Regulatory effects of hedgehog pathway on intestinal epithelial barrier function under hypoxia

Methods  The IEC-6 cells of rats were divided into 3 groups: the normoxia group (21% oxygen concentration), the hypoxia group (2% oxygen concentration) and the hypoxia+cyclopamine group (cells pretreated by 5 mmol/L of cyclopamine, and then exposed in an atmosphere with 2% oxygen concentration . The mRNA expressions of IHH, PTCH and GLI-1 were detected, and the transepithelial electrical resistance (TER) was determined. The protein expressions of tight junction proteins (ZO-1, Occludin, Claudin-1) and IHH were assayed by using the Western blot. All data were analyzed using the one-way analysis of variance or LSD-t test.

Results  The relative mRNA expressions of IHH, PTCH and GLI-1 were 0.056±0.009, 0.459±0.087, 0.142±0.023 in the normoxia group, and 0.303±0.052, 0.678±0.073, 0.483±0.061 in the hypoxia group, with significant difference between the 2 groups (t=-14.05,-11.85,-6.52, P<0.05). The relative protein expressions of IHH in the normoxia group and the hypoxia group were 0.39±0.06 and 0.91±0.15, with a significant difference between the 2 groups (t=-8.08, P<0.05). The TERs of the normoxia group, the hypoxia group and the hypoxia+cyclopamine group were (134±5)Ohm/cm3, (100±6)Ohm/cm3 and (118±5)Ohm/cm3 with significant difference between the 3 groups (F=1.04, P<0.05). Compared with the normoxia group, the TER of the hypoxia group was decreased by 27.7% (t=7.84, P<0.05); compared with the hypoxia group, the TER of the hypoxia+cyclopamine group were increased by 16.4%, but it was still significantly lower than the normoxia group (t=4.23, P<0.05). The expressions of ZO-1, Occludin and Claudin-1 were 1.18±0.24, 0.80±0.13 and 0.90±0.09 in the normoxia group, and 0.58±0.08, 0.32±0.05 and 0.50±0.09 in the hypoxia group, and 0.92±0.21, 0.43±0.10 and 0.82±0.11 in the hypoxia+cyclopamine group, with significant difference between the 3 groups (F=4.95, 2.88, 10.09, P<0.05). The expressions of ZO-1, Occludin and Claudin-1 in hypoxia group were decreased by 48.7%, 40.0% and 55.6% when compared with the normoxia group (t=12.86, 9.35, 18.90, P<0.05). The expressions of ZO-1, Occludin and Claudin-1 in the hypoxia+cyclopamine group were increased by 59.9%, 35.2% and 65.1% when compared with the hypoxia group (t=5.63, 2.92, 6.66, P<0.05). Conclusion  Hedgehog signal pathway could be activated under hypoxia, and then the expressions of tight junction proteins are decreased, which finally induces the injury of intestinal epithelial barrier function.