The Study Of Astragalus Polysaccharides Regulate The Migration Of IEC-6 Cells Through Polyamines And [Ca²⁺]_cyt

Research BackgroundGastrointestinal mucosal lesion is one of the pathogenesis basis of digestive diseases.A variety of gastrointestinal diseases exist pathological changes of gastrointestinal mucosal injuries.Replenishing qi and invigorating spleen method have exact therapeutic effects on chronic gastritis,peptic ulcer,inflammatory bowel disease and other digestive diseases patients who have spleen deficiency syndromes.The therapeutic effects of Yi-qi-jian-pi herbs on gastrointestinal related diseases may be closely related to their protect effect on gastrointestinal mucosal barrier intergrity and their promote effect on mucosal repair after injuries.Gastrointestinal mucosa is a rapidly self-renewing tissue in the body.The process that gastrointestinal mucosal reseal wounds consists of restitution phase and subsequent repair phase.The rapid restitution phase is identified by remaining active epithelial cell migrate into the defect and reseal superfical wounds,thus this process does not require cell proliferation.Studies on IEC-6 cells have shown that polyamines are necessary for cell migration.Increased cellular polyamines could improve the expression of Kv1.1,therefore hypolarize membrane potential and enhance driving force of Ca²⁺influx.Store-operated Channe?SOC?is one of the main channels for Ca²⁺ influx.TRPC1 function as SOC channel,it play important role in Ca²⁺ influx.STIM1 is known as a Ca²⁺ sensor that plays essential role in the activation of SOCs.In response to Ca²⁺ store depletion,STIM1 translocates to the plasma membrane and interact with TRPC1,then induce the formation of STIM1/TRPC1 complex,which improving TRPC1 mediated Ca²⁺ signaling and promoting cell migration.STIM2 is an inhibitor of SOC.Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca²⁺ signaling by altering ratio of STIM1 to STIM2.Acativation of Rho-GTPase followed by elevation of[Ca²⁺]cyt,increasing phosphorylation of MLC,stimulating stress fiber formation and cytoskeleton rearrangement,finally promoting cell migration.Our previous studies found that extracts?polysaccharide,flavonoid and so on?from Chinese herbs,which replenishing qi and invigorating spleen,such as Atractylodes,Codonopsis,Astragalus and Licorice and polysaccharide extracted from Sijunzi Decoction could promote the migration of IEC-6 cell,the mechanism of which was related to its effect on the polyamine-mediated signaling pathway and the regulation of Ca²⁺was the key factor.ObjectiveTo investigate the mechanism and material basis of the repair effect of Astragalus on gastrointestinal mucosal injury,the Astragalus polysaccharide component with high pharmacological activity was selected as the drug and its effects on the polyamine-mediated signaling pathway(especially the regulation of Ca²⁺)was observed during the migration of intestinal epithelial cells?IEC-6?,so as to provide evidence for its clinical treatment of gastrointestinal mucosal injury.Methods1.Extraction,isolation and purification of Astragalus polysaccharides:Polysaccharides samples No.1-No.4 was obtained from herbal medicine Astragalus Membranaceus by water extraction and alcohol precipitation,sevag de-protein,DEAE-52 cellulose column chromatography and sephadex dextran column chromatography.Phenol-sulfuric acid method was applied to determine the content of various polysaccharide samples from Astragalus.High performance liquid chromatography was used to analyzethe purity of the samples No.3 and No.4 of Astragalus polysaccharides.2.Selection of the testing drug:The effects of four samples of Astragalus polysaccharides on cell migration were investigated by the IEC-6 cell scratching migration model,and the polysaccharide samples with the best pharmacological activity were selected for the following experiments.3.Methods used to measure the relevant indicators of the mechanism of action:?The expression of TRPC1,STIM1 and STIM2 mRNA was quantited by real-time quantitative PCR.?The expression of Kv1.1?TRPC1?STIM1?STIM2?RhoA?Rac1?Cdc42 proteins was determined by western blot.?The cell membrane potential?Em?,intracellular free calcium level([Ca²⁺]cyt)was measured by flow cytometry.The expression