CN116785277B - 染料木黄酮在制备调控肠道菌丰度的制剂中的应用 - Google Patents
染料木黄酮在制备调控肠道菌丰度的制剂中的应用 Download PDFInfo
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Abstract
本发明公开了染料木黄酮在制备调控肠道菌丰度的制剂中的应用。通过灌胃活的Marvinbryantia formatexigens菌而不是热灭活的该菌能够通过增加粪便中短链脂肪酸浓度缓解DSS诱导的急性结肠炎。说明染料木黄酮能够通过提高肠道Marvinbryantia formatexigens相对丰度来增加短链脂肪酸生成来缓解结肠炎。这些发现为染料木黄酮介导的IBD预防提供了新的见解,并将促进肠道炎症和损伤疾病的预防策略的发展。
Description
技术领域
本发明涉及药物技术领域,具体涉及染料木黄酮在制备调控肠道菌丰度的制剂中的应用。
背景技术
染料木黄酮是是大豆生长过程中产生的一类次生代谢产物,属于黄酮类化合物中异黄酮类成分,具有抗癌、抗衰老、抗炎症、调节更年期等作用,具有开发成为功能食品、饲料添加剂的潜力。
染料木黄酮是一种酪氨酸激酶抑制剂,具有化学结构4',5,7- 三羟基异黄酮。染料木黄酮是一种主要的大豆异黄酮以其各种生物活性和治疗特性而受到广泛关注。在未加工的大豆中存在少量染料木黄酮单体,更多的是以葡糖苷的形式存在;加工后的大豆中含有丰富的染料木黄酮。染料木黄酮同时具有类雌激素和拮抗雌激素的特性,从而抑制酪氨酸激酶。染料木黄酮具有抗脂肪生成、抗蠕虫、抗炎、降血脂,对缺血的神经保护作用损伤,在治疗高血压、骨质疏松症和调节绝经后症状方面的作用有雌激素尤其是在染料木黄酮化学结的相似性。它也能清除自由基,从而具有抗氧化性能。染料木黄酮在癌症的化学预防中也发挥着重要作用治疗以及减少脂肪组织的沉积,也用作治疗常见病的关键分子障碍。在大豆植物中,染料木黄酮以糖苷结合物的形式存在浓度范围约为 0.2-1 mg/kg。其各种药理活性中,染料木黄酮还包括防止DNA氧化损伤,抑制过程中血管生成、拓扑异构酶 II 和影响信号转导分子。染料木黄酮还参与管理胰腺β细胞增殖和胰岛素分泌,尤其是在绝经后状况。它具有保护作用细胞凋亡并影响调节肥胖的许多信号通路以及人体生理学中的各种代谢综合征。
大豆异黄酮的生物利用度完全取决于降解能力、微生物菌群、采食剂量、年龄、性别、药代动力学属性及其化学形式以及饮食也可能会影响。染料木黄酮存在于两种形式,称为染料木苷的糖基化形式和另一种形式是称为染料木黄酮的苷元部分。染料木黄酮糖基化偶联物作为染料木苷口服更有利于生物利用与染料木黄酮相比,由于染料木黄酮的水溶性较低。但染料木黄酮在肠道中的生物利用度高于染料木苷,因为染料木苷的亲水性及其较高的分子量。染料木黄酮在每个个体之间存在差异代谢、肠道转运时间和肠道菌群差异,主要涉及染料木黄酮代谢的氧化、还原和结合过程。涉及的主要信号通路在染料木黄酮代谢中是硫酸化和葡萄糖醛酸化。染料木黄酮在肝脏和肠道的代谢率较高。除了肝脏、肾脏、心脏和肺的组织也能够代谢染料木黄酮。肠道微生物能够将染料木黄酮转化为双氢染料木黄酮和 6-羟基-O-去甲基安哥拉紫檀素。
