Research Progress on Regulatory Effects and Mechanism of Akkermansia muciniphila on Glycolipid Metabolism and Intestinal Health
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摘要: 嗜黏蛋白阿克曼菌（Akkermansia muciniphila，A. muciniphila）是一种功能性肠道共生菌，其在消化道中的分布及丰度的失衡与糖脂代谢紊乱及肠炎等多种疾病的发生密切相关。本文旨在对A. muciniphila在调节糖脂代谢、缓解肠道炎症和增强肠道屏障方面的益生功能及其中分子作用机制进行简要综述，同时针对目前A. muciniphila应用存在安全且有效的补充剂量范围待确定、低成本高密度工业化生产工艺待挖掘等问题展望未来，为进一步将A. muciniphila开发成为新型益生菌添加剂提供参考依据。Abstract: Akkermansia muciniphila (A. muciniphila) is a functional gut commensal bacterium. The imbalance in the distribution and abundance of A. muciniphila in the gastrointestinal tract is closely related to the occurrence of various diseases, including glucolipid metabolic disorders and enteritis. This review aims to provide a concise overview of the probiotic function of A. muciniphila in regulating glucose and lipid metabolism, alleviating intestinal inflammation, and enhancing intestinal barrier function, as well as its molecular mechanism of action. Meanwhile, to tackle the challenges of industrial applying A. muciniphila, such as defining a safe and effective supplemental dosage and creating a cost-efficient, high-density production process, new research directions are proposed to guide the development of A. muciniphila as a novel probiotic additive.
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肠道微生物菌群是存在于肠道中复杂多样且庞大的微生物群落，各细菌种与宿主保持着复杂的相互联系，彼此之间相互作用、相互影响，最终形成一种动态平衡的共生互利关系[1−2]。肠道微生物菌群是调节宿主健康的关键因素之一。嗜黏蛋白阿克曼菌（Akkermansia muciniphila，A.muciniphila）是荷兰科学家于2004年在健康人粪便中发现的一种新的肠道共生菌，隶属疣微菌门（Verrucomicrobia）疣微菌纲（Verrucomicrobiae）疣微菌科（Verrucomicrobiales）中的阿克曼属（Akkermansia）[3−4]。A. muciniphila是一种呈卵圆形、有菌毛、无芽孢、无动力的革兰氏阴性厌氧菌，单细胞长轴约0.6~1.0 μm，可单独或成对生长[3]，定植于人类的小肠（空肠和回肠）和大肠（盲肠和结肠），并以结肠段为主要栖息地[5]。
近年来，大量研究表明A. muciniphila与机体健康及免疫和代谢状态有关，在人和动物出生后和生命最初的几个月内就参与调控α-L-岩藻糖苷酶、exo-α-唾液酸苷酶、β-半乳糖苷酶和β-己糖胺酶等糖类化合物相关降解酶的表达，利用母乳低聚糖（human milk oligosaccharides，HMOs）在婴幼儿肠道内实现早期存活和定植[5−6]。在婴儿出生一年内，该菌在肠道中的比例可达到成人水平，约占人体肠道微生物总数的1%～3%[7]。正常情况下，随着年龄增长，A. muciniphila在人体内的丰度随着年龄的增长而逐渐降低[8]，而在长寿老人肠道中水平较高[9]。定植在动物消化道中的A. muciniphila参与调控消化道微生物菌群的稳态，其在消化道中分布及丰度的失衡与糖脂代谢紊乱（如肥胖症、糖尿病、非酒精性脂肪性肝病）及肠炎等疾病的发生密切相关。另外，A. muciniphila能够刺激肠道黏蛋白（Mucin，MUC）的降解和产生，不仅具有维持黏液层厚度和肠道屏障完整性等重要作用，并且还可以介导宿主肠道炎症反应，表现出开发成为具有肠道免疫调节作用益生菌的巨大潜力。
本综述旨在对A. muciniphila在调节糖脂代谢、缓解肠道炎症和增强肠道屏障方面的益生功能及其中分子作用机制进行简要综述，同时针对目前A. muciniphila应用中存在的问题与挑战，提出未来研究方向，为进一步将A. muciniphila开发成保障肠道健康的新型益生菌添加剂提供参考依据。
1. Akkermansia muciniphila的益生功能
1.1 调节糖脂代谢
糖脂代谢紊乱（glucolipid metabolic disorders，GLMD）引发的血糖和血脂异常、肥胖、非酒精性脂肪肝等疾病在人类和动物群体中发病率较高，不仅影响机体正常代谢功能，通常还会导致多种并发症，严重威胁健康。多项研究表明，A. muciniphila在健康人的肠道微生物群中含量丰富，而在GLMD发生和发展过程中该菌水平均出现下降[10−12]。另外，肥胖症患者体内的该菌水平与其空腹血糖数值、腰臀比和皮下脂肪细胞直径呈负相关[13]。以上研究结果显示A. muciniphila与机体的糖脂代谢存在紧密关系，且GLMD与该菌丰度降低有关。同时，A. muciniphila与GLMD之间的互作关系也反映在给药治疗方面，有研究发现在GLMD药物治疗和恢复的过程中，A. muciniphila水平也出现恢复。二甲双胍是治疗糖尿病的常用药物，使用二甲双胍使肠道A. muciniphila水平上升，并能使饮食诱导肥胖小鼠的葡萄糖稳态得到改善[14]。当2型糖尿病（type 2 diabetes，T2DM）患者体内的A. muciniphila水平较高时，二甲双胍的疗效也会增强[15]，这反映出A. muciniphila和二甲双胍存在协同效应，但确切原因尚不清楚。万古霉素是一种专门针对革兰氏阳性菌的糖肽类抗生素，使用万古霉素能够增加非肥胖糖尿病（non-obese diabete，NOD）小鼠肠道A. muciniphila数量[16]。上述结果表明，GLMD的药物缓解及治疗与肠道中A. muciniphila数量增加有紧密关联。
GLMD引发的肥胖、高血糖、血脂异常等又可以通过单核细胞浸润和增加巨噬细胞分化诱发肝脏炎症，导致代谢相关脂肪肝（metabolic-associated fatty liver disease，MAFLD）并加剧肝脏脂肪变性和纤维化，而持续三个月每天口服补充A. muciniphila活菌，可以降低肝功能障碍和炎症的相关血液标志物水平，并且不影响肠道微生物组整体结构[17]。另外，对链脲佐菌素诱导的糖尿病SD大鼠服用A. muciniphila，可以降低炎症指标如肿瘤坏死因子-α（TNF-α）、脂多糖（LPS）和丙二醛的水平，而且A. muciniphila还能选择性地促进宿主动物肠道有益微生物群的生长，在治疗糖尿病方面显示出与二甲双胍相当的疗效[18]。补充A. muciniphila还能通过调节γδT17细胞和进一步巨噬细胞极化来预防非酒精性脂肪性肝炎（nonalcoholic steatohepatitis，NASH）[19]。