CN117143766A - 一株修复肠神经的副干酪乳杆菌及其应用 - Google Patents
一株修复肠神经的副干酪乳杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株修复肠神经的副干酪乳杆菌及其应用,属于微生物领域。本发明提供的副干酪乳杆菌CCFM1321能够显著增加肠神经胶质细胞数量,提高胶质细胞源性神经营养因子GDNF的水平及其受体GFRα1/RET的表达,修复小鼠受损的肠神经系统。下调Toll样受体2(TLR2)的过度激活,降低抑制性神经递质的表达,提高血清素5‑HT及其受体5‑HT2B的表达,起到保护肠神经系统的效果。并且能够降低由肠神经受损导致的结肠组织中炎症水平,改善结肠组织病理损伤,缓解肠道动力障碍。因此针对肠神经系统异常的胃肠道疾病可以采用更加个性化的治疗方案,具有可观的应用前景。
Description
技术领域
本发明涉及一株修复肠神经的副干酪乳杆菌及其应用,属于微生物领域。
背景技术
胃肠道对于维持宿主的正常生理活动起重要作用,例如食物和废物的运输,营养物质的消化和吸收;信号分子和抗菌物质的分泌;保持肠道屏障的完整性;维持肠道菌群平衡以及防止病原体的侵入等。这些关键过程的调节主要依赖于肠神经系统(entericnervous system,ENS),ENS是胃肠道消化和防御功能调控的核心。
ENS由肠神经元和众多的肠神经胶质细胞(enteric glail cells,EGCs)组成,可独立于中枢神经系统起到自主调节肠道稳态的作用,因此被称为“胃肠道的大脑”、“人体第二大脑”等。EGCs是ENS的重要组成部分,具有支持、营养和保护肠神经元、调节肠动力、维持肠道稳态的作用。EGCs与肠神经元之间活跃的信号机制能够调节胃肠道反射:在生理状态下,EGCs可以分泌多种神经营养因子如胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)、亚硝基谷胱甘肽、转化生长因子-β等对肠神经以及肠上皮细胞产生保护作用。其中GDNF-RET信号通路是在ENS正常发育中重要的信号通路,RET是一种跨膜酪氨酸激酶,在GDNF与辅助受体GDNF家族受体(GFRα1)结合时被激活。GDNF-GFRα1-RET信号是ENS祖细胞存活、增殖和迁移所必需的。EGCs与免疫细胞之间存在双向沟通,EGCs可以响应免疫调节信号,如细胞外环境中的细胞因子,细菌和神经递质。通过自分泌或旁分泌调控EGCs分泌的神经胶质细胞系衍生的神经营养因子(GDNF)可以抑制其凋亡,减少促炎细胞因子的释放,发挥免疫抑制和抗炎作用,并参与免疫屏障的功能。因此EGCs的神经--免疫调节作用在胃肠道功能中扮演重要角色,上述任何一个或多个功能受损都会导致广泛且明显的肠神经病变。以长期服用刺激性泻药(番泻苷、大黄素等)导致的顽固性便秘——泻剂结肠为例:泻剂结肠在组织病理学上表现出严重的肠神经损伤,主要表现为肠神经胶质细胞的数量丢失以及功能障碍、并伴随着肠神经元退行性变化,也有部分研究观察到结肠粘膜病变。
ENS退行性病变可导致胃肠道功能异常进而造成肠道运动障碍。肠道运动障碍分为动力减弱型及动力增加型,临床上常表现为便秘和腹泻症状。而目前有关于肠神经系统异常病因及诊疗机制研究尚不完善。针对ENS退行性病变可能的治疗靶点有:增加保护性物质,即可以通过增加某些神经营养因子(GDNF)或5-HT4受体激动剂(普芦卡必利)发挥对ENS的保护作用;调节肠道菌群,肠道微生物已被证明对ENS的发展和维持有着深远的影响。缺乏肠道微生物的小鼠的ENS是不成熟的。研究表明,在无菌小鼠中,微生物定植诱导的ENS的成熟依赖于5-HT4受体(5-HT4R)信号,取消内源性5-HT4或阻断5-HT4R可阻止这一配对过程。此外,5-HT4R激动剂对无菌小鼠的治疗使得肠神经细胞的分化和成熟。肠道微生物也可能通过微生物的模式识别受体(如TLR2、TLR4等)调节ENS的发育和功能。EGCs能差异性的应答益生菌(副乳杆菌F19)和有害菌(大肠杆菌)的刺激,这也提示EGCs可能是肠道菌群的主要靶细胞。
因此深入研究了解微生物和EGCs间的相互影响和作用,揭示共生微生物与宿主的神经和免疫系统的联系,有助于阐明其发病机制和致病性,为胃肠道疾病提供新的治疗策略。
发明内容
技术问题