染料木黄酮已被广泛用作各种抗氧化剂具有清除活性氧 (ROS) 和活性氮(RNS)的生物作用。它抑制Fe2+、NADPH 引发微粒体脂质过氧化、腺苷二磷酸 (ADP) 复合物、NADH氧化酶和啮齿动物肝脏线粒体中的呼吸链。在对 18种巨噬细胞的研究中,染料木黄酮抑制氧化应激引起的核因子kB(NF-kB) 的激活,调控与免疫相关的基因表达和炎症反应。染料木黄酮提升表达抗氧化酶,例如谷胱甘肽过氧化物酶在人类前列腺恶性肿瘤细胞中并保护这些细胞免受体外氧化性 DNA 危害。
肠道炎症是常发于畜牧养殖过程中的常见疾病,轻者导致腹泻,体重下降,重者导致动物死亡,严重影响畜牧业经济效益。人类医学中易见炎症性肠病(Inflammatory BowelDisease,IBD),它是一种慢性非特异性肠道炎症性疾病,其中包括溃疡性结肠炎和克罗恩病。这两种疾病的临床表现通常有腹胀、腹痛、有血便等。全球约有600万至800万人受到影响。作为一种慢性、进行性和复发性的肠道疾病,IBD严重影响患者的生活质量和日常生活,增加医疗负担。肠道炎症的诱导因素有很多,目前有研究证明,营养因素及病原微生物是引起养殖动物肠道炎症的最主要因素。肠炎性疾病受到多种环境因素影响,如致病菌,毒素,免疫缺陷等,不同肠炎模型的发病机制不同。
研究证据显示,染料木黄酮能够有效地缓解更年期综合征、骨质疏松、老年痴呆等激素性依赖疾病,但确切作用机制至今仍不清楚。
肠道是机体最大的消化器官,由于其消化食物的生理特性,胃肠道不断受到外来抗原的侵扰。外来抗原主要来源于共生微生物及食物,少量来自入侵的细菌、病毒及肿瘤抗原。因此,肠道免疫系统形成了一种独特的免疫机制,既具有适当促进免疫反应的能力,又有抑制免疫反应的平衡机制。这种微妙的平衡变化通常与肠道疾病的发展有关。
近年来大量的研究证据显示,肠道菌群在代谢性疾病的发病机制中起着重要的作用,而且膳食是改变肠道菌群的关键因素。而目前染料木黄酮对于肠道菌群调控及机制的研究还不多。
发明内容
本发明公开了染料木黄酮在制备调控肠道菌丰度的制剂中的应用。
本发明公开了染料木黄酮显著改善肠炎中肠道菌群的结构及组成。
本发明采用如下技术方案:
本发明在小鼠日粮中添加染料木黄酮400mg/kg, 提前饲喂含染料木黄酮日粮4周后用葡聚糖硫酸钠(DSS)诱导小鼠结肠炎(试验流程见图1中a),结果表明染料木黄酮能够增加炎症组Marvinbryantia formatexigens菌群相对丰度。
本发明用购自DSMZ保藏中心(No. 14469)Marvinbryantia formatexigens菌代替染料木黄酮进行试验(试验流程见图5中a),结果表明该菌能够替代染料木黄酮发挥相同的功能,并且改善结肠炎作用机制及对肠道细胞的调控与染料木黄酮相同。
本发明公开了通过日粮补充染料木黄酮能够增加Marvinbryantia formatexigens菌相对丰度,其发酵产生短链脂肪酸缓解急性结肠炎。
因此本发明提供染料木黄酮在制备调控肠道菌丰度的制剂中的应用,所述肠道菌包括Marvinbryantia formatexigens。
具体地,所述肠道菌包括异普氏菌属(Alloprevotella)菌以及Marvinbryantia、反刍真杆菌群(Eubacterium ruminantium group)、瘤胃球菌科 UCG-013(Ruminococcaceae UCG-013)、瘤胃梭菌5(Ruminiclostridium 5)、艾森贝氏菌属(Eisenbergiella)、Romboutsia、链球菌属(Streptococcus)、棒状杆菌1(Corynebacterium 1)、不动杆菌属(Acinetobacter)、气球菌属(Aerococcus)、葡萄球菌(Staphylococcus)中的一种或几种。