另外，A. muciniphila可以通过恢复肠道屏障，改善代谢性内毒素血症诱发的炎症，从而减轻Apoe−/−小鼠动脉粥样硬化病变[20]。有研究发现服用A. muciniphila可减少高脂饮食（high-fat-diet，HFD）诱导的体重增加、脂肪量增加和葡萄糖不耐受，同时增强肠道屏障功能[11,21]，这可能与补充A. muciniphila可适度增加循环中能量代谢和食物摄入的关键调节因子—胰高血糖素样肽-1（Glucagon-like peptide-1，GLP-1）有关。Yoon等[22]从A. muciniphila中鉴定出了一种P9蛋白，并发现P9是促进糖脂代谢紊乱小鼠GLP-1分泌的原因，因此推测P9可能是A. muciniphila调节糖脂代谢的关键蛋白。上述结果表明，A. muciniphila活菌的补充可以替代药物缓解或治疗GLMD的发生与发展。
研究发现，不仅有活性的A. muciniphila可以改善GLMD，巴氏灭活后的嗜黏蛋白阿克曼菌（pasteurized Akkermansia muciniphila，PAKK）仍能发挥此效果。PAKK能改善脂质代谢，抑制脂肪酸合成并促进脂肪酸氧化和转运，从而改善肝脂肪变性，减少脂质积累[23]。HFD诱导肥胖小鼠饲喂PAKK可增加粪便中能量，减少碳水化合物的吸收和促进肠上皮细胞的新陈代谢[24]。PAKK可以改善高能量、低蛋白饮食诱发的脂肪肝出血性综合征（FLHS）蛋鸡脂质代谢，提高鸡蛋质量和蛋黄的脂质含量[25]。值得一提的是，在糖代谢紊乱的鲤鱼中，PAKK在降低血清TG和促进糖酵解方面的作用比活菌和无细胞上清液更强[26]。
上述结果表明，无论是直接补充A. muciniphila活菌，还是补充其灭活菌或特定菌体成分都具有缓解甚至治疗GLMD的作用，显示出A. muciniphila源后生元“Postbiotics”产品（即对宿主健康有益的无生命微生物和/或其成分的制剂[27]）具有广泛的应用前景。
1.2 缓解肠道炎症
肠道菌群失衡是炎症性肠炎（inflammatory bowel disease，IBD）发生发展的主要诱因[28]，有研究显示，在IBD患者的肠道中A. muciniphila的水平出现下降甚至接近于零，表明IBD的发生可能与肠道中A. muciniphila的丢失有关[29−31]，但是在口服补充A. muciniphila后可以缓解甚至预防肠道炎症[32−34]。Bian等[32]报道补充A. muciniphila能减轻葡聚糖硫酸钠（dextran sulfate sodium，DSS）诱导的小鼠结肠炎。纪漫萍[33]也发现在抗生素处理伪无菌小鼠模型下，连续A. muciniphila灌胃3周可以显著预防DSS诱导的急性结肠炎症状。杨鑫[34]基于沙门氏菌侵染HT-29细胞构建体外结肠炎症模型，发现A. muciniphila预防组对黏膜屏障功能（Occludin、ZO-1和MUC2）和肠免疫水平（TNF-α、IL-17A和IL-10）均有显著积极效应。除此之外，体外实验发现A. muciniphila细胞外囊泡（A. muciniphila extracellular vesicles，AmEVs）可以被LPS诱导的RAW264.7巨噬细胞吸收并发挥抗炎作用[35]。另外，还有研究发现仅口服AmEVs也能保护DSS诱导的结肠炎小鼠免受IBD表型（如体重下降、结肠长度和结肠壁炎症细胞浸润）的影响，表明A. muciniphila衍生EVs也对肠炎具有保护作用[36]，这些研究结果为A. muciniphila及其EVs预防结肠炎提供了一种潜在的干预方法。以上研究表明，通过补充A. muciniphila及其衍生物能有效减缓急性结肠肠炎症状，降低IBD易感性，对预防IBD发生、发展具有重要意义。
