方晶，薛博瑜，方南元,2

(1.南京中醫(yī)藥大學(xué)，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(xì)菌，其分類(lèi)至少有500～1 000種，數(shù)量可達(dá)100億～1 000億。在正常情況下，腸道細(xì)菌與宿主和平共生，細(xì)菌不會(huì)進(jìn)入體內(nèi)導(dǎo)致病理變化，這主要是因?yàn)槟c內(nèi)壁存在腸黏膜屏障，阻隔腸道細(xì)菌的穿越。腸黏膜屏障是指選擇性保證基本營(yíng)養(yǎng)物質(zhì)、水及電解質(zhì)等通過(guò)腸黏膜，而防止腸腔中有害的物質(zhì)(如細(xì)菌、病毒、毒素等)穿過(guò)腸黏膜進(jìn)入機(jī)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(lèi)：機(jī)械屏障、化學(xué)屏障、生物屏障及免疫屏障[1]。其中腸黏膜機(jī)械屏障在保護(hù)腸道中起到至關(guān)重要的作用，它由腸上皮細(xì)胞(intestinal epithelial cell)、上皮細(xì)胞間緊密連接(tight junction, TJ)及覆蓋在上皮細(xì)胞表面的黏液層共同構(gòu)成。腸黏膜屏障功能失?？蓪?dǎo)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(dǎo)致全身多系統(tǒng)慢性低度炎癥，進(jìn)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(jī)械屏障破壞機(jī)制目前尚不完全明確，但越來(lái)越多的研究證明腸道細(xì)菌在腸黏膜機(jī)械屏障功能障礙中起重要作用，現(xiàn)作者對(duì)它們之間關(guān)系綜述如下。

1 腸道細(xì)菌與腸道黏液層的關(guān)系

腸上皮細(xì)胞表面覆有黏液層，它是腸腔與上皮細(xì)胞間的物理屏障，也是腸黏膜機(jī)械屏障的第1道防線(xiàn)。腸道的黏液層主要由腸道杯狀細(xì)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(xì)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(xì)菌可進(jìn)入，內(nèi)層較稠厚、結(jié)構(gòu)致密，細(xì)菌不易穿透。正常人結(jié)腸中細(xì)菌不會(huì)與腸上皮細(xì)胞直接接觸[2]，而飲食結(jié)構(gòu)改變可導(dǎo)致腸道菌群失調(diào)和腸道炎癥，使黏液層變薄或消失，細(xì)菌或其代謝產(chǎn)物穿過(guò)腸黏膜表面黏液層，黏附于腸上皮細(xì)胞，直接或間接誘導(dǎo)腸上皮細(xì)胞的損傷，導(dǎo)致腸黏膜機(jī)械屏障功能下降[3- 4]。多種細(xì)菌及其代謝產(chǎn)物可影響?zhàn)ひ簩有纬?，破壞黏液層結(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(luò)，使黏液層變薄，黏液素降解細(xì)菌Akkermansia municiphila可穿透黏液層并在其中生長(zhǎng)，進(jìn)一步破壞黏液層的網(wǎng)絡(luò)結(jié)構(gòu)。脆弱類(lèi)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(lái)Hansson教授及其團(tuán)隊(duì)[7]發(fā)現(xiàn)一種分布于結(jié)腸腸腺頂部的“哨兵”杯狀細(xì)胞，當(dāng)細(xì)菌或其代謝產(chǎn)物穿過(guò)黏液層時(shí)可被這種細(xì)胞上的Toll樣受體所感知，促使其他杯狀細(xì)胞分泌MUC2黏蛋白，將入侵的細(xì)菌及其代謝物沖回到腸腔內(nèi)并形成新的黏液層保護(hù)腸上皮細(xì)胞。小腸隱窩內(nèi)的潘氏細(xì)胞可在細(xì)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(xì)胞表面黏液層，同時(shí)也分泌大量抗菌肽和溶菌酶共同抵抗腸道細(xì)菌入侵，保護(hù)腸上皮細(xì)胞[8- 10]。當(dāng)潘氏細(xì)胞功能異常，如Nod2基因突變、UPR轉(zhuǎn)錄因子X(jué)BP- 1功能異常等，可導(dǎo)致細(xì)菌對(duì)潘氏細(xì)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(xì)菌侵襲上皮細(xì)胞破壞腸黏膜屏障[11- 12]。腸道細(xì)菌代謝產(chǎn)生的短鏈脂肪酸對(duì)腸黏膜黏液層具有保護(hù)作用。研究發(fā)現(xiàn)丁酸可改善因黏液素缺失而導(dǎo)致的腸黏膜屏障功能缺失[13]，同樣，利用乙酸或丁酸干預(yù)人腸道杯狀細(xì)胞后可上調(diào)黏液素MUC2的表達(dá)與分泌[14]。

