血小板活化因子致肠黏膜机械屏障损伤的机制及肠三叶因子的保护作用
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摘要
前言
肠黏膜屏障是机体最重要的免疫防御屏障,将机体与肠道内的外源性物质隔离开来,避免病原微生物的侵袭和抗原分子的损伤。肠黏膜屏障主要包括以下三个方面:①肠道连续的黏膜上皮构成的机械屏障,②由肠道淋巴样组织细胞网提供的细胞及体液免疫系统,也称免疫屏障,③正常的大量的肠道厌氧菌群,防止致病微生物的过度增生及在肠道黏膜的定植,构成微生物屏障。
肠黏膜的机械屏障由肠表面黏液,微绒毛,肠黏膜上皮细胞及其间的连接以及黏膜的特殊结构组成,是肠黏膜屏障的结构基础。上皮细胞的形态改变、连接缝隙增宽或开放数目增多都会影响上皮细胞屏障功能,使通透性增加。目前研究发现肠黏膜通透性增加参与了多种疾病的发生发展,如:炎症性肠病、败血症、烧伤、终末期肝病、重症胰腺炎等,进一步引起全身炎症反应综合征甚至多器官功能衰竭。近期研究发现肠黏膜屏障的破坏在儿科急慢性疾病的发生发展中亦具有显著的意义:急性期,细菌移位可引起败血症及多器官功能衰竭;而婴儿时期的肠黏膜屏障破坏将会成为随后产生的部分变应性疾病的始发因素,如:湿疹,食物过敏,谷胶病,1型糖尿病,哮喘,炎症性肠病及孤独症等。因此肠黏膜屏障功能及其完整性这一课题在儿科领域也受到越来越多的关注。
肠上皮屏障通透性增加与多种因素有关,不仅包括疾病的起始致病因子如感染、缺血等,也包括机体继发产生的内源性免疫介质。寻找这个炎症连锁反应中的关键因素一直是我们努力的方向。有趣的是,一直在循环系统中倍受关注的血小板活化因子(PAF)同样可以由肠上皮细胞产生,存在于正常的小肠组织中,并调节黏膜的通透性。更为重要的是实验发现,给予小剂量PAF,在不足以引起肠损伤的情况下,即可以增加肠黏膜的通透性,触发细胞因子和转录因子的活化。因此,PAF被认为是肠上皮屏障通透性增加的关键因素,但具体作用机制和方式还不明了。
本实验中我们应用Caco-2细胞株建立体外肠上皮细胞屏障模型,观察PAF对肠上皮细胞屏障通透性的影响,确定其作用位点,并在此基础上进一步探讨PAF对维持肠黏膜机械屏障正常通透性的细胞骨架蛋白F-actin,构成细胞紧密连接的重要蛋白Occludin,Claudin-1,ZO-1,以及在肠道的自我保护和修复中具有重要意义的三叶肽ITF在基因、蛋白、定位等方面的影响,系统探讨PAF对肠黏膜机械屏障的损伤机制。
肠三叶因子(ITF)属三叶肽家族,是近年来被人们注意到的对胃肠道粘膜屏障有重要保护和修复作用的多肽,是一种新型的生长因子类多肽物质。它的生理功能主要体现在两个方面:首先ITF可与粘液糖蛋白相互作用或交联,形成粘弹性的粘液凝胶层,对肠道粘液层起固定和支持作用,防止有害物质对肠粘膜细胞的损伤,从而增强胃肠道粘膜屏障的保护能力;其次ITF具有很强的促进细胞增殖与移行的能力,被认为是粘膜损伤的快速反应肽,在损伤早期即可表达,促进受损区域上皮细胞重建并加快上皮细胞移行速度,因而在肠道的自我保护机制中占据重要地位。大量的动物实验也证明肠三叶因子在维持肠上皮细胞的完整性,恢复肠黏膜的正常通透性方面均起到重要作用。近期的体外实验也发现ITF可以调控紧密连接蛋白Claudin-1和Claudin-2的表达而影响肠上皮的跨上皮阻力。但PAF是否可以影响内源性ITF的表达?外源性给予基因重组ITF是否可以起到保护作用?其机制如何?这些问题的回答也为临床应用ITF治疗胃肠黏膜损伤性疾病提供了可靠的理论依据。
材料和方法
一、细胞培养
Caco-2细胞株,来源于人大肠癌细胞,可分化成有极性的肠上皮细胞。应用含有150 ml/L胎牛血清和青霉素-链霉素双抗液的RPMI 1640高糖培养液,在37℃、50 ml/L CO_2条件下进行培养,每7 d按1:2的比例传代。将细胞接种于transwell、盖玻片、六孔板上或培养瓶中,待细胞生长融合至80%左右,换无血清培养液,按实验分组加药,进行后续实验。每种实验均选取至少三组非同代细胞进行。
二、实验分组
分别设正常对照组、实验组(含模型对照组)、ITF预防组、ITF治疗组。
正常对照组:不加刺激物及干预因素。
实验组:加入PAF,终浓度分别为50ng╱L、100ng/L和200ng╱L,并把终浓度100ng╱L的组作为模型对照组,分别作用2,4,8,12,24,48h。
ITF预防组:先加入ITF0.3g╱L,30min后加入PAF100ng╱L,共培养24h。
ITF治疗组:先加入PAF100ng/L,30min后加入ITF0.3g╱L,共培养24h。
三、实验方法及检测指标
1、体外肠上皮细胞屏障模型的建立
应用Caco-2细胞株在transwell上建立体外肠上皮细胞屏障模型,并应用跨上皮细胞电阻(TEER)和荧光黄透过率两种指标检测肠上皮细胞屏障的形成情况。
2、PAF对细胞增殖和凋亡的影响
(1)MTT比色法检测细胞活力,反映细胞增殖情况。
(2)用Annexin V-EGFP和Hoechst试剂盒,应用流式细胞技术和免疫荧光染色技术,通过细胞膜和细胞核染色联合检测细胞凋亡的发生。
3、PAF对肠上皮细胞屏障通透性的影响和ITF的保护作用
(1)TEER的测定:应用EVOM测定Caco-2单层上皮细胞的TEER。
(2)荧光黄透过量的检测:应用荧光黄作为标记物,检测PAF或ITF对Caco-2细胞细胞旁转运的影响。用荧光分光光度计测定荧光黄吸光度(激发波长427nm,发射波长536 nm),根据标准曲线计算荧光黄浓度。
4、PAF和ITF对肠上皮细胞间紧密连接的影响
透射电镜下观察加入PAF或/和ITF不同时间后,Caco-2细胞间紧密连接形态学的改变。
5、PAF和ITF对肠上皮细胞骨架蛋白F-actin的影响
应用罗丹明-鬼笔环肽(TRITC-phalloidin)特异性荧光探针直接标记F-actin,流式细胞技术测定F-actin的含量;直接免疫荧光技术观察F-actin的定位和重排情况。
6、PAF对内源性ITF表达的影响
给予不同浓度PAF作用不同时间后,应用实时定量PCR技术和免疫组化技术检测PAF对ITFmRNA和蛋白定位的影响。
7、PAF和ITF对肠上皮细胞紧密连接蛋白Occludin,Claudin-1,ZO-1表达和定位的影响
应用免疫荧光、蛋白印迹杂交(Western blot)和实时定量PCR技术检测肠上皮细胞紧密连接蛋白Occludin,Claudin-1,ZO-1定位和蛋白、基因表达的变化情况。
四、统计学分析
所有实验均重复3次以上,结果用Mean±SD表示,统计由SPSS13.0软件完成。采用One-Way ANOVA法比较总体和组间差异,P＜0.05认为有显著差异。
结果
1、利用Caco-2细胞建立肠上皮细胞屏障
Caco-2细胞随着培养时间的延长,逐渐融合成片,成单层生长,光镜下呈多角形、不规则多边形或鹅卵石样,相邻细胞连接紧密,可见致密的反光带;TEER值逐渐升高,至21天左右,细胞形成紧密单层,TEER值达到300～400Ω/cm~2,对荧光黄透过量很小。电镜下发现细胞呈高分化,细胞间形成完整的紧密连接,绒毛整齐,极性形成,成单层排列。体外肠上皮细胞屏障建立。
2、PAF对细胞增殖和凋亡的影响
MTT染色发现,即使大剂量的PAF也未影响到细胞的增殖和细胞活力,各加药浓度与对照组相比无显著性差异(P＞0.05)。
应用Annexin V和Hoechst染色试剂盒,应用荧光显微镜和流式细胞仪检测,均未发现给予PAF后出现明显的细胞凋亡增加(P＞0.05)。
3、PAF对肠上皮细胞屏障通透性的影响和ITF的保护作用
与对照组相比,50ng╱LPAF即可引起TEER的下降和荧光黄透过量的增加,以PAF100ng/L组作用最明显(P＜0.01);在作用时间上,PAF100ng/L作用4h后,TEER值开始下降(P＜0.01),荧光黄透过量开始升高(P＜0.01),24h这种作用达到最强(P＜0.01)。预防和治疗性给予ITF后,与模型对照组相比,TEER有所恢复,荧光黄透过量减少,预防组作用更明显(P＜0.05)。
4、PAF和ITF对肠上皮细胞间紧密连接的影响
正常Caco-2细胞,紧密连接结构完整,呈致密的带状结构。加入PAF100ng/L作用24h后,电镜下可见紧密连接结构破坏、断裂,细胞间隙增宽。加入ITF0.3 g/L共同作用24h,紧密连接及细胞极性均部分恢复。
5、PAF和ITF对肠上皮细胞骨架蛋白F-actin的影响
在正常Caco-2细胞,F-actin主要环绕于细胞周边,细胞中央偶尔可见不规则纤维丝。细胞间F-actin排列紧密圆滑,轮廓完整连续,无明显间隙,细胞界限清晰。
PAF作用8-12h后,就可见细胞骨架F-actin出现重排,外周致密带边缘变的毛糙不规整,细胞中央有弥散actin及少量应力纤维形成。作用24h,出现明显横跨细胞的应力纤维结构,48h后周边肌动蛋白丝带变细,断断续续,胞浆内仍可见少量应力纤维。给予不同浓度的PAF发现,以PAF100ng╱L破坏最为明显。
