Résumé
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, SPD), alpha-difluoromethylornithine (2.5 mmol·L, DFMO), 4-Aminopyridine (40 μmol·L, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca ([Ca]) 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] 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], 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 RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.
Sujets)
Animaux , Rats , Astragalus membranaceus , Chimie , Atractylodes , Chimie , Lignée cellulaire , Mouvement cellulaire , Médicaments issus de plantes chinoises , Chimie , Pharmacologie , Cellules épithéliales , Biologie cellulaire , Métabolisme , Intestins , Biologie cellulaire , Génétique , Métabolisme , Polyamines , Métabolisme , Polyosides , Chimie , Pharmacologie , Rhizome , Chimie , Transduction du signal , Protéine G RhoA , MétabolismeRésumé
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, SPD), alpha-difluoromethylornithine (2.5 mmol·L, DFMO), 4-Aminopyridine (40 μmol·L, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca ([Ca]) 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] 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], 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 RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.
Sujets)
Animaux , Rats , Astragalus membranaceus , Chimie , Atractylodes , Chimie , Lignée cellulaire , Mouvement cellulaire , Médicaments issus de plantes chinoises , Chimie , Pharmacologie , Cellules épithéliales , Biologie cellulaire , Métabolisme , Intestins , Biologie cellulaire , Génétique , Métabolisme , Polyamines , Métabolisme , Polyosides , Chimie , Pharmacologie , Rhizome , Chimie , Transduction du signal , Protéine G RhoA , MétabolismeRésumé
<p><b>OBJECTIVE</b>To study the effect of reserpine (RSP) for changing salivary protein secretion in Pi-deficient rats and to explore its possible mechanism.</p><p><b>METHODS</b>Twenty rats allocated in the RSP group were given subcutaneous injection of RSP [0.4 mg/(kg x d)] for 9 successive days, while the other 20 rats in the control group were injected with same volume of saline instead. On the 10th day, ten rats randomly selected from each group were subjected for extracting saliva to detect salivary amylase activity (sAA) before and after an acid stimulation; and drawing blood from the orbital vein to measure the contents of vasoactive intestinal peptide (VIP) and cyclic adenosine monophosphate (cAMP). Then they were sacrificed and their parotids were taken out for pathological examination with HE staining, as well as for VIP and cAMP measuring, and zymogen granules counting under a transmission electron microscope. The remainder animals were stopped injecting and normally fed to 40 days, then subjected to be detected as above-mentioned.</p><p><b>RESULTS</b>Food intake and body weight reduction were more significantly in the RSP group than in the control group. On the 10th day, the ratio of sAA before/after stimulation in the RSP group was 0.39 +/- 0.18, significantly lower than that in the control group (0.80 +/- 0.21, P < 0.01), but it was restored rapidly, reaching the normal range on the 25th day, on the 40th day, it became significantly different to the level on the 10th day (P < 0.05) and approached the level in the control group (P > 0.05). No significant pathological change of parotid was found in both groups; but the number of zymogen granules in the RSP group was remarkably more than that in the control group (41.4 +/- 4.9 vs 34.6 +/- 5.2, P < 0.01). Serum level of VIP in the RSP group was significantly less while that of cAMP was higher than that in the control group (22.5 +/- 13.1 mg/L vs 38.5 +/- 14.1 mg/L, and 125.8 +/- 15.5 micromol/L vs 105.3 +/- 16.7 micromol/L, both P < 0.05), but no inter-group difference was found in parotid tissue contents of both VIP and cAMP. All the indices detected became equivalent in the two groups on the 40th day.</p><p><b>CONCLUSION</b>The reduction of salivary protein in Pi-deficient rats induced by RSP may be related to the regulatory pathway of VIP and cAMP.</p>