The role of myosin light chain kinase in intestinal epithelial barrier dysfunction due to hypoxia / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the role of myosin light chain kinase (MLCK) in intestinal epithelial barrier dysfunction after hypoxia.</p><p><b>METHODS</b>The Caco-2 monolayers developed with Transwell inserts were exposed to hypoxia for 0 h (NC group), 2, 6, 8, 12 and 24 h (H group), and 6 h hypoxic specimens were treated with 100 mol/L ML-9 (T group). The transepithelial electrical resistance (TER) of monolayers was measured with an ohmmeter. The tight junction protein ZO-1 of monolayers was analyzed by immunofluorescence assay. The protein expressions of phosphorylated myosin light chain (p-MLC) and MLCK were detected by Western blotting.</p><p><b>RESULTS</b>The TER of monolayers in H group at 6, 8, 12 and 24 h was 422 +/- 17, 427 +/- 27, 403 +/- 40 and 426 +/- 22 ohms respectively, which was significantly lower than that of NC group (451 +/- 27 ohms, P < 0.05). The TER of monolayers in T group was 558 +/- 110 ohms, which was significantly higher than that in H group at each time point ( P < 0.01). The ZO-1 of monolayers in H group at 6 h was irregular in arrangement, with interruptions and rugae, and sawtooth. These abnormalities were ameliorated in T group (regular in arrangement, with little or without ruga and sawtooth). The protein expressions of p-MLC and MLCK in H group at each time point were higher than those in NC group.</p><p><b>CONCLUSIONS</b>Intestinal epithelial barrier dysfunction after hypoxia can be mediated by MLCK.</p>

Effect of hypoxia on hypoxia inducible factor 1alpha (HIF-1alpha) activation in intestinal epithelial cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effect of hypoxia on HIF-1alpha activation in intestinal epithelial cells.</p><p><b>METHODS</b>Intestinal epithelial cells were randomly divided into normal control group, hypoxia group and hypoxia plus oligomycin group (oligomycin group). In hypoxia group, the cells were exposed to hypoxia for 1, 2, 6, 12 and 24 h. In oligomycin group, the cells were treated with oligomycin in concentration of 5, 10, 20 and 40 microg/mL for 1 h prior to 6-hour hypoxic exposure. HIF-1alpha protein expression was assayed by western blot method. Nuclear translocation of HIF-1alpha was detected by immunofluorescence analysis.</p><p><b>RESULTS</b>Compared with that in control group (0.08 +/- 0.07), HIF-1alpha protein expression in hypoxia group increased significantly at 1 h (0.52 +/- 0.30, P < 0.05), and reached the peak value (2.37 +/- 1.08, P < 0.05) at 6 h. Nuclear translocation of HIF-1alpha was also induced by hypoxia. HIF-1alpha protein expression in oligomycin group in the concentration of 5, 10, 20 and 40 microg/mL of oligomycin was 1.62 +/- 0.96, 1.48 +/- 0.56, 1.08 +/- 0.36 and 0.58 +/- 0.11 respectively, which was significantly lower than that only after exposure to hypoxia for 6 h (2.67 +/- 1.38, P < 0.05). The nuclear translocation of HIF-1alpha induced by hypoxia was also obviously inhibited by oligomycin pretreatment.</p><p><b>CONCLUSION</b>Oligomycin, a specific inhibitor of respiratory chain, inhibits HIF-1alpha activation, which suggests that mitochondria respiratory chain may play an important role in aforementioned process.</p>