肠道细菌与肠黏膜机械屏障损伤关系的研究进展

方晶，薛博瑜，方南元,2

(1.南京中医药大学，江苏 南京 210046； 2.江苏省中医院，江苏 南京 210029)

人体肠道内存在大量细菌，其分类至少有500～1 000种，数量可达100亿～1 000亿。在正常情况下，肠道细菌与宿主和平共生，细菌不会进入体内导致病理变化，这主要是因为肠内壁存在肠黏膜屏障，阻隔肠道细菌的穿越。肠黏膜屏障是指选择性保证基本营养物质、水及电解质等通过肠黏膜，而防止肠腔中有害的物质(如细菌、病毒、毒素等)穿过肠黏膜进入机体其它组织器官的功能与结构的总和。肠黏膜屏障可分为4类：机械屏障、化学屏障、生物屏障及免疫屏障[1]。其中肠黏膜机械屏障在保护肠道中起到至关重要的作用，它由肠上皮细胞(intestinal epithelial cell)、上皮细胞间紧密连接(tight junction, TJ)及覆盖在上皮细胞表面的黏液层共同构成。肠黏膜屏障功能失常可导致肠道通透性增加，肠道内的病原微生物及其代谢产物易位入血，导致全身多系统慢性低度炎症，进而引起多种疾病(如：炎症性肠病、代谢性疾病、帕金森病等)的发生发展。肠黏膜机械屏障破坏机制目前尚不完全明确，但越来越多的研究证明肠道细菌在肠黏膜机械屏障功能障碍中起重要作用，现作者对它们之间关系综述如下。

1 肠道细菌与肠道黏液层的关系

肠上皮细胞表面覆有黏液层，它是肠腔与上皮细胞间的物理屏障，也是肠黏膜机械屏障的第1道防线。肠道的黏液层主要由肠道杯状细胞分泌的黏蛋白经高度糖基化聚合形成，但其在小肠和结肠的结构形式略有不同，小肠表面的黏液层呈单层、不连续状，并未完全覆盖小肠上皮细胞表面，而结肠黏液层分内外两层，外层较稀稠、结构疏松，细菌可进入，内层较稠厚、结构致密，细菌不易穿透。正常人结肠中细菌不会与肠上皮细胞直接接触[2]，而饮食结构改变可导致肠道菌群失调和肠道炎症，使黏液层变薄或消失，细菌或其代谢产物穿过肠黏膜表面黏液层，黏附于肠上皮细胞，直接或间接诱导肠上皮细胞的损伤，导致肠黏膜机械屏障功能下降[3- 4]。多种细菌及其代谢产物可影响黏液层形成，破坏黏液层结构。有研究[5]发现硫酸盐还原菌所产生的硫化物能溶解黏液素聚合物网络，使黏液层变薄，黏液素降解细菌Akkermansia municiphila可穿透黏液层并在其中生长，进一步破坏黏液层的网络结构。脆弱类杆菌(bacteriodes fragilis)分泌的BFT毒素具有蛋白水解酶样作用，可降解黏液素蛋白，破坏黏液层结构[6]。近来Hansson教授及其团队[7]发现一种分布于结肠肠腺顶部的“哨兵”杯状细胞，当细菌或其代谢产物穿过黏液层时可被这种细胞上的Toll样受体所感知，促使其他杯状细胞分泌MUC2黏蛋白，将入侵的细菌及其代谢物冲回到肠腔内并形成新的黏液层保护肠上皮细胞。小肠隐窝内的潘氏细胞可在细菌代谢物脂多糖、胞壁酸、胞壁酰二肽等作用下分泌MUC2黏蛋白形成小肠上皮细胞表面黏液层，同时也分泌大量抗菌肽和溶菌酶共同抵抗肠道细菌入侵，保护肠上皮细胞[8- 10]。当潘氏细胞功能异常，如Nod2基因突变、UPR转录因子XBP- 1功能异常等，可导致细菌对潘氏细胞的刺激作用减弱，减少黏蛋白及抗菌物质的产生，大量细菌侵袭上皮细胞破坏肠黏膜屏障[11- 12]。肠道细菌代谢产生的短链脂肪酸对肠黏膜黏液层具有保护作用。研究发现丁酸可改善因黏液素缺失而导致的肠黏膜屏障功能缺失[13]，同样，利用乙酸或丁酸干预人肠道杯状细胞后可上调黏液素MUC2的表达与分泌[14]。

