Nitric oxide and butyrate affect HIF-1 activity and modulate function of tight junction in intestinal epithelial cell monolayers

背景：低氧条件下肠道细菌的产物与肠道上皮细胞之间的相互作用尚不完全清楚。低氧诱导因子-1(HIF-1)是细胞适应低氧条件的关键转录因子之一。我们研究了肠道细菌产物丁酸盐对HIF-1转录活性和HIF-1介导的几个基因转录的影响，以及对上皮细胞屏障功能的影响。方法：以Caco-2细胞作为人肠上皮细胞模型。在低氧实验中，我们将Caco-2细胞放入一个5%二氧化碳、1%氧气和氮气平衡的室内。对于荧光素酶的检测，我们构建了报告载体pHREpgk1-Luc，该报告载体在最小SV40启动子的上游含有磷酸甘油酸激酶-1(PGK-1)5‘侧翼区的缺氧反应元件(HRE)。用无处不在的方法对PGK-1、iNOS或GAPDH进行逆转录分析。为了研究Caco-2细胞的屏障功能，测定了跨皮细胞电阻(TEER)。结果：丁酸可降低HIF-1转录活性。在Caco-2细胞中，HIF-1介导的几个基因的转录受到抑制。丁酸盐也降低了低氧条件下的TEER。讨论：由于HIF-1被认为是细胞对低氧适应的关键转录因子，相关基因的活性和转录的抑制可能表明丁酸盐在低氧条件下对肠上皮细胞的有害影响。这种屏障功能的丧失可能与丁酸在低氧条件下诱导的上皮细胞紊乱有关。

Background : Interaction between the product of the bacteria reside in the intestine and the intestinal epithelial cells under hypoxic conditions is not yet to be fully understood. Hypoxia inducible factor-1 (HIF-1) is one of the pivotal transcriptional factor by which cells may adapt to hypoxic conditions. We investigated the effect of intestinal bacterial product, butyrate on HIF-1 transcriptional activity and transcription of several genes mediated by HIF-1, as well as the barrier function of epithelial cells. Methods : We used the Caco-2 cells as a model of human intestinal epithelial cells. For hypoxic experiments, we put the Caco-2 cells into a chamber with 5%CO2, 1%O2 and N2 balance. For luciferase assay, we constructed the reporter plasmid pHREpgk1-Luc which has hypoxia response element (HRE) from 5'-flanking region of phosphoglycerate kinase-1 (PGK-1) at just upstream of minimum SV40 promoter. Reverse transcription analysis was performed about PGK-1, iNOS, or GAPDH with ubiquitous protocols. To study the barrier functions of Caco-2 cells, Transepithelial electronic resistance (TEER) was measured. Results : Butyrate reduced HIF-1 transcriptional activity. The transcription of several HIF-1 mediated genes was inhibited in Caco-2 cells. Butyrate also reduced TEER under the hypoxic condition. Discussion : As HIF-1 is regarded to be critical transcriptional factor for cellular adaptation to hypoxia, the inhibition of the activity as well as transcription of the related genes might suggest the deleterious effect of butyrate on intestinal epithelium under hypoxia. The loss of the barrier function might be associated with butyrate-induced epithelial perturbation under hypoxia.