本发明要解决的技术问题是,提供一株通过调控GDNF/RET信号通路修复肠神经的副干酪乳杆菌,并且提供该菌株的应用。
技术方案
为了解决上述技术问题,本发明提供了一株副干酪乳杆菌(Lacticaseibacillusparacasei),能够有效的修复肠神经系统。该菌株且与CCFM1164菌株相比,肠神经胶质细胞标志物(GFAP和S100β)的表达上调,胶质细胞源性神经营养因子(GDNF)含量增加38.25%,炎症水平和结肠病理损伤也明显改善,表明该菌株能够修复肠神经系统并缓解由肠神经受损导致的多种不利影响,从而给肠神经系统异常患者带来帮助。
本发明提供了一株副干酪乳杆菌(Lacticaseibacillus paracasei),所述副干酪乳杆菌于2023年8月4日保藏于广东省微生物菌种保藏中心,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC No:63717。
所述副干酪乳杆菌(Lacticaseibacillus paracasei)来自于浙江省金华市一女童的粪便样品中,该菌株经测序分析,将测序得到的序列在NCBI Standard NucleotideBLAST中进行核酸序列比对,结果显示与副干酪乳杆菌属的核酸序列相似度为100%;结果显示菌株为副干酪乳杆菌,将其命名为副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321。
所述副干酪乳杆菌CCFM1321具有下述生物学特性:
(1)菌体特征:呈革兰氏染色阳性,不形成孢子,非运动性的细菌。
(2)菌落特征:呈圆形凸起、光滑、边缘整齐。
(3)生长特性:在37℃恒温条件下,在MRS培养基培养约18h到达对数末期。
(4)对模拟胃肠液具有较强耐受能力。
(5)显著提高结肠组织中肠神经胶质细胞数量,增加结肠组织中胶质细胞源性神经营养因子及其受体的含量或表达量,下调TLR2的过度激活,降低抑制性神经递质的表达,提高血清素及其受体5-HT2B的表达,并且能够改善结肠组织的病理损伤,降低肠道炎症水平,缓解动力减弱型肠道运动障碍。
本发明还提供了一种含有上述副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321的微生物制剂。
本发明还提供了一种食品,所述食品中含有上述副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321的微生物制剂。
在本发明的一种实施方式中,所述食品为发酵食品,所述发酵食品为使用副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321发酵生产制得,所述发酵食品包括固态食品、液态食品、半固态食品。
在本发明的一种实施方式中,所述副干酪乳杆菌在食品中的添加量至少为108CFU/mL或108CFU/g。
在本发明的一种实施方式中,所述发酵食品包括乳制品、豆制品或果蔬制品。
在本发明的一种实施方式中,所述乳制品为发酵乳制品,包括发酵乳、发酵乳饮料、奶油、乳酪或乳粉;所述豆制品包括豆奶、豆乳饮料、豆乳粉;所述果蔬制品包括以白菜、白萝卜、黄瓜、甜菜、黄桃或杨梅为原料发酵的发酵果蔬饮品或食品。
本发明还提供了一种药品,所述药品中含有上述副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321或上述微生物菌剂。
在本发明的一种实施方式中,所述药品包含副干酪乳杆菌CCFM1321以及能够在药学上允许的载体。
在本发明的一种实施方式中,所述载体包括医学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂、矫味剂中的一种或多种。
在本发明的一种实施方式中,所述药品的剂型包括颗粒剂、胶囊剂、片剂、丸剂或口服液。
在本发明的一种实施方式中,所述副干酪乳杆菌(Lacticaseibacillusparacasei)CCFM1321在药品中的添加量至少为108CFU/mL或108CFU/g。
本发明还提供了上述副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321或上述微生物菌剂在制备有助于润肠通便的保健品中的应用。
