Department of Morphology, Faculty of Medicine, Federal University of Ceará, Delmiro de Farias, 60416-030 Fortaleza, CE, Brazil.
Biomed Res Int. 2013;2013:152052. doi: 10.1155/2013/152052. Epub 2012 Dec 27.
Clostridium difficile is a major cause of antibiotic-associated colitis and is associated with significant morbidity and mortality. Glutamine (Gln) is a major fuel for the intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. IEC-6 cells were used in the in vitro experiments. Cell morphology was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cell proliferation was assessed by WST-1 and Ki-67 and apoptosis was assessed by TUNEL. Cytoskeleton was evaluated by immunofluorescence for RhoA and F-actin. RhoA was quantified by immunoblotting. TcdA induced cell shrinkage as observed by AFM, SEM, and fluorescent microscopy. Additionally, collapse of the F-actin cytoskeleton was demonstrated by immunofluorescence. TcdA decreased cell volume and area and increased cell height by 79%, 66.2%, and 58.9%, respectively. Following TcdA treatment, Ala-Gln and Gln supplementation, significantly increased RhoA by 65.5% and 89.7%, respectively at 24 h. Ala-Gln supplementation increased cell proliferation by 137.5% at 24 h and decreased cell apoptosis by 61.4% at 24 h following TcdA treatment. In conclusion, TcdA altered intestinal cell morphology and cytoskeleton organization, decreased cell proliferation, and increased cell apoptosis. Ala-Gln and Gln supplementation reduced intestinal epithelial cell damage and increased RhoA expression.
艰难梭菌是抗生素相关性结肠炎的主要病因，与发病率和死亡率显著相关。谷氨酰胺（Gln）是肠道细胞群的主要燃料。丙氨酰-谷氨酰胺（Ala-Gln）是一种二肽，具有很高的溶解性和良好的耐受性。在体外实验中使用了 IEC-6 细胞。通过原子力显微镜（AFM）和扫描电子显微镜（SEM）评估细胞形态。通过 WST-1 和 Ki-67 评估细胞增殖，通过 TUNEL 评估细胞凋亡。通过免疫荧光法评估 RhoA 和 F-肌动蛋白评估细胞骨架。通过免疫印迹法定量 RhoA。TcdA 诱导细胞收缩，通过 AFM、SEM 和荧光显微镜观察到。此外，免疫荧光显示 F-肌动蛋白细胞骨架崩溃。TcdA 分别使细胞体积和面积减少 66.2%和 58.9%，使细胞高度增加 79%。在 TcdA 处理后，Ala-Gln 和 Gln 补充分别使 RhoA 在 24 小时时增加 65.5%和 89.7%。Ala-Gln 补充可使 TcdA 处理后 24 小时的细胞增殖增加 137.5%，细胞凋亡减少 61.4%。总之，TcdA 改变了肠道细胞的形态和细胞骨架组织，降低了细胞增殖，增加了细胞凋亡。Ala-Gln 和 Gln 补充减少了肠道上皮细胞损伤并增加了 RhoA 的表达。