2 腸道細(xì)菌與腸上皮細(xì)胞的關(guān)系

腸上皮細(xì)胞是腸黏膜機(jī)械屏障的重要組成部分，當(dāng)腸道細(xì)菌及其毒性產(chǎn)物穿過(guò)黏液層后可直接黏附于腸上皮細(xì)胞，而腸黏膜表面的sIgA可識(shí)別腸道菌群尤其是G- 桿菌并包裹細(xì)菌，封閉細(xì)菌與腸上皮細(xì)胞結(jié)合的特異部位，阻止其與腸上皮細(xì)胞黏附，同時(shí)也能中和腸道內(nèi)的毒素，并結(jié)合抗原分子形成免疫復(fù)合物介導(dǎo)吞噬細(xì)胞清除病原微生物[15]。sIgA主要由漿細(xì)胞產(chǎn)生的IgA和腸上皮細(xì)胞產(chǎn)生的分泌片段(secretory component)組裝而成。大腸上皮細(xì)胞可特異性地表達(dá)Lypd8蛋白，后者和革蘭陰性桿菌鞭毛結(jié)合可抑制細(xì)菌運(yùn)動(dòng)，阻止細(xì)菌進(jìn)入內(nèi)部黏液層侵襲上皮細(xì)胞，保護(hù)腸黏膜屏障。如各種原因?qū)е碌膕IgA、Lypd8蛋白表達(dá)、功能障礙，最終都將導(dǎo)致細(xì)菌與腸上皮細(xì)胞的黏附增多[16]。細(xì)菌可直接與腸上皮細(xì)胞膜上相應(yīng)受體結(jié)合，或通過(guò)其代謝產(chǎn)物激活細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)引發(fā)細(xì)胞骨架重排，將細(xì)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(xì)胞膜上的整合素緊密結(jié)合，激活酪氨酸激酶引起細(xì)胞骨架肌動(dòng)蛋白重排，細(xì)菌隨即陷入細(xì)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?；?，從而破壞腸上皮細(xì)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(yù)后可降低毒素A導(dǎo)致的細(xì)胞毒性與炎癥反應(yīng)，改善腸黏膜屏障功能。

短鏈脂肪酸(short- chain fatty acids，SCFAs)在保護(hù)腸黏膜屏障中發(fā)揮重要作用。SCFAs是人結(jié)腸細(xì)菌對(duì)碳水化合物發(fā)酵的主要代謝產(chǎn)物，其本質(zhì)是含6個(gè)或6個(gè)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(guò)被動(dòng)擴(kuò)散和載體轉(zhuǎn)運(yùn)蛋白進(jìn)入腸道細(xì)胞內(nèi)[19- 20]，也可由單羧酸轉(zhuǎn)運(yùn)蛋白1(monocarboxylate transporter 1，MCT- 1)、鈉耦合單羧酸轉(zhuǎn)運(yùn)蛋白1(sodium- coupled monocarboxylate transporter 1，SMCT- 1)受體介導(dǎo)進(jìn)入腸上皮細(xì)胞發(fā)揮其生理功能[21]。丁酸是結(jié)腸上皮細(xì)胞重要的能量來(lái)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(xì)胞能量代謝障礙，可導(dǎo)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(wú)菌小鼠，發(fā)現(xiàn)該菌可恢復(fù)其結(jié)腸上皮細(xì)胞的能量代謝，進(jìn)一步利用丁酸干預(yù)無(wú)菌小鼠原代結(jié)腸上皮細(xì)胞可恢復(fù)細(xì)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(hù)結(jié)腸上皮細(xì)胞的完整性。此外，SCFAs也可通過(guò)調(diào)控TLRs表達(dá)、激活炎癥小體產(chǎn)生大量保護(hù)性細(xì)胞因子，維持腸上皮細(xì)胞完整性、細(xì)胞自我修復(fù)[24- 25]。