本发明还提供了上述副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321或上述微生物菌剂在制备缓解肠道神经受损的药品中的应用,其特征在于,所述的缓解肠道受损包括以下至少一种:
(a)增加肠神经胶质细胞数量,修复受损的肠神经系统;
(b)提高胶质细胞源性神经营养因子(GDNF)的水平及其受体GFRα1/RET的表达;
(c)下调Toll样受体2(TLR2)的过度激活,降低神经元型一氧化氮合酶(nNOS)表达;
(d)提高血清素(5-HT)及其受体5-HT2B的表达;
(e)降低结肠组织中炎症水平,改善结肠组织病理损伤;
(f)缓解动力减弱型肠道运动障碍。
本发明还提供含有所述副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321的菌剂。
在本发明的一种实施方式中,所述菌剂中副干酪乳杆菌(Lacticaseibacillusparacasei)CCFM1321的活菌数量≥108cfu/g或108cfu/mL。
在本发明的一种实施方式中,所述菌剂是将含有副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321的菌液干燥得到的活菌数量≥108cfu/g或108cfu/mL的粉剂。
在本发明的一种实施方式中,所述干燥是指真空冷冻干燥。
有益效果
(1)本发明的副干酪乳杆菌GDMCC No:63717活性较好,相较于模型组,CCFM1321干预组能使结肠组织中肠神经胶质细胞标志蛋白基因S100β表达量显著提高49.98%(P<0.05),结肠组织中胶质细胞源性神经营养因子含量增加185.66%(P<0.01)及其RET受体表达量提高48.68%(P<0.01),Toll样受体2(TLR2)的过度激活下调43.15%(p<0.01),神经元型一氧化氮合酶(nNOS)的表达降低70.21%(p<0.01),血清素(5-HT)的表达量提高151.35%(p<0.05),降低结肠组织中炎症水平,改善结肠组织的病理损伤,并能够缓解动力减弱型肠道运动障碍。重要的是,副干酪乳杆菌CCFM1321在EGC数量、GDNF含量及其受体RET表达量等指标上效果要明显优于普芦卡必利药物,且与CCFM1164相比,GDNF含量能够明显增加38.25%,且对于EGC数量的增多更具有显著性。因此,此菌株对于肠神经的修复效果更为明显。
(2)本发明可以视为一种缓解或治疗动力减弱型肠道运动障碍的药物,还可以应用于药品或者是一些发酵食品及功能性食品中,从而广泛发挥其作用,具有非常有价值的应用前景。
生物材料保藏
一株副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321,其分类学命名为:Lacticaseibacillus paracasei,已于2023年8月4日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:63717,保藏地址为广州市先烈中路100号大院59号楼5楼,广东省微生物研究所。
附图说明
图1:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321对肠神经系统的影响,(a)S100β相对表达量;(b)GFAP相对表达量。
图2:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321对GDNF/GFRα1/RET信号通路的影响,(a)GDNF浓度;(c)GFRα1相对表达量;(c)RET相对表达量。
图3:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321对TLR2和nNOS的影响,(a)TLR2相对表达量;(b)nNOS相对表达量。
图4:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321对5-HT和5-HT2B的影响,(a)5-HT相对表达量;(b)5-HT2B相对表达量。
图5:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321对炎症因子及肠道机械屏障的影响,(a)IL-1β相对表达量;(b)IL-6相对表达量;(c)结肠组织H&E染色。
