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3,5,7,3′,4′-Pentamethoxyflavone Enhances the Barrier Function through Transcriptional Regulation of the Tight Junction in Human Intestinal Caco-2 Cells
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2021-08-27 , DOI: 10.1021/acs.jafc.1c04295 Yunika Mayangsari 1, 2 , Natsumi Sugimachi 3 , Wenxi Xu 4 , Chinatsu Mano 5 , Yuki Tanaka 5 , Osamu Ueda 5 , Tomohiro Sakuta 5 , Yoshiharu Suzuki 5 , Yoshinari Yamamoto 4 , Takuya Suzuki 1, 4
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2021-08-27 , DOI: 10.1021/acs.jafc.1c04295 Yunika Mayangsari 1, 2 , Natsumi Sugimachi 3 , Wenxi Xu 4 , Chinatsu Mano 5 , Yuki Tanaka 5 , Osamu Ueda 5 , Tomohiro Sakuta 5 , Yoshiharu Suzuki 5 , Yoshinari Yamamoto 4 , Takuya Suzuki 1, 4
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The intestinal tight junction (TJ) barrier plays a pivotal role in the regulation of intestinal homeostasis. This study investigated the effects of 3,5,7,3′,4′-pentamethoxyflavone (PMF), a major polymethoxyflavone found in black ginger, on TJ barrier regulation using intestinal Caco-2 cells. PMF treatment enhanced the TJ barrier integrity in Caco-2 cells, indicated by increased transepithelial electrical resistance (control, 1261 ± 36 Ω·cm2; 100 μM PMF, 1383 ± 55 Ω·cm2 at 48 h, p < 0.05) and decreased permeability to fluorescein-conjugated dextran (control, 24.2 ± 1.8 pmol/(cm2 × h); 100 μM PMF, 18.6 ± 1.0 pmol/(cm2 × h), p < 0.05). Immunoblot analysis revealed that PMF increased the cytoskeletal association and cellular expression of the TJ proteins, zonula occludens-1, claudin-3, and claudin-4 (e.g., occludin; control, 1.00 ± 0.2; 100 μM PMF, 3.69 ± 0.86 at 48 h, p < 0.05). Quantitative reverse transcriptase-polymerase chain reaction analysis and a luciferase promoter assay showed that PMF enhanced the transcription of occludin, claudin-3, and claudin-4. The promoter assay with site-directed mutagenesis indicated that PMF-induced occludin and claudin-3 transcription was mediated by transcription factors, KLF5 and EGR1, respectively, while PMF activated claudin-4 transcription through GATA1 and AP1. Taken together, the transcriptional regulation of TJ proteins is involved in PMF-mediated promotion of the intestinal barrier in vitro.
中文翻译：
3,5,7,3',4'-五甲氧基黄酮通过转录调控人肠 Caco-2 细胞紧密连接增强屏障功能
肠道紧密连接 (TJ) 屏障在调节肠道稳态中起着关键作用。本研究使用肠道 Caco-2 细胞研究了 3,5,7,3',4'-五甲氧基黄酮 (PMF)（一种在黑姜中发现的主要多甲氧基黄酮）对 TJ 屏障调节的影响。PMF 处理增强了 Caco-2 细胞中 TJ 屏障的完整性，表现为跨上皮电阻增加（对照，1261 ± 36 Ω·cm 2；100 μM PMF，1383 ± 55 Ω·cm 2在 48 小时，p < 0.05）和降低对荧光素偶联葡聚糖的渗透性（对照，24.2 ± 1.8 pmol/（cm 2 × h）；100 μM PMF，18.6 ± 1.0 pmol/（cm 2 × h），p< 0.05)。免疫印迹分析显示，PMF 增加了 TJ 蛋白、小带 occludens-1、claudin-3 和 claudin-4（例如，occludin；对照，1.00 ± 0.2；100 μM PMF，48 时为 3.69 ± 0.86）的细胞骨架关联和细胞表达h, p < 0.05)。定量逆转录酶-聚合酶链反应分析和荧光素酶启动子测定表明 PMF 增强了 occludin、claudin-3 和 claudin-4 的转录。定点突变的启动子分析表明 PMF 诱导的 occludin 和 claudin-3 转录分别由转录因子 KLF5 和 EGR1 介导，而 PMF 通过 GATA1 和 AP1 激活 claudin-4 转录。总之，TJ 蛋白的转录调控参与 PMF 介导的肠屏障促进体外。
更新日期：2021-09-08
中文翻译：
3,5,7,3',4'-五甲氧基黄酮通过转录调控人肠 Caco-2 细胞紧密连接增强屏障功能
肠道紧密连接 (TJ) 屏障在调节肠道稳态中起着关键作用。本研究使用肠道 Caco-2 细胞研究了 3,5,7,3',4'-五甲氧基黄酮 (PMF)（一种在黑姜中发现的主要多甲氧基黄酮）对 TJ 屏障调节的影响。PMF 处理增强了 Caco-2 细胞中 TJ 屏障的完整性，表现为跨上皮电阻增加（对照，1261 ± 36 Ω·cm 2；100 μM PMF，1383 ± 55 Ω·cm 2在 48 小时，p < 0.05）和降低对荧光素偶联葡聚糖的渗透性（对照，24.2 ± 1.8 pmol/（cm 2 × h）；100 μM PMF，18.6 ± 1.0 pmol/（cm 2 × h），p< 0.05)。免疫印迹分析显示，PMF 增加了 TJ 蛋白、小带 occludens-1、claudin-3 和 claudin-4（例如，occludin；对照，1.00 ± 0.2；100 μM PMF，48 时为 3.69 ± 0.86）的细胞骨架关联和细胞表达h, p < 0.05)。定量逆转录酶-聚合酶链反应分析和荧光素酶启动子测定表明 PMF 增强了 occludin、claudin-3 和 claudin-4 的转录。定点突变的启动子分析表明 PMF 诱导的 occludin 和 claudin-3 转录分别由转录因子 KLF5 和 EGR1 介导，而 PMF 通过 GATA1 和 AP1 激活 claudin-4 转录。总之，TJ 蛋白的转录调控参与 PMF 介导的肠屏障促进体外。
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