Normoxia group: the FHC cells were cultured in 95% air and 5% CO2 at 37 centigrade.
Hypoxia (H) group: the cells were cultured with a mixed anaerobic gas of 1% O2, 5% CO2 and 94% N2 at 37 centigrade 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-α (pIΚB-α), phosphorylation NF-ΚBp65 (pNF-ΚBp65) and their downstream target gene
cyclooxygenase-2 (COX-2), and
hypoxia-inducible factor-1α (HIF-1α) 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-1α 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-α, 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, F value was 4.614, 1.652, 5.970, 2.076, and P value 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-1α 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: