腸道細菌與腸黏膜機械屏障損傷關(guān)系的研究進展

方晶，薛博瑜，方南元,2

(1.南京中醫(yī)藥大學，江蘇 南京 210046； 2.江蘇省中醫(yī)院，江蘇 南京 210029)

人體腸道內(nèi)存在大量細菌，其分類至少有500～1 000種，數(shù)量可達100億～1 000億。在正常情況下，腸道細菌與宿主和平共生，細菌不會進入體內(nèi)導致病理變化，這主要是因為腸內(nèi)壁存在腸黏膜屏障，阻隔腸道細菌的穿越。腸黏膜屏障是指選擇性保證基本營養(yǎng)物質(zhì)、水及電解質(zhì)等通過腸黏膜，而防止腸腔中有害的物質(zhì)(如細菌、病毒、毒素等)穿過腸黏膜進入機體其它組織器官的功能與結(jié)構(gòu)的總和。腸黏膜屏障可分為4類：機械屏障、化學屏障、生物屏障及免疫屏障[1]。其中腸黏膜機械屏障在保護腸道中起到至關(guān)重要的作用，它由腸上皮細胞(intestinal epithelial cell)、上皮細胞間緊密連接(tight junction, TJ)及覆蓋在上皮細胞表面的黏液層共同構(gòu)成。腸黏膜屏障功能失常可導致腸道通透性增加，腸道內(nèi)的病原微生物及其代謝產(chǎn)物易位入血，導致全身多系統(tǒng)慢性低度炎癥，進而引起多種疾病(如：炎癥性腸病、代謝性疾病、帕金森病等)的發(fā)生發(fā)展。腸黏膜機械屏障破壞機制目前尚不完全明確，但越來越多的研究證明腸道細菌在腸黏膜機械屏障功能障礙中起重要作用，現(xiàn)作者對它們之間關(guān)系綜述如下。

1 腸道細菌與腸道黏液層的關(guān)系

腸上皮細胞表面覆有黏液層，它是腸腔與上皮細胞間的物理屏障，也是腸黏膜機械屏障的第1道防線。腸道的黏液層主要由腸道杯狀細胞分泌的黏蛋白經(jīng)高度糖基化聚合形成，但其在小腸和結(jié)腸的結(jié)構(gòu)形式略有不同，小腸表面的黏液層呈單層、不連續(xù)狀，并未完全覆蓋小腸上皮細胞表面，而結(jié)腸黏液層分內(nèi)外兩層，外層較稀稠、結(jié)構(gòu)疏松，細菌可進入，內(nèi)層較稠厚、結(jié)構(gòu)致密，細菌不易穿透。正常人結(jié)腸中細菌不會與腸上皮細胞直接接觸[2]，而飲食結(jié)構(gòu)改變可導致腸道菌群失調(diào)和腸道炎癥，使黏液層變薄或消失，細菌或其代謝產(chǎn)物穿過腸黏膜表面黏液層，黏附于腸上皮細胞，直接或間接誘導腸上皮細胞的損傷，導致腸黏膜機械屏障功能下降[3- 4]。多種細菌及其代謝產(chǎn)物可影響?zhàn)ひ簩有纬桑茐酿ひ簩咏Y(jié)構(gòu)。有研究[5]發(fā)現(xiàn)硫酸鹽還原菌所產(chǎn)生的硫化物能溶解黏液素聚合物網(wǎng)絡，使黏液層變薄，黏液素降解細菌Akkermansia municiphila可穿透黏液層并在其中生長，進一步破壞黏液層的網(wǎng)絡結(jié)構(gòu)。脆弱類桿菌(bacteriodes fragilis)分泌的BFT毒素具有蛋白水解酶樣作用，可降解黏液素蛋白，破壞黏液層結(jié)構(gòu)[6]。近來Hansson教授及其團隊[7]發(fā)現(xiàn)一種分布于結(jié)腸腸腺頂部的“哨兵”杯狀細胞，當細菌或其代謝產(chǎn)物穿過黏液層時可被這種細胞上的Toll樣受體所感知，促使其他杯狀細胞分泌MUC2黏蛋白，將入侵的細菌及其代謝物沖回到腸腔內(nèi)并形成新的黏液層保護腸上皮細胞。小腸隱窩內(nèi)的潘氏細胞可在細菌代謝物脂多糖、胞壁酸、胞壁酰二肽等作用下分泌MUC2黏蛋白形成小腸上皮細胞表面黏液層，同時也分泌大量抗菌肽和溶菌酶共同抵抗腸道細菌入侵，保護腸上皮細胞[8- 10]。當潘氏細胞功能異常，如Nod2基因突變、UPR轉(zhuǎn)錄因子XBP- 1功能異常等，可導致細菌對潘氏細胞的刺激作用減弱，減少黏蛋白及抗菌物質(zhì)的產(chǎn)生，大量細菌侵襲上皮細胞破壞腸黏膜屏障[11- 12]。腸道細菌代謝產(chǎn)生的短鏈脂肪酸對腸黏膜黏液層具有保護作用。研究發(fā)現(xiàn)丁酸可改善因黏液素缺失而導致的腸黏膜屏障功能缺失[13]，同樣，利用乙酸或丁酸干預人腸道杯狀細胞后可上調(diào)黏液素MUC2的表達與分泌[14]。

