Department of General Physiology, St. Petersburg State University, St. Petersburg, 197374, Russia.
Faculty of Medicine, St. Petersburg State University, St. Petersburg, 197374, Russia.
Pflugers Arch. 2019 Sep;471(9):1183-1189. doi: 10.1007/s00424-019-02294-z. Epub 2019 Jun 20.
Cholera toxin is commonly known to induce chloride secretion of the intestine. In recent years, effects on epithelial barrier function have been reported, indicating synergistic co-regulation of transporters and tight junction proteins. Our current study focused on the analysis of cholera toxin effects on transepithelial resistance and on tight junction proteins, the latter known as structural correlates of barrier function. Ligated segments of the rat jejunum were injected with buffered solution containing cholera toxin (1 μg/ml) and incubated for 4 h. Subsequently, selfsame tissue specimens were mounted in Ussing chambers, and cholera toxin (1 μg/ml) was added on the apical side. Transepithelial resistance and permeability of sodium fluorescein (376 Da) were analyzed. Subsequently, tissues were removed, expression and localization of claudins were analyzed, and morphological studies were performed employing transmission electron microscopy and confocal laser scanning microscopy. Cholera toxin induced a marked decrease in transepithelial resistance in the rat jejunal epithelium and an increase in paracellular permeability for sodium fluorescein. Immunoblotting of tight junction proteins revealed an increase in claudin-2 signals, which was verified by confocal laser scanning immunofluorescence microscopy, and a decrease in tricellulin, whereas other tight junction proteins remained unchanged. Transmission electron microscopy showed a reduction in the number of microvilli after incubation with cholera toxin. Moreover, cholera toxin led to a widening of the intercellular space between enterocytes. In accordance with the commonly known prosecretory effect of cholera toxin, our study revealed a complementary effect on small intestinal barrier function and integrity, which might constitute a pathomechanism with high relevance for prevention and therapeutic approaches.
霍乱毒素通常被认为可诱导肠道的氯离子分泌。近年来，有报道称其对上皮屏障功能也具有影响，表明转运蛋白和紧密连接蛋白存在协同调控作用。本研究重点分析了霍乱毒素对跨上皮电阻和紧密连接蛋白的影响，后者被认为是屏障功能的结构相关物。用含霍乱毒素（1μg/ml）的缓冲溶液对大鼠空肠结扎段进行注射，并孵育 4 小时。随后，将相同的组织标本置于 Ussing 室中，并在顶端加入霍乱毒素（1μg/ml）。分析跨上皮电阻和 376Da 钠荧光素的通透性。随后，取出组织，分析紧密连接蛋白的表达和定位，并采用透射电子显微镜和共聚焦激光扫描显微镜进行形态学研究。霍乱毒素诱导大鼠空肠上皮细胞的跨上皮电阻明显降低，对钠荧光素的细胞旁通透性增加。免疫印迹分析显示紧密连接蛋白 claudin-2 信号增加，这一结果通过共聚焦激光扫描免疫荧光显微镜得到验证，同时 tricellulin 减少，而其他紧密连接蛋白无变化。透射电镜显示，用霍乱毒素孵育后微绒毛数量减少。此外，霍乱毒素导致肠细胞间的细胞间隙增宽。与霍乱毒素常见的促分泌作用一致，本研究揭示了其对小肠屏障功能和完整性的补充作用，这可能构成具有高度预防和治疗意义的发病机制。