其中,所述调控包括上调Marvinbryantia formatexigens菌的丰度。
进一步的,所述调控包括上调Marvinbryantia formatexigens菌和/或异普氏菌属菌的丰度。
更进一步地,所述调控包括下调反刍真杆菌群、瘤胃球菌科 UCG-013、瘤胃梭菌5、艾森贝氏菌属、Romboutsia、链球菌属、棒状杆菌1、不动杆菌属、气球菌属和/或葡萄球菌的丰度。
本发明还提供调控肠道菌群丰度的制剂在制备提高肠道中短链脂肪酸含量的药物中的应用,所述肠道菌包括Marvinbryantia formatexigens、异普氏菌属以及反刍真杆菌群、反刍真杆菌群、瘤胃球菌科 UCG-013、瘤胃梭菌5、艾森贝氏菌属、Romboutsia、链球菌属、棒状杆菌1、不动杆菌属、气球菌属和葡萄球菌中的一种或几种。
优选地,所述短链脂肪酸为乙酸和/或丁酸。
具体地,所述制剂是食品、饲料的添加剂。
本发明研究表明,灌胃活的Marvinbryantia formatexigens菌而不是热灭活的该菌通过增加粪便中短链脂肪酸浓度能够缓解DSS诱导的急性结肠炎。这说明染料木黄酮能够通过提高肠道Marvinbryantia formatexigens相对丰度来增加短链脂肪酸生成来缓解结肠炎。不仅如此,该缓解过程涉及促进COX-2通路导致炎症反应。这些发现为染料木黄酮介导的IBD预防提供了新的见解,并将促进肠道炎症和损伤疾病的预防策略的发展。
附图说明
图1.染料木黄酮缓和DSS诱导的结肠炎症;其中,a为实验流程,b为染料木黄酮缓解DSS诱导导致的体重损失,c为染料木黄酮缓解DSS诱导导致的结肠长度缩短,d为染料木黄酮缓解DSS诱导导致的病理评分增加,e为肠道通透性分析结果,f为病理分析结果。
图2.染料木黄酮减轻DSS诱导的炎症和炎性小体;其中,a为小鼠结肠中磷酸化的NFκB和IκB以及iNOS和cox-2蛋白水平表达情况,b为ASC和Caspase-1蛋白的表达,c为TLR4基因表达、PGEs-1和MCP-1基因表达情况,d为TNF-α,INOS-1,IL-6和NLRP3基因表达。
图3.染料木黄酮调控肠道菌群组成及促进短链脂肪酸产生;其中,a为日粮补充染料木黄酮能够扭转结肠炎导致的α多样性指数,b为各组菌群在门水平上的相对丰度,c为各组菌群在属水平上的相对丰度,d为染料木黄酮显著增加Marvinbryantia和异普氏菌属菌的相对丰度。
图4.肠道菌群耗竭能够废除染料木黄酮的益处,其中,a为具体的试验示意图,b为抗生素耗竭肠道菌群并补充染料木黄酮并不能缓解由DSS诱导的结肠炎引起的体重损失,c为抗生素耗竭肠道菌群并补充染料木黄酮并不能缓解由DSS诱导的结肠炎引起结肠缩短,d为抗生素耗竭肠道菌群并补充染料木黄酮并不能缓解由DSS诱导的结肠炎引起的炎症病理评分升高。
图5.Marvinbryantia formatexigens菌缓解DSS诱导的结肠炎症,其中,a为模型建立示意图,b为灌胃Marvinbryantia formatexigens菌后诱导DSS结肠炎能够缓解体重下降,c为灌胃Marvinbryantia formatexigens菌后诱导DSS结肠炎结肠缩短,d为灌胃Marvinbryantia formatexigens菌后诱导DSS结肠炎结肠病理评分升高,e为灌胃Marvinbryantia formatexigens活菌能够降低肠道炎症病理评分。
