肌球蛋白轻链激酶介导缺氧后肠上皮屏障功能紊乱的研究

The role of myosin light chain kinase in intestinal epithelial barrier dysfunction due to hypoxia

目的了解肌球蛋白轻链激酶（MLCK）在缺氧后肠上皮屏障功能损害中的作用。方法建立单层肠上皮细胞缺氧模型，根据缺氧时间不同将细胞分为正常对照组（缺氧0h），缺氧2、6、8、12、24h组，（5-氯萘-1-磺酰）-1-六氢-1，4-双苯妥英钠（ML-9）处理组（缺氧6h并用终浓度为100μmol／L的MLCK特异性抑制剂ML-9处理）。用电阻仪测定单层肠上皮细胞跨细胞电阻（TER）；免疫荧光法检测单层肠上皮细胞紧密连接蛋白ZO-1的变化；蛋白质印迹法检测肠上皮细胞磷酸化肌球蛋白轻链（p-MLC）及MLCK蛋白表达。结果缺氧6、8、12、24h组单层肠上皮细胞TER值分别为（422±17）、（427±27）、（403±40）、（426±22）Q，均低于正常对照组[（451±27）Ω，P〈0．05]，ML-9处理组为（558±110）Ω，显著高于各单纯缺氧组（P〈0．01）。缺氧6h组单层肠上皮细胞紧密连接蛋白ZO-1发生明显改变，主要表现为不规则，出现明显皱褶、圆滑排列程度减弱，甚至有断裂及锯齿等不连续现象。ML-9组肠上皮细胞紧密连接蛋白ZO-1近似于正常对照组，排列较规则，皱褶及锯齿状改变较单纯缺氧组减少或消失。各单纯缺氧组肠上皮细胞MLCK、p-MLC蛋白表达水平均较正常对照组增加。结论MLCK可介导缺氧引起的肠上皮屏障功能紊乱。

Objective To study the role of myosin light chain kinase （MLCK） in intestinal epithe- lial barrier dysfunction after hypoxia. Methods The Caco-2 monolayers developed with Transwell inserts were exposed to hypoxia for 0 h（ NC group） , 2, 6, 8, 12 and 24 h（ H group） , and 6 h hypoxic specimens were treated with 100 mol/L ML-9 （T group）. The transepithelial electrical resistance （TER） of monolayers was measured with an ohmmeter. The tight junction protein ZO-1 of monolayers was analyzed by immunofluo- rescence assay. The protein expressions of phosphorylated myosin light chain （p-MLC） and MLCK were de- tected by Western blotting. Results The TER of monolayers in H group at 6, 8, 12 and 24 h was 422±17, 427±27, 403±40 and 426±22Ω respectively, which was significantly lower than that of NC group （451±27Ω, P〈0.05）. The TER of monolayers in T group was 558 ± 110Ω, which was significantly high- er than that in H group at each time point （ P 〈0.01 ）. The ZO-1 of monolayers in H group at 6 h was irregular in arrangement, with interruptions and rugae, and sawtooth. These abnormalities were amelioated in T group（ regular in arrangement, with little or without ruga and sawtooth）. The protein expressions of p-MLC and MLCK in H group at each time point were higher than those in NC group. Conclusions Intestinal ep- ithelial barrier dysfunction after hypoxia can be mediated by MLCK.