生理浓度丁酸钠对Caco-2细胞肠屏障作用中JNK通路的效应  

Role of c-Jun N-terminal Kinase in the intestinal barrier of Caco-2 cell under physiological concentrations of sodium butyrate

作　　者：戴丽娜[1] 侯龙龙[1] 黄璜[1] 黄晓忠[1] 朱利斌[1] 林锦[2] 李仲荣[1] 

机构地区：[1]温州医科大学附属第二医院育英儿童医院小儿外科,325027 [2]美国纽约大学西奈山Icahn医学院Kravis儿童医院

出　　处：《中华小儿外科杂志》2014年第11期859-864,共6页Chinese Journal of Pediatric Surgery

基　　金：浙江省自然科学基金（LY12H04005）;浙江省医药卫生支撑学科基金（11-ZC27）

摘　　要：目的 观察生理浓度丁酸钠作用Caco-2细胞肠屏障时JNK细胞信号通路的作用及与AMPK通路的关系.方法 建立Caco-2细胞单层肠屏障模型,分成不加药物的对照组（Con组）、仅添加2 mmol/L丁酸钠的生理浓度丁酸钠组（2BA组）、仅加入JNK特异性抑制剂的20μmol/LSP600125组（SP组）、20 μmol/L SP600125＋生理浓度丁酸钠组（Mix组）.检测各组处理后24、48、72 h跨上皮细胞电阻（TER）、CCK-8法吸光度及72 h菊粉-FITC透过率、流式细胞仪检测细胞凋亡水平、吖啶橙（AO）/碘化丙啶（PI）双染法荧光显微镜检测凋亡率、Western blot法检测JNK、AMPK、ACC等蛋白的表达.结果 2BA组TER在药物作用后24、48、72 h均明显高于Con组,以72 h时最显著,此时2BA组为（509.64±12.62）Ω·cm2,而Con组为（459.30±7.62）Ω·cm2 （P＜0.05）;Mix组TER为（261.01±9.22）Ω·cm2较2BA组（509.64±12.62）Ω·cm2低.Mix组72 h CCK-8吸光度为0.47±0.13、72 h菊粉-FITC为（7.89±0.80）cm/h、流式细胞仪凋亡水平为（61.23±1.54）％及AO/PI双染荧光显微镜凋亡结果为9.7±1.3,均比2BA组的0.98±0.08、（4.05±1.38）cm/h、（15.90±5.95）％、5.5±1.3高（P＜0.05）.2BA组p-AMPK、p-ACC水平较Con组和Mix组上调（P＜0.05）.Mix组p-JNK水平与2BA组相比显著下调（P＜0.01）.结论 生理浓度丁酸钠可增强肠屏障功能,而JNK特异性抑制剂可破坏这一作用,提示JNK在生理浓度丁酸钠增强肠屏障功能起协同作用,这一作用可能与AMPK活化有关.Objective To explore the impact of JNK under concentrations of sodium butyrate on the Caco-2 cell intestinal barrier model and the relationship with AMP-activated protein kinase （AMPK）.Methods A monolayer model of intestinal barrier was established by culturing Caco-2 cell.There were four experimental groups of control （Con）,20 mol/L SP600125 （SP）,physiological concentrations of sodium butyrate （2BA） and 20 μmol/L SP600125 ＋ 2 mmol/L sodium butyrate （mix）.We determined the levels of transepithelial electrical resistance （TER） at 24 h,48h,72 h and the permeability of inulin-FITC,Caco-2 cells vigor by CCK-8 assay,AO/PI fluorescent dye,apoptosis by flow cytometer and the expression of JNK,AMPK and ACC at 72 h.SP600125 was administered 1h before sodium butyrate.Results 2BA group＇s TER was higher than that of Con group （P＜0.05） while that of mix group was lower than 2BA group （P＜0.05）.The absorbance,permeability of inulin-FITC,apoptotic cells of AO/PI fluorescent dye and apoptotic level of mix group were all higher than those of 2BA group （P＜0.05）.In contrast to Con and mix groups,the expressions of p-AMPK and p-ACC of 2BA group were higher （P＜0.05）.p-JNK of mix group was lower than that of 2BA group （P＜0.01）.Conclusions Physiological concentration of sodium butyrate promotes intestinal barrier function while JNK may act synergistically in this process.And it is probably related with the activation of AMPK signal pathway.

关 键 词：小肠结肠炎 坏死性 凋亡 动力传导 细胞 

分 类 号：R725.7[医药卫生—儿科]