图6. Marvinbryantia formatexigens减轻DSS诱导的炎症和炎性小体,其中, a为相关蛋白表达情况,b为相关蛋白表达情况,c为相关基因表达情况。
图7.Marvinbryantia formatexigens菌促进肠道短链脂肪酸产生。
具体实施方式
以下结合附图对本发明的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
抗生素试验和灌菌试验
在抗生素处理试验,32只6周龄雄性小鼠被随机分为4组都用葡聚糖硫酸钠 (DSS)诱导结肠炎模型,分别为补充Abx(新霉素:30mg/L,万古霉素:10mg/L,甲硝唑:50mg/L,氨苄青霉素:50mg/L)耗竭肠道菌群组饲喂普通日粮在第10周龄用DSS诱导结肠炎(Abx+DSS),补充Abx耗竭肠道菌群并添加染料木黄酮(GEN)组饲喂添加染料木黄酮400mg/kg日粮在第10周龄用DSS诱导结肠炎(Abx+DSS+GEN),DSS诱导结肠炎组饲喂普通日粮在第10周龄用DSS诱导结肠炎(DSS),DSS诱导结肠炎并添加染料木黄酮组饲喂添加染料木黄酮400mg/kg日粮在第10周龄用DSS诱导结肠炎(DSS+GEN)。Abx处理耗竭肠道菌群通过在饲喂抗生素饮水4周根据前人的方法。所有小鼠自由饮水和采食。抗生素处理4周后紧接着构建结肠炎模型通过饮水中添加2%的DSS诱导,饮水5天后更换为正常饮水恢复3天后所有小鼠被处死收集结肠样品和结肠内容物液氮冷冻后-80度保存用于以后分析。
灌菌试验,40只6周龄雄性小鼠被随机分为5组,分别为对照组饲喂普通日粮(CON),DSS诱导结肠炎组第10周龄用DSS诱导结肠炎(DSS),灌服活菌组(LB),DSS诱导结肠炎并灌服活菌组(LB+DSS),DSS诱导结肠炎灌服热灭活菌组(HK+DSS)。小鼠活菌灌服量为100微升的10-9CFU/ml的Marvinbryantia formatexigens菌,每天灌服1次,连续灌服到处死。热灭活菌是细菌被85℃热处理10分钟。结肠炎模型通过饮水中添加2%的DSS诱导,饮水5天后更换为正常饮水恢复3天后所有小鼠被处死收集结肠样品和结肠内容物液氮冷冻后-80度保存用于以后分析。
组织病理染色和免疫荧光分析
甲醛固定的结肠组织经梯度酒精脱水后用石蜡包埋。组织被切为5微米厚度,经苏木精伊红染色后用于病理评分。病理评分被评估根据之前的评价标准。杯状细胞分泌的碱性多糖被染色用过碘雪夫氏染色。
OTC包埋的冷冻肠道被切为7微米厚度的切片,抗原被修复采用柠檬酸缓冲液。之后组织被通透30分钟使用含0.5%曲那通100 的磷酸盐缓冲液。组织被封闭利用5%山羊血清的PBS.组织在4℃一抗抗体孵育过夜。之后,切片组织用PBS洗3次,在避光条件下荧光标记二抗孵育2小时。切片组织用PBS洗3次,用含有DAPI的抗猝灭剂封片。肠道增殖细胞被染色使用在处死前2小时小鼠被注射EDU以40mg/kg体重,肠道组织增殖细胞被检测使用EDU检测试剂盒(碧云天,上海中国)。杯状细胞被标记FITC的uea-1染色。荧光信号被采集使用激光共聚焦显微镜(徕卡,德国)。所有抗体信息及稀释倍数被展示在下表1。
表1:免疫印迹及免疫荧光用到的抗体信息
实时荧光定量PCR
结肠组织总RNA使用trizol试剂盒提取根据说明书。cDNA通过逆转录合成。QPCR在一个20微升的反应体系中进行,包含10微升的mixed, 上游引物和下游引物各1微升,无菌无酶水7.5微升,模板0.5微升。反应程序为95 ℃ 5分钟,40个循环的95℃10秒和60℃30秒。β-actin被作为内参基因。所有被检测基因的引物序列展示在表2中。