当预防或治疗性给予ITF并共同培养24h后,正常F-actin染色的细胞比例增加,周边肌动蛋白丝带逐渐清晰,胞浆内应力纤维减少,但外周致密带边缘仍毛糙不规整,环点状断裂未完全修复。比较而言,预防性给药恢复细胞骨架正常结构的能力更强。
流式细胞仪检测发现:PAF作用8h后,F-actin含量开始减少,峰左移;24h后明显下降,峰左移明显,48h与24h相比无明显差异。PAF100ng╱L组下降最为明显,与对照组相比差异显著(P＜0.01)。给予ITF作用24h,F-actin的含量有所增加,与模型组相比差异显著(P＜0.01),但仍低于对照组;峰右移,但未移回原位。
6、PAF对内源性ITF表达的影响
给予50ng╱L PAF 8h后,ITFmRNA表达开始下降,24h降到最低,48h有所恢复,但与对照组比较没有统计学差异。PAF100ng╱L组作用4h ITFmRNA即开始下降,24h降到低谷(P＜0.05),48h有所恢复。进一步加大PAF浓度(200ng╱L),与100ng/L组无明显差异。
免疫组化显示在正常Caco-2细胞中,ITF阳性表达呈棕黄色颗粒,位于胞浆内,胞膜也有少量表达。给予不同浓度PAF(50,100,200ng╱L)作用24h,ITF表达减少,染色变淡,以100ng/L组改变最明显。
7、PAF和ITF对肠上皮细胞紧密连接蛋白Occludin,Claudin-1,ZO-1表达和定位的影响
(1)紧密连接蛋白Occludin,Claudin-1,ZO-1在肠上皮细胞的定位改变
正常Caco-2细胞Occludin,Claudin-1,ZO-1主要分布在细胞内近胞膜处,细胞间连接紧密。PAF50ng╱L作用24h后,Occludin边缘粗糙呈锯齿状,荧光信号减弱,向膜下转移。随PAF浓度加大(100ng╱L)改变更加明显,细胞间出现间隙,Occludin环断裂,崩解,胞浆内见阳性染色。但PAF浓度继续加大(200ng/L),改变并未继续加重。Claudin-1,ZO-1表达位置和变化规律与Occludin基本一致。预防和治疗性给予ITF后,可明显减轻Occludin,Claudin-1,ZO-1的破坏,边缘变得连续,但没有完全恢复。ITF预防组效果更明显。
(2)Occludin,Claudin-1,ZO-1 mRNA表达的改变
利用标准曲线对样品中的Occludin,Claudin-1,ZO-1基因和GAPDH基因进行定量,发现不同浓度PAF组与正常对照组比较Occludin,Claudin-1,ZO-1 mRNA的水平均有降低,以PAF 100ng/L组改变最明显(P＜0.01),三者变化趋势一致;基因水平从4h开始降低,到24h降到低谷,48h有所恢复。以PAF 100ng/L组作为模型组,预防或治疗性给予ITF0.3g╱L作用24h,预防和对照组Occludin,Claudin-1,ZO-1基因表达均较模型组增强,但未恢复到对照组水平,预防组作用更明显(P＜0.05),但两组之间无统计学差异。
(3)Occludin,Claudin-1,ZO-1蛋白表达的改变
Occludin和Claudin-1发挥功能需要丝氨酸和苏氨酸残基磷酸化,磷酸化会使分子量上调。
应用Western blot法对Occludin进行蛋白杂交,发现两条蛋白条带,分别在65kDa和80kDa,灰度分析发现各组标本65kDa的蛋白含量无统计学差异,但是对于80kDa的蛋白含量,PAF组较对照组明显下降并呈剂量依赖性(P＜0.01);
对Claudin-1进行蛋白杂交,发现25kDa和20kDa两条蛋白条带,20kDa的蛋白含量无统计学差异(P＞0.05),但是25kDa的蛋白含量,PAF组较对照组明显下降并呈剂量依赖性(P＜0.01);
对ZO-1进行蛋白杂交,发现在220kDa处出现特异性蛋白条带,其灰度随PAF给药浓度的增加而降低,以PAF 100ng╱L作用24h降到最低,与对照组相比P＜0.01。
预防或治疗性给予ITF后,三者的蛋白表达较模型组增强,但未恢复到对照组水平,与mRNA表达的变化趋势具有一致性。
结论
1、培养2-3w的Caco-2细胞可作为体外研究肠上皮细胞屏障的工具。
2、PAF可直接破坏肠上皮细胞屏障,其增加肠上皮细胞通透性的作用是凋亡非依赖性的,其作用位点在旁细胞途径。
3、ITF可以通过恢复紧密连接的结构降低肠通透性,从而发挥其保护作用。
4、PAF引起细胞骨架F-actin的重排和再分布以及蛋白含量的减少,是PAF引起肠上皮细胞屏障通透性增加的机制之一。
5、PAF使内源性ITF基因和蛋白表达减少,抑制了肠道的自我保护和修复能力。
6、外源性重组ITF部分逆转细胞骨架F-actin的重排和再分布,可以作为一个肠道保护因素。
7、PAF可以引起紧密连接蛋白Occludin,Claudin-1和ZO-1的基因和蛋白水平的改变以及异常分布,具有剂量和时间依赖性,且三者的改变具有协同性,这可能是PAF引起肠上皮细胞屏障破坏的内在机制。
8、ITF可以部分恢复紧密连接蛋白Occludin,Claudin-1和ZO-1的基因和蛋白水平改变以及异常分布,恢复紧密连接的正常结构,可能是其保护肠黏膜屏障的一个重要机制。
Objective
In addition to being the organ responsible for digestion and absorption of nutrients, the intestine serves a barrier function that is a critical component of the innate immune system.Only a single layer of epithelial cells separates the luminal contents from effector immune cells in the lamina propria and the internal milieu of the body. Complete intestinal barrier is comprised of 1.The mechanical barrier is comprised of continual epithelial of gut mucosal;2.Cellular and humoral immunity provided by lymphoid tissue of intestinal tract;3.Normal and massive commensal intestinal bacterial clusters,preventing the over proliferating and permanent planting in gut mucosal of pathogenic microorganism.
The mechanical barrier is comprised of mucus,microvilli,epithelial cells and junctions among them and other particular constructions,which is the structural foundation of intestinal mucosal barrier.Not only the morphous change of epithelial cells but also the wideness of junction gap and the increasing of opened cell-cell junctions could affect the function of intestinal epithelial barrier and increase the permeability.The disruption of barrier integrity has been implicated in the pathogenesis of a wide variety of gastrointestinal and systemic disorders such as inflammatory bowel disease,liver failure,acute severe pancreases and multiple organ system failure.In addition to these,many acute and chronic gastrointestinal and systemic disorders in paediatrics such as sepsis,eczema,food allergies,celiac enteropathy,type 1 diabetes, asthma,and so on were also implicated in,Hense studying the mechanisms of high permeability and to explore the effectual treatment become the goal of this experiment.
The increase of intestinal epithelial barrier permeability implicated in several factors,including initiatal causative agent as infection or ischemia and secondum inflammatory mediator.In recent years,the roles of platelet activating factor(PAF)to impairment of gastrointestinal mucosa has been paid increasing attention.PAF can be produced by intestinal epithelial cells and constitutively present in normal small intestinal tissue regulating the mucosal permeability.A large body of evidence suggests that PAF is involved in the pathogenesis of intestinal injury in various diseases.Moreover,PAF at doses insufficient to cause bowel necrosis triggers the expression of cytokines,and activates transcription factors in the intestine.So PAF is regarded as central amplify mediator and key factor of increasing of intestinal epithelial barrier permeability.
The mechanisms of PAF-related epithelial breakdown are unclear,but regulation of paracellular pathways,especially via rearranging cytoskeleton F-actin and changing interepithelial tight junction(TJ)proteins such as Occludin,Claudin-1,ZO-1 is likelyto play a significant role.Therefore,we detect the effect of PAF on intestinal epithelial F-actin and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells to study the mechanisms involved in disruption of intestinal epithelium mechanical barrier induced by PAF.
Intestinal trefoil factor(ITF)is a member of trefoil peptide family,which is important in maintenance and reparation of the intestinal mucosal barrier.It can not only stimulate cell migrating and proliferating,promote epithelial cell reparation,but also interact with the mucus,stabilize the mucus gel by perhaps interacting with intestinal mucin and increasing the viscosity.So it is important in the self-protection mechanism of intestine.Many researches in vivo also demonstrated ITF was important in the maintenance of intestinal epithelium barrier integrity and in the rescovery of mucosal normal permeability.Recent in vitro experiment find ITF can regulate the expression of TJ proteins of Claudin-1 and Claudin-2,then to affect TEER.But whether PAF can affect the expression of ITF? Administer of rITF can protect the intestinal mucosal barrier? What is the mechanisms? To answer these questions and to provide theory base for clinical application of rITF were the basic consideration of this study.
Materials and methods
1.Cell cultures
Caco-2 cells were grown in a culture medium composed of RPMI 1640 with 4.5 mg/ml glucose,50 U/ml penicillin,50 U/ml streptomycin,and 15%FBS in an atmosphere of 5%CO_2 and 90%relative humidity maintained at 37℃.Parental cells were split at a ratio of 1:3 on reaching confluency,and set up on Transwell filters,6 well plates or T-50 flasks for propagation and experiments.
2.PAF and ITF treatment
Cell monolayers were exposed in RPMI 1640 without FBS for 24hs then PAF of different concentrations(0,50,100,200ng/L)was administered,incubating for different time(0,2,4,8,12,24,48h);ITF(0.3g/L)was administered 30mins before or after PAF(100ng/L)was given and incubated for 24hs in 6 well plates,T-50 flasks or transwell Cell Culture Inserts to undertake different experiments.
3.Experimental methods and analysis index
(1)Establishing intestinal epithelial barrier model in vitro
Caco-2 cells were plated on Transwell filters and monitored regularly by visualization with an inverted microscope and by TEER and unidirectional flux of lucifer yellow measurements to ensure the form of intestinal epithelial barrier.
(2)Cell proliferation and apoptosis
MTT was used to detect cell vigor;Annexin V-EGFP and Hoechst were used to detect cell apoptosis through immunofluorescence and FCM.
