ITF对PAF引起的肠上皮细胞骨架F-actin破坏的抑制作用

Treatment with ITF attenuates PAF-induced disruption of the F-actin cytoskeleton in an in vitro model of intestinal epithelium

目的:探讨血小板活化因子(PAF)对肠上皮细胞骨架F-actin的影响以及肠三叶因子(ITF)对此影响的抑制作用.方法:体外培养人结肠腺癌细胞株Caco-2,分为4组.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0、50、100和200nmol/L,作用24h;PAF100nmol/L,分别作用0、2、4、8、12、24、48h;ITF预防组:先加入ITF0.3mol/L,30min后加入PAF100nmol/L;ITF治疗组:先加入PAF100nmol/L,30min后加入ITF0.3mol/L,24h后进行实验.应用跨上皮电阻(TEER)反映肠上皮细胞屏障通透性;免疫荧光染色法观察F-actin的定位、重排以及形态学变化;流式细胞术对F-actin蛋白进行定量分析.结果:给予50nmol/LPAF,作用8-12h即可引起TEER的下降,PAF100nmol/L作用24h,TEER降到最低点,与对照组相比,差异显著(232.75/cm2±15.74/cm2vs346.75/cm2±26.69/cm2,P<0.01);预防或治疗性给予ITF,TEER有所恢复,与模型组相比,差异显著(313.75/cm2±18.28/cm2,299/cm2±13.16/cm2vs232.75/cm2±15.74/cm2,均P<0.01).TRITC-phalloidin直接免疫荧光染色显示正常Caco-2细胞,F-actin主要环绕于细胞周边,排列紧密圆滑,无明显间隙,细胞界限清晰.PAF100nmol/L作用24h后,F-actin出现重排,断裂,周边肌动蛋白丝带模糊,部分细胞出现横跨细胞的应力纤维结构.预防或治疗性给予ITF后,具有正常F-actin染色的细胞比例增加,周边肌动蛋白丝带逐渐清晰,胞质内应力纤维减少,但环点状断裂未完全修复.预防组作用更强.以TRITC-phalloidin的相对平均荧光强度表示F-actin的含量,经流式细胞仪检测发现PAF100nmol/L作用24h后,F-actin含量明显减少(218.56±23.18vs425.35±40.31,P<0.01),给予ITF后F-actin的含量有所增加,与模型组相比差异显著(391.76±58.57,360.86±8.68vs218.56±23.18,均P<0.01),但仍低于对照组.结论:PAF可以改变肠上皮细胞骨架F-actin的定位及定量,从而影响肠上皮细胞屏障功能;ITF可以通过抑制F-actin重排,恢复F-actin蛋白定量而稳定细胞骨架.

AIM： To explore whether treatment with plateletactivating factor （PAF） disrupts the intestinal epithelial barrier by altering the F-actin cytoskeleton and, if so, whether treatment with intestinal trefoil factor （ITF） exerts a protective effect against PAF- induced disruption of the F-actin cytoskeleton. METHODS： An in vitro model of intestinal epithelium was established with Caco-2 cells. PAF at different concentrations （0, 50, 100, 200 nmol/L） was incubated with postconfluent monolayers of Caco-2 cells for 24 h. Moreover, 100 nmol/L PAF was administered for different periods （0, 2, 4, 8, 12, 24, 48 h）. ITF （0.3 tool/L） was administered 30 min before or after PAF treatment （100 nmol/L）. Transendothelial electrical resistance （TEER） was measured to evaluate the permeability of intestinal epithelial cell monolayers. Immunofluorescent staining and flow cytometry were used to observe the morphological alterations and conduct protein quantitation of the F-actin cytoskeleton. RESULTS： Compared with the control group, TEER decreased in cells treated with PAF （100 nmol/L） for 24 h （232.75 Ω/cm^2 ± 15.74 Ω/cm^2 vs 346.75 Ω/cm^2±26.69 Ω/cm2, P 〈 0.01）. While compared with the model group, TEER had little enhancement in cells treated with ITF （313.75 Ω/cm^2 ± 18.28Ω/cm^2, 299 Ω/cm^2 ± 13.16 Ω/cm^2 vs 232.75 Ω/cm^2 ± 15.74 Ω/cm2, both P 〈 0.01）. Treatment with PAF induced a significant decline in paracellular permeability. After treatment with PAF （100 nmol/L） for 24 h, extensive disorganization, kinking, condensation, and beading of the F-actin ring could be seen and the mean fluorescent intensity of F-actin had a significant decline （218.56 ± 23.18 vs 425.35 ± 40.31, P 〈 0.01）. Treatment with ITF could reverse the high permeability of intestinal epithelial cell monolayers partly by recovering the normal structure of F-actin and increasing the content of F-actin （391.76 ± 58.57, 360.86 ± 8.68 vs 218.56 ± 23.18, both P 〈 0.01）. CONCLUSION： PAF plays an important role in the regulation of intestinal mucosal permeability and induces the structural alterations of the F-actin cytoskeleton. Treatment with ITF can protect intestinal epithelium by restricting the rearrangement of the F-actin cytoskeleton.