表2: RT-qPCR引物序列
其中,Salprobe 的5`和3`端分别修饰连接FAM和MGB。
肠道通透性检测
小鼠在处死前8个小时绝食但不停止饮水。在绝食4个小时的时候每只小鼠灌胃200微升4 kDa FITC-dextran1mg/ml PBS。接着绝食4小时后处死小鼠采集血液样品。血清被分离通过血液离心5000g离心10分钟。使用多功能酶标仪测定血清中的FITC荧光值被。同时,根据建立的标准曲线计算FITC的浓度。
肠道菌群分析
1)肠道细菌基因组DNA提取
准确称取200mg小鼠盲肠内容物,使用QIAamp DNA Stool Mini Kit试剂盒,按照试剂盒说明书进行操作。使用1%琼脂糖凝胶和荧光光度计检测DNA浓度和纯度。
2)肠道细菌16SrRNA基因的PCR扩增及Miseq测序
细菌16SrRNA基因的V3和V4区进行PCR扩增采用常规的细菌引物(515 F 5‘-GTGCCAGCMGCCGCGGTAA-3’(SEQ ID NO.42)和806 R 5‘-CCGTCAATTCMTTTGAGTTT-3’ (SEQID NO.43))该引物还包含Illumina 5‘悬置的适配器序列,按照试剂盒说明书的操作规程采用两步法扩增文库。最初的PCR反应在25μL反应体积内进行,使用1-2μL DNA模板、250mMdNTPs、每个引物0.25mM、1×反应缓冲液和0.5μPhusion DNA 聚合酶。PCR反应条件:94℃预变性2 min;94℃变性30s,56℃退火30s,72℃延伸30s,共25个循环;最后72℃延伸5min。第二步PCR采用双8个碱基序列标签进行复接。8个周期PCR反应用于在16S扩增子的两端加入两个独特的标签。PCR反应条件:94℃预变性3 min;94℃变性30s,56℃退火30s,72℃延伸30s,共8个循环;最后72℃延伸5min。在建库前,使用DNA凝胶提取试剂盒对PCR产物进行回收纯化,并使用FTC-3000TM Real-time PCR仪对PCR产物进行定量测定。基因文库混合后,使用Miseq v3Reagent Kit试剂盒,按照Illumina Miseq高通量高通量测序仪的操作规程,进行2×300bp配对末端的测序。
实施例1、各种模型小鼠的建立
在染料木黄酮缓解结肠炎的试验中,32只6周龄雄性小鼠被随机分为4组,分别为对照组饲喂普通日粮(CON),DSS诱导结肠炎组饲喂普通日粮在第10周龄用DSS诱导结肠炎(DSS),染料木黄酮组饲喂添加染料木黄酮400mg/kg日粮(GEN),DSS诱导结肠炎并添加染料木黄酮组饲喂添加染料木黄酮400mg/kg日粮在第10周龄用DSS诱导结肠炎(DSS+GEN)。所有小鼠被维持在中国农业大学SPF动物设备内,每笼4只。自由饮水和采食。结肠炎模型通过饮水中添加1%的DSS诱导,饮水5天后更换为正常饮水恢复3天后所有小鼠被处死收集结肠样品和结肠内容物液氮冷冻后-80度保存用于以后分析。大约0.5厘米结肠的近肛门端被截取用多聚甲醛固定用于切片染色,同样,大约0.5厘米结肠的近肛门端被截取并被OTC包埋冷冻-80度保存用于冷切片的免疫荧光检测。
实施例2、染料木黄酮对结肠炎的减轻作用