(3)Determination of epithelial monolayer resistance and paracellular permeability
The transepithelial electrical resistance(TEER)of the filter-grown Caco-2 intestinal monolayers was measured by using an epithelial voltohm-meter(EVOM); the unidirectional flux of paracellular marker lucifer yellow was measured to determination the Caco-2 monolayer paracellular permeability using fluorescence spectrophotometer.
(4)Transmission electron microscopy
Cells were treated with PAF or ITF and ultrathin sections were made and stained with saturated uranyl acetate and Reynold's lead citrate.Ultrastructure of junctional complexes was observated by transmission electron microscopy.
(5)F-actin rearrangment and assays
When treated by PAF and ITF with different dose and different time,TRITC-phalloidin staining was performed.Direct immunofluorescent staining observe cytoskeleton F-actin rearrangement,Flow cytometry was used to assays F-actin contents.
(6)Assessment of ITF protein localization and expression
Cells were treated with PAF and the localization of ITF protein were detected by immunohistochemistry and ITFmRNA expression was measured useing SYBR Green I based real-time quantitative PCR.
(7)Assessment of Occludin,Claudin-1,ZO-1 protein localization and expression
Cells were treated with PAF or ITF for different time,the localization and expression of occluding,Claudin-1,ZO-1 protein were detected by immunofluorescence and Western blot analysis.SYBR Green I based real-time quantitative PCR was used to measure the expression of mRNA.
4.Statistics
Data was expressed as Mean±SD.SPSS version 13.0 was used to perform statistical analysis.Results were analysed with one-way ANOVA,using LSD test for inter-group comparison,p＜0.05 was regarded as significant difference.
Results
1.Establishing intestinal epithelial barrier model using Caco-2 cell
Cultured Caco-2 cell confluencd as monolayer with time passed.When visualized with an inverted microscope,like polygon、irregular polygon or cobble cells presented, there were tight junctions amomg cells.When cultured about 21days,TEER reached 300-400Ω·cm~2 and there was little flux of lucifer yellow,transmission electron microscopy also found cells differentiated better,had well-arranged villi and polarity alined as monolayer,which was the marker of intestinal epithelial barrier model in vitro.
2.PAF not affects the Cell proliferation and apoptosis
There was no effect on the Caco-2's vigor and proliferation by PAF even very high dose(P＞0.05)and no positive results of apoptosis.
3.PAF increases intestinal epithelial paracellular permeability and abolished by ITF
PAF treatment of Caco-2 monolayers produced a concentration and time-dependent drop in TEER and increase of paracellular markers luminal yellow, when PAF100ng/L incubated with Caco-2 monolayers for 24h,significant difference was calculated(P＜0.01).When incubated with ITF(0.3g/L),there was a significant increase of TEER values from 2h and the peak protection effect was at 24h,the variation of LY flux was paralleled to that of TEER.
4.PAF disrupts the interepithelial tight junction
The presence of electron-dense material in the space between cells near the brush border reflects the TJ.In cells without PAF,the TJ displayed an intact structure.When the cells were incubated with PAF 100ng/L for 24 hours,the TJ complex appeared reduced and contained less electron-dense material.
5.Alterations in F-actin induced by PAF and ITF
Control cells exhibits an intact actin network—a normal and smooth architecture of the F-actin "ring" at the areas associated with the plasma membrane(i.e.areas of cell-cell contact).PAF reduced the percentage of cells displaying normal actin.Following exposure to PAF,especially100ng/L for 24h,extensive disorganization, kinking,condensation,and beading of the actin ring could be seen.Preincubation with ITF protected the appearance of F-actin under PAF insult.
Measurement of fluorescence intensity was investigated in a large number of cells (10000 cells per sample)by FACS.PAF induced a dose and time-dependent decline in fluorescent phalloidin and there were significant changes at 24h when treated with PAF of 100ng/L,at the same time,when pretreated with ITF and incubated 24h, fluorescence intensity recovered,which indicated ITF had protection effect definitely.
6.Effect on ITF induced by PAF
ITF mRNA decreased begain with 8h and lower at 24h,recovered partly at 48h, but there was no significant difference when given PAF of 50ng/L.Group of PAF 100ng/L compared with control,ITF mRNA decreased at 4h and at a low ebb at 24h, P＜0.05,recovered partly at 48h.High dose PAF(200ng/L)has no significant difference with PAF 100ng/L.
Immunohistochemistry showed that in normal Caco-2 cells,ITF positive stain sited in cytoplasm and some little part in cytomembrane.It decreased after PAF administered and reached the lowest point with PAF 100ng/L(P＜0.01).
7.Tight junction protein occludin,and ZO-1 distribution changed, mRNA and protein expression decreased caused by PAF and the protection of ITF
Indirect immunofluorescent staining showed in the control Caco-2 monolayers, Occludin,Claudin-1 and ZO-1 proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders.PAF caused a progressive disturbance in the continuity of Occludin,Claudin-1 and ZO-1 localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contact,rITF can partly recover the disruption of distribution of these proteins.The three kinds of proteins were almost paralleled to each other in location and changing rule.
Real time qPCR demonstrated that the mRNA expression of Occludin,Claudin-1 and ZO-1 in PAF treatment groups with different concentrations(50,100 and 200 ng/L) especilly100ng/L for 24 hours was significantly different from the corresponding time normal groups(P＜0.05),preincubated with rITF,the expression of mRNA increased,but treatment with rITF the expression of mRNA was not changed.
Using Westem blot analysis found PAF produced a progressive decrease in higher molecular form,a phosphorylated form(80 kDa)of Occludin and 25 kDa of Claudin-1 protein expression(P＜0.01),whereas the low molecular form,a nonphosphorylated form (65 kDa and 20 kDa),was no significant change.ZO-1 protein was 220 kDa,when treatment with PAF of 100ng/L for 24h,there was a significant difference compared with control group(P＜0.01).Like the expression of mRNA,preincubated with rITF, protein expression also increased(P＜0.01).
Conclusion
1.Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro.
2.PAF disrupts the intestinal epithelial barrier directly,the increasing of intestinal epithelial permeability is independent of apoptosis and its target locates at paracellular pathway.
3.ITF can protect intestinal epithelium by recover the normal structure of tight junction,and then decrease the intestinal epithelial permeability.
4.PAF induces the structural alterations and the decrease of protein quantity of the cytoskeleton F-actin,which is one of the mechanisms for PAF increases intestinal epithelial permeability.
5.PAF causes a significant decrease of ITF mRNA and protein expression, inhibiting the self-protection and reparation of the intestinal mucosal barrier.
6.ITF could reverse the hyper-permeability by recover the normal structural of F-actin.It is an important evidence contributing to the function of ITF in epithelial protection.
7.PAF disrupts the intestinal epithelial barrier,the mechanism of which would correlate to the decrease of Occludin,Claudin-1 and ZO-1 action proteins and mRNA expression and abnormal distribution.
8.Pretreatly and therapeutically interventing of ITF may protect intestinal barrier function by recovering Occludin,Claudin-1 and ZO-1 action proteins and mRNA expression and abnormal distribution partly.
肠黏膜屏障是机体最重要的免疫防御屏障,将机体与肠道内的外源性物质隔离开来,避免病原微生物的侵袭和抗原分子的损伤。肠黏膜屏障主要包括以下三个方面:①肠道连续的黏膜上皮构成的机械屏障,②由肠道淋巴样组织细胞网提供的细胞及体液免疫系统,也称免疫屏障,③正常的大量的肠道厌氧菌群,防止致病微生物的过度增生及在肠道黏膜的定植,构成微生物屏障。
肠黏膜的机械屏障由肠表面黏液,微绒毛,肠黏膜上皮细胞及其间的连接以及黏膜的特殊结构组成,是肠黏膜屏障的结构基础。上皮细胞的形态改变、连接缝隙增宽或开放数目增多都会影响上皮细胞屏障功能,使通透性增加。目前研究发现肠黏膜通透性增加参与了多种疾病的发生发展,如:炎症性肠病、败血症、烧伤、终末期肝病、重症胰腺炎等,进一步引起全身炎症反应综合征甚至多器官功能衰竭。近期研究发现肠黏膜屏障的破坏在儿科急慢性疾病的发生发展中亦具有显著的意义:急性期,细菌移位可引起败血症及多器官功能衰竭;而婴儿时期的肠黏膜屏障破坏将会成为随后产生的部分变应性疾病的始发因素,如:湿疹,食物过敏,谷胶病,1型糖尿病,哮喘,炎症性肠病及孤独症等。因此肠黏膜屏障功能及其完整性这一课题在儿科领域也受到越来越多的关注。
肠上皮屏障通透性增加与多种因素有关,不仅包括疾病的起始致病因子如感染、缺血等,也包括机体继发产生的内源性免疫介质。寻找这个炎症连锁反应中的关键因素一直是我们努力的方向。有趣的是,一直在循环系统中倍受关注的血小板活化因子(PAF)同样可以由肠上皮细胞产生,存在于正常的小肠组织中,并调节黏膜的通透性。更为重要的是实验发现,给予小剂量PAF,在不足以引起肠损伤的情况下,即可以增加肠黏膜的通透性,触发细胞因子和转录因子的活化。因此,PAF被认为是肠上皮屏障通透性增加的关键因素,但具体作用机制和方式还不明了。
本实验中我们应用Caco-2细胞株建立体外肠上皮细胞屏障模型,观察PAF对肠上皮细胞屏障通透性的影响,确定其作用位点,并在此基础上进一步探讨PAF对维持肠黏膜机械屏障正常通透性的细胞骨架蛋白F-actin,构成细胞紧密连接的重要蛋白Occludin,Claudin-1,ZO-1,以及在肠道的自我保护和修复中具有重要意义的三叶肽ITF在基因、蛋白、定位等方面的影响,系统探讨PAF对肠黏膜机械屏障的损伤机制。
肠三叶因子(ITF)属三叶肽家族,是近年来被人们注意到的对胃肠道粘膜屏障有重要保护和修复作用的多肽,是一种新型的生长因子类多肽物质。它的生理功能主要体现在两个方面:首先ITF可与粘液糖蛋白相互作用或交联,形成粘弹性的粘液凝胶层,对肠道粘液层起固定和支持作用,防止有害物质对肠粘膜细胞的损伤,从而增强胃肠道粘膜屏障的保护能力;其次ITF具有很强的促进细胞增殖与移行的能力,被认为是粘膜损伤的快速反应肽,在损伤早期即可表达,促进受损区域上皮细胞重建并加快上皮细胞移行速度,因而在肠道的自我保护机制中占据重要地位。大量的动物实验也证明肠三叶因子在维持肠上皮细胞的完整性,恢复肠黏膜的正常通透性方面均起到重要作用。近期的体外实验也发现ITF可以调控紧密连接蛋白Claudin-1和Claudin-2的表达而影响肠上皮的跨上皮阻力。但PAF是否可以影响内源性ITF的表达?外源性给予基因重组ITF是否可以起到保护作用?其机制如何?这些问题的回答也为临床应用ITF治疗胃肠黏膜损伤性疾病提供了可靠的理论依据。
材料和方法
一、细胞培养
Caco-2细胞株,来源于人大肠癌细胞,可分化成有极性的肠上皮细胞。应用含有150 ml/L胎牛血清和青霉素-链霉素双抗液的RPMI 1640高糖培养液,在37℃、50 ml/L CO_2条件下进行培养,每7 d按1:2的比例传代。将细胞接种于transwell、盖玻片、六孔板上或培养瓶中,待细胞生长融合至80%左右,换无血清培养液,按实验分组加药,进行后续实验。每种实验均选取至少三组非同代细胞进行。
二、实验分组
分别设正常对照组、实验组(含模型对照组)、ITF预防组、ITF治疗组。
正常对照组:不加刺激物及干预因素。
实验组:加入PAF,终浓度分别为50ng╱L、100ng/L和200ng╱L,并把终浓度100ng╱L的组作为模型对照组,分别作用2,4,8,12,24,48h。
ITF预防组:先加入ITF0.3g╱L,30min后加入PAF100ng╱L,共培养24h。
ITF治疗组:先加入PAF100ng/L,30min后加入ITF0.3g╱L,共培养24h。
三、实验方法及检测指标
1、体外肠上皮细胞屏障模型的建立
应用Caco-2细胞株在transwell上建立体外肠上皮细胞屏障模型,并应用跨上皮细胞电阻(TEER)和荧光黄透过率两种指标检测肠上皮细胞屏障的形成情况。
2、PAF对细胞增殖和凋亡的影响
(1)MTT比色法检测细胞活力,反映细胞增殖情况。
(2)用Annexin V-EGFP和Hoechst试剂盒,应用流式细胞技术和免疫荧光染色技术,通过细胞膜和细胞核染色联合检测细胞凋亡的发生。
3、PAF对肠上皮细胞屏障通透性的影响和ITF的保护作用
(1)TEER的测定:应用EVOM测定Caco-2单层上皮细胞的TEER。
(2)荧光黄透过量的检测:应用荧光黄作为标记物,检测PAF或ITF对Caco-2细胞细胞旁转运的影响。用荧光分光光度计测定荧光黄吸光度(激发波长427nm,发射波长536 nm),根据标准曲线计算荧光黄浓度。
4、PAF和ITF对肠上皮细胞间紧密连接的影响
透射电镜下观察加入PAF或/和ITF不同时间后,Caco-2细胞间紧密连接形态学的改变。
5、PAF和ITF对肠上皮细胞骨架蛋白F-actin的影响
应用罗丹明-鬼笔环肽(TRITC-phalloidin)特异性荧光探针直接标记F-actin,流式细胞技术测定F-actin的含量;直接免疫荧光技术观察F-actin的定位和重排情况。
6、PAF对内源性ITF表达的影响
给予不同浓度PAF作用不同时间后,应用实时定量PCR技术和免疫组化技术检测PAF对ITFmRNA和蛋白定位的影响。
7、PAF和ITF对肠上皮细胞紧密连接蛋白Occludin,Claudin-1,ZO-1表达和定位的影响
应用免疫荧光、蛋白印迹杂交(Western blot)和实时定量PCR技术检测肠上皮细胞紧密连接蛋白Occludin,Claudin-1,ZO-1定位和蛋白、基因表达的变化情况。
四、统计学分析
所有实验均重复3次以上,结果用Mean±SD表示,统计由SPSS13.0软件完成。采用One-Way ANOVA法比较总体和组间差异,P＜0.05认为有显著差异。
结果
1、利用Caco-2细胞建立肠上皮细胞屏障
Caco-2细胞随着培养时间的延长,逐渐融合成片,成单层生长,光镜下呈多角形、不规则多边形或鹅卵石样,相邻细胞连接紧密,可见致密的反光带;TEER值逐渐升高,至21天左右,细胞形成紧密单层,TEER值达到300～400Ω/cm~2,对荧光黄透过量很小。电镜下发现细胞呈高分化,细胞间形成完整的紧密连接,绒毛整齐,极性形成,成单层排列。体外肠上皮细胞屏障建立。
2、PAF对细胞增殖和凋亡的影响
MTT染色发现,即使大剂量的PAF也未影响到细胞的增殖和细胞活力,各加药浓度与对照组相比无显著性差异(P＞0.05)。
应用Annexin V和Hoechst染色试剂盒,应用荧光显微镜和流式细胞仪检测,均未发现给予PAF后出现明显的细胞凋亡增加(P＞0.05)。
3、PAF对肠上皮细胞屏障通透性的影响和ITF的保护作用
与对照组相比,50ng╱LPAF即可引起TEER的下降和荧光黄透过量的增加,以PAF100ng/L组作用最明显(P＜0.01);在作用时间上,PAF100ng/L作用4h后,TEER值开始下降(P＜0.01),荧光黄透过量开始升高(P＜0.01),24h这种作用达到最强(P＜0.01)。预防和治疗性给予ITF后,与模型对照组相比,TEER有所恢复,荧光黄透过量减少,预防组作用更明显(P＜0.05)。
4、PAF和ITF对肠上皮细胞间紧密连接的影响
正常Caco-2细胞,紧密连接结构完整,呈致密的带状结构。加入PAF100ng/L作用24h后,电镜下可见紧密连接结构破坏、断裂,细胞间隙增宽。加入ITF0.3 g/L共同作用24h,紧密连接及细胞极性均部分恢复。
5、PAF和ITF对肠上皮细胞骨架蛋白F-actin的影响
在正常Caco-2细胞,F-actin主要环绕于细胞周边,细胞中央偶尔可见不规则纤维丝。细胞间F-actin排列紧密圆滑,轮廓完整连续,无明显间隙,细胞界限清晰。
PAF作用8-12h后,就可见细胞骨架F-actin出现重排,外周致密带边缘变的毛糙不规整,细胞中央有弥散actin及少量应力纤维形成。作用24h,出现明显横跨细胞的应力纤维结构,48h后周边肌动蛋白丝带变细,断断续续,胞浆内仍可见少量应力纤维。给予不同浓度的PAF发现,以PAF100ng╱L破坏最为明显。
当预防或治疗性给予ITF并共同培养24h后,正常F-actin染色的细胞比例增加,周边肌动蛋白丝带逐渐清晰,胞浆内应力纤维减少,但外周致密带边缘仍毛糙不规整,环点状断裂未完全修复。比较而言,预防性给药恢复细胞骨架正常结构的能力更强。
流式细胞仪检测发现:PAF作用8h后,F-actin含量开始减少,峰左移;24h后明显下降,峰左移明显,48h与24h相比无明显差异。PAF100ng╱L组下降最为明显,与对照组相比差异显著(P＜0.01)。给予ITF作用24h,F-actin的含量有所增加,与模型组相比差异显著(P＜0.01),但仍低于对照组;峰右移,但未移回原位。
6、PAF对内源性ITF表达的影响
给予50ng╱L PAF 8h后,ITFmRNA表达开始下降,24h降到最低,48h有所恢复,但与对照组比较没有统计学差异。PAF100ng╱L组作用4h ITFmRNA即开始下降,24h降到低谷(P＜0.05),48h有所恢复。进一步加大PAF浓度(200ng╱L),与100ng/L组无明显差异。
免疫组化显示在正常Caco-2细胞中,ITF阳性表达呈棕黄色颗粒,位于胞浆内,胞膜也有少量表达。给予不同浓度PAF(50,100,200ng╱L)作用24h,ITF表达减少,染色变淡,以100ng/L组改变最明显。
7、PAF和ITF对肠上皮细胞紧密连接蛋白Occludin,Claudin-1,ZO-1表达和定位的影响
(1)紧密连接蛋白Occludin,Claudin-1,ZO-1在肠上皮细胞的定位改变
正常Caco-2细胞Occludin,Claudin-1,ZO-1主要分布在细胞内近胞膜处,细胞间连接紧密。PAF50ng╱L作用24h后,Occludin边缘粗糙呈锯齿状,荧光信号减弱,向膜下转移。随PAF浓度加大(100ng╱L)改变更加明显,细胞间出现间隙,Occludin环断裂,崩解,胞浆内见阳性染色。但PAF浓度继续加大(200ng/L),改变并未继续加重。Claudin-1,ZO-1表达位置和变化规律与Occludin基本一致。预防和治疗性给予ITF后,可明显减轻Occludin,Claudin-1,ZO-1的破坏,边缘变得连续,但没有完全恢复。ITF预防组效果更明显。
(2)Occludin,Claudin-1,ZO-1 mRNA表达的改变
利用标准曲线对样品中的Occludin,Claudin-1,ZO-1基因和GAPDH基因进行定量,发现不同浓度PAF组与正常对照组比较Occludin,Claudin-1,ZO-1 mRNA的水平均有降低,以PAF 100ng/L组改变最明显(P＜0.01),三者变化趋势一致;基因水平从4h开始降低,到24h降到低谷,48h有所恢复。以PAF 100ng/L组作为模型组,预防或治疗性给予ITF0.3g╱L作用24h,预防和对照组Occludin,Claudin-1,ZO-1基因表达均较模型组增强,但未恢复到对照组水平,预防组作用更明显(P＜0.05),但两组之间无统计学差异。
(3)Occludin,Claudin-1,ZO-1蛋白表达的改变
Occludin和Claudin-1发挥功能需要丝氨酸和苏氨酸残基磷酸化,磷酸化会使分子量上调。
应用Western blot法对Occludin进行蛋白杂交,发现两条蛋白条带,分别在65kDa和80kDa,灰度分析发现各组标本65kDa的蛋白含量无统计学差异,但是对于80kDa的蛋白含量,PAF组较对照组明显下降并呈剂量依赖性(P＜0.01);
对Claudin-1进行蛋白杂交,发现25kDa和20kDa两条蛋白条带,20kDa的蛋白含量无统计学差异(P＞0.05),但是25kDa的蛋白含量,PAF组较对照组明显下降并呈剂量依赖性(P＜0.01);
对ZO-1进行蛋白杂交,发现在220kDa处出现特异性蛋白条带,其灰度随PAF给药浓度的增加而降低,以PAF 100ng╱L作用24h降到最低,与对照组相比P＜0.01。
预防或治疗性给予ITF后,三者的蛋白表达较模型组增强,但未恢复到对照组水平,与mRNA表达的变化趋势具有一致性。
结论
1、培养2-3w的Caco-2细胞可作为体外研究肠上皮细胞屏障的工具。
2、PAF可直接破坏肠上皮细胞屏障,其增加肠上皮细胞通透性的作用是凋亡非依赖性的,其作用位点在旁细胞途径。
3、ITF可以通过恢复紧密连接的结构降低肠通透性,从而发挥其保护作用。
4、PAF引起细胞骨架F-actin的重排和再分布以及蛋白含量的减少,是PAF引起肠上皮细胞屏障通透性增加的机制之一。
5、PAF使内源性ITF基因和蛋白表达减少,抑制了肠道的自我保护和修复能力。
6、外源性重组ITF部分逆转细胞骨架F-actin的重排和再分布,可以作为一个肠道保护因素。
7、PAF可以引起紧密连接蛋白Occludin,Claudin-1和ZO-1的基因和蛋白水平的改变以及异常分布,具有剂量和时间依赖性,且三者的改变具有协同性,这可能是PAF引起肠上皮细胞屏障破坏的内在机制。
8、ITF可以部分恢复紧密连接蛋白Occludin,Claudin-1和ZO-1的基因和蛋白水平改变以及异常分布,恢复紧密连接的正常结构,可能是其保护肠黏膜屏障的一个重要机制。
Objective
In addition to being the organ responsible for digestion and absorption of nutrients, the intestine serves a barrier function that is a critical component of the innate immune system.Only a single layer of epithelial cells separates the luminal contents from effector immune cells in the lamina propria and the internal milieu of the body. Complete intestinal barrier is comprised of 1.The mechanical barrier is comprised of continual epithelial of gut mucosal;2.Cellular and humoral immunity provided by lymphoid tissue of intestinal tract;3.Normal and massive commensal intestinal bacterial clusters,preventing the over proliferating and permanent planting in gut mucosal of pathogenic microorganism.
The mechanical barrier is comprised of mucus,microvilli,epithelial cells and junctions among them and other particular constructions,which is the structural foundation of intestinal mucosal barrier.Not only the morphous change of epithelial cells but also the wideness of junction gap and the increasing of opened cell-cell junctions could affect the function of intestinal epithelial barrier and increase the permeability.The disruption of barrier integrity has been implicated in the pathogenesis of a wide variety of gastrointestinal and systemic disorders such as inflammatory bowel disease,liver failure,acute severe pancreases and multiple organ system failure.In addition to these,many acute and chronic gastrointestinal and systemic disorders in paediatrics such as sepsis,eczema,food allergies,celiac enteropathy,type 1 diabetes, asthma,and so on were also implicated in,Hense studying the mechanisms of high permeability and to explore the effectual treatment become the goal of this experiment.
The increase of intestinal epithelial barrier permeability implicated in several factors,including initiatal causative agent as infection or ischemia and secondum inflammatory mediator.In recent years,the roles of platelet activating factor(PAF)to impairment of gastrointestinal mucosa has been paid increasing attention.PAF can be produced by intestinal epithelial cells and constitutively present in normal small intestinal tissue regulating the mucosal permeability.A large body of evidence suggests that PAF is involved in the pathogenesis of intestinal injury in various diseases.Moreover,PAF at doses insufficient to cause bowel necrosis triggers the expression of cytokines,and activates transcription factors in the intestine.So PAF is regarded as central amplify mediator and key factor of increasing of intestinal epithelial barrier permeability.
The mechanisms of PAF-related epithelial breakdown are unclear,but regulation of paracellular pathways,especially via rearranging cytoskeleton F-actin and changing interepithelial tight junction(TJ)proteins such as Occludin,Claudin-1,ZO-1 is likelyto play a significant role.Therefore,we detect the effect of PAF on intestinal epithelial F-actin and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells to study the mechanisms involved in disruption of intestinal epithelium mechanical barrier induced by PAF.
Intestinal trefoil factor(ITF)is a member of trefoil peptide family,which is important in maintenance and reparation of the intestinal mucosal barrier.It can not only stimulate cell migrating and proliferating,promote epithelial cell reparation,but also interact with the mucus,stabilize the mucus gel by perhaps interacting with intestinal mucin and increasing the viscosity.So it is important in the self-protection mechanism of intestine.Many researches in vivo also demonstrated ITF was important in the maintenance of intestinal epithelium barrier integrity and in the rescovery of mucosal normal permeability.Recent in vitro experiment find ITF can regulate the expression of TJ proteins of Claudin-1 and Claudin-2,then to affect TEER.But whether PAF can affect the expression of ITF? Administer of rITF can protect the intestinal mucosal barrier? What is the mechanisms? To answer these questions and to provide theory base for clinical application of rITF were the basic consideration of this study.
Materials and methods
1.Cell cultures
Caco-2 cells were grown in a culture medium composed of RPMI 1640 with 4.5 mg/ml glucose,50 U/ml penicillin,50 U/ml streptomycin,and 15%FBS in an atmosphere of 5%CO_2 and 90%relative humidity maintained at 37℃.Parental cells were split at a ratio of 1:3 on reaching confluency,and set up on Transwell filters,6 well plates or T-50 flasks for propagation and experiments.
2.PAF and ITF treatment
Cell monolayers were exposed in RPMI 1640 without FBS for 24hs then PAF of different concentrations(0,50,100,200ng/L)was administered,incubating for different time(0,2,4,8,12,24,48h);ITF(0.3g/L)was administered 30mins before or after PAF(100ng/L)was given and incubated for 24hs in 6 well plates,T-50 flasks or transwell Cell Culture Inserts to undertake different experiments.
3.Experimental methods and analysis index
(1)Establishing intestinal epithelial barrier model in vitro
Caco-2 cells were plated on Transwell filters and monitored regularly by visualization with an inverted microscope and by TEER and unidirectional flux of lucifer yellow measurements to ensure the form of intestinal epithelial barrier.
(2)Cell proliferation and apoptosis
MTT was used to detect cell vigor;Annexin V-EGFP and Hoechst were used to detect cell apoptosis through immunofluorescence and FCM.
(3)Determination of epithelial monolayer resistance and paracellular permeability
The transepithelial electrical resistance(TEER)of the filter-grown Caco-2 intestinal monolayers was measured by using an epithelial voltohm-meter(EVOM); the unidirectional flux of paracellular marker lucifer yellow was measured to determination the Caco-2 monolayer paracellular permeability using fluorescence spectrophotometer.
(4)Transmission electron microscopy
Cells were treated with PAF or ITF and ultrathin sections were made and stained with saturated uranyl acetate and Reynold's lead citrate.Ultrastructure of junctional complexes was observated by transmission electron microscopy.
(5)F-actin rearrangment and assays
When treated by PAF and ITF with different dose and different time,TRITC-phalloidin staining was performed.Direct immunofluorescent staining observe cytoskeleton F-actin rearrangement,Flow cytometry was used to assays F-actin contents.
(6)Assessment of ITF protein localization and expression
Cells were treated with PAF and the localization of ITF protein were detected by immunohistochemistry and ITFmRNA expression was measured useing SYBR Green I based real-time quantitative PCR.
(7)Assessment of Occludin,Claudin-1,ZO-1 protein localization and expression
Cells were treated with PAF or ITF for different time,the localization and expression of occluding,Claudin-1,ZO-1 protein were detected by immunofluorescence and Western blot analysis.SYBR Green I based real-time quantitative PCR was used to measure the expression of mRNA.
4.Statistics
Data was expressed as Mean±SD.SPSS version 13.0 was used to perform statistical analysis.Results were analysed with one-way ANOVA,using LSD test for inter-group comparison,p＜0.05 was regarded as significant difference.
Results
1.Establishing intestinal epithelial barrier model using Caco-2 cell
Cultured Caco-2 cell confluencd as monolayer with time passed.When visualized with an inverted microscope,like polygon、irregular polygon or cobble cells presented, there were tight junctions amomg cells.When cultured about 21days,TEER reached 300-400Ω·cm~2 and there was little flux of lucifer yellow,transmission electron microscopy also found cells differentiated better,had well-arranged villi and polarity alined as monolayer,which was the marker of intestinal epithelial barrier model in vitro.
2.PAF not affects the Cell proliferation and apoptosis
There was no effect on the Caco-2's vigor and proliferation by PAF even very high dose(P＞0.05)and no positive results of apoptosis.
3.PAF increases intestinal epithelial paracellular permeability and abolished by ITF
PAF treatment of Caco-2 monolayers produced a concentration and time-dependent drop in TEER and increase of paracellular markers luminal yellow, when PAF100ng/L incubated with Caco-2 monolayers for 24h,significant difference was calculated(P＜0.01).When incubated with ITF(0.3g/L),there was a significant increase of TEER values from 2h and the peak protection effect was at 24h,the variation of LY flux was paralleled to that of TEER.
4.PAF disrupts the interepithelial tight junction
The presence of electron-dense material in the space between cells near the brush border reflects the TJ.In cells without PAF,the TJ displayed an intact structure.When the cells were incubated with PAF 100ng/L for 24 hours,the TJ complex appeared reduced and contained less electron-dense material.
5.Alterations in F-actin induced by PAF and ITF
Control cells exhibits an intact actin network—a normal and smooth architecture of the F-actin "ring" at the areas associated with the plasma membrane(i.e.areas of cell-cell contact).PAF reduced the percentage of cells displaying normal actin.Following exposure to PAF,especially100ng/L for 24h,extensive disorganization, kinking,condensation,and beading of the actin ring could be seen.Preincubation with ITF protected the appearance of F-actin under PAF insult.
Measurement of fluorescence intensity was investigated in a large number of cells (10000 cells per sample)by FACS.PAF induced a dose and time-dependent decline in fluorescent phalloidin and there were significant changes at 24h when treated with PAF of 100ng/L,at the same time,when pretreated with ITF and incubated 24h, fluorescence intensity recovered,which indicated ITF had protection effect definitely.
6.Effect on ITF induced by PAF
ITF mRNA decreased begain with 8h and lower at 24h,recovered partly at 48h, but there was no significant difference when given PAF of 50ng/L.Group of PAF 100ng/L compared with control,ITF mRNA decreased at 4h and at a low ebb at 24h, P＜0.05,recovered partly at 48h.High dose PAF(200ng/L)has no significant difference with PAF 100ng/L.
Immunohistochemistry showed that in normal Caco-2 cells,ITF positive stain sited in cytoplasm and some little part in cytomembrane.It decreased after PAF administered and reached the lowest point with PAF 100ng/L(P＜0.01).
7.Tight junction protein occludin,and ZO-1 distribution changed, mRNA and protein expression decreased caused by PAF and the protection of ITF
Indirect immunofluorescent staining showed in the control Caco-2 monolayers, Occludin,Claudin-1 and ZO-1 proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders.PAF caused a progressive disturbance in the continuity of Occludin,Claudin-1 and ZO-1 localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contact,rITF can partly recover the disruption of distribution of these proteins.The three kinds of proteins were almost paralleled to each other in location and changing rule.
Real time qPCR demonstrated that the mRNA expression of Occludin,Claudin-1 and ZO-1 in PAF treatment groups with different concentrations(50,100 and 200 ng/L) especilly100ng/L for 24 hours was significantly different from the corresponding time normal groups(P＜0.05),preincubated with rITF,the expression of mRNA increased,but treatment with rITF the expression of mRNA was not changed.
Using Westem blot analysis found PAF produced a progressive decrease in higher molecular form,a phosphorylated form(80 kDa)of Occludin and 25 kDa of Claudin-1 protein expression(P＜0.01),whereas the low molecular form,a nonphosphorylated form (65 kDa and 20 kDa),was no significant change.ZO-1 protein was 220 kDa,when treatment with PAF of 100ng/L for 24h,there was a significant difference compared with control group(P＜0.01).Like the expression of mRNA,preincubated with rITF, protein expression also increased(P＜0.01).
Conclusion
1.Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro.
2.PAF disrupts the intestinal epithelial barrier directly,the increasing of intestinal epithelial permeability is independent of apoptosis and its target locates at paracellular pathway.
3.ITF can protect intestinal epithelium by recover the normal structure of tight junction,and then decrease the intestinal epithelial permeability.
4.PAF induces the structural alterations and the decrease of protein quantity of the cytoskeleton F-actin,which is one of the mechanisms for PAF increases intestinal epithelial permeability.
5.PAF causes a significant decrease of ITF mRNA and protein expression, inhibiting the self-protection and reparation of the intestinal mucosal barrier.
6.ITF could reverse the hyper-permeability by recover the normal structural of F-actin.It is an important evidence contributing to the function of ITF in epithelial protection.
7.PAF disrupts the intestinal epithelial barrier,the mechanism of which would correlate to the decrease of Occludin,Claudin-1 and ZO-1 action proteins and mRNA expression and abnormal distribution.
8.Pretreatly and therapeutically interventing of ITF may protect intestinal barrier function by recovering Occludin,Claudin-1 and ZO-1 action proteins and mRNA expression and abnormal distribution partly.
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