缺氧/再复氧与脂多糖激活肠上皮细胞核转录因子-κB和低氧诱导因子-1α信号通路以及大黄素对其的干预作用  

Hypoxia/reoxygenation and lipopolysaccharide induced nuclear factor-κB and hypoxia-inducible factor-1α signaling pathways in intestinal epithelial cell injury and the interventional effect of emodin

作　　者：祁蕾[1] 苑博[1] 傅强[2] 

机构地区：[1]天津医科大学,300070 [2]天津市第四中心医院,300140

出　　处：《中华危重病急救医学》2014年第6期409-414,共6页Chinese Critical Care Medicine

基　　金：国家重点基础研究发展计划（973）项目（2009CB522703）

摘　　要：目的 以缺氧/再复氧（H/R）和脂多糖（LPS）刺激人结肠上皮细胞株（FHC）模拟体内肠上皮细胞遭受缺血、缺血/再灌注和炎症打击的病理过程,探讨大黄素干预的可能作用靶点.方法 常氧组：在37 ℃下用95％空气和5％CO2培养.缺氧（H）组：于37℃下用1％O2、5％CO2和94％N2的混合厌氧气体使细胞缺氧1、2、3、4h.H＋LPS组：在H组基础上给予LPS 1 mg/L刺激.H/R组：于缺氧3h后分别复氧1、2、3、4h.H/R＋LPS组：在H/R基础上给予LPS 1 mg/L刺激.大黄素干预组：在H3 h/R2 h＋LPS基础上给予20、40、60、80 μmol/L大黄素进行干预.用蛋白质免疫印迹试验（Western Blot）检测磷酸化核转录因子-κB（NF-κB）抑制蛋白-α（pIκB-α）、磷酸化NF-κBp65（pNF-κBp65）、环氧化酶-2（COX-2）、低氧诱导因子-1α（HIF-1α）蛋白表达.相差显微镜下观察各组肠上皮细胞的形态学改变;用四甲基偶氮唑盐（MTT）法检测大黄素对肠上皮细胞增殖情况的影响.结果 ①H组：pIκB-α、pNF-κBp65、COX-2表达量均于H1 h达峰值,分别为0.350±0.018、1.083±0.054、0.903±0.045,然后递减（F值分别为3.011、7.247、5.754,P值分别为0.013、0.000、0.005）;HIF-1α在H3 h表达最高（1.511±0.076）,但各时间点间比较无差异（F=1.881,P=0.062）.H＋LPS组：pIκB-α、pNF-κBp65、COX-2、HIF-1α表达量均随缺氧时间延长而增加,H3 h达峰值,分别为0.504±0.025、1.255±0.063、0.812±0.041、1.209±0.075（F值分别为2.683、8.774、9.765、2.432,P值分别为0.011、0.000、0.000、0.026）.H/R组：随着再复氧时间延长,pIκB-α、pNF-κBp65、COX-2表达递减,于H3 h/R4 h降至最低,分别为0.712±0.034、1.202±0.048、0.691±0.042（F值分别为1.923、6.765、2.719,P值分别为0.063、0.000、0.016）;与H组相比,H/R组HIF-1α在再复氧过程中表达明显减少,但各时间点间无差异（F=1.280,P=0.081）.H/R＋LPS组：随着再复氧时间延长,pIκB-α、pNF-κBp65、COXObjective To observe pathological process of intestinal epithelial cells subjected to ischemia,ischemia/reperfusion injury and inflammation simulated hypoxia/reoxygenation （H/R） and lipopolysaccharide （LPS） challenged human fetal normal colonic cell （FHC） line in vivo,and to observe the changes when the assaulted intestinal epithelial cells were treated with emodin,in order to explore the possible intervention targets of emodin.Methods Normoxia group：the FHC cells were cultured in 95％ air and 5％ CO2 at 37 ℃.Hypoxia （H） group：the cells were cultured with a mixed anaerobic gas of 1％ O2,5％ CO2 and 94％ N2 at 37 ℃ for 1,2,3,4 hours.H ＋ LPS group：the cells were cultured in hypoxic condition as H group with simultaneous challenge of LPS （1 mg/L）.H/R group：the cells were cultured in hypoxia for 3 hours followed by reoxygenation for 1,2,3 and 4 hours,respectively.H/R ＋ LPS group：the cells were cultured in H/R as H/R group and LPS （1 mg/L） simultaneously.Emodin intervention group：the cells were cultured in H3 h/R2 h ＋ LPS and emodin （20,40,60,80 μmol/L） simultaneously.The variation trends of phosphorylation nuclear factor-κB profilin-o （pIκB-α）,phosphorylation NF-κBp65 （pNF-κBp65） and their downstream target gene cyclooxygenase-2 （COX-2）,and hypoxia-inducible factor-1α （HIF-1 α） were determined by Western Blot.The morphological changes in intestinal epithelium in different groups were observed using light microscope.The effect of emodin on the proliferation of intestinal epithelial cell was measured by methyl thiazolyl tetrazolium （MTT） assay.Results ① H group：the expressions of pIκB-α,pNF-κBp65 and COX-2 were upregulated,peaking at H1 h （0.350 ± 0.018,1.083 ± 0.054,0.903 ± 0.045）,and then they gradually lowered （F value was 3.011,7.247,5.754,P value was 0.013,0.000,0.005,respectively）.The expression of HIF-1 α peaked at H3 h （1.511 ± 0.076）,but there was no significant difference among different groups （F=1.88

关 键 词：脂多糖 低氧诱导因子-1Α 肠上皮细胞 环氧化酶-2 核转录因子-ΚB 大黄素 

分 类 号：R459.7[医药卫生—急诊医学]