2 肠道细菌与肠上皮细胞的关系

肠上皮细胞是肠黏膜机械屏障的重要组成部分，当肠道细菌及其毒性产物穿过黏液层后可直接黏附于肠上皮细胞，而肠黏膜表面的sIgA可识别肠道菌群尤其是G- 杆菌并包裹细菌，封闭细菌与肠上皮细胞结合的特异部位，阻止其与肠上皮细胞黏附，同时也能中和肠道内的毒素，并结合抗原分子形成免疫复合物介导吞噬细胞清除病原微生物[15]。sIgA主要由浆细胞产生的IgA和肠上皮细胞产生的分泌片段(secretory component)组装而成。大肠上皮细胞可特异性地表达Lypd8蛋白，后者和革兰阴性杆菌鞭毛结合可抑制细菌运动，阻止细菌进入内部黏液层侵袭上皮细胞，保护肠黏膜屏障。如各种原因导致的sIgA、Lypd8蛋白表达、功能障碍，最终都将导致细菌与肠上皮细胞的黏附增多[16]。细菌可直接与肠上皮细胞膜上相应受体结合，或通过其代谢产物激活细胞信号转导系统引发细胞骨架重排，将细菌包裹内吞入胞。耶尔森菌有一种外膜蛋白，可与肠上皮细胞膜上的整合素紧密结合，激活酪氨酸激酶引起细胞骨架肌动蛋白重排，细菌随即陷入细胞内。有研究[17]发现艰难梭形芽孢杆菌产生的毒素A可使tubulin蛋白去乙酰化，从而破坏肠上皮细胞微管结构而影响肠黏膜屏障完整性，而予乙酸钾干预后可降低毒素A导致的细胞毒性与炎症反应，改善肠黏膜屏障功能。

短链脂肪酸(short- chain fatty acids，SCFAs)在保护肠黏膜屏障中发挥重要作用。SCFAs是人结肠细菌对碳水化合物发酵的主要代谢产物，其本质是含6个或6个以下碳原子的饱和脂肪酸，包括乙酸、丙酸、丁酸等[18]。肠道中的SCFAs可通过被动扩散和载体转运蛋白进入肠道细胞内[19- 20]，也可由单羧酸转运蛋白1(monocarboxylate transporter 1，MCT- 1)、钠耦合单羧酸转运蛋白1(sodium- coupled monocarboxylate transporter 1，SMCT- 1)受体介导进入肠上皮细胞发挥其生理功能[21]。丁酸是结肠上皮细胞重要的能量来源之一，人体肠道内存在大量产丁酸菌，如Clostridium、Eubacterium、Butyrivibrio等，如菌群失常，产丁酸菌比例减少，丁酸生成减少，结肠上皮细胞能量代谢障碍，可导致肠黏膜屏障功能下降[22]。有研究者[23]将产丁酸菌Butyrivibrio fibrisolvens(B. Fibrisolvens)移植无菌小鼠，发现该菌可恢复其结肠上皮细胞的能量代谢，进一步利用丁酸干预无菌小鼠原代结肠上皮细胞可恢复细胞氧化磷酸化和ATP水平，保持能量稳态并抑制自噬，保护结肠上皮细胞的完整性。此外，SCFAs也可通过调控TLRs表达、激活炎症小体产生大量保护性细胞因子，维持肠上皮细胞完整性、细胞自我修复[24- 25]。

肠上皮细胞起源于肠隐窝的干细胞，2～5 d可全部更新脱落1次，脱落的上皮细胞由肠隐窝中的干细胞不断增殖、分化、迁移来补充。在某些细菌及其毒性产物的刺激下肠上皮细胞的增殖、脱落可加快，从而保护肠黏膜屏障完整性。Sellin等发现,被细菌感染的肠上皮细胞可通过Wnt/β cateni信号通路的介导增强肠上皮细胞的增殖，且肠上皮细胞通过脱落形式起到自我保护作用[26- 27]。而某些高毒性致病菌则抑制肠上皮细胞的增殖、脱落，破坏肠黏膜屏障功能，如致贺菌(Shigella)效应分子IpaB可诱导肠上皮细胞的增殖过程停滞在G2/M期[28]。大肠杆菌(Escherichiacoli)产生的NleB直接以死亡受体信号复合物为作用目标，其结合到包括TNF受体、FAS、RIPK1、TRADD和FADD在内的多种含DD的蛋白的“死亡域”上，最终使肠上皮细胞停滞于G1/2细胞周期，延迟细胞增殖[29- 30]。