图6:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321对肠道运动功能的影响,(a)首粒黑便时间;(b)5h粪便粒数;(c)小肠推进率;(d)粪便含水量。
具体实施方式
下述实施例中所涉及的雄性C57BL/6J小鼠购自浙江维通利华实验动物技术有限公司。
下述实施例中所涉及的菌株信息如下:
副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321,分类命名为Lacticaseibacillus paracasei,已于2023年8月4日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC NO:63717,保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所。
副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1164,其分类学命名为:Lacticaseibacillus paracasei,已公开于专利CN112877260A,保藏编号为GDMCC No:61479。
下述实施例中所涉及的培养基如下:
MRS液体培养基:牛肉膏10g;胰蛋白胨10g;酵母粉5g;葡萄糖20g;无水乙酸钠5g;MgSO4·7H2O 0.1g;MnSO4·H2O 0.05g;柠檬酸氢二铵2g;K2HPO4·3H2O 2.6g;吐温80 1mL;调节pH为6.8±0.2;定容至1L。高压灭菌115℃20min。
MRS固体培养基:在MRS液体培养基的基础上添加2%琼脂粉。
下述实施例中所涉及的副干酪乳杆菌菌悬液的制备
将副干酪乳杆菌CCFM1321和副干酪乳杆菌CCFM1164分别接种至MRS固体培养基中,在37℃条件下培养48h得到单菌落,将制备得到的单菌落分别接种至MRS液体培养基中,在37℃条件下培养24h进行活化。
将活化3代后的菌液以2%体积比的接种量分别接种至1L MRS液体培养基中,振荡混匀后于恒温培养箱中37℃培养18h。在8000g/min,4℃的条件下离心10min,去上清后,用无菌生理盐水进行清洗2次,同样以相同条件进行离心,去上清后,用质量分数为10%的灭菌脱脂乳液重悬菌体,分别制备得到终浓度均为5×109CFU/mL的副干酪乳杆菌CCFM1321菌悬液、副干酪乳杆菌CCFM1164菌悬液,至此获得灌胃前备用菌液,-80℃冰箱冻存一周。
在进行动物实验前,将冰箱中冻存的菌液取出,用平板涂布法测定初始和冻存一周后的活菌数量。初始活菌数和1周后活菌数的数量级并没有产生变化,说明将菌液冻存后不会对实验产生影响,可用于动物实验。
下述实施例中所涉及的S100β/GFAP基因、GFRα1/RET基因、TLR2/nNOS基因和IL-1β/IL-6基因的表达量的检测方法
采用实时荧光定量聚合酶链反应RT-qPCR)来测定S100β/GFAP基因、GFRα1/RET基因、TLR2/nNOS基因和IL-1β/IL-6基因的表达量,首先需要从新鲜组织中提取RNA,具体方法如下:
将小鼠解剖后取出的新鲜结肠组织0.2g在加有液氮的研钵(180℃,4h高温灭酶)中反复研磨,再向研钵中加入1mL TrizoL试剂,继续研磨,待液体基本澄清后,收集至1.5mL无酶离心管中,室温静置15min,向离心管中加入200μL三氯甲烷溶液,轻摇15s,室温静置10min,4℃、12000r/min离心15min,取600μL上层无色水相至另一只无酶离心管中,加入500μL异丙醇。上下颠倒混匀,室温下静置10min,静置结束后,4℃、12000r/min离心10min,弃去上清,留下RNA在离心管底部形成的白色沉淀,加入1mL用DEPC水配制的75%的乙醇溶液,漩涡震荡重悬,4℃、7500r/min离心5min,弃去上清,室温自然挥发干燥。向干燥的RNA中加入30μL RNase free water,待RNA溶解后,以Nanodrop测定RNA浓度及纯度,并通过琼脂糖凝胶电泳检测RNA的质量。以提取的总RNA为模板,根据康维世纪公司的HiFiScript gDNARemovaL RT MasterMix反转录试剂盒说明书操作步骤逆转录合成cDNA,-20℃下保存。