腸上皮細(xì)胞起源于腸隱窩的干細(xì)胞，2～5 d可全部更新脫落1次，脫落的上皮細(xì)胞由腸隱窩中的干細(xì)胞不斷增殖、分化、遷移來(lái)補(bǔ)充。在某些細(xì)菌及其毒性產(chǎn)物的刺激下腸上皮細(xì)胞的增殖、脫落可加快，從而保護(hù)腸黏膜屏障完整性。Sellin等發(fā)現(xiàn),被細(xì)菌感染的腸上皮細(xì)胞可通過(guò)Wnt/β cateni信號(hào)通路的介導(dǎo)增強(qiáng)腸上皮細(xì)胞的增殖，且腸上皮細(xì)胞通過(guò)脫落形式起到自我保護(hù)作用[26- 27]。而某些高毒性致病菌則抑制腸上皮細(xì)胞的增殖、脫落，破壞腸黏膜屏障功能，如致賀菌(Shigella)效應(yīng)分子IpaB可誘導(dǎo)腸上皮細(xì)胞的增殖過(guò)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(hào)復(fù)合物為作用目標(biāo)，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(xì)胞停滯于G1/2細(xì)胞周期，延遲細(xì)胞增殖[29- 30]。

3 腸道細(xì)菌與TJ的關(guān)系

TJ是細(xì)胞間最重要的連接方式，它位于上皮細(xì)胞頂端，呈箍狀圍繞在細(xì)胞的周?chē)?，將相鄰上皮?xì)胞緊密連接在一起，阻止毒性大分子及微生物通過(guò)，保護(hù)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(xì)菌可通過(guò)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(xì)胞TJs。細(xì)菌的分泌系統(tǒng)(secretion system)是將細(xì)菌合成的毒性蛋白轉(zhuǎn)運(yùn)到細(xì)菌外或宿主細(xì)胞內(nèi)的轉(zhuǎn)運(yùn)系統(tǒng)，腸道細(xì)菌可通過(guò)這一方式將其產(chǎn)生的毒性蛋白傳遞至腸上皮細(xì)胞內(nèi)，影響TJ蛋白的表達(dá)和定位。如大腸桿菌可通過(guò)3型分泌系統(tǒng)(T3SS)將NleA蛋白轉(zhuǎn)運(yùn)至細(xì)胞內(nèi)導(dǎo)致TJ蛋白的破壞[31]。福氏志賀菌(Shigella flexnerS.Flexneri)同樣可通過(guò)T3SS干擾TJ蛋白ZO- 1、Cldn1、occludin的表達(dá)[32]。幽門(mén)螺旋桿菌(Helicobacterpylori，HP)可導(dǎo)致胃、十二指腸潰瘍及胃癌，研究表明HP可通過(guò)4型分泌系統(tǒng)(T4SS)將細(xì)胞毒性相關(guān)基因A(CagA)編碼的效應(yīng)蛋白傳遞至胃腸上皮細(xì)胞[33]，下調(diào)ERK、蛋白酶激活受體- 1(Par1)信號(hào)通路，干擾TJ的表達(dá)與定位[34]。腸道細(xì)菌還可直接分泌一些酶或毒性蛋白至細(xì)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(xì)胞內(nèi)信號(hào)通路導(dǎo)致TJ蛋白的錯(cuò)誤定位，破壞腸黏膜屏障[35- 36]。

乙醇、乙醛是腸道細(xì)菌的代謝產(chǎn)物，飲食攝入的糖類(lèi)物質(zhì)通過(guò)腸道細(xì)菌酵解產(chǎn)生乙醇，再由細(xì)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(xì)胞，發(fā)現(xiàn)在上調(diào)CLOCK、PER2蛋白表達(dá)的同時(shí)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(dǎo)致的腸屏障高通透。乙醛則被證實(shí)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(jìn)而導(dǎo)致TJ蛋白重新分布[40- 41]，并使之從細(xì)胞骨架上脫離[42]。上文提到的細(xì)菌代謝產(chǎn)物SCFAs同樣也會(huì)對(duì)TJ蛋白產(chǎn)生影響，有研究[43]表明丁酸可通過(guò)活化AMPK通路上調(diào)腸上皮細(xì)胞ZO- 1、occludin的表達(dá)，增強(qiáng)上皮細(xì)胞跨膜電阻(TER)，保護(hù)腸屏障完整性。此外，致病細(xì)菌或細(xì)菌代謝產(chǎn)物刺激炎癥細(xì)胞產(chǎn)生大量炎癥因子，如IFNγ、TNFα等，這些炎癥因子既能通過(guò)MLCK和ROCK介導(dǎo)途徑使腸上皮細(xì)胞的TJ蛋白從細(xì)胞骨架上脫離[44]，也可使TJ蛋白的表達(dá)下調(diào)[45]。

