CN110771897A - 一种干预慢性饮酒诱导的小肠损伤的功能性食品及应用 - Google Patents
一种干预慢性饮酒诱导的小肠损伤的功能性食品及应用 Download PDFInfo
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Abstract
本发明公开了一种干预慢性饮酒诱导的小肠损伤的功能性食品及应用。本发明中壳寡糖对慢性饮酒诱导的小肠损伤的干预作用不但体现为增加小肠绒毛长度、隐窝深度和肌层厚度,还可以表现为提高紧密连接蛋白Occludin的基因表达量、降低Claudin4的基因表达量,降低血浆中D‑乳酸含量和二胺氧化酶活性,能够降低丙二醛和蛋白质羰基含量,从而缓解酒精引起的肠组织的氧化损伤,同时表现为能够降低小肠炎性因子IL‑6、IL‑1β和TNF‑α的表达,从而降低酒精引起的小肠组织的炎症反应。
Description
技术领域
本发明涉及一种干预慢性饮酒诱导的小肠损伤的功能性食品及应用,属于功能食品技术领域。
背景技术
饮酒是造成200多种疾病和损伤病症的一个因素,大部分酗酒者会出现组织损伤或器官功能障碍。20%~30%的重度酗酒者患有酒精性脂肪肝炎和肝硬化等酒精性肝病。慢性饮酒会对生长因子,细胞因子和免疫功能产生破坏性影响,通过炎症反应损害机体器官,因此确定导致疾病进展的炎症来源至关重要。有报道称,胃肠道可能是酒精介导的器官损伤的重要炎症来源。
壳寡糖是由壳聚糖经化学或酶解后获得,研究发现壳寡糖具有抗肿瘤、降胆固醇、降血压等多种活性,在食品中作为抗氧化剂和抗菌剂等应用,壳寡糖与胃、十二指肠粘膜细胞亲和,可以通过活化细胞作用,促进受损的粘膜修复,利于溃疡的愈合。壳寡糖的分子量与应用技术效果之间关系密切,但壳寡糖对慢性饮酒诱导的小肠损伤的干预作用及其作用机制尚未报道,影响其进一步的推广应用。
发明内容
为解决上述技术问题,本发明提供一种壳寡糖在制备干预慢性饮酒诱导的小肠损伤的功能性食品中的应用,本发明采用相对分子量为1299,脱乙酰度为85%的壳寡糖提高紧密连接蛋白Occludin的基因表达量、降低Claudin4的基因表达量,降低血浆中D-乳酸含量和二胺氧化酶活性,能够降低丙二醛和蛋白质羰基含量,从而缓解酒精引起的肠组织的氧化损伤,同时表现为能够降低小肠炎性因子IL-6、IL-1β和TNF-α的表达,从而降低酒精引起的小肠组织的炎症反应。
本发明的第一个目的是提供一种干预慢性饮酒诱导的小肠损伤的功能性食品,所述功能性食品包含相对分子量为1200-1300的壳寡糖。
进一步地,所述的壳寡糖的聚合度为6-7。
进一步地,所述的壳寡糖的脱乙酰度为80-90%。
进一步地,所述的壳寡糖的用量,以质量百分比计为0.05%-0.08%。
进一步地,所述的干预慢性饮酒诱导的小肠损伤的功能性食品为提高紧密连接蛋白Occludin的表达量、降低Claudin4的表达量的功能性食品。
进一步地,所述的干预慢性饮酒诱导的小肠损伤的功能性食品为降低血浆中D-乳酸含量和二胺氧化酶活性的功能性食品。
进一步地,所述的干预慢性饮酒诱导的小肠损伤的功能性食品为降低丙二醛和蛋白质羰基含量的功能性食品。
进一步地,所述的干预慢性饮酒诱导的小肠损伤的功能性食品为降低小肠炎性因子IL-6、IL-1β和TNF-α的表达的功能性食品。
本发明的第二个目的是提供壳寡糖在制备干预慢性饮酒诱导的小肠损伤的功能性食品中的应用,所述的壳寡糖的相对分子量为1200-1300。