小鼠S100β/GFAP基因、GFRα1/RET基因、TLR2/nNOS基因、IL-1β/IL-6基因和内参基因GAPDH基因引物如表1所示,
表1引物序列
RT-qPCR反应体系及条件:
用CFX96TM实时荧光定量PCR仪进行PCR扩增,并读取荧光信号。
RT-qPCR反应体系为:
表2RT-qPCR反应体系
RT-qPCR反应条件为:
95℃30s;95℃10s,60℃30s,共40个循环。以GAPDH基因作为内参基因,通过CFX96Manager软件分析结果。
下述实施例中所涉及的结肠组织中GDNF的含量的检测方法
采用ELISA方法对结肠组织中GDNF表达量进行定量。具体方法如下:用预冷的PBS冲洗结肠组织,去除残留血液,剔除周围脂肪组织,称重后将其剪碎。将剪碎的组织与PBS溶液按照1:9的重量体积比,在高通量组织破碎仪上进行破碎,最后将匀浆与5000×g离心510分钟,取上清检测,参照相应试剂盒说明书进行实验,根据标准曲线计算中组织中GDNF。
实施例1:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321的获得
1、副干酪乳杆菌的分离筛选:
(l)使用一次性无菌取便器采集浙江省金华市一女童的粪便样品,置于厌氧环境下37℃培养72小时;观察记录菌落形态,挑取菌落划线纯化;在MRS液体培养基中,37℃培养48小时,所得菌落进行革兰氏染色,记录菌落形态。弃除菌落中的革兰氏阴性菌菌株和革兰氏阳性球菌,挑选得到革兰氏阳性杆菌。
(2)过氧化氢酶分析后,弃除过氧化氢酶阳性菌株,保留过氧化氢酶阴性菌株。
2、副干酪乳杆菌的分子生物学鉴定:
(l)单菌基因组抽提:将步骤1所筛选得到的过氧化氢酶阴性菌株培养过夜,取培养过夜的菌悬液l mL于1.5mL离心管,10000rpm离心2min,弃上清得菌体;用l mL无菌水吹洗菌体后,10000rpm离心2min,弃上清得菌体;加入200μL SDS裂解液,80℃水浴30min;加入酚-氯仿溶液200μL于菌体裂解液中,其中酚-氯仿溶液的组成成分及体积比为Tris饱和酚:氯仿:异戊醇=25:24:1,颠倒混匀后,12000rpm离心5~10min,取上清200μL;加入400μL冰乙醇或冰异丙醇于200uL上清中,-20℃静置1h,12000rpm离心5~10min,弃上清;加入500μL70%(体积百分数)冰乙醇重悬沉淀,12000rpm离心1~3min,弃上清;60℃烘箱烘干,或者自然晾干;50μL ddH2O重溶沉淀以备PCR。
(2)16S rDNA PCR:
A.细菌16S rDNA50μLPCR反应体系:10×Taq buffer,5μL;dNTP,5μL;27F,0.5μL;1492R,0.5μL;Taq酶,0.5μL;模板,0.5μL;ddH2O,38μL。
B.PCR条件:95℃5min;95℃10s;55℃30s;72℃30s;step2-4 30×;72℃5min;12℃2min。
C.制备1%琼脂糖凝胶,之后将PCR产物与10000×loading buffer混合,上样量2μL,120V跑30min,然后进行凝胶成像。
D.将得到PCR产物送专业测序公司,将得到的测序结果与使用BLAST在GeneBank中进行搜索和相似性比对,鉴定为副干酪乳杆菌,命名为副干酪乳杆菌(Lacticaseibacillusparacasei)CCFM1321,保藏于广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC NO:63717,-80℃保藏备用。
实施例2:副干酪乳杆菌CCFM1321提高肠神经受损小鼠结肠组织中肠神经胶质细胞数量具体步骤如下:
(1)副干酪乳杆菌(Lacticaseibacillus paracasei)菌悬液的制备
将副干酪乳杆菌CCFM1321菌种于-80℃冰箱取出后,划线于MRS固体培养基中,37℃培养48h,挑取单菌落于MRS液体培养基中,37℃培养24h,制备得到种子液;
将制备得到的种子液以2%(v/v)的接种量接种于新的MRS液体培养基中,于37℃培养18h,按照同样的方式再次培养一代,制备得到副干酪乳杆菌CCFM1321发酵液;
然后将制备得到副干酪乳杆菌CCFM1321发酵液在8000r/min、4℃条件下离心10min,然后用质量分数10%的脱脂乳水溶液进行重悬,制得菌悬液,用于动物实验。
