Amelioration Of Hypoxia And LPS-Induced Intestinal Epithelial Barrier Dysfunction By Emodin Via Suppression Of NF-κB And HIF-1a Signaling Pathways

Paper oneThe intervention of emodin on LPS-induced HIF-1a and COX-2expression in intestinal epithelial cells[Abstract] Objective To observe the expression of HIF-la and its downstream target gene COX-2in LPS-treated intestinal epithelial cells, and to explore the possible intervention targets of emodin. Methods Human intestinal epithelial cells were cultured in vitro by being treated with LPS to establish the experimental model.1. The expression trend of HIF-la and COX-2was measured by Western blot in LPS dose-dependent and time-dependent experiments. The expression trend of HIF-1a, COX-2, Phospho-IκB-a and Phospho-NF-κB p65was measured in LPS plus various concentrations of emodin treated groups.2. The mRNA levels of HIF-la were detected by PCR after cells were treated with LPS or LPS plus emodin respectively.3. The effect of emodin on the proliferation of intestinal epithelial cells was measured by MTT assay in each group. Data were analyzed with ANOVA, and P<0.05was considered significant. Results1. LPS induced the expression of HIF-1a in a dose-dependent and a time-dependent manner. With increasing concentrations of LPS, HIF-la increased to the peak when cells were treated with LPS at10-3mg/ml (P<0.05), and then gradually decreased. HIF-la reached the peak at0.5h after treatment, and then decreased to the lowest level at4h (P<0.05), finally returned to a high level. The expression trend of COX-2was similar to HIF-1a (P<0.05). Emodin inhibited the expression of LPS-induced HIF-la, COX-2, Phospho-IκB-a and Phospho-NF-KB p65with a significant dose-effect relationship (P<0.05).2. The PCR showed that emodin inhibited the increasing mRNA levels of HIF-1a after LPS treatment.3. MTT assay showed emodin (0-80μmol/L)had no significant effect on cell proliferation (P>0.05). Although emodin produced biological effect at this concentration range, it had no drug toxicity to cells. Conclusion LPS induces HIF-1a-COX-2signaling pathway in a time-dependent and a dose-dependent manner in intestinal epithelial cells. Emodin block the hypoxia pathway of LPS-HIF-1a-COX-2and the inflammatory pathway of LPS-IκB-a-NF-κB-COX-2, which may play a protective effect on intestinal epithelial cells. Paper twoHypoxia and LPS induced NF-κB and HIF-1a signaling pathway in intestinal epithelial cells and the intervention efficacy of emodin[Abstract] Objective To observe the expression changes of Hypoxia-inducible factor (HIF)-1a, Nuclear factor (NF)-κB and their downstream target gene Cyclooxygenase-2(COX-2) when the intestinal epithelial cells were treated with hypoxia (H), hypoxia/reoxygenation (HR) and lipopolysaccharide (LPS), and to explore the possible intervention targets of emodin. Methods Human fetal normal colonic cell (FHC) line was cultured by being treated with H±LPS, HR±LPS in vitro to simulate the pathological process of ischemia, ischemia/reperfusion and inflammation injury in intestinal epithelial cell in vivo. Normoxia group:the cells were treated with95%air and5%CO2at37℃. H group:the cells were treated with a mixed anaerobic gas of1%O2,5%CO2and94%N2at37℃for1,2,3,4h. H+LPS group: the cells were treated with hypoxia as H group and LPS (1μg/mL) simultaneously. HR group:the cells were treated with hypoxia for3h and then treated with reoxygenation for1,2,3and4h respectively. HR+LPS group: the cells were treated with HR as HR group and LPS (1μg/mL) simultaneously. Emodin intervention group:the cells were treated with H3hR2h+LPS and emodin (20,40,60,80/μmol/L) simultaneously. The variation trend of HIF-1a, IκB-a, NF-κB and COX-2was measured by Western blot; and the morphological changes of intestinal epithelial in different groups were observed by microscopy. Results1. H group:Compared with normoxia, the expression of all the three proteins of NF-κB pathway increased. The trend of the proteins was similar: they increased to the peak at Hih and then decreased (P=0.013,0.000,0.005). The expression of HIF-1a reached to the peak at H3h, but there was no significant difference among groups (P=0.062). H+LPS group: the trend of all the four proteins (pIκB-a, pNF-KBp65, COX-2, HIF-1a) was similar. As the duration of hypoxia increased, there was an increase at Hih; a maximal induction was attained at H3h; and there was a decrease thereafter (P=0.011,0.000,0.000,0.026). HR group: With the prolonged duration of reoxygenation, the expression of NF-κB signaling pathway proteins decreased and dropped to the lowest at H3HR4h (P=0.063,0.000,0.016).HIF-1a decreased with the prolonged duration of reoxygenation, but there was no significant difference among groups (P=0.081). HR±LPS group:the four proteins didn’t degrade with the prolonged duration of reoxygenation, and their similar expression increased to the top at R2h-3h (P=0.037,0.001,0.067,0.000). Emodin group:Emodin which was co-treated with HR+LPS inhibited the expression of HIF-la and NF-κB pathways with a dose-effect relationship(P<0.05or P<0.01).2. Being treated with HR+LPS, there were morphological of cells: vacuoles deformation and fusion. Conclusion Both hypoxia and inflammation can activate the hypoxia pathway of HIF-1a and the inflammation pathway of NF-κB, but different stimuli cause varying degrees of activation of these two pathways. In HR group, both pathways weakened during reoxygenation. However, in HR±LPS group, the proteins remained relative 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 works together to destroy the intestinal epithelial cells and induces gut-derived sepsis. Emodin may inhibit inflammation by blocking HIF-1a/NF-κKB-COX-2signaling pathways.