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

背景：在缺氧条件下，肠道细菌产物与肠上皮细胞之间的相互作用尚未完全了解。缺氧诱导因子-1 （Hypoxia inducible factor-1, HIF-1）是细胞适应缺氧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介导的几种基因转录以及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细胞置于5%CO2, 1%O2和N2平衡的腔室中。为了进行荧光素酶检测，我们构建了报告质粒pHREpgk1-Luc，该质粒在最小SV40启动子上游的磷酸甘油酸激酶-1 （PGK-1）的5'侧区具有缺氧反应元件（HRE）。采用通用方案对PGK-1、iNOS或GAPDH进行反转录分析。为了研究Caco-2细胞的屏障功能，我们测量了Caco-2细胞的上皮电阻抗（TEER）。结果：丁酸降低HIF-1转录活性。多个HIF-1介导基因的转录在Caco-2细胞中受到抑制。在低氧条件下，丁酸盐也能降低TEER。讨论：HIF-1被认为是细胞适应缺氧的关键转录因子，抑制相关基因的活性和转录可能提示丁酸盐对缺氧条件下肠上皮的有害作用。屏障功能的丧失可能与缺氧下丁酸盐诱导的上皮扰动有关。

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.