A. muciniphila对肠炎的缓解作用与宿主自身免疫水平有关，有研究发现向IL-10−/−基因敲除诱导的慢性结肠炎小鼠饲喂A. muciniphila反而会扰乱小鼠结肠黏液平衡，加剧伤寒沙门氏菌感染引起的肠道炎症[37]。Seregin等[38]研究发现，IL-10−/−NLRP6−/−基因共敲除小鼠所诱导的慢性结肠炎程度显著高于IL-10−/−基因敲除小鼠，并且前者肠道会富集更多的黏液降解菌A. muciniphila。其原因是由于NLRP6炎性小体活化后促进分泌的IL-18能够抑制A. muciniphila在肠黏膜的定植，敲除NLRP6−/−基因导致的IL-18缺失，从而解除了IL-18对A. muciniphila的抑制作用，促进了肠黏液层分解，增加了肠黏膜通透性和细菌异位引起炎症风险；而对NLRP6−/−基因敲除小鼠腹腔注射重组IL-18能够减少肠黏膜的A. muciniphila丰度，表明NLRP6炎性小体的活化参与调控A. muciniphila在肠黏膜的定植。另外，A. muciniphila过度定植会降低结肠炎和结肠癌小鼠肠道MUC含量和肠上皮细胞之间ZO-1、Claudin-4等的转录和蛋白表达水平，打破了黏蛋白分泌和降解之间的动态平衡，从而使肠黏液层厚下降，最终导致小鼠的肠道屏障受到破坏[39]。因此，A. muciniphila对肠炎的缓解作用与宿主健康状态存在关联，在宿主免疫缺陷或免疫稳态被破坏情况下，补充A. muciniphila反而会加剧肠炎，所以直接将A. muciniphila作为益生菌补充时需评估宿主肠黏膜健康状态。
1.3 增强肠黏膜屏障功能
A. muciniphila作为定植于肠道黏液层的主要微生物之一，优先选择肠上皮杯状细胞分泌的黏蛋白作为能量来源，参与黏蛋白的表达，改变黏液层厚度，从而影响肠道屏障功能[40]。全基因组测序结果显示A. muciniphila约有14%的基因能编码黏蛋白降解酶，可生成蛋白酶、糖苷酶、唾液酸酶和硫酸酯酶等78种[4]，通过黏蛋白的发酵和降解释放硫酸盐和短链脂肪酸（short-chain fatty acids，SCFAs），其中所产生的SCFAs能够支持A. muciniphila本身和其他肠道菌（如普通拟杆菌和粪厌氧棒状菌[41−42]）的生存。无菌小鼠在直接补充A. muciniphila活菌制剂后，该菌能在回肠、盲肠和结肠黏膜中距离上皮细胞约50 µm处聚集、定植和生长，其中在盲肠段的丰度高达1010 CFU/g，且能显著影响结肠调节（免疫）细胞增殖和分化的生长因子（Igf1等）、细胞表面分子（Cd19、Cd37）、磷酸酶（Ptprc或Cd45）、蛋白激酶（Lck、Fyn）等的表达[43]。另外，给DSS诱导急性结肠炎小鼠灌胃A. muciniphila，在缓解肠炎的同时也能增加肠黏膜杯状细胞数量并促进黏蛋白的表达，并且发现A. muciniphila缓解肠道的作用与NLRP3炎性小体通路激活有关[44]。另外，还有研究发现，补充A. muciniphila可防止与年龄相关的结肠黏液层厚度下降，并减轻老年时的炎症和免疫相关过程[45]，甚至能适度延长小鼠的寿命[46]，这表明补充A. muciniphila有助于促进健康老龄化。Caco-2与HT-29细胞模型上的研究也同样证实了A. muciniphila具有增强肠道黏膜屏障的作用，显著增加跨上皮间电阻，促进肠细胞增殖、保障上皮细胞完整性和增强体外细胞的屏障功能，并促进损伤部位黏膜的修复[47]。除此之外，Chelakkot等[48]研究发现，与健康个体相比，T2DM患者粪便中AmEVs数量降低，并且给HFD诱导T2DM的小鼠补充AmEVs可以增强其肠道屏障的紧密连接功能，减少体重增加并改善葡萄糖耐量。Ashrafian等[49]对比A. muciniphila和AmEVs对HFD模型小鼠的影响，结果表明喂养此两种物质都可以改善小鼠肠道屏障的完整性、炎症、能量平衡和血液参数（即血脂和葡萄糖水平），但与A. muciniphila本身相比，AmEVs导致小鼠的身体和脂肪重量损失更显著。以上研究结果表明A. muciniphila及其EVs与肠道稳态存在紧密联系，突出了它们在相关疾病治疗中的积极作用，表现AmEVs在未来作为新的治疗策略来增强肠道屏障功能的可能性。
2. Akkermansia muciniphila益生作用的分子机制