腸上皮細(xì)胞表達(dá)的某些TJs有細(xì)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(dāng)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(jī)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(jìn)而發(fā)揮其細(xì)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(dǎo)C.Difficile對(duì)細(xì)胞的黏附并使細(xì)胞骨架坍塌，最終導(dǎo)致細(xì)胞死亡[48]。脂解刺激的脂蛋白受體(LSR)被認(rèn)為是CDT的識(shí)別受體，而LSR被證明是存在于3個(gè)上皮細(xì)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(dǎo)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(jī)械屏障，還可通過(guò)刺激免疫細(xì)胞誘導(dǎo)炎癥反應(yīng)，或通過(guò)microRNAs在轉(zhuǎn)錄后水平影響腸黏膜屏障相關(guān)細(xì)胞、蛋白的功能，導(dǎo)致屏障功能缺失。但目前關(guān)于腸道菌群對(duì)腸黏膜機(jī)械屏障影響的研究還存在一些問(wèn)題：(1)研究多集中于某些特殊種類(lèi)細(xì)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(duì)腸黏膜機(jī)械屏障的作用；(2)由于目前宏基因組檢測(cè)技術(shù)及細(xì)菌基因庫(kù)的不完善，只能測(cè)到屬一級(jí)的細(xì)菌，無(wú)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(xì)菌存在種群差異、個(gè)體差異，因此細(xì)胞及動(dòng)物研究尚不能完全反映人體腸道菌群的實(shí)際變化。

[參考文獻(xiàn)]

[1] 黃蓉,歐希龍.腸道黏膜屏障功能損傷機(jī)制及其防治的研究進(jìn)展[J].現(xiàn)代醫(yī)學(xué),2015,43(5):659- 662.

[2] JOHANSSON M E V,PHILLIPSON M,PETERSSON J,et al.The inner of the two Muc2 mucin- dependent mucus layers in colon is devoid of bacteria[J].PNAS,2008,105(39):15064- 15069.

[3] SOUZA H S P,TORTORI C J A,CASTELO B M T L,et al.Apoptosis in the intestinal mucosa of patients with inflammatory bowel disease:evidence of altered expression of FasL and perforin cytotoxic pathways[J].Int J Colorectal Dis,2005,20(3):277- 286.

[4] JOHANSSON M E V,GUSTAFSSON J K,HOLMEN L J,et al.Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis[J].Gut,2013,63(2):281- 291.

[5] IJSSENNAGGER N,BELZER C,HOOIVELD G J,et al.Gut microbiota facilitates dietary heme- induced epithelial hyperproliferation by opening the mucus barrier in colon[J].PNAS,2015,112(32):10038- 10043.

[6] RHEE K J,WU S,WU X,et al.Induction of persistent colitis by a human commensal,enterotoxigenic bacteroides fragilis,in wild- type C57BL/6 mice[J].Infection and Immunity,2009,77(4):1708- 1718.

[7] GEORGE M H,BIRCHENOUGH E E L N,MALIN E V,et al.A sentinel goblet cell guards the colonic crypt by triggering Nlrp6- dependent Muc2 secretion[J].Science,2016,352(6):1535- 1542.

[8] SALZMAN N H,GHOSH D,HUTTNER K M,et al.Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin[J].Nature,2003,4(422):522- 526.

[9] BARKEP N,VANES J H,KUIPERS J,et al.Identification of stem cells in small intestine and colon by marker gene Lgr5[J].Science,2007,449(6):1003- 1007.

[10] MCGUCKIN M A,LINDEN S K,SUTTON P,et al.Keeping bacteria at a distance[J].Microbiol,2011,9(4):265- 278.

[11] KOBAYASHI K S,CHAMAILLARD M,OGURA Y,et al.Nod2- dependent regulation of innate and adaptive immunity in the intestinal tract[J].Science,2005,307(5710):727- 731.

