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Atractylodes macrocephala Koidz promotes intestinal epithelial restitution via the polyamine—Voltage-gated K+ channel pathway

Song, Hou-Pan, Li, Ru-Liu, Chen, Xu, Wang, Yi-Yu, Cai, Jia-Zhong, Liu, Jia, Chen, Wei-Wen
Journal of ethnopharmacology 2014 v.152 no.1 pp. 163-172
Atractylodes macrocephala, Oriental traditional medicine, Western blotting, alpha-difluoromethylornithine, calcium, cell movement, computer software, digestive system diseases, drugs, flow cytometry, high performance liquid chromatography, intestinal mucosa, membrane potential, messenger RNA, models, polystyrenes, potassium channels, protein content, protein synthesis, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, signal transduction, spermidine
Atractylodes macrocephala Koidz (AMK) has been used widely as a digestive and tonic in traditional Chinese medicine. AMK has shown noteworthy promoting effect on intestinal epithelial cell migration, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via IEC-6 cell migration model.A wounding model of IEC-6 cells was induced by a single-edge razor blade along the diameter of six-well polystyrene plates. The cells were grown in control cultures and in cultures containing spermidine (5μmol/L, SPD, reference drug), alpha-difluoromethylornithine (2.5mmol/L, DFMO, polyamine inhibitor), AMK (50, 100, and 200μg/mL), DFMO plus SPD and DFMO plus AMK for 24h. The membrane potential (MP) and cytosolic free Ca²⁺ concentration ([Ca²⁺]cyt) were detected by flow cytometry, and polyamines content was determined via high-performance liquid chromatography (HPLC). The expression of Kv1.1 mRNA and protein levels were assessed by RT-qPCR and Western blot analysis, respectively. Cell migration assay was carried out using the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK.(1) Treatment with AMK caused significant increases in cellular polyamines content, membrane hyperpolarization, an elevation of [Ca²⁺]cyt and an acceleration of cell migration in IEC-6 cells, as compared to control group. (2) AMK not only reversed the inhibitory effects of DFMO on the polyamines content, MP, and [Ca²⁺]cyt but also restored IEC-6 cell migration to control levels. (3) The Kv1.1 mRNA and protein expression were significantly increased by AMK treatment in control and polyamine-deficient IEC-6 cells.The results of our current studies revealed that treatment with AMK significantly stimulates the migration of intestinal epithelial cells through polyamine-Kv1.1 channel signaling pathway, which could promote the healing of intestinal injury. These results suggest the potential usefulness of AMK to cure intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.