The Study On The Mechanism Of Astragalus Polysaccharides Promoting IEC-6 Cell Migration From The Perspective Of Ca²⁺ Regulation Of Polyamine Signaling Pathway

BackgroundSpleen deficiency is one of the main TCM Syndromes of many gastrointestinal diseases;The pathological changes of gastrointestinal mucosa injury can be seen in patients with spleen deficiency.Clinical and experimental studies show that Jianpi Yiqi prescription has the effect of repairing gastrointestinal mucosa injury.The gastrointestinal mucosa is connected with the outside,and the structure of the mucosa is easily damaged a.The repair and renewal of the gastrointestinal mucosa plays an important role in maintaining the gastrointestinal homeostasis.The repair of gastrointestinal mucosa after injury includes two stages:early stage of restoration and subsequent repair stage.The migration of intestinal epithelial cells on the early stage of restoration is the main formation.The subsequent repair includes cell migration,proliferation,differentiation,mucosal reconstruction and other processes.Polyamine plays an important role in the repair of gastrointestinal mucosa after injury.In the intestine epithelial cell migration pathway,polyamine regulates the free Ca²⁺concentration([Ca²⁺]cyt)in the cytoplasm to affect the cell migration;TRPC1 is a non voltage dependent cationic channel protein on the cell membrane,which is mainly