(2)取8周龄的健康雄性C57BL/6J小鼠30只,适应环境1周,随机分为5组:对照组、模型组、普芦卡必利组(阳性药物组)、副干酪乳杆菌CCFM1164组(阳性菌组)(已公开于专利CN112877260A)、副干酪乳杆菌CCFM1321组,每组含小鼠6只,灌胃菌悬液的剂量为5×109CFU/mL,每天早上9点开始灌胃,每次0.2mL。
实验动物分组及处理方法见表3:
表3实验动物分组
实验结束后,用小鼠结肠中肠神经胶质细胞特征性蛋白S100β和GFAP的基因表达水平来表征肠神经中胶质细胞的数量。
黏膜的胶质细胞主要参与上皮屏障功能,神经节内的胶质细胞主要作用于神经修复,与神经元密切互动,支持这些细胞和胶质细胞的分化、参与神经的发生和形成。胶质细胞的数量可以很好地反应肠神经的健康状况,S100β和GFAP和是神经胶质细胞的特征性蛋白,根据S100β和GFAP的量可以反映胶质细胞的数量。由图1可知,模型组小鼠S100β和GFAP表达量相比于对照组分别减少了49.96%和43.79%(p<0.01),表明模型小鼠存在肠神经损伤,肠神经受损模型构建成功。灌胃CCFM1321后,标志蛋白基因S100β和GFAP表达水平较模型组分别提高了49.98%和60.67%(p<0.05),GFAP表达量较CCFM1164组提高了25.95%,表明副干酪乳杆菌组CCFM1321参与了肠神经重建,可恢复肠神经受损小鼠的肠神经健康。
实施例3:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321可提高肠神经受损小鼠结肠组织胶质细胞源性神经营养因子(GDNF)的含量
C57BL/6J小鼠分组、造模及处理方法同实施例2。采用ELISA方法对结肠组织中GDNF表达量进行定量。具体方法如下:
用预冷的PBS冲洗结肠组织,去除残留血液,剔除周围脂肪组织,称重后将其剪碎。将剪碎的组织与PBS溶液按照1:9的重量体积比,在高通量组织破碎仪上进行破碎,最后将匀浆于5000×g离心5-10分钟,取上清检测,参照相应试剂盒说明书进行实验,根据标准曲线计算结肠组织中GDNF。结果如图2a所示。
GDNF是一种多肽类的神经营养因子,对损伤的神经细胞有营养和保护作用,在退变性神经元中表达降低,提示神经元损伤。由图2a可知,模型组肠神经受损小鼠GDNF水平(20.16pg/mg)较对照组(44.09pg/mg)降低了54.28%(p<0.01),提示存在肠神经受损。药物普芦卡必利组(27.53pg/mg)相较于模型组的GDNF水平提高36.56%,CCFM1164组(41.65pg/mg)相较于模型组的GDNF水平提高106.56%(p<0.05),灌胃CCFM1321后GDNF水平(57.59pg/mg)较模型组显著提高了185.66%(p<0.001)。CCFM1321组GDNF水平比CCFM1164组提高了38.25%,比药物普芦卡必利组提高了109.19%。
副干酪乳杆菌CCFM1321通过提高GDNF表达量,达到滋养肠神经的效果,恢复正常的肠神经功能。
实施例4:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321可提高肠神经受损小鼠结肠组织中GDNF下游受体GFRα1/RET的表达量
C57BL/6J小鼠分组、造模及处理方法同实施例2。具体方法如下:
采用实时荧光定量聚合酶链反应(RT-qPCR)来测定GFRα1和RET基因的表达量。神经胶质细胞系衍生的神经营养因子(GDNF)是一种配体,通过共受体GDNF家族受体α-1(GFRα1)和受体酪氨酸激酶“RET”激活几种信号通路,这些信号通路对多个神经元群体的发育和维持至关重要。由图2b/c可知,肠神经受损小鼠GFRα1基因表达量相比于对照组明显减少了45.92%(p<0.01),RET基因表达量相比于对照组明显减少了58.93%(p<0.001),表明肠神经受损小鼠GDNF/GFRα1/RET信号通路存在异常,肠神经系统功能异常。灌胃CCFM1321后,GFRα1基因和RET基因的表达水平较模型组分别提高了49.36%和48.68%(p<0.01),趋向于空白对照组,优于CCFM1164组和阳性药物组,其中CCFM1321组较CCFM1164组RET基因表达量提高了23.42%。
