| BackgroundsIn Traditional Chinese Medicine(TCM), the spleen is a major organ that play a pivotal role in digesting and absorbing foods and drinks in order to transformate them to the essence and nutrition, and it is also responsible for transporting the essence and nutrition from absorbed food and drinking to the whole body, it is called "spleen governing transportation and transformation". So, once pathogenic factors harm the spleen, the function of digestive system in human body can be abnormal. In other words, the model of spleen-qi deficiency, due to the disfunction of transportation and transformation of spleen and stomach, is analogous to abnormity of the digestive system fuction. Meanwhile, the disfunction of digestive system is a substantial part of due to the damage of gastrointestinal mucosa.The maintenance of the integrity of the epithelium of mammalian Gastrointestinal mucosa depends on a complex interplay that included two processes—The rapid repair and the following repair. The rapid repair may occur within an hour of the damage, consisted of the sloughing of the damaged epithelial cells and the migration of the remaining viable cells within the crypts to the luminal surface of the colon and the villous tips in the small intestine. The following repair may occur after 24 hours of the damage, consisted of cell proliferation, differentiation, and apoptosis. So, the early epithelia restitution is very important to the repair of the gastrointestinal mucosa and occurs as a consequence of epithelial cells migration over the damaged area after superficial injury.Objectivethe objective of this acticle is to clarify that what is the structural analysis of the polysaccharide from the Sijunzi decoction and how its purified polysaccharide can promote the migration of the IEC-6 cells via the polyamine-regulated pathway.Methods1.We obtained the Sijunzi decoction total polysaccharides by the water extraction and the alcohol precipitation method, and got the Sijunzi decoction crude polysaccharides by the Sevag method, then the "crude polysaccharides" were isolated and purified by the DEAE-52 and the Sephadex G-100.2.We observed the effects of the different the Sijunzi decoction polysaccharide samples metioned above on the migration of the cells by the in vitro IEC-6 cell migration respectively, and took the cell migration images by the fluorescence inverted microscope, then counted the number of the cell migration near the scratch by Image pro-plus 6.0 software.3.The chemical structures of the polysaccharides through isolating and purifying that had the bioactivity of the cell migration were characterized by the HPLC,GC-MS and IR analysis.4.We incubated the IEC-6 cells that were migrating by the fluorescent dye-DiBAC4(3), then test their average fluorescence intensity of membranepotential(Em) by the flow cytometry.5. We incubated the IEC-6 cells that were migrating by the fluorescent dye-Fluo-3, AM, then observed the intracellular free calcium concentration [Ca2+]cyt and take their photos by the Laser scanning confocal microscope. After that, analysed the average fluorescence intensity of every photo by Image pro-plus 6.0 software.6.We tested the effect of the Sijunzi decoction polysaccharides on the mRNA and proteins expression levels of Kv1.1, TRPC1, PLC-y1, RhoA, Rac1 and Cdc42 in the process of the cells migration by the technology of the RT-Qpcr and Western-blot. Result1.The pharmaceutical chemicals of the Sijunzi Decoction were extracted and isolated by heating reflux and ethanol precipitation, removal of protein by sevag method, DEAE-52 fiber column and Sephadex G-100 gel column chromatography to get the Sijunzi Decoction total polysaccharides, the Sijunzi Decoction crude polysaccharides, D1 and D149, respectively. The suCar content (glucose) of 4 kinds of polysaccharide samples were 71.4%,75.5%,81.0% and 98.5% respectively by Phenol-sulfuric acid method, the purity of D1 and D149 were 86.43% and 98.62%, respectively by HPLC-GPC, and the lowest effective doses of promoting cell migration were 200,100,50 and 30 mg/L, respectively. The results show that after different purification steps, the suCar content and purity of the Sijunzi Decoction were increased, and the effect of the drug was enhanced.2.The data from our research demonstrated that the Sijunzi decoction polysacchride(D149) were a sort of homogeneous polysaccharide with 4199 in molecular weight, and was composed of glucose, robose, xylose, Calactose, mannose, arabinose in molar ratio of 18.7:2.1:1.3:1.0:7.4:6.7. FT-IR showed that D149 processed the characteristic absorption peak of polysacchrides, and proved that it had the furanose and pyranose. The results preliminarily characterized the structure of the Si-jun-zi decoction polysacchrides(D 149).3.We found the mechanism that D149(30 mg/L-120 mg/L) promoted cell migration by the polyamine-mediated signaling pathway:①D149 could promote the cells migration through increasing the expression of Kv1.1 mRNA and proteins, causing Em hyperpolarized; in the presence of DFMO, D149 could reserve the expression of Kv 1.1 mRNA and proteins to the normal, then Em repolarized. ②D149 could increase the expression of TRPC1 and PLC-γ1 mRNA and proteins, and [Ca2+]cyt elevated in the process of cells migration; in the presence of DFMO, D149 could reserve the expression of TRPC1 and PLC-yl mRNA and proteins to the normal, then [Ca2+]cyt elevated to the normal; In free calcium medium, the number of cells migration obviously decreased, compared with the control medium. The number of cells migration obvious increased by adding ionomycin in free calcium medium. D149 could promote cells migration, but the numbers were obviously lower then the control medium group. ③D149 could increase the expression of Rho GTPase(RhoA, Racl and Cdc42)mRNA and proteins in the process of cells migration; in the presence of DFMO, D149 could reserve the expression of Rho GTPase(RhoA, Racl and Cdc42) mRNA and proteins to the normal. ConclusionThe findings above suggested the mechanism that D149 promoted gastrointestinal mucosal early reconstruction could be related to the polyamine content in cells after wounding. This study provided evidences for exploring pharmacodynamic materials of the Sijunzi and mechanism action in improving cells migration abilities. |
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