[12] KASER A,LEE A,FRANKE A,et al.XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[J].Cell,2008,134(5):743- 756.

[13] FERREIRA T M,LEONEL A J,MELO M A,et al.Oral Supplementation of butyrate reduces mucositis and intestinal permeability associated with 5- fluorouracil administration[J].Lipids,2012,47(5):669- 678.

[14] BURGER- VAN P N,VINCENT A,PUIMAN P J,et al.The regulation of intestinal mucin MUC2 expression by short- chain fatty acids:implications for epithelial protection[J].Biochemical Journal,2009,420(2):211- 219.

[15] CORTHESY B.Secretory immunoglobulin A:well beyond immune exclusion at mucosal surfaces[J].Immunopharmacol Immunotoxicol,2009,31(2):174- 179.

[16] OKUMURA R,KURAKAWA T,NAKANO T,et al.Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia[J].Nature,2016,532(7597):117- 121.

[17] LU L F,KIM D H,LEE I H,et al.Potassium acetate blocks clostridium difficile toxin A- induced microtubule disassembly by directly inhibiting histone deacetylase 6,thereby ameliorating inflammatory responses in the gut[J].J Microbiol Biotechn,2016,26(4):693- 699.

[18] TAN J,MCKENZIE C,POTAMITIS M,et al.The Role of short- chain fatty acids in health and disease[J].Advances in Immunology,2014,121(1):91- 120.

[19] YANASE H,TAKEBE K,NIO- KOBAYASHI J,et al.Cellular expression of a sodium- dependent monocarboxylate transporter(Slc5a8) and the MCT family in the mouse kidney[J].Histochemistry and Cell Biology,2008,130(5):957- 966.

[20] MIYAUCHI S,GOPAL E,FEI Y J,et al.Functional identification of SLC5A8,a tumor suppressor down- regulated in colon cancer,as a Na- coupled transporter for short- chain fatty acids[J].The Journal of Biological Chemistry,2004,279(4):13293- 13296.

[21] HALESTRAP A P,WILSON M C.The monocarboxylate transporter family- role and regulation[J].Iubmb Life,2012,64(2):109- 119.

[22] BARCENILLA A,PRYDE S E,MARTIN J C,et al.Phylogenetic relationships of butyrate- producing bacteria from the human gut[J].Applied and Environmental Microbiology,2000,66(4):1654- 1661.

[23] DONHOE D R,GARGE N,ZHANG X,et al.The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon[J].Cell Metabolism,2011,13(5):517- 526.

[24] THINWA J,SEGIVIA J A,BOSE S,et al.Integrin- mediated first signal for inflammasome activation in intestinal epithelial cells[J].The Journal of Immunology,2014,193(3):1373- 1382.

[25] YONEZAWA T,HAGA S,KOBYASHI Y,et al.Short- chain fatty acid signaling pathways in bovine mammary epithelial cells[J].Regulatory Peptides,2009,153(1- 3):30- 36.

[26] SELLIN J H,WANG Y,SINGH P,et al.β- Catenin stabilization imparts crypt progenitor phenotype to hyperproliferating colonic epithelia[J].Experimental Cell Research,2009,315(1):97- 109.

[27] SELLIN M E,MULLER A A,FELMY B,et al.Epithelium- intrinsic NAIP/NLRC4 inflammasome drives infected enterocyte expulsion to restrict salmonella replication in the intestinal mucosa[J].Cell Host & Microbe,2014,16(8):237- 248.

[28] IWAI H,KIM M,YOSHIKAWA Y,et al.A bacterial effector targets Mad2L2,an APC inhibitor,to modulate host cell cycling[J].Cell,2007,130(8):611- 623.

[29] LI S,ZHANG L,YAO Q,et al.Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains[J].Nature,2013,501(7466):242- 246.

[30] MORIKAWA H,KIM M,MIMUOR H,et al.The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of cullin1[J].Biochem Bioph Res Co,2010,401(2):268- 274.

[31] THANABALASURIAR A,KIM J,GRUENHEID S.The inhibition of COPⅡ trafficking is important for intestinal epithelial tight junction disruption during enteropathogenic escherichia coli and citrobacter rodentium infection[J].Institut Pasteur,2013,15(6):738- 744.

[32] SAKAGUCHI T,KOHLER H,GU X,et al.Shigella flexneri regulates tight junction- associated proteins in human intestinal epithelial cells[J].Cell Microbiol,2002,4(6):367- 381.