本发明的有益效果是:
本发明中壳寡糖对慢性饮酒诱导的小肠损伤的干预作用不但体现为增加小肠绒毛长度、隐窝深度和肌层厚度,还可以表现为提高紧密连接蛋白Occludin的基因表达量、降低Claudin4的基因表达量,降低血浆中D-乳酸含量和二胺氧化酶活性,能够降低丙二醛和蛋白质羰基含量,从而缓解酒精引起的肠组织的氧化损伤,同时表现为能够降低小肠炎性因子IL-6、IL-1β和TNF-α的表达,从而降低酒精引起的小肠组织的炎症反应。
附图说明
图1为壳寡糖对慢性饮酒后大鼠小肠绒毛长度、隐窝深度和肌层厚度的影响结果图。
图2为壳寡糖对慢性饮酒后大鼠小肠紧密连接蛋白Occludin和Claudin4基因表达的影响结果图。
图3为壳寡糖对慢性饮酒诱导的大鼠血液中D-乳酸(D-LA)和二胺氧化酶(DAO)的影响结果图。
图4为壳寡糖对慢性饮酒诱导的大鼠小肠丙二醛(MDA)和蛋白质羰基含量的影响结果图。
图5为壳寡糖对慢性饮酒诱导的大鼠小肠炎性因子IL-6、IL-1β和TNF-α的基因表达的影响结果图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实验材料:
药品与试剂:
壳寡糖(分子量1299Da,脱乙酰度85%):自制;D-Lactic Acid ELISA kit、二胺氧化酶(DAO)测定试剂盒、蛋白质羰基含量测定试剂盒、丙二醛试剂盒:南京建成生物工程研究所;Oligo(β-actin、Occludin、Claudin4、IL-6、IL-1β、TNF-α):苏州金唯智生物科技有限公司,引物序列见表1;AMV第一链cDNA合成试剂盒、2xSG Fast qPCR Master Mix(HighRox):生工生物工程(上海)股份有限公司。
表1 RT-PCR引物序列
仪器:
4K15冷冻型大容量离心机:德国Sigma公司;M5酶标仪:美国Molecular Devices公司;StepOnePlus实时荧光定量PCR仪:美国应用生物系统公司。
实验方法:
选取6周龄SPF级Wistar雄性大鼠30只(购自格瑞斯威动物中心),预饲养7d后,根据体重随机分为3组:对照组(Control),酒精组(ETOH),酒精+壳寡糖组(ETOH+COS)。大鼠自由进食进水。每周对大鼠体重进行记录,并对其进食量进行记录和分析。环境温度控制在22±2℃,湿度60-70%。饲养6周后大鼠禁食不禁水15h,心脏取血,3500r/min,离心20min分离血浆,ELISA法测定D-LA含量,分光光度法测定DAO含量;取大鼠小肠组织,其中一部分用4%的多聚甲醛固定,4℃保存以备后续石蜡切片的制作用,另一部分4℃生理盐水漂洗干净后,在-80℃的冰箱内保存备用。
病理检测:
对小鼠肝脏切片进行HE染色并显微镜观察小肠绒毛、隐窝和肌层的情况:
在4%多聚甲醛溶液中固定24h后,将组织块移至包埋盒中经乙醇梯度脱水、透明、浸蜡、包埋、切片、烤片,HE染色后,于光学显微镜下观察。
紧密连接蛋白Occludin、Claudin4和炎性因子IL-6、IL-1β、TNF-α的表达:
取100mg小肠组织置于1mL Trizol试剂中,于玻璃研磨器中进行破碎处理,加200μL氯仿震荡,静置;12000r/min,4℃下离心15min,取上清200μL,加异丙醇200μL震荡,静置5min;12000r/min,4℃下离心15min,弃上清,加入100%乙醇1mL,12000r/min,4℃下离心7min,弃上清后再加入75%乙醇1mL,12000r/min,4℃下离心7min,弃上清;加入DEPC水30μL,待完全溶解后,测定RNA浓度。按照试剂盒说明书,将提取的小肠组织RNA反转录为cDNA,使用SYBR Green Mix进行基因扩增检测,其扩增程序为:95℃变性30s;95℃变性3s,60℃退火30s为第一个循环,共40个循环。检测各模板的Ct值,实验以β-actin为内参基因,以正常组为对照组,使用2~△△Ct法进行分析计算。
统计学方法:
采用SPSS 17.0统计软件中的one-way ANOVA方法对实验数据进行多重比较、差异显著性检验分析。
实施例1:壳寡糖对慢性饮酒后大鼠小肠绒毛、隐窝和肌层的影响
对大鼠进行6周的酒精和壳寡糖饲喂后,取其小肠,制作病理学组织切片,进行HE染色,参考图1可知,本发明灌胃壳寡糖后能够改善酒精诱导的大鼠小肠绒毛变短、尖端脱落,形态不明显、隐窝深度增加和肌层厚度降低的问题。
实施例2:壳寡糖对慢性饮酒后大鼠小肠紧密连接蛋白基因表达的影响
相邻的上皮细胞的间通过多种蛋白及分子构成的连接复合体,从顶端到基膜依次为紧密连接(TJ)、粘附连接、桥粒连接和缝隙连接等,共同起着加强细胞间连接功能、组成传递信息通道的作用,其中以TJ最为重要。炎症性肠病在发病时,肠粘膜所产生的大量炎症因子等会损伤肠上皮细胞,并影响肠上皮细胞TJ蛋白的表达及分布,破坏TJ结构,造成肠粘膜屏障功能受损。参考图2所示,与正常组相比,6周的酒精灌胃使小肠组织的紧密连接蛋白Occludin的基因表达显著降低(P<0.01),且使紧密连接蛋白Claudin4的基因表达显著提高(P<0.01)。与模型组相比,壳寡糖干预的大鼠,其小肠Claudin4的基因表达恢复至正常水平,与对照组相比无显著性差异(P<0.01);同时,与模型组相比,壳寡糖干预可显著提高Occludin的基因表达(P<0.05),缓解了由于灌胃酒精导致的小肠紧密连接蛋白的表达降低及紧密连接结构的破坏。
实施例3:壳寡糖对慢性饮酒后大鼠血液中D-乳酸和二胺氧化酶的影响
当肠黏膜通透性增加时,肠道细菌产生的大量D-乳酸(D-LA)就可以进入血液循环,因此可被作为细菌感染和肠通透性增加的可靠指标。参考图3可知,与对照组相比,灌胃6周酒精的大鼠血浆中D-LA含量显著上升(P<0.01),可见酒精损伤肠道屏障,增加了小肠粘膜的通透性,而壳寡糖的干预可以显著降低血浆中的D-LA含量(P<0.01),使其恢复至与正常组无显著性差异的水平(P<0.01)。
二胺氧化酶(DAO)主要催化组胺的二胺氧化过程,其存在于人小肠黏膜、胎盘、肾脏和胸腺组织中,但血液中的DAO主要来自于小肠。目前,DAO的检测在实验研究中经常被作为一种很好的肠黏膜受损标志物。参考图3可知,与对照组相比,灌胃6周酒精的大鼠血浆中DAO活性显著上升(P<0.05),可见酒精损伤肠道屏障,增加了小肠粘膜的通透性,而壳寡糖的干预可以显著降低血浆中的D-LA活性(P<0.05),使其恢复至与正常组无显著性差异的水平(P<0.05)。综上,与模型组相比,壳寡糖可以显著降低大鼠血浆中D-LA含量和DAO活性,减缓慢性饮酒造成的小肠粘膜损伤。
实施例4:壳寡糖对慢性饮酒后大鼠小肠氧化损伤的影响
