg/100g，锰100μg/100g，碘100μg/100g，硒12.3μg/100g，维生素A190国际单位/100g，维生素D 380国际单位/100g，维生素E国际单位/100g，维生素K1 54μg/100g，维生素C 80mg/100g,维生素B1 660μg/100g，维生素B21100μg/100g，维生素B6 400μg/100g，维生素B12 1.5μg/100g，烟酸5000μg/100g，泛酸2600μg/100g，叶酸76μg/100g，生物素20μg/100g，胆碱80mg/100g，核苷酸58mg/100g，能量510kcal/100g。其中所用的人乳寡糖为实施例1所述制备方法制备得到的人乳寡糖，其添加量为20mg/ml。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种人乳寡糖的制备方法，其特征在于，所述制备方法包括以下步骤：
A.将人乳离心去除脂肪，收集的乳清，并将乳清经超滤膜超滤去除蛋白质及残余脂肪，滤液经无水乙醇处理，过夜沉淀，离心去除沉淀后，最终收集到的上清液经冻干得到粗寡糖；
B.粗寡糖经Luna HILIC column分离去除乳糖，色谱条件：A液超纯水，B液色谱纯乙睛，0–40min,80％–50％B；40–45min,20％B；45–55min,80％B，每分钟回收一管滤液，滤液经HPLC-MS/MS-QTOF进行鉴定，色谱柱为Luna HILIC column，色谱条件同上，质谱条件为负离子模式，碰撞电压30V，扫描范围m/z 300-2000(MS)，m/z 50-2000(MS/MS)，鞘气温度350℃，鞘气流速8L/min，经鉴定后去除含有乳糖的组分，剩余滤液冻干，即为纯化后的人乳寡糖。
2.一种人乳寡糖，其特征在于，所述人乳寡糖由权利要求1所述的制备方法制备得到。
3.一种人乳寡糖在制备用于通过缓解肠道缺氧损伤而治疗或预防NEC的药物或食品中的用途，其中所述人乳寡糖为权利要求2所述的人乳寡糖。
4.根据权利要求3所述的用途，其特征在于，所述药物和食品均为组合物。
5.根据权利要求4所述的用途，其特征在于，所述食品包括奶粉、酸奶制品，调制乳饮料。
6.根据权利要求4所述的用途，其特征在于，所述药物中人乳寡糖的浓度为10mg/mL～20mg/mL。
7.根据权利要求4或5所述的用途，其特征在于，所述功能性食品中人乳寡糖的浓度为10mg/mL～20mg/mL。
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Application publication date: 20191122 |