and distribution of STIM1 protein were detected by immunofluorescence.The expression of the complex protein of STIM1/TRPC1 and STIM1/STIM2 were determined by co-immunoprecipitation.Results1.Extraction,isolation and purification of Astragalus polysaccharides:? The yield of Astragalus polysaccharides 1,2,3 and 4 were 5.64%,3.34%,1.50%and 6.5 ‰respectively.? The polysaccharide content in Astragalus polysaccharides 1,2,3 and 4 were 31.0%,50.9%,66.3%and 40%,respectively.Polysaccharide content in the 3rd sample of Astragalus polysaccharide is highest.2.Effects of Astragalus polysaccharides on cell migration:All the four samples of Astragalus polysaccharides were able to promote cell migration.The 3rd sample of Astragalus polysaccharide showed the best effect on promoting cell migration,therefore 3rd sample of Astragalus polysaccharide was selected as testing drug.The experimental dose is 20mg/L to 160mg/L.3.Effects of the 3rd sample of Astragalus polysaccharide?here in after referred to as "Astragalus Polysaccharides"?on cell migration:Astragalus Polysaccharides could promote cell migration and reverse the inhibitory effect of DFMO?a polyamine synthesis inhibitor?on cell migration,which suggested that the promoting effect of Astragalus Polysaccharides on cell migration was related to the increased intracellular polyamine content.4.Effects of Astragalus Polysaccharides on regulation of Ca²⁺ in polyamine signaling pathway during cell migration?1?Effect on the upstream factors of Ca²⁺ regulation:? Increase the expression of potassium channel protein Kv1.1;reverse the inhibiting effect of DFMO on Kv1.1;?Increase the cell membrane potential,induced the membrane potential hyperpolarization,and reverse the DFMO-induced membrane potential depolarization.It is indicated that Astragalus Polysaccharides enhance the driving force for Ca²⁺ influx through activating voltage-dependent potassium channel and elevating membrane potential.?2?Effect on the factors regulated by Ca²⁺:?Promote Ca²⁺ sensitor STIM1 translocate to plasma,reverse the inhibitory effect of DFMO on the translocation of STIM1 and increase the expression of STIM1 in the membrane;? Enhance the expression of STIM1 mRNA and protein,reverse the inhibitory effect of DFMO on the expression of STIM1 mRNA and protein;Decrease the expression of STIM2 mRNA and protein,and inhibit the enhenced expression of STIM2 mRNA and protein induced by DFMO.?Increase the expression of TRPC1/STIM1 protein complex and decrease the expression of STIM1/STIM2 protein complex.Increase the expression of TRPC1 and STIM1 protein in TRPC1/STIM1 complex when DFMO loaded.Reduce the expression of STIM2 protein in STIM1/STIM2 complex when DFMO loaded.Suggesting that Astragalus polysaccharides affect TRPC1-mediated Ca²⁺ signaling by regulating Ca²⁺sensitors.?3?Effects on calcium chaniel protein and[Ca²⁺]cyt:? Encence the expression of mRNA and protein of calcium channel TRPC1.Decrease the inhibitory effect of DFMO on the expression of mRNA and protein of TRPC1.? Increase[Ca²⁺]cyt,reverse the reduction of[Ca²⁺]cyt induced by DFMO.If extracellular Ca²⁺removed?culture without calcium?,not only the cell migration number in control group decreased significantly,the effect of Astragalus polysaccharide on promoting cell migration was also significantly weakened.Suggesting that Astragalus Polysaccharides promote cell migration by enhancing TRPC1-mediated Ca²⁺ influx and increasing[Ca²⁺]cyt.?4?Effect on the downstream factors regulated by Ca²⁺:Promote the expression of Rho-GTPase protein?RhoA,Rac1,Cdc42?.Reverse the inhibiting effect of DFMO on the protein expression of RhoA,Racl and Cdc42.It suggested that Astragalus polysaccharides could promote cell migration by increasing Rho-GTPase.ConclusionAstragalus polysaccharide is one of the effective components in Astragalus that promoting cell migration.The mechanism of its action mechanism is related to its effect on polyamine-mediated signaling pathway during cell migration,in which Ca²⁺ is one of the key regulator that regulating the upstream and downstream targets of[Ca²⁺]cyt could promote cell migration.The results of the study can provide reference for the mechanism of Yi-qi-jian-pi herb Astragalus of protecting gastrointestinal mucosa from injury.