IL-22改善脂多糖诱导的Caco-2单层肠上皮紧密连接损伤

IL-22 ameliorates lipopolysaccharide-induced intestinal epithelial tight junction injury in Caco-2 monolayers

目的紧密连接(TJ)屏障功能障碍在炎症性肠病(IBD)中起重要作用。据报道IL-22对IBD有直接的保护作用。研究IL-22对TJ屏障功能的保护作用。方法选择肠上皮Caco-2单层细胞,分为对照组、脂多糖(LPS)组、IL-22组、LPS+IL-22组。采用LPS(100μg/mL)或IL-22(100 ng/mL)处理肠上皮Caco-2单层细胞0、12、24、48 h。Western blot检测IL-22受体IL-22R1和TJ蛋白(ZO-1和Occludin)表达。检测Caco-2单层细胞膜跨膜电阻(TEER)。分析Caco-2单层细胞被LPS、IL-22处理48 h后的细胞膜TEER、TJ蛋白变化和免疫荧光方法观察TJ蛋白在细胞膜的分布。结果LPS可上调Caco-2细胞中IL-22R1的表达。LPS可显著降低Caco-2单层细胞膜TEER(刺激24 h,下降水平接近50%),差异有显著统计学意义(P<0.001)。IL-22组ZO-1、Occludin表达水平高于LPS组、LPS+IL-22组,差异有显著统计学意义(P<0.01)。LPS+IL-22组ZO-1、Occludin表达水平高于LPS组,差异有显著统计学意义(P<0.01)。免疫荧光法检测可见,对照组和IL-22组ZO-1、Occludin位于细胞膜,呈明亮连续条带;LPS组,位于细胞膜的ZO-1、Occludin明亮带变暗,显示强度和间断度降低;LPS+IL-22组细胞膜可见ZO-1、Occludin明亮带,但明亮带略弱于对照组和IL-22组。结论IL-22在LPS诱导的肠屏障功能紊乱中起保护作用,并为IL-22在IBD中如何促进肠上皮屏障的完整性提供了新的证据。

Objective To study the protective effect of interleukin(IL)-22 on tight junction(TJ)barrier function,based on the fact that TJ barrier dysfunction plays an important role in inflammatory bowel disease(IBD)and IL-22 shows direct protective effect on IBD.Methods Intestinal epithelial Caco-2 monolayer cells were selected and divided into control group,lipopolysaccharide(LPS)group,IL-22 group and LPS+IL-22 group.LPS(100μg/mL)or IL-22(100 ng/mL)was used to treat Caco-2 monolayers for 0,12,24 and 48 hours,respectively.Western blot was used to detect IL-22 receptor IL-22 R1 and TJ protein(ZO-1 and Occludin)expression.The Caco-2 monolayer cell membrane trans-epithellal electric resistance(TEER)was detected.The changes of TEER and TJ proteins in Caco-2 monolayer cell treated with LPS and IL-22 for 48-hour were analyzed,and immunofluorescence was used to observe distribution of TJ protein in cell membrane.Results LPS up-regulated IL-22 R1 expression in Caco-2 cell.LPS significantly reduced TEER of Caco-2 monolayers(stimulated for 24-hour and decreased close to 50%),and the difference was statistically significant(P<0.001).The expression levels of ZO-1 and Occludin in IL-22 group were higher than those in LPS group and LPS+IL-22 group,and the differences were statistically significant(P<0.01).The expression levels of ZO-1 and Occludin in LPS+IL-22 group were higher than those in LPS group,and the differences were statistically significant(P<0.01).The immunofluorescence detection results showed that ZO-1 and Occludin in control group and IL-22 group were located on cell membrane with bright continuous bands.ZO-1 and Occludin bright bands in cell membrane of LPS group was darkened,the intensity and discontinuity were reduced;ZO-1 and Occludin bright bands were observed in LPS+IL-22 group on cell membrane,but the bright bands were slightly darker than that in control group and IL-22 group.Conclusion It is demonstrated that IL-22 plays protective role in disruption of intestinal barrier function induced by LPS,which provides new evidence for IL-22 promoting integrity of intestinal epithelial barrier in IBD.