据报道,GDNF/GFRα1/RET三元复合物发出的信号在肠神经系统的自我更新中起至关重要作用。因此推断CCFM1321菌株修复肠神经或依赖于GDNF/GFRα1/RET信号通路。
实施例5:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321调节肠神经受损小鼠结肠组织中TLR2的过度激活,降低神经元型一氧化氮合酶(nNOS)的表达
C57BL/6J小鼠分组、造模及处理方法同实施例2。具体方法如下:
采用实时荧光定量聚合酶链反应(RT-qPCR)来测定TLR2和nNOS基因的表达量。研究表明TLR2的激活能够促进氮能神经元的增殖,对于肠动力具有调节作用。由图3可知,肠神经受损小鼠TLR2基因表达量相比于对照组明显增加了44.59%(p<0.05),nNOS基因表达量相比于对照组增加了131.42%(p<0.01),表明模型小鼠存在TLR2过度激活现象,导致抑制性神经递质的过度释放,对肠神经产生毒害作用。灌胃CCFM1321后,将TLR2基因的过度表达下调了43.15%(p<0.01),将神经元型一氧化氮合酶的表达降低了70.21%(p<0.001)。
推测CCFM1321菌株修复肠神经或跟TLR2-nNOS信号通路有关。
实施例6:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321提高肠神经受损小鼠结肠组织中5-HT及其受体5-HT2B的表达
C57BL/6J小鼠分组、造模及处理方法同实施例2。具体方法如下:
采用实时荧光定量聚合酶链式反应(RT-qPCR)来测定5-HT和5-HT2B基因的表达量。
血清素(5-HT)主要由肠嗜铬细胞释放,并且通过作用于相应受体才能产生作用。其中,5-HT能够通过作用于5-HT2B,从而激活乙酰胆碱或降钙素基因相关肽,进而增加近端平滑肌的收缩,5-HT2B也能够调节Cajal-间质细胞的数量进而调节肠蠕动。结果如图4所示,肠神经受损小鼠5-HT基因表达量相比于对照组明显减少了65.39%(p<0.05),5-HT2B基因表达量相比于对照组明显减少了30.11%(p<0.01),表明肠神经受损小鼠结肠组织兴奋性神经递质减少。灌胃CCFM1321后,5-HT基因和5-HT2B基因表达水平较模型组分别提高了151.35%(p<0.05)和48.49%,较CCFM1164组分别提高了44.53%和47.01%,趋向于空白对照组,优于阳性药物组。
因此,从结果中可以发现,副干酪乳杆菌CCFM1321能够通过促进结肠组织中5-HT和5-HT2B基因的表达来修复肠神经系统。
实施例7:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321降低小鼠肠神经受损所导致的炎症水平和结肠组织病理损伤
C57BL/6J小鼠分组、造模及处理方法同实施例2。具体方法如下:
采用实时荧光定量聚合酶链式反应(RT-qPCR)来测定IL-1β和IL-6基因的表达量,采用H&E染色小鼠结肠组织切片来表征小鼠结肠组织病理损伤。小鼠解剖后取新鲜组织放入多聚甲醛固定液,浸泡后,过夜冲洗。将样品依次经70%、80%、90%各级乙醇溶液脱水,各30min,再放入95%、100%各2次,每次20min进行脱水。放入1/2纯酒精,1/2二甲苯等量混合液15min,二甲苯前洗液15min、二甲苯后洗液15min至透明为止。放入二甲苯和石蜡各半的混合液15min,再放入石蜡Ⅰ、石蜡Ⅱ透蜡各50-60分钟来除去透明剂。将处理好的样品进行包埋切片、展片、烤片、H&E染色与封片。
IL-1β和IL-6具有较强的促炎活性,可诱导多种促炎介质,并最终导致广泛的炎症事件。结果如图5a/b所示,模型组与对照组相比两种促炎因子都显著升高(p<0.01),表明肠神经受损小鼠存在肠道炎症。灌胃CCFM1321后,IL-1β和IL-6基因表达水平较模型组显著降低(p<0.05)。观察小鼠结肠组织的病理切片,如图5c所示,模型组与对照组相比,模型组小鼠的结肠组织存在黏液层变薄,隐窝结构缩短,杯状细胞减少,并且存在炎症浸润的现象。但是灌胃副干酪乳杆菌CCFM1321后,小鼠结肠组织中的黏液层变厚,杯状细胞的界限更加清晰明了,同时平滑肌结构也较为完整。而灌胃普芦卡必利药物与副干酪乳杆菌CCFM1164后,小鼠的黏液层厚度增加,但增加的程度不如CCFM1321,杯状细胞与隐窝结构更加清晰,炎症浸润的情况也有所减少。