3 肠道细菌与TJ的关系

TJ是细胞间最重要的连接方式，它位于上皮细胞顶端，呈箍状围绕在细胞的周围，将相邻上皮细胞紧密连接在一起，阻止毒性大分子及微生物通过，保护肠黏膜屏障。TJs分为结构蛋白和功能蛋白，结构蛋白主要有occludin、Claudin和JAM等；功能蛋白主要有ZO- 1、ZO- 2、ZO- 3、Cingulin和Zonulin等。肠道细菌可通过其分泌系统或直接分泌的方式释放毒性蛋白，破坏肠上皮细胞TJs。细菌的分泌系统(secretion system)是将细菌合成的毒性蛋白转运到细菌外或宿主细胞内的转运系统，肠道细菌可通过这一方式将其产生的毒性蛋白传递至肠上皮细胞内，影响TJ蛋白的表达和定位。如大肠杆菌可通过3型分泌系统(T3SS)将NleA蛋白转运至细胞内导致TJ蛋白的破坏[31]。福氏志贺菌(Shigella flexnerS.Flexneri)同样可通过T3SS干扰TJ蛋白ZO- 1、Cldn1、occludin的表达[32]。幽门螺旋杆菌(Helicobacterpylori，HP)可导致胃、十二指肠溃疡及胃癌，研究表明HP可通过4型分泌系统(T4SS)将细胞毒性相关基因A(CagA)编码的效应蛋白传递至胃肠上皮细胞[33]，下调ERK、蛋白酶激活受体- 1(Par1)信号通路，干扰TJ的表达与定位[34]。肠道细菌还可直接分泌一些酶或毒性蛋白至细胞外隙，如血凝素蛋白酶(hemagglutinin/protease，HA/P)、occluden小带毒素(zonula occluden toxin, ZOT)，它们可激活细胞内信号通路导致TJ蛋白的错误定位，破坏肠黏膜屏障[35- 36]。

乙醇、乙醛是肠道细菌的代谢产物，饮食摄入的糖类物质通过肠道细菌酵解产生乙醇，再由细菌中的乙醇脱氢酶将乙醇转化为乙醛[37- 38]。有体外研究[39]用0.2%低浓度乙醇刺激caco2细胞，发现在上调CLOCK、PER2蛋白表达的同时肠屏障通透性也升高，而特异性沉默CLOCK、PER2后能显著抑制乙醇导致的肠屏障高通透。乙醛则被证实可抑制酪氨酸磷酸酯酶(PTPase)的活性，使TJ蛋白ZO- 1和黏附蛋白的酪氨酸磷酸化水平下降，进而导致TJ蛋白重新分布[40- 41]，并使之从细胞骨架上脱离[42]。上文提到的细菌代谢产物SCFAs同样也会对TJ蛋白产生影响，有研究[43]表明丁酸可通过活化AMPK通路上调肠上皮细胞ZO- 1、occludin的表达，增强上皮细胞跨膜电阻(TER)，保护肠屏障完整性。此外，致病细菌或细菌代谢产物刺激炎症细胞产生大量炎症因子，如IFNγ、TNFα等，这些炎症因子既能通过MLCK和ROCK介导途径使肠上皮细胞的TJ蛋白从细胞骨架上脱离[44]，也可使TJ蛋白的表达下调[45]。

肠上皮细胞表达的某些TJs有细菌毒素受体的作用。claudin3和claudin4是最早被发现的产气荚膜杆菌肠毒素(C.perfringens enterotoxin, CPE)受体，当CPE与claudin3/4结合后可使其从TJ蛋白链上脱落，破坏肠黏膜机械屏障完整性[46]。也有研究[47]表明,CPE与Claudin家族蛋白结合进而发挥其细胞毒素和穿孔素作用。艰难梭状芽孢杆菌转移酶(C.Difficile transferase，CDT)可诱导C.Difficile对细胞的黏附并使细胞骨架坍塌，最终导致细胞死亡[48]。脂解刺激的脂蛋白受体(LSR)被认为是CDT的识别受体，而LSR被证明是存在于3个上皮细胞交接处的一种TJs相关蛋白[49- 50]。

环境、饮食、遗传等因素均可导致肠道菌群紊乱，致病菌比例增高，其毒性代谢产物不仅可直接破坏肠黏膜机械屏障，还可通过刺激免疫细胞诱导炎症反应，或通过microRNAs在转录后水平影响肠黏膜屏障相关细胞、蛋白的功能，导致屏障功能缺失。但目前关于肠道菌群对肠黏膜机械屏障影响的研究还存在一些问题：(1)研究多集中于某些特殊种类细菌及其代谢产物，在肠道菌群中所占比例较小，尚不能全面反映肠道菌群对肠黏膜机械屏障的作用；(2)由于目前宏基因组检测技术及细菌基因库的不完善，只能测到属一级的细菌，无法确定关键菌群的种株，研究的精确性不足；(3)由于肠道细菌存在种群差异、个体差异，因此细胞及动物研究尚不能完全反映人体肠道菌群的实际变化。

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