大量研究证明了A. muciniphila在调节糖脂代谢和维持肠道健康方面的益生功能，其中作用机制主要是以下四个方面（图1）：调节AMP活化蛋白激酶（AMP-activated protein kinase，AMPK）信号通路；调节内源性大麻素系统（endogenous cannabinoid system，ECS）；介宿主炎症反应。
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2.1 调节AMP活化蛋白激酶（AMPK）信号通路
AMPK作为葡萄糖和脂质代谢的关键调节因子，是葡萄糖生成的重要抑制因子，激活AMPK可影响葡萄糖生成关键酶的表达以及其他多种代谢途径的发生，并在调节葡萄糖和脂质代谢中发挥作用[50]。AMPK不仅是调节糖脂代谢酶活性和促进糖原生成的关键转录因子，还是能量平衡的调节因子，能够通过协调各种代谢途径（如脂质代谢[51]、葡萄糖代谢[52]和线粒体稳态[53]）来平衡机体的营养供应和能量需求。补充A. muciniphila可以使小鼠肝脏和肌肉中胰岛素下游信号通路分子磷酸化蛋白激酶的蛋白水平增加，并抑制葡萄糖-6-磷酸酶（glucose-6-phosphatase，G6Pase）、磷酸烯醇式丙酮酸羧激酶（phosphoenolpyruvate carboxykinase，PEPCK）等糖异生关键酶的表达水平有关，从而使肝脏和肌肉的胰岛素敏感性和对血糖的处理能力增加[54]，A. muciniphila对糖脂代谢的调控可能与糖异生的关键酶（如G6Pase、PEPCK）有关。这一观点在A. muciniphila亚型（A. muciniphilasub，即I亚型GP01株）改善奥氮平诱导的代谢综合征研究中得到了证实，发现A. muciniphila GP01能缓解奥氮平导致的高血糖，并降低关键酶G6Pase和PEPCK的水平[55]。此外，连续4周对T2DM大鼠进行黑豆壳和黑米花青素提取物灌胃，在增加A. muciniphila丰度的同时能抑制G6Pase和PEPCK表达，并激活AMP活化蛋白激酶（AMPK）信号通路[56]，说明A. muciniphila可能通过AMPK途径调节G6Pase、PEPCK和其他糖原生成关键酶表达的。在Caco-2细胞模型中的研究证明了无论是A. muciniphila活菌还是经过巴氏杀菌灭活的A. muciniphila能够通过TLR2/AMPK/NFκB缓解由LPS诱导肠道屏障功能紊乱[57]。AMPK磷酸化抑制下游分子调控CREB调控转录辅激活因子（TORC2），从而阻止其与cAMP反应元件结合蛋白结合转录表达过氧化物酶体增殖激活受体γ辅激活因子1-α（PGC1-α），最终降低PGC1-α对G6Pase和PEPCK的转录表达，从而抑制肝糖原生成[58−61]。此外，AMPK的激活可诱导体内糖原合成酶激酶3（GSK3）磷酸化水平升高，导致调节糖原合成最后一步的关键酶—糖原合成酶表达水平升高[62]。长期激活AMPK还能降低固醇调节元件结合蛋白-1c（SREBP-1c）的转录活性，下调脂肪酸合成酶（FAS）、丙酮酸激酶（PK）和其他脂肪生成相关基因的表达，从而通过促进脂肪氧化来调节脂肪沉积[63]。与此同时，有研究发现二甲双胍[15,64]、鹰茶[65]等物质在预防或治疗代谢综合征（MetS）方面的作用机制与激活AMPK途径有关，但在这些物质作用的同时也出现A. muciniphila丰度的增加，这些物质可能是通过促进A. muciniphila的生长作用于AMPK途径，从而实现对糖脂代谢的调节。
2.2 调节内源性大麻素系统
