Hedgehog信号通路在缺氧肠上皮屏障功能调控的作用
Regulatory effects of hedgehog pathway on intestinal epithelial barrier function under hypoxia
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摘要:目的 探讨Hedgehog(Hh)信号通路在缺氧肠上皮屏障功能调控的作用。方法 大鼠小肠上皮细胞系IEC-6细胞分为3组：常氧组(21%氧浓度)、缺氧组(2%氧浓度)、缺氧+环巴胺组(用5 mmol/L的环巴胺预处理30 min后，再给予2%氧浓度进行缺氧处理)。RT-PCR检测Hh信号通路IHH、PTCH、GLI-1 mRNA的表达变化，电阻测定仪检测跨上皮电阻(TER)，Western blot检测IHH、紧密连接蛋白经典表达分子胞质附着蛋白(ZO-1)、咬合蛋白(Occludin)、闭合蛋白(Claudin-1)的表达情况。组间比较采取单因素方差分析，两两比较采用LSD-t检验。结果 RT-PCR检测结果表明：常氧组Hh信号通路IHH、PTCH、GLI-1 mRNA的相对表达量分别为0.056±0.009、0.459±0.087、0.142±0.023；缺氧组分别为0.303±0.052、0.678±0.073、0.483±0.061，两组比较，差异有统计学意义(t=-14.05，-11.85，-6.52，P<0.05)。Western blot检测结果显示：常氧组和缺氧组的IHH相对蛋白表达量分别为0.39±0.06和0.91±0.15，两组比较，差异有统计学意义(t=-8.08，P<0.05)。常氧组、缺氧组和缺氧+环巴胺组的TER分别为(134±5) Ohm/cm2、(100±6) Ohm/cm2、(118±5) Ohm/cm2，3组比较，差异有统计学意义(F=1.04，P<0.05)。与常氧组比较，缺氧组下降约27.7%(t=7.84，P<0.05)；与缺氧组比较，缺氧+环巴胺组回升约16.4%，但仍低于常氧组(t=4.23，P<0.05)。常氧组胞质附着蛋白-1、咬合蛋白、闭合蛋白-1的表达分别为1.18±0.24、0.80±0.13、0.90±0.09，缺氧组分别为0.58±0.08、0.32±0.05、0.50±0.09，缺氧+环巴胺组分别为0.92±0.21、0.43±0.10、0.82±0.11，3组比较，差异有统计学意义(F=4.95，2.88，10.09，P<0.05)。缺氧组较常氧组分别降低48.7%、40.0%、55.6%(t=12.86，9.35，18.90，P<0.05)；缺氧+环巴胺组较缺氧组分别提高59.9%、35.2%、65.1%(t=5.63，2.92，6.66，P<0.05)。结论 缺氧条件可刺激激活Hh信号通路，从而引起紧密连接蛋白表达降低，导致肠上皮屏障功能损害。Abstract:Objective To investigate the 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.
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