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
10.3760/cma.j.issn.2095-4352.2014.06.009
- VernacularTitle: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
- Author:
Lei QI
;
Bo YUAN
;
Qiang FU
- Publication Type:Journal Article
- Keywords:
Lipopolysaccharide;
Hypoxia-inducible factor-1 α;
Intestinal epithelial cell;
Cyclooxygenase-2;
Nuclear factor-κB;
Emodin
- From:
Chinese Critical Care Medicine
2014;26(6):409-414
- CountryChina
- Language:Chinese
-
Abstract:
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.