2 腸道細菌與腸上皮細胞的關(guān)系

腸上皮細胞是腸黏膜機械屏障的重要組成部分，當腸道細菌及其毒性產(chǎn)物穿過黏液層后可直接黏附于腸上皮細胞，而腸黏膜表面的sIgA可識別腸道菌群尤其是G- 桿菌并包裹細菌，封閉細菌與腸上皮細胞結(jié)合的特異部位，阻止其與腸上皮細胞黏附，同時也能中和腸道內(nèi)的毒素，并結(jié)合抗原分子形成免疫復合物介導吞噬細胞清除病原微生物[15]。sIgA主要由漿細胞產(chǎn)生的IgA和腸上皮細胞產(chǎn)生的分泌片段(secretory component)組裝而成。大腸上皮細胞可特異性地表達Lypd8蛋白，后者和革蘭陰性桿菌鞭毛結(jié)合可抑制細菌運動，阻止細菌進入內(nèi)部黏液層侵襲上皮細胞，保護腸黏膜屏障。如各種原因?qū)е碌膕IgA、Lypd8蛋白表達、功能障礙，最終都將導致細菌與腸上皮細胞的黏附增多[16]。細菌可直接與腸上皮細胞膜上相應受體結(jié)合，或通過其代謝產(chǎn)物激活細胞信號轉(zhuǎn)導系統(tǒng)引發(fā)細胞骨架重排，將細菌包裹內(nèi)吞入胞。耶爾森菌有一種外膜蛋白，可與腸上皮細胞膜上的整合素緊密結(jié)合，激活酪氨酸激酶引起細胞骨架肌動蛋白重排，細菌隨即陷入細胞內(nèi)。有研究[17]發(fā)現(xiàn)艱難梭形芽孢桿菌產(chǎn)生的毒素A可使tubulin蛋白去乙?；瑥亩茐哪c上皮細胞微管結(jié)構(gòu)而影響腸黏膜屏障完整性，而予乙酸鉀干預后可降低毒素A導致的細胞毒性與炎癥反應，改善腸黏膜屏障功能。