实施例8:副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321对肠神经受损导致的小鼠肠道动力障碍相关症状的缓解
C57BL/6J小鼠分组、造模及处理方法同实施例2。具体方法如下:
第16周灌胃结束后,将小鼠单只放入垫有吸水纸的笼盒中,收集粪便,称重即为湿重,冻干后,即为干重,按照如下公式计算粪便含水量。
粪便含水量=(粪便湿重-粪便干重)/粪便湿重×100%
分别向每只小鼠灌胃0.2mL墨汁,从灌胃开始,记录每只小鼠排首粒黑便时间和5小时内排黑便粒数。
小鼠处死之前,每只小鼠灌胃0.2mL墨汁,30min后处死解剖,剪取上端自幽门下端至盲肠,测量小肠全长为“小肠总长度”,从幽门到墨汁前沿为“墨汁推进长度”,按照以下公式计算小肠推进率。
小肠推进率=(墨汁推进长度(cm))/(小肠总长度(cm))×100%
粪便含水量、首粒黑便时间、5h粪便粒数、小肠推进率结果如图6所示,由图可知,相比于对照组,造模后小鼠粪便含水量下降了10.98%(p<0.01),首粒黑便时间延长至空白对照组的1.38倍(p<0.01),5h内粪便粒数减少了30.26%(p<0.01),而小肠推进率无显著变化,即模型小鼠存在由肠神经受损导致的结肠转运障碍。相较于模型组,灌胃副干酪乳杆菌CCFM1321处理可将肠神经受损小鼠的首粒黑便时间缩短至模型的70.38%(p<0.05),5h粪便粒数较模型组提高了66.67%(p<0.01),增强肠神经受损小鼠的结肠蠕动能力,且效果优于药物普芦卡必利组和阳性菌株对照组。说明灌胃副干酪乳杆菌CCFM1321可以缓解肠神经受损导致的小鼠肠道动力障碍的症状。
因此,从结果中可以看出,副干酪乳杆菌CCFM1321能够有效修复受损的肠神经系统,改善由肠神经受损引发的肠道炎症和结肠病理损伤,并且能够缓解肠神经受损导致的肠道动力障碍。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一株副干酪乳杆菌(Lacticaseibacillus paracasei)CCFM1321,其特征在于,所述副干酪乳杆菌CCFM1321于2023年8月4日保藏于广东省微生物菌种保藏中心,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC No:63717。
2.含有权利要求1所述的副干酪乳杆菌的微生物菌剂。
3.含权利要求1所述的副干酪乳杆菌或权利要求2所述的微生物菌剂的食品。
4.如权利要求3所述的食品,其特征在于,所述食品包括发酵食品。
5.如权利要求3或4所述的食品,其特征在于,所述副干酪乳杆菌在食品中的添加量至少为108CFU/mL或108CFU/g。
6.含有权利要求1所述的副干酪乳杆菌或权利要求2所述的微生物菌剂的药品。
7.如权利要求6所述的药品,其特征在于,所述药品的剂型包括颗粒剂、胶囊剂、片剂、丸剂或口服液。
8.如权利要求6或7所述的药品,其特征在于,所述副干酪乳杆菌在药品中的添加量至少为108CFU/mL或108CFU/g。
9.权利要求1所述的副干酪乳杆菌或权利要求2所述的微生物菌剂在制备有助于润肠通便的保健品中的应用。
10.权利要求1所述的副干酪乳杆菌或权利要求2所述的微生物菌剂在制备缓解肠道神经受损的药品中的应用,其特征在于,所述的缓解肠道受损包括以下至少一种:
(a)增加肠神经胶质细胞数量,修复受损的肠神经系统;
(b)提高胶质细胞源性神经营养因子GDNF的水平及其受体GFRα1/RET的表达;
(c)下调Toll样受体2的过度激活,降低神经元型一氧化氮合酶nNOS表达;
(d)提高血清素5-HT及其受体5-HT2B表达;
(e)降低结肠组织中炎症水平,改善结肠组织病理损伤;
(f)缓解动力减弱型肠道运动障碍。
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CN117701477A (zh) * | 2024-02-05 | 2024-03-15 | 中科微智(北京)生物科技有限公司 | 一株副干酪乳酪杆菌及其在改善肠道疾病中的应用 |
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