内源性大麻素系统（endogenous cannabinoid system，ECS）可对抗炎症和胃肠道异常分泌，在肠道和脂肪组织的生理学中发挥重要作用，是许多消化系统疾病，如肠道疾病、肠易激综合征和分泌相关疾病的治疗靶点[66−67]。ECS是一种内源性的复杂稳态调控信号网络系统，由大麻素受体（CBRs）、其内源性配体以及参与内源性大麻素合成和降解的酶组成[68−69]。CBRs主要包括1型大麻素受体（cannabinoid receptor type 1，CB1）、2型大麻素受体（cannabinoid receptor type 2，CB2）、过氧化物酶体增殖激活受体（peroxisome proliferator-activated receptors，PPARs）和其他G蛋白偶联受体（GPCRs）[67,70]。CB1的激活可增加SREBP-1c的表达，进而形成与脂肪肝和高甘油三酯血症相关的表型[71−72]。CB2主要在免疫细胞和造血细胞中表达，激活CB2可改善脂肪变性和抗纤维化反应[71,73]。PPARs（分为PPARα、PPARβ/δ和PPARγ共3种不同的亚型）参与调节涉及脂肪酸摄取和氧化、脂质和碳水化合物代谢、炎症和细胞增殖的基因[74−75]，一旦被多种合成和内源性配体激活，它们就会调节下游靶基因的转录[76]。因此，在生理和病理条件下被激活的核受体被认为是内源性大麻素的新靶点[77]。最新研究表明，肠道微生物群与PPARs之间存在直接的相互作用，肠道微生物群可能会诱导PPARs的表达和激活[78−79]。对HFD诱导的NASH大鼠饲喂白藜芦醇通过调节ECS维持肠道屏障的完整性和抑制肠道炎症，结果显示白藜芦醇增加了肠道A. muciniphila相对丰度，说明A. muciniphila与ECS之间可能存在关联[80]。直接灌胃A. muciniphila可以增加HFD模型小鼠回肠中2-OG、2-AG和2-PG的水平，并起到预防肥胖的效果[11]，这可能与A. muciniphila通过影响CBRs（尤其是PPARs）调节机体能量平衡有关[74]。肖琳等[81]研究表明A. muciniphila活菌灌胃能上调结肠组织中CB2 mRNA的含量和表达量，并起到改善肠道活动功能的作用以及改善肠道因素对内脏高敏的影响，但在不同条件下对CB2表达的影响不同[32]。进一步研究发现，A. muciniphila活菌、灭活菌及其菌体成分衍生物对CBRs基因表达的影响并不完全同，但全都对PPARs基因的转录表现积极效应[82]，以上研究结果说明主要是通过影响PPARs基因的表达水平来调节ECS。另外，有研究提出乙酸通过肝脏中的AMPK介导上调PPARα基因和脂肪酸氧化相关蛋白来抑制体内脂肪和肝脏脂质的积累[83]，而乙酸作为细菌代谢物SCFAs的主要组成，这说明A. muciniphila可能主要是通过产生SCFAs来促进PPARs相关基因的表达从而调节ECS。以上研究表明，A. muciniphila可能是通过AMPK途径间接或直接的影响PPARs基因的表达水平来调节ECS，从而进一步发挥益生功能，但具体作用机制仍有待研究。
2.3 介导炎症反应
肠道微生物群还能刺激宿主免疫力，防止病原体定植和健康微生物群落被破坏后导致的本地病原菌过度生长[84]。A. muciniphila是肠道中参与免疫调节和增强肠道屏障功能的微生物之一，它能增强宿主先天性免疫反应，并在增强肠道屏障功能方面发挥作用[11,85]。基于不同模型背景下，研究结果均显示A. muciniphila具有良好的介导炎症的反应特性，而其主要是通过2种模式介导炎症反应：激活TLR2受体和调节肠道免疫细胞组成。
2.3.1 激活TLR2受体
在肠道中，最有代表性的模式识别受体是肠道中的Toll样受体（Toll-like receptor，TLRs），它可以通过识别特定生物保守分子成分激活相应信号通路诱导机体炎症反应的天然免疫受体，是连接特异性和非特异性免疫的桥梁。在炎症性疾病发生发展过程中，有研究显示A. muciniphila可特异性激活TLR2受体[85−86]。
TLR2作为TLRs主要类型之一，能够通过介导相关信号途径来抑制下游核转录因子-κB（nuclear transcription factor κB，NF-κB）的表达。TLR2/NF-κB信号通路在等炎症发生、发展等方面起关键作用，与多种细胞的活动、代谢调节等密切相关[87]。有研究表明A. muciniphila可以通过激活TLR2受体抑制NF-κB信号通路[61]，其中作用机制可能是与A. muciniphila处理会增加小鼠肝脏中NF-κB抑制蛋白—IκBα的蛋白水平有关[88]，此炎症信号通路的抑制能降低炎症因子的表达，避免炎症性反应持续性的发生。