Soleiman Amal Ali, Thameem Farook, Khan Islam
Department of Biochemistry, Faculty of Medicine, Kuwait University, Jabriya, Kuwait.
PLoS One. 2017 May 11;12(5):e0176767. doi: 10.1371/journal.pone.0176767. eCollection 2017.
The Na-H exchanger [NHE] performs an electroneutral uptake of NaCl and water from the lumen of the gastrointestinal tract. There are several distinct NHE isoforms, some of which show an altered expression in the inflammatory bowel diseases (IBD). In this study, we examined a role of NHE-2 in experimental colitis.
Colitis was induced in male Sprague-Dawley rats by intra-rectal administration of trinitrobenzenesulphonic acid (TNBS). On day 6 post-TNBS, the animals were sacrificed, colonic and ileal segments were taken out, cleaned with phosphate buffered saline and used in this study.
There was a significant decrease in the level of NHE-2 protein as measured by ECL western blot analysis and confocal immunofluorescence microscopy. The levels of NHE-2 mRNA and heteronuclear RNA measured by an end-point RT-PCR and a real time PCR were also decreased significantly in the inflamed colon. However, there was no change in the level of NHE-2 protein in response to in vitro TNF-α treatment of uninflamed rat colonic segment. These changes were selective and localized to the colon as actin, an internal control, remained unchanged. Confocal immunofluorescence microscopy revealed co-localization of NHE-2 and NHE-3 in the brush borders of colonic epithelial cells. Inflamed colon showed a significant increase in myeloperoxidase activity and colon hypertrophy. In addition, there was a significant decrease in body weight and goblet cells' mucin staining in the TNBS treated colon. These changes were not conspicuous in the non-inflamed ileum.
These findings demonstrate suppression of NHE-2 expression on the brush borders in the colonic epithelial cells which is regulated transcriptionally. However a role of TNF-α in the regulation of NHE-2 is discounted in the present model of colitis. This decrease in the NHE-2 expression will lead to a loss of electrolyte and water uptake thus contributing to the symptoms associated with IBD.
钠-氢交换体[NHE]介导胃肠道管腔中氯化钠和水的电中性摄取。存在几种不同的NHE亚型，其中一些在炎症性肠病（IBD）中表达改变。在本研究中，我们研究了NHE-2在实验性结肠炎中的作用。
通过直肠内给予三硝基苯磺酸（TNBS）诱导雄性Sprague-Dawley大鼠发生结肠炎。TNBS处理后第6天，处死动物，取出结肠和回肠段，用磷酸盐缓冲盐水清洗后用于本研究。
通过增强化学发光（ECL）蛋白质印迹分析和共聚焦免疫荧光显微镜检测发现，NHE-2蛋白水平显著降低。通过终点逆转录聚合酶链反应（RT-PCR）和实时PCR检测的NHE-2信使核糖核酸（mRNA）和核不均一RNA水平在发炎的结肠中也显著降低。然而，未发炎的大鼠结肠段经体外肿瘤坏死因子-α（TNF-α）处理后，NHE-2蛋白水平没有变化。这些变化具有选择性，且局限于结肠，作为内参的肌动蛋白水平保持不变。共聚焦免疫荧光显微镜显示NHE-2和NHE-3在结肠上皮细胞刷状缘共定位。发炎的结肠髓过氧化物酶活性显著增加，结肠肥大。此外，TNBS处理的结肠中体重和杯状细胞黏蛋白染色显著降低。这些变化在未发炎的回肠中不明显。
这些发现表明结肠上皮细胞刷状缘上NHE-2的表达受到转录调控而被抑制。然而，在本结肠炎模型中，TNF-α在NHE-2调节中的作用被排除。NHE-2表达的降低将导致电解质和水摄取的丧失，从而导致与IBD相关的症状。
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