Polysaccharide extracts of Astragalus membranaceus and Atractylodes macrocephala promote intestinal epithelial cell migration by activating the polyamine-mediated K^+ channel  

作　　者：ZENG Dan HU Can LI Ru-Liu LIN Chuan-Quan CAI Jia-Zhong WU Ting-Ting SUI Jing-Jing LU Wen-Biao CHEN Wei-Wen 

机构地区：[1]Pi-wei Institute,Guangzhou University of Chinese Medicine [2]College of Traditional Chinese Medicine,Guangzhou University of Chinese Medicine

出　　处：《Chinese Journal of Natural Medicines》2018年第9期674-682,共9页中国天然药物（英文版）

基　　金：supported by the National Natural Science Foundation of China(Nos.30772753,81173254,and 81673940);the Science and Technology Program of Guangzhou(No.20160701335);the First-class discipline construction major project of Guangzhou University of Chinese Medicine(Guangzhou University of Chinese Medicine Planning,2018-No.6)

摘　　要：Astragalus membranaceus(Radix Astragali, RA) and Atractylodes macrocephala(Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control--media or media containing spermidine(5 μmol·L^(-1), SPD), alpha-difluoromethylornithine(2.5 mmol·L^(-1), DFMO), 4-Aminopyridine-(40 μ-mol·L^(-1), 4-AP), the polysaccharide extracts of RA or RAM(50, 100, or 200 mg·L^(-1)), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca^(2+)([Ca^(2+)]cyt) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca^(2+)]cyt and accelerated migration of IEC-6 cells, compared with the controls(P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca^(2+)]cyt, but also restored IEC-6 cell migration to control level(P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased(P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0:14.1:0.3:19.9:181.3:6.3 in RA and 1.0:4.3:0.1:5.7:2.8:2.2 in Astragalus membranaceus（Radix Astragali, RA） and Atractylodes macrocephala（Rhizoma Atractylodis Macrocephalae, RAM） are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control--media or media containing spermidine（5 μmol·L^-1 SPD）, alpha-difluoromethylornithine（2.5 mmol·L^-1 DFMO）, 4-Aminopyridine-（40 μ-mol·L^-1, 4-AP）, the polysaccharide extracts of RA or RAM（50, 100, or 200 mg·L^-1, DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca^2＋（[Ca^2＋]cyt） was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca^2＋]cyt and accelerated migration of IEC-6 cells, compared with the controls（P 〈 0.01）. Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca^2＋]cyt, but also restored IEC-6 cell migration to control level（P 〈 0.01 or 〈 0.05）. Kv1.1 mRNA and protein expressions were increased（P 〈 0.05） after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0：14.1：0.3：19.9：181.

关 键 词：Intestinal epithelial CELL (IEC-6) CELL MIGRATION POLYSACCHARIDE EXTRACTS Astragalus membranaceus Atractylodes macrocephala Polyamines K^+ channel 

分 类 号：R965[医药卫生—药理学]