另外，A. muciniphila通过调节该信号通路也能降低TNF-α、IL-1β、IL-2和IFN-γ等促炎因子的表达，增强转化生长因子-β（TGF-β）和IL-10等抗炎因子的表达[57]。基于以上结果，表明A. muciniphila能够通过调节TLR2/NF-κB信号通路从而发挥抗炎作用，达到抑制炎症的发生发展的效果。除此之外，仅A. muciniphila的外膜蛋白Amuc_1100也可以激活TLR2，并提高TJ蛋白表达，使血浆脂多糖浓度正常化，最终改善高胆固醇血症，减轻高脂血症小鼠的炎症反应[21]。同时，Amuc_1100与肠上皮细胞表面的TLR2结合后可以激活AMPK通路，从而增加ZO-1的表达，并同时抑制孔隙生成蛋白Claudin-2的表达[57]，而这种双重调控作用在介导炎症反应的同时还可以维护肠道屏障的完整性。总之，A. muciniphila通过激活TLR2不仅能抑制NF-κB信号通路，还可以调节AMPK信号通路，且A. muciniphila激活TLR2受体的关键可能是在于其外膜蛋白Amuc_1100。
2.3.2 调节肠道免疫细胞组成
肠道中CD4+T细胞向辅助性T细胞（helper T cell，Th）1/Th2、Th17/Treg的分化平衡能够维持肠道免疫稳态，Th1/Th2、Th17/Treg比值升高引起的免疫稳态失衡则会诱导肠道炎症反应[89−90]。在肠炎疾病的发展过程中，A. muciniphila能促进CD4+T细胞向Treg细胞分化，并将CD4+T细胞重新编程为Treg谱系，从而增加Treg的绝对数量和占CD4+T细胞的百分比，从而调节机体免疫功能，缓解肠道炎性损伤[15,91]。Treg细胞表达转录因子Foxp3在限制肠道炎症反应方面起关键作用[92]，而口服A. muciniphila可以诱导内脏脂肪组织中的Foxp3+Treg细胞，从而显著增强HFD小鼠糖耐量并减轻脂肪组织炎症[15]。
A. muciniphila不仅可以影响CD4+T细胞的分化方向，还能通过调节免疫细胞组成来影响机体免疫系统。Katiraei等[93]研究发现补充A. muciniphila可以增加肠系膜淋巴结（mesenteric lymph node，mLN）的B细胞总数，显著减少中性粒细胞和T细胞总数以及树突状细胞（dendritic cells，DC）上抗原肽复合体MHCII的表达以及B细胞上共刺激信号CD86的表达，其中MHCII和CD86都参与T细胞介导的免疫反应，最终减少mLN中促炎性T细胞介导的免疫反应，这表明A. muciniphila可影响mLN中免疫细胞的组成并可能降低免疫细胞的活化状态，从而发挥免疫调节特性。除此之外，有研究发现SCFAs也能调节多种与免疫相关细胞的增殖、分化和激活，直接影响免疫细胞和免疫调节器来维持内环境稳态[94]，比如抑制单核细胞（Monocyte）、巨噬细胞（Macrophage）和DC的成熟，降低促炎细胞因子的产生[95−96]，同时SCFAs还可以直接与免疫细胞相互作用，减少全身炎症[97]，这说明A. muciniphila调节免疫细胞的组成可能与其代谢物有关。以上研究结果表明，在机体正常健康状态时，体内免疫细胞分化处于动态平衡，而当机体处于炎症状态时，此平衡被破坏而外源性补充A. muciniphila活菌或利用植物提取物[98−99]、天然生物活性肽[100]等生物活性物质提高该菌丰度后，A. muciniphila的增加可以通过调节相关免疫细胞的增殖、分化，维持免疫稳态，从而进一步缓解机体炎症情况。综上所述，A. muciniphila与机体免疫过程有密切的联系，往往可以影响相关免疫细胞和免疫因子介导炎症反应从而维持机体肠道健康状态。
3. Akkermansia muciniphila应用存在的问题
3.1 安全且有效的活菌补充剂量范围
A. muciniphila活菌和巴氏灭活菌已被证明安全性较好且以进入临床试验阶段[18,101]，结果显示短期内无不良反应。但是，Shin等[15]发现补充4.0×106 CFU活菌并不能改善高脂饮食小鼠的糖耐量受损。由此可见，A. muciniphila益生菌效应的产生可能与补充剂量有关。而且，有研究显示在宿主免疫缺陷或免疫稳态被破坏情况下，补充A. muciniphila反而会加剧肠炎[37−39]，所以直接将A. muciniphila作为益生菌补充时需对宿主肠黏膜健康状态进行评估从而保证活菌在安全补充剂量范围内。另外，A. muciniphila在制备、储存和胃肠道通过过程中的存活率及最终在肠道内的定植率也均未知，所以如何提供安全且有效的A. muciniphila活菌补充剂量是一个亟需解决的问题。
3.2 工业化生产工艺及成本
A. muciniphila工业化生产工艺和成本是将其商品化开发过程中亟待解决的关键问题。目前体外培养A. muciniphila的传统方法是将脑心浸液肉汤或其与猪胃粘蛋白混合液置于厌氧环境中进行静态培养[42,102−103]，但这种方法面临底物利用率和生物量低的问题。随着研究的深入，考虑到培养基中所使用的黏蛋白复杂且成本较高，有人提出使用葡萄糖和N-乙酰葡糖胺作为碳源来发酵培养A. muciniphila[104]。然而，与其他益生菌的培养密度相比，这种方法在工业上仍相对不经济，所以大规模、低成本、高效率地培养A. muciniphila仍值得进一步探索。近年，Wu等[105]优化了摇瓶中的培养条件，并采用pH控制策略在5升生物反应器中进行了放大培养，结果显示在最佳条件下，生物反应器中的最大OD600可以达到13.03（1.03×1010 CFU/mL），这是目前报道的使用唯一碳源（葡萄糖）的最高水平。该研究方法在不影响细菌的生物功能和降低工业化生产成本的前提下，实现了A. muciniphila的高细胞密度培养，但其可行性仍需在实践中进一步确定。
此外，A. muciniphila还未列于我国国家卫生健康委颁布的《可用于食品的菌种名单》和《可用于婴幼儿食品的菌种名单》。鉴于目前A. muciniphila活菌应用存在的问题及挑战，A. muciniphila来源的后生元产品或通过外源性添加营养活性物质提高该菌在体内的丰度表现出优越性。多项研究表明，A. muciniphila的成分或其代谢物也能在动物机体中发挥与活菌相似甚至更优越的益生作用。近年来，一种工程菌株Lactococcus lactis NZP9成功地表达和分泌了来自A. muciniphila BAA-835的P9蛋白。此外，Lactococcus lactis NZP9上清液中的P9蛋白使肠道L细胞分泌的GLP-1增加了5.5倍[106]，这表明A. muciniphila源的后生元产品在未来取代A. muciniphila活菌投入生产的潜力巨大。
4. 结论与展望
嗜黏蛋白阿克曼菌A. muciniphila作为一种在婴幼儿肠道内早期存活和定植的肠道菌，与婴幼儿早期肠道菌群平衡及后期机体免疫功能的完善密切相关，其在消化道中分布及丰度影响着机体健康。另外，该菌在调节糖脂代谢、缓解肠道炎症、增强肠道屏障功能等方面的重要益生功能已得到证实，其中分子作用机制包括A. muciniphila可以直接或间接的调控AMPK信号通路来维持血糖平衡、激活CBRs从而调节ECS来增强肠道屏障功能、激活TLR2抑制NF-κB信号通路和调节肠道免疫细胞组成来介导肠道炎症反应。
A. muciniphila展现出开发为新型益生菌的巨大潜力，但是目前该菌在活菌应用方面还存在安全且有效的补充剂量范围待确定、低成本高密度工业化生产工艺待挖掘、与宿主健康状态之间相互作用机制待阐明等问题，故当前应用如AmEVs、Amuc_1100、P9蛋白等A. muciniphila来源的后生元产品在安全及高效生产方面表现出优越性。
综上所述，未来可从以下4个方面继续开展将A. muciniphila开发为益生菌制剂的研究探索：a.开发A. muciniphila源后生元产品；b.确定A. muciniphila活菌安全且有效的补充剂量范围；c.实现低成本高密度的培养A. muciniphila；d.阐明A. muciniphila与宿主健康状态之间相互作用机制。
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