Study Of Protective Effect Of Emodin On Damage Of Tight-junction Protein Of Intestinal Epithelial Cells

Objective: To observe the changes in expression and distribution of tight-junction protein and the expression changes of hypoxia-inducible factor(HIF)-1a, Nuclear factor(NF)-kB and their downstream target gene of intestinal epithelial cells in inflammatory injury induced by hypoxia/reoxygenation and LPS, and to explore the influence of emodin. Methods: Human fetal normal colonic cell line(FHC) was treated with HR+LPS in vitro to simulate the injury of ischemia/reperfusion and inflammation in intestinal epithelial cell in vivo. It was divided into four groups: Normoxia group:the cells were and were cultured in 1:1 mixture of Ham F-12 medium and Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum at 37℃ with 95% air and 5%CO2 as control. H/R+LPS group: the cells were treated with a mixed anaerobic gas of 1%O2, 5%CO2 and 94%N2 at 37 for 3℃ h and then reoxygenation for 2h and LPS(1mg·L-1) simultaneously.Emodin intervention group: the cells were treated with HR+LPS and emodin(80μmol·L-1)simultaneously. DMOG treatment group:the cells were treated with 95% air 和 5%CO2 at 37 ℃and dimethyloxaloylglycine(DMOG,10mM) for 5h.The variation trend of HIF-1a,IkB-a,NF-kB,COX-2,ZO-1, occludin and vascular endothelial growth factor(VEGF) was measured by Western blot; The distribution of ZO-1 was observed by indirect immunofluorescent assay. Results:?Compared with normoxia group,the expression of ZO-1 decreased in the other three groups(p<0.05),the expression of ZO-1 increased in the emodin intervention group compared with H/R+LPS group(p<0.05); while the expression of occludin also decreased in the other three groups compared with normoxia group but there were no significant differences(p>0.5);The results of indirect immunofluorescent assay showed that ZO-1 was distributed along the cell membrane continuous smoothly under the normoxic condition,an obvious loss expression of ZO-1 and discontinuously distribution could be seen in the DMOG treatment group.In the H/R+LPS group, expression of ZO-1 was less than it was in the normoxia group and ZO-1 distributed discontinuously and irregularly with rupture and serration appearance. Expression of ZO-1 in the emodin intervention group is increased and the discontinuously and Irregularly distribution was in remission compared with the H/R+LPS group;?Compared with normoxia group, the expression of proteins of HIF-1a/NF-kB-COX-2 pathway increased in the other three groups(p<0.05).The expression of these proteins decreased in the emodin intervention group compared with H/R+LPS group(p<0.05);? The expression of VEGF increased in the other three groups compared with normoxia group,(p<0.05),it decreased in the emodin intervention group compared with H/R +LPS group(p<0.05). Conclusions:?Stimulating with hypoxia/reoxygenation and LPS can lead to a reduced expression of the tight junction protein and irregular and discontinuous distribution of the tight junction protein in the intestinal epithelial cells;? Stimulating with hypoxia/reoxygenation and LPS can activate the HIF-1a/NF-kB-COX-2 signaling pathway.DMOG can also lead to a damage of the tight junction protein and a activation of NF-kB and COX-2,which providing an evidence that HIF-1a play a role in the damage of the tight junction protein and regulation of NF-kB and COX-2 in the intestinal epithelial cells;?VEGF,a downstream target gene of HIF-1a may play a role in the change of tight junction protein of intestinal epithelial cells in inflammatory injury induced by hypoxia/reoxygenation and LPS;④Emodin can attenuate the harmful changes in the tight-junction protein in the intestinal epithelial cells by blocking HIF-1a/NF-kB-COX-2 signaling pathways and decreasing the expression of VEGF,a downstream target gene of HIF-1a.