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

一氧化氮和丁酸盐影响 HIF-1 活性并调节肠上皮细胞单层紧密连接的功能

• 批准号: 12671151
• 负责人: UNNO Naoki
• 金额: $ 1.47万
• 依托单位: Hamamatsu University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671151/
• 关键词: HIF-1; Hypoxia; Intestinal epithelial cell; Butyrate; Permeability; Nitric oxide; 腸上皮細胞; Inos; 酪酸; iNOS; HIF; 低酸素; peroxynitrite

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.

背景资料：在缺氧条件下，肠道内细菌产物与肠道上皮细胞之间的相互作用尚未完全了解。低氧诱导因子-1（Hypoxia inducible factor-1，HIF-1）是细胞适应低氧环境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转录活性和由HIF-1介导的几个基因转录的影响，以及上皮细胞屏障功能。方法：采用Caco-2细胞作为人小肠上皮细胞的模型。缺氧实验中，我们将Caco-2细胞置于5%CO2、1%O2和N2平衡的小室中。为了进行荧光素酶检测，我们构建了报告质粒pHREpgk 1-Luc，该质粒在SV 40最小启动子上游的磷酸甘油酸激酶-1（PGK-1）5 '侧翼区具有缺氧反应元件（HRE）。用普遍存在的方案进行关于PGK-1、iNOS或GAPDH的逆转录分析。为了研究Caco-2细胞的屏障功能，测量了跨上皮电阻（TEER）。结果：丁酸盐降低HIF-1的转录活性。在Caco-2细胞中，HIF-1介导的几个基因的转录被抑制。丁酸也降低了缺氧条件下的TEER。讨论内容：由于HIF-1被认为是细胞适应缺氧的关键转录因子，因此丁酸盐对HIF-1活性的抑制以及相关基因转录的抑制可能提示丁酸盐对缺氧条件下肠上皮细胞的损伤作用。屏障功能的丧失可能与缺氧条件下丁酸诱导的上皮扰动有关。

项目成果

Miki K., Unno N, Nagata T, Mitsuoka H, Saito T, Ishimaru K, Koide Y, Nakamura S: "Butyrate suppress HIF-1 activity in intestinal epithelial cells under hypoxic conditions"Shock. 17. 9 (2002)

Miki K.、Unno N、Nagata T、Mitsuoka H、Saito T、Ishimaru K、Koide Y、Nakamura S：“丁酸盐在缺氧条件下抑制肠上皮细胞中的 HIF-1 活性”休克。

三鬼慶太, 海野直樹, 永田 年, 内嶋真人, 三岡 博, 斉藤孝晶, 中村 達, 小出幸夫: "腸管上皮細胞内のHIF-1活性とバリアー機能に対する酷酸の効果についての実験的検討"日本ショック学会誌. 18. (2003)

Keita Miki、Naoki Unno、Toshi Nagata、Masato Uchijima、Hiroshi Mioka、Takaaki Saito、Tatsu Nakamura、Yukio Koide：《强酸对肠上皮细胞 HIF-1 活性和屏障功能影响的实验研究》综述《Journal of日本休克协会。18。（2003）

Miki K., Unno N, Nagata T, Mitsuoka H, Saito T, Ishimaru K, Koide Y, Nakamura S.: "Butyrate suppress HIF-1 activity in intestinal epithelial cells under hypoxic conditions"Shock. 17. (2002)

Miki K.、Unno N、Nagata T、Mitsuoka H、Saito T、Ishimaru K、Koide Y、Nakamura S.：“丁酸盐在缺氧条件下抑制肠上皮细胞中的 HIF-1 活性”休克。