[33] GERLACH R G,HENSEL M.Protein secretion systems and adhesins:the molecular armory of Gram- negative pathogens[J].Int J Med Microbiol,2007,297(1):401- 415.

[34] BACKERT S,CLYNEl M,TEGTMEYER N.Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA byHelicobacterpylori[J].Cell Commun Signal,2011,9:28.

[35] di PIERRO M,LU R,UZZAU S,et al.Zonula occludens toxin structure- function analysis.Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain[J].The Journal of Biological Chemistry,2001,276(6):19160- 19165.

[36] SCHMIDTA E,KELLYB S M,van der WALLEA C F.Tight junction modulation and biochemical characterisation of the zonula occludens toxin C- and N- termini[J].Federation of European Biochemical Societies,2007,581(5):2974- 2980.

[37] FERRIER L,BERARD F,DEBRAUWER L,et al.Impairment of the intestinal barrier by ethanol involves enteric microflora and mast cell activation in rodents[J].The American Journal of Pathology,2006,168(4):1148- 1154.

[38] SALASPURO M.Bacteriocolonic pathway for ethanol oxidation:characteristics and implications[J].Ann Med,1996,28(3):195- 200.

[39] SWANSON G,FORSYTH C B,TANG Y,et al.Role of intestinal circadian genes in alcohol- induced gut leakiness[J].Alcoholism:Clinical and Experimental Research,2011,35(7):1305- 1314.

[40] ATKINSON K J,RAO R K.Role of protein tyrosine phosphorylation in acetaldehyde- induced disruption of epithelial tight junctions[J].Am J Physiol Gastrointest Liver Physiol,2001,280(6):G1280- G1288.

[41] SHETH P,SETH A,ATKINSON K J,et al.Acetaldehyde dissociates the PTP1B-E- cadherin-β- catenin complex in Caco- 2 cell monolayers by a phosphorylation- dependent mechanism[J].Biochemical Journal,2007,402(2):291- 300.

[42] SUZUKI T,SETH A,RAO R.Role of phospholipase Cγ- induced activation of protein kinase C∈(PKC∈) and PKCβI in epidermal growth factor- mediated protection of tight junctions from acetaldehyde in Caco- 2 cell monolayers[J].Journal of Biological Chemistry,2008,283(6):3574- 3583.

[43] VOLTOLINI C,BATTERSBY S,ETHERINGTON S L,et al.A novel antiinflammatory role for the short- chain fatty acids in human labor[J].Endocrinology,2012,153(1):395- 403.

[44] UTECH M,IVANOV A I,SAMARIN S N,et al.Mechanism of IFN- gamma- induced endocytosis of tight junction proteins:myosin Ⅱ- dependent vacuolarization of the apical plasma membrane[J].Molecular Biology of the Cell,2005,16(10):5040- 5052.

[45] ZEISSIG S,BURGEL N,GUNZEL D,et al.Changes in expression and distribution of claudin 2,5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn’s disease[J].Gut,2007,56(1):61- 72.

[46] KATAHIRA J,SUGIYAMA H,INOUEI N,et al.Clostridium perfringens enterotoxin utilizes two structurally related membrane proteins as functional receptorsinvivo[J].The Journal of Biological Chemistry,1997,272(10):26652- 26658.

[47] VESHNYAKOVA A,PIONTEK J,PROTZE J,et al.Mechanism of clostridium perfringens enterotoxin interaction with claudin- 3/- 4 protein suggests structural modifications of the toxin to target specific claudins[J].Journal of Biological Chemistry,2012,287(3):1698- 1708.

[48] KUEHNE S A,COLLERY M M,KELLY M L,et al.Importance of toxin A,toxin B,and CDT in virulence of an epidemic clostridium difficile strain[J].Journal of Infectious Diseases,2013,209(1):83- 86.

[49] MASUDA S,ODA Y,SASAKI H,et al.LSR defines cell corners for tricellular tight junction formation in epithelial cells[J].J Cell Sci,2011,124(Pt 4):548- 555.

[50] PAPATHEODOROU P,CARETTE J E,BELL G W,et al.Lipolysis- stimulated lipoprotein receptor(LSR) is the host receptor for the binary toxin Clostridium difficile transferase(CDT)[J].Proceedings of the National Academy of Sciences,2011,108(39):16422- 16427.