短鏈脂肪酸(short- chain fatty acids，SCFAs)在保護腸黏膜屏障中發(fā)揮重要作用。SCFAs是人結(jié)腸細菌對碳水化合物發(fā)酵的主要代謝產(chǎn)物，其本質(zhì)是含6個或6個以下碳原子的飽和脂肪酸，包括乙酸、丙酸、丁酸等[18]。腸道中的SCFAs可通過被動擴散和載體轉(zhuǎn)運蛋白進入腸道細胞內(nèi)[19- 20]，也可由單羧酸轉(zhuǎn)運蛋白1(monocarboxylate transporter 1，MCT- 1)、鈉耦合單羧酸轉(zhuǎn)運蛋白1(sodium- coupled monocarboxylate transporter 1，SMCT- 1)受體介導進入腸上皮細胞發(fā)揮其生理功能[21]。丁酸是結(jié)腸上皮細胞重要的能量來源之一，人體腸道內(nèi)存在大量產(chǎn)丁酸菌，如Clostridium、Eubacterium、Butyrivibrio等，如菌群失常，產(chǎn)丁酸菌比例減少，丁酸生成減少，結(jié)腸上皮細胞能量代謝障礙，可導致腸黏膜屏障功能下降[22]。有研究者[23]將產(chǎn)丁酸菌Butyrivibrio fibrisolvens(B. Fibrisolvens)移植無菌小鼠，發(fā)現(xiàn)該菌可恢復其結(jié)腸上皮細胞的能量代謝，進一步利用丁酸干預無菌小鼠原代結(jié)腸上皮細胞可恢復細胞氧化磷酸化和ATP水平，保持能量穩(wěn)態(tài)并抑制自噬，保護結(jié)腸上皮細胞的完整性。此外，SCFAs也可通過調(diào)控TLRs表達、激活炎癥小體產(chǎn)生大量保護性細胞因子，維持腸上皮細胞完整性、細胞自我修復[24- 25]。

腸上皮細胞起源于腸隱窩的干細胞，2～5 d可全部更新脫落1次，脫落的上皮細胞由腸隱窩中的干細胞不斷增殖、分化、遷移來補充。在某些細菌及其毒性產(chǎn)物的刺激下腸上皮細胞的增殖、脫落可加快，從而保護腸黏膜屏障完整性。Sellin等發(fā)現(xiàn),被細菌感染的腸上皮細胞可通過Wnt/β cateni信號通路的介導增強腸上皮細胞的增殖，且腸上皮細胞通過脫落形式起到自我保護作用[26- 27]。而某些高毒性致病菌則抑制腸上皮細胞的增殖、脫落，破壞腸黏膜屏障功能，如致賀菌(Shigella)效應分子IpaB可誘導腸上皮細胞的增殖過程停滯在G2/M期[28]。大腸桿菌(Escherichiacoli)產(chǎn)生的NleB直接以死亡受體信號復合物為作用目標，其結(jié)合到包括TNF受體、FAS、RIPK1、TRADD和FADD在內(nèi)的多種含DD的蛋白的“死亡域”上，最終使腸上皮細胞停滯于G1/2細胞周期，延遲細胞增殖[29- 30]。

3 腸道細菌與TJ的關(guān)系

TJ是細胞間最重要的連接方式，它位于上皮細胞頂端，呈箍狀圍繞在細胞的周圍，將相鄰上皮細胞緊密連接在一起，阻止毒性大分子及微生物通過，保護腸黏膜屏障。TJs分為結(jié)構(gòu)蛋白和功能蛋白，結(jié)構(gòu)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。腸道細菌可通過其分泌系統(tǒng)或直接分泌的方式釋放毒性蛋白，破壞腸上皮細胞TJs。細菌的分泌系統(tǒng)(secretion system)是將細菌合成的毒性蛋白轉(zhuǎn)運到細菌外或宿主細胞內(nèi)的轉(zhuǎn)運系統(tǒng)，腸道細菌可通過這一方式將其產(chǎn)生的毒性蛋白傳遞至腸上皮細胞內(nèi)，影響TJ蛋白的表達和定位。如大腸桿菌可通過3型分泌系統(tǒng)(T3SS)將NleA蛋白轉(zhuǎn)運至細胞內(nèi)導致TJ蛋白的破壞[31]。福氏志賀菌(Shigella flexnerS.Flexneri)同樣可通過T3SS干擾TJ蛋白ZO- 1、Cldn1、occludin的表達[32]。幽門螺旋桿菌(Helicobacterpylori，HP)可導致胃、十二指腸潰瘍及胃癌，研究表明HP可通過4型分泌系統(tǒng)(T4SS)將細胞毒性相關(guān)基因A(CagA)編碼的效應蛋白傳遞至胃腸上皮細胞[33]，下調(diào)ERK、蛋白酶激活受體- 1(Par1)信號通路，干擾TJ的表達與定位[34]。腸道細菌還可直接分泌一些酶或毒性蛋白至細胞外隙，如血凝素蛋白酶(hemagglutinin/protease，HA/P)、occluden小帶毒素(zonula occluden toxin, ZOT)，它們可激活細胞內(nèi)信號通路導致TJ蛋白的錯誤定位，破壞腸黏膜屏障[35- 36]。