丙二醛(MDA)作为脂质过氧化物的代表,机体缺血缺氧时产生自由基的多少与MDA水平呈正相关,反映了氧自由基对细胞损伤的程度,它的产生能加剧黏膜的损伤,破坏细胞骨架,最终导致肠屏障的损害,细菌移位。因此可通过MDA了解脂质过氧化程度,以间接反映大鼠小肠黏膜的损伤程度及肠功能。蛋白质羰基含量的增加表明蛋白质的氧化变化和化学修饰可能导致许多酶的结构改变和功能失活,组织中的蛋白质羰基含量能够反映机体蛋白质的氧化损伤的情况。
参考图4可知,与对照组相比,连续6周灌胃酒精(模型组)显著提高了大鼠小肠组织的MDA含量(P<0.01)和蛋白质羰基含量(P<0.01),而与模型组相比,连续6周的壳寡糖干预,可以显著降低大鼠小肠组织中MDA含量(P<0.01)和蛋白质羰基含量(P<0.01),降低灌胃酒精造成的氧化损伤。
实施例5:壳寡糖对慢性饮酒后大鼠小肠炎性因子的基因表达的影响
参考图5可知,与正常组相比,连续6周灌胃酒精的大鼠小肠组织中的IL-6、IL-1β、TNF-α的基因表达均显著增高(P<0.01),说明灌胃酒精能够提高炎性因子的表达,造成肠道炎症的发生。与模型组相比,灌胃壳寡糖能够降低小肠组织中IL-6、IL-1β、TNF-α的转录水平(P<0.01),且恢复至与对照组无显著性差异的水平(P<0.01),说明壳寡糖可以改善灌胃酒精造成的炎症反应,从而发挥抗炎作用。
综上,本发明壳寡糖添加后能够显著增加小肠绒毛长度、隐窝深度和肌层厚度;本发明壳寡糖添加后能够显著提高紧密连接蛋白Occludin的基因表达量、降低Claudin4的基因表达量;本发明壳寡糖添加后能够显著降低血浆中D-LA和DAO的含量;本发明壳寡糖添加后能明显降低MDA和蛋白质羰基含量;本发明壳寡糖添加后能够明显降低小肠炎性因子IL-6、IL-1β和TNF-α的表达,从而降低酒精引起的小肠组织的炎症反应。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (9)
1.一种干预慢性饮酒诱导的小肠损伤的功能性食品,其特征在于,包含相对分子量为1200-1300的壳寡糖。
2.根据权利要求1所述的功能性食品,其特征在于,所述的壳寡糖的聚合度为6-7。
3.根据权利要求1所述的功能性食品,其特征在于,所述的壳寡糖的脱乙酰度为80-90%。
4.根据权利要求1所述的功能性食品,其特征在于,所述的壳寡糖的用量,以质量百分比计为0.05%-0.08%。
5.根据权利要求1所述的功能性食品,其特征在于,所述的干预慢性饮酒诱导的小肠损伤的功能性食品为提高紧密连接蛋白Occludin的表达量、降低Claudin4的表达量的功能性食品。
6.根据权利要求1所述的功能性食品,其特征在于,所述的干预慢性饮酒诱导的小肠损伤的功能性食品为降低血浆中D-乳酸含量和二胺氧化酶活性的功能性食品。
7.根据权利要求1所述的功能性食品,其特征在于,所述的干预慢性饮酒诱导的小肠损伤的功能性食品为降低丙二醛和蛋白质羰基含量的功能性食品。
8.根据权利要求1所述的功能性食品,其特征在于,所述的干预慢性饮酒诱导的小肠损伤的功能性食品为降低小肠炎性因子IL-6、IL-1β和TNF-α的表达的功能性食品。
9.壳寡糖在制备干预慢性饮酒诱导的小肠损伤的功能性食品中的应用,其特征在于,所述的壳寡糖的相对分子量为1200-1300。
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