为了研究染料木黄酮对结肠炎的减轻作用,小鼠被提前饲喂4周含染料木黄酮400mg/kg日粮,之后通过饮用含2%DSS的饮水5天建立结肠炎模型;然后,换回正常饮水恢复5天取样(图1中a)。与DSS结肠组相比,口服染料木黄酮能够显著减轻DSS诱导的结肠炎,主要表现为染料木黄酮能够显著缓解DSS诱导导致的体重损失,结肠长度缩短和病理评分增加(图1中b,c,d)。肠道通透性分析进一步说明染料木黄酮能够缓解DSS诱导的肠道屏障功能损伤(图1中e)。不仅如此,病理分析显示口服染料木黄酮能够缓解炎症细胞浸润和黏膜损伤(图1中f)。
为了进一步评估染料木黄酮对肠道炎症反应的影响,结肠中炎症及炎性小体的蛋白水平及基因表达被检测通过WB和qPCR。口服染料木黄酮,经 DSS 处理的小鼠结肠中磷酸化的NFκB和IκB显着降低,其下游的iNOS和cox-2蛋白水平也显著降低(图2中a)。DSS诱导的结肠炎能显著增加炎症蛋白NFκB和IκB的心酸化水平,以及iNOS和cox-2蛋白表达(图2中a)。不仅如此,DSS处理能够显著增加NLRP3炎性小体,以及与炎性小体结合的ASC和Caspase-1蛋白的表达。与此对比,日粮补充染料木黄酮能够抑制由DSS诱导的NLRP3炎性小体,ASC和Caspase-1蛋白的表达(图2中b)。与蛋白结果相一致的是日粮补充染料木黄酮能够抑制由DSS处理导致的NFκB上游TLR4基因表达升高和Cox-2下游的PGEs-1和MCP-1基因表达上升(图2中c)。同时,DSS诱导得结肠炎能够增肌TNF-α,INOS-1,IL-6和NLRP3基因表达,补充染料木黄酮能够废除DSS诱导的炎症反应和炎性小体升高(图2中d)。
实施例3、染料木黄酮对肠道菌群的调控
染料木黄酮对肠道菌群的调控被探究。在α多样性指数中,DSS诱导的结肠炎显著降低Chao1,observed species, PD—whole-tree和Shannon指数,日粮补充染料木黄酮能够扭转结肠炎导致的α多样性指数下降(图3中a)。
在门水平上,厚壁菌门和拟杆菌门是结肠内容物的优势菌门(图3中b)。在属水平上,乳酸菌,瘤胃球菌和蝽螺菌科是主要的优势菌属(图3中c)。与DSS诱导的结肠炎组相比,预先饲喂染料木黄酮的肠炎组能够显著增加蝽螺菌科和阿克曼菌。不仅如此,本发明比较了结肠炎组和添加染料木黄酮的结肠炎组主要差异菌属,后者能够显著增加Marvinbryantia和异普氏菌属菌的相对丰度,并抑制其他10个菌属,如反刍真杆菌群、瘤胃球菌科 UCG-013、瘤胃梭菌5、艾森贝氏菌属、Romboutsia、链球菌属、棒状杆菌1、不动杆菌属、气球菌属和/或葡萄球菌的丰度。并且Marvinbryantia菌比异普氏菌属菌具有更大的差异,在两个组中相对丰度的比值分别为14.5和5.8(图3中d)。
基于 Bray-Curtis 距离的主坐标分析 (PCoA) 显示在4个分组之间的肠道微生物群结构存在差异(图3中e)。
为了进一步研究肠道菌群在发酵产生短链脂肪酸的差异,主要的短链脂肪酸乙酸,丙酸和丁酸的含量被检测。结果显示DSS诱导的结肠炎能够显著减少粪便中乙酸,丁酸和总短链脂肪酸的含量(图3中f)。预先饲喂染料木黄酮能够扭转短链脂肪酸的降低。在各处理组之间丙酸浓度差异不显著。因此,肠道菌群结构的变化可能是染料木黄酮发挥缓解结肠炎的重要介导体。
实施例4、染料木黄酮通过肠道菌介导发挥缓解结肠炎的作用
为了验证染料木黄酮通过肠道菌介导发挥缓解结肠炎的作用,抗生素耗竭肠道菌群被实施。具体的试验示意图被显示在图4中a。肠道菌群被抗生素耗竭后,染料木黄酮对结肠炎的效果被抑制。表现为抗生素耗竭肠道菌群并补充染料木黄酮并不能缓解由DSS诱导的结肠炎引起的体重损失,结肠缩短和炎症病理评分升高(图4中b,c,d)。病理染色显示,肠道抗生素耗竭肠道菌群后,添加或不添加染料木黄酮在DSS诱导结肠延后都表现为炎性细胞浸润和上皮损伤,这与未添加抗生素的组有相反的结果(图4中e)。
实施例5、Marvinbryantia formatexigens菌缓解DSS诱导的结肠炎症
本研究中抗生素耗竭肠道菌群已经证明染料木黄酮通过肠道菌群缓解结肠炎。在结肠炎模型组中,添加染料木黄酮显著增加Marvinbryantia formatexigens菌的相对丰度。因此,Marvinbryantia formatexigens菌被用于验证染料木黄酮是否通过上调该菌来缓解结肠炎,试验示意图如图5中a。灌服Marvinbryantia formatexigens活菌能够缓解由DSS诱导的结肠炎症。表现为,与DSS诱导结肠炎组相比,灌胃Marvinbryantia formatexigens菌后能够缓解DSS诱导结肠炎的体重下降、结肠缩短和结肠病理评分升高(图5中b,c,d)。不仅如此,中性黏液被PAS染色显示DSS诱导炎症组的阳性细胞数量显著低于对照组,而灌胃Marvinbryantia formatexigens菌能够缓解由结肠炎引起中性黏液分泌减少。灌胃热灭活的Marvinbryantia formatexigens对结肠炎没有缓解作用。这说明只有活菌具有染料木黄酮的缓解功能。灌服活菌能够降低肠道炎症病理评分,缓解DSS诱导肠道损伤导致的杯状细胞数量减少(图5中e)。
实施例6、Marvinbryantia formatexigens减轻DSS诱导的炎症和炎性小体以及对肠道脂肪酸水平的影响
与染料木黄酮缓解DSS结肠炎的分子机制类似,灌胃Marvinbryantia formatexigens菌能够抑制结肠炎小鼠NFκB和IκB蛋白的磷酸化,其下游的iNOS和cox-2蛋白水平也显著降低(图6中a)。与染料木黄酮结果类似,DSS诱导的结肠炎能显著增加炎症蛋白NFκB和IκB的磷酸化水平,以及iNOS和cox-2蛋白表达(图6中a)。不仅如此,DSS处理能够显著增加NLRP3炎性小体,以及与炎性小体结合的ASC和Caspase-1蛋白的表达。与此对比,小鼠灌胃Marvinbryantia formatexigens菌能够抑制由DSS诱导的NLRP3炎性小体,ASC和Caspase-1蛋白表达上升(图6中b)。与蛋白结果相一致的是Marvinbryantia formatexigens能够抑制由DSS处理导致的NFκB上游TLR4基因表达升高和Cox-2下游的PGEs-1和MCP-1基因表达上升(图6中c)。抗生素耗竭肠道菌群能够废止染料木黄酮对结肠炎的缓解作用,表现为抗生素耗竭肠道菌群并饲喂染料木黄酮不能抑制炎性蛋白NFκB和IκB的磷酸化水平升高由DSS诱导的结肠炎。
Marvinbryantia formatexigens能够促进粪便中短链脂肪酸含量增加(参见图7)。
本发明通过上述实施例来说明本发明的工艺方法,但本发明并不局限于上述工艺步骤,即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (2)
1.染料木黄酮在制备调控肠道菌丰度的制剂中的应用,其特征在于,所述肠道菌为Marvinbryantia formatexigens。
2.根据权利要求1所述的应用,其特征在于,所述调控为上调Marvinbryantia formatexigens菌的丰度。
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