乙醇、乙醛是腸道細菌的代謝產(chǎn)物，飲食攝入的糖類物質(zhì)通過腸道細菌酵解產(chǎn)生乙醇，再由細菌中的乙醇脫氫酶將乙醇轉(zhuǎn)化為乙醛[37- 38]。有體外研究[39]用0.2%低濃度乙醇刺激caco2細胞，發(fā)現(xiàn)在上調(diào)CLOCK、PER2蛋白表達的同時腸屏障通透性也升高，而特異性沉默CLOCK、PER2后能顯著抑制乙醇導致的腸屏障高通透。乙醛則被證實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，進而導致TJ蛋白重新分布[40- 41]，并使之從細胞骨架上脫離[42]。上文提到的細菌代謝產(chǎn)物SCFAs同樣也會對TJ蛋白產(chǎn)生影響，有研究[43]表明丁酸可通過活化AMPK通路上調(diào)腸上皮細胞ZO- 1、occludin的表達，增強上皮細胞跨膜電阻(TER)，保護腸屏障完整性。此外，致病細菌或細菌代謝產(chǎn)物刺激炎癥細胞產(chǎn)生大量炎癥因子，如IFNγ、TNFα等，這些炎癥因子既能通過MLCK和ROCK介導途徑使腸上皮細胞的TJ蛋白從細胞骨架上脫離[44]，也可使TJ蛋白的表達下調(diào)[45]。

腸上皮細胞表達的某些TJs有細菌毒素受體的作用。claudin3和claudin4是最早被發(fā)現(xiàn)的產(chǎn)氣莢膜桿菌腸毒素(C.perfringens enterotoxin, CPE)受體，當CPE與claudin3/4結(jié)合后可使其從TJ蛋白鏈上脫落，破壞腸黏膜機械屏障完整性[46]。也有研究[47]表明,CPE與Claudin家族蛋白結(jié)合進而發(fā)揮其細胞毒素和穿孔素作用。艱難梭狀芽孢桿菌轉(zhuǎn)移酶(C.Difficile transferase，CDT)可誘導C.Difficile對細胞的黏附并使細胞骨架坍塌，最終導致細胞死亡[48]。脂解刺激的脂蛋白受體(LSR)被認為是CDT的識別受體，而LSR被證明是存在于3個上皮細胞交接處的一種TJs相關(guān)蛋白[49- 50]。

環(huán)境、飲食、遺傳等因素均可導致腸道菌群紊亂，致病菌比例增高，其毒性代謝產(chǎn)物不僅可直接破壞腸黏膜機械屏障，還可通過刺激免疫細胞誘導炎癥反應，或通過microRNAs在轉(zhuǎn)錄后水平影響腸黏膜屏障相關(guān)細胞、蛋白的功能，導致屏障功能缺失。但目前關(guān)于腸道菌群對腸黏膜機械屏障影響的研究還存在一些問題：(1)研究多集中于某些特殊種類細菌及其代謝產(chǎn)物，在腸道菌群中所占比例較小，尚不能全面反映腸道菌群對腸黏膜機械屏障的作用；(2)由于目前宏基因組檢測技術(shù)及細菌基因庫的不完善，只能測到屬一級的細菌，無法確定關(guān)鍵菌群的種株，研究的精確性不足；(3)由于腸道細菌存在種群差異、個體差異，因此細胞及動物研究尚不能完全反映人體腸道菌群的實際變化。

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