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
결양/재복양여지다당격활장상피세포핵전록인자-κB화저양유도인자-1α신호통로이급대황소대기적간예작용
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
  • 万方数据
    中华危重病急救医学 중화위중병급구의학
    Chinese Critical Care Medicine
    2014年 6期 409-414 ,共6页

    祁蕾%苑博%傅强

    기뢰%원박%부강

    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 脂多糖%低氧誘導因子-1α%腸上皮細胞%環氧化酶-2%覈轉錄因子-κB%大黃素
    지다당%저양유도인자-1α%장상피세포%배양화매-2%핵전록인자-κB%대황소
    Lipopolysaccharide%Hypoxia-inducible factor-1 α%Intestinal epithelial cell%Cyclooxygenase-2%Nuclear factor-κB%Emodin
    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、COX-2、HIF-1α并无降解,于R2～3 h达峰值,分别为3.302±0.061、2.315±0.055、2.017±0.043、2.413±0.098(F值分别为4.614、1.652、5.970、2.076,P值分别为0.001、0.067、0.000、0.037).大黄素共同干预组:大黄素可以抑制NF-κB和HIF-1α通路蛋白表达,存在量效关系(P＜0.05或P＜0.01).80 μmol/L大黄素可使pIκB-α、pNF-κBp65、COX-2、HIF-1α蛋白表达量降至最低,分别为2.599±0.130、1.772±0.089、2.590±0.129、2.518±0.125;大黄素后处理细胞则无此效果.②经过H/R+LPS处理的细胞内发生空泡样变、形态改变、细胞融合,相比H组生长速度缓慢.③MTT法检测结果显示,20～ 80 μmol/L大黄素对细胞增殖无明显影响,说明大黄素在此浓度范围不仅产生了生物学效应,且对细胞无药物毒性.结论 缺氧或炎症均可激活HIF-1α的缺氧通路和NF-κB的炎症通路,在H/R过程中,这两条通路蛋白随着再复氧时间延长表达降低,但在H/R+LPS过程中蛋白仍相对高表达,缺血/再灌注损伤可能与内毒素共同作用破坏肠上皮细胞,导致肠源性脓毒症的发生.在炎症早期阶段而非晚期阶段,用大黄素可以阻断NF-κB/HIF-1 α-COX-2信号通路,从而发挥抗炎作用.
    목적 이결양/재복양(H/R)화지다당(LPS)자격인결장상피세포주(FHC)모의체내장상피세포조수결혈、결혈/재관주화염증타격적병리과정,탐토대황소간예적가능작용파점.방법 상양조:재37 ℃하용95％공기화5％CO2배양.결양(H)조:우37℃하용1％O2、5％CO2화94％N2적혼합염양기체사세포결양1、2、3、4h.H+LPS조:재H조기출상급여LPS 1 mg/L자격.H/R조:우결양3h후분별복양1、2、3、4h.H/R+LPS조:재H/R기출상급여LPS 1 mg/L자격.대황소간예조:재H3 h/R2 h+LPS기출상급여20、40、60、80 μmol/L대황소진행간예.용단백질면역인적시험(Western Blot)검측린산화핵전록인자-κB(NF-κB)억제단백-α(pIκB-α)、린산화NF-κBp65(pNF-κBp65)、배양화매-2(COX-2)、저양유도인자-1α(HIF-1α)단백표체.상차현미경하관찰각조장상피세포적형태학개변;용사갑기우담서염(MTT)법검측대황소대장상피세포증식정황적영향.결과 ①H조:pIκB-α、pNF-κBp65、COX-2표체량균우H1 h체봉치,분별위0.350±0.018、1.083±0.054、0.903±0.045,연후체감(F치분별위3.011、7.247、5.754,P치분별위0.013、0.000、0.005);HIF-1α재H3 h표체최고(1.511±0.076),단각시간점간비교무차이(F=1.881,P=0.062).H+LPS조:pIκB-α、pNF-κBp65、COX-2、HIF-1α표체량균수결양시간연장이증가,H3 h체봉치,분별위0.504±0.025、1.255±0.063、0.812±0.041、1.209±0.075(F치분별위2.683、8.774、9.765、2.432,P치분별위0.011、0.000、0.000、0.026).H/R조:수착재복양시간연장,pIκB-α、pNF-κBp65、COX-2표체체감,우H3 h/R4 h강지최저,분별위0.712±0.034、1.202±0.048、0.691±0.042(F치분별위1.923、6.765、2.719,P치분별위0.063、0.000、0.016);여H조상비,H/R조HIF-1α재재복양과정중표체명현감소,단각시간점간무차이(F=1.280,P=0.081).H/R+LPS조:수착재복양시간연장,pIκB-α、pNF-κBp65、COX-2、HIF-1α병무강해,우R2～3 h체봉치,분별위3.302±0.061、2.315±0.055、2.017±0.043、2.413±0.098(F치분별위4.614、1.652、5.970、2.076,P치분별위0.001、0.067、0.000、0.037).대황소공동간예조:대황소가이억제NF-κB화HIF-1α통로단백표체,존재량효관계(P＜0.05혹P＜0.01).80 μmol/L대황소가사pIκB-α、pNF-κBp65、COX-2、HIF-1α단백표체량강지최저,분별위2.599±0.130、1.772±0.089、2.590±0.129、2.518±0.125;대황소후처리세포칙무차효과.②경과H/R+LPS처리적세포내발생공포양변、형태개변、세포융합,상비H조생장속도완만.③MTT법검측결과현시,20～ 80 μmol/L대황소대세포증식무명현영향,설명대황소재차농도범위불부산생료생물학효응,차대세포무약물독성.결론 결양혹염증균가격활HIF-1α적결양통로화NF-κB적염증통로,재H/R과정중,저량조통로단백수착재복양시간연장표체강저,단재H/R+LPS과정중단백잉상대고표체,결혈/재관주손상가능여내독소공동작용파배장상피세포,도치장원성농독증적발생.재염증조기계단이비만기계단,용대황소가이조단NF-κB/HIF-1 α-COX-2신호통로,종이발휘항염작용.
    Objective 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=1.881,P=0.062).H + LPS group:the expressions of pIκB-α,pNF-κBp65,COX-2,HIF-1α were increased with elongation of duration of hypoxia,and a maximal induction was observed at H3 h (0.504 ± 0.025,1.255 ± 0.063,0.812 ± 0.041,1.209 ± 0.075,F value was 2.683,8.774,9.765,2.432,and P value was 0.011,0.000,0.000,0.026,respectively).H/R group:with the prolonged duration of reoxygenation,the expressions of NF-κB signaling pathway proteins (pIκB-α,pNF-κBp65,COX-2) were decreased and dropped to nadir at H3 h/R4 h (0.712 ± 0.034,1.202 ± 0.048,0.691 ± 0.042,F value was 1.923,6.765,2.719,and P value was 0.063,0.000,0.016,respectively).Compared with H group,HIF-1α was decreased with a prolonged duration of reoxygenation in H/R group,but there was no significant difference in value among different time points (F=1.280,P=0.081).H/R + LPS group:pIκB-o,pNF-κBp65,COX-2,HIF-1α showed no sign of degradation with the prolonged duration of reoxygenation,and their expression increased to maximum analogously at R2-3 h (3.302 ± 0.061,2.315 ± 0.055,2.017 ± 0.043,2.413 ± 0.098,Fvalue was 4.614,1.652,5.970,2.076,and Pvalue was 0.001,0.067,0.000,0.037,respectively).Emodin group:emodin when co-treated with H/R + LPS inhibited the expression of HIF-1o and NF-κB pathways with a dose-effect relationship (P＜0.05 or P＜0.01).Emodin at the dose of 80 μmol/L showed most marked inhibition (2.599 ± 0.130,1.772 ± 0.089,2.590 ± 0.129,2.518 ± 0.125).However,after treatment of emodin did not show such effect.② After treatment with H/R + LPS,there were morphological changes in cells:vacuoles,deformation and fusion.The speed of cell growth became much slower compared with H group.③ Emodin (20-80 μmol/L) had no significant effect on cell proliferation.Although emodin produced biological effect in this concentration range,it had no cellular toxicity.Conclusions Both hypoxia and inflammation can activate the hypoxia pathway of HIF-1α and the pro-inflammatory pathway of NF-κB,but different stimuli cause varying degrees of activation in these two pathways.In H/R group,both pathways were weakened during reoxygenation.However,in H/R + LPS group,the proteins remained to show a relatively high expression during the process of reoxygenation.This may be related to the pathophysiological mechanism of intestinal ischemia/reperfusion injury:hypoxia/reperfusion injury and LPS act together to destroy the intestinal epithelial cells and induce gut-derived sepsis.Emodin may inhibit inflammation by blocking HIF-1α/NF-κB-COX-2 signaling pathways.
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