沉默信息调节因子1对缺氧条件下肠上皮屏障功能的保护作用及机制

Role of SIRT1 in the protection of intestinal epithelial barrier under hypoxia and its mechanism

目的观察沉默信息调节因子1（SIRT1）对肠缺氧上皮Caco-2细胞屏障功能的影响并探讨其分子机制。方法对Caco-2细胞分别进行1％O2浓度缺氧6h（缺氧组）和常规处理（常规组）及加浓度为40μmol／L Resveratrol（SIRT1激动剂）预处理6h后再缺氧6h（观察组），分别检测3组肠上皮细胞跨上皮电阻（TER），PCR及Western blot分别检测3组SIRT1及紧密连接蛋白ZO—1、Occludin和Claudin-1的mRNA与蛋白表达。结果缺氧组SIRT1mRNA及其蛋白的相对表达量均明显低于常规组（分别为0．40±0．02比0．70＋0．07，P=0．001；0．37±0．03比0．76±0．03，P=0．001），也明显低于观察组（0．50±0．02，P=0．026；0．54±0．02，P=0．011）。缺氧组3种紧密连接蛋白mRNA表达均低于常规组（P〈0．05），也低于观察组（P〈0．05），观察组的ZO-1及Claudin-1表达与常规组比较，差异无统计学意义（P〉0．05）。3种紧密连接蛋白的表达水平常规组最高，观察组次之，缺氧组最低，两两比较，差异均具有统计学意义（均P〈0．05）。常规组、缺氧组和观察组的TER分别为（142±7）Ohm／cm^2、（94±3）Ohm／cm^2和（119±7）Ohm／cm^2；两两比较，差异均具有统计学意义（均P〈0．05）。结论缺氧条件下SIRT1水平降低，而SIRT1的激活可通过增加肠道紧密连接蛋白ZO-1、Occludin和Claudin-1mRNA与蛋白的表达，从而减轻缺氧对肠上皮屏障功能的损伤。

Objective To observe the effect of SIRT1 on intestinal barrier function of epithelial Caco-2 cells under hypoxia and investigate its mechanism. Methods Caco-2 cells were randomly divided into three groups： normoxia group （Nx）, hypoxia group （Hx, 1%O2 for 6 h） and hypoxia plus 40 μmol/L Resveratrol （ agonist of SIRT1 ） group （Hx＋Res）. Transepithelial electrical resistance （TER） was determined, mRNA and protein expressions of SIRT1 and tight junctions（ZO-1, Oecludin, Claudin- 1 ） were examined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. Results Both mRNA and protein expressions of SIRT1 were significantly reduced in Hx group as compared with Nx group （0.40±0.02 vs. 0.70±0.07, P=0.001; 0.37±0.03 vs. 0.76±0.03, P=0.001）. The mRNA and protein expressions of SIRT1 were significantly increased in Hx ＋Res group as compared with Hx group（0.50±0.02 vs. 0.40±0.02, P=0.026; 0.54±0.02 vs. 0.37±0.03, P=0.011）. The expression levels of ZO-1, Occludin and Claudin-1 in Hx group were lower than those in Nx group （P〈0.05）, however, pretreatment with Resveratrol could attenuate the decreased expression of above 3 molecules under hypoxia （P〈0.05）. TERs of Nx group, Hx group and Hx＋Res group were （142±7） Ohm/cm2, （94±3） Ohm/cm2 and （119±7） Ohm/cm2 respectively. Compare with the Nx group, the TER of Hx group was significantly decreased （P〈0.05）. TER of Hx＋Res group was significantly increased compare with Hx group, but it was still significantly lower than that in Nx group （P〈0.05）. Conclusions Expression of SIRT1 is significantly reduced under hypoxia. Activation of SIRT1 can maintain the epithelial barrier function through regulating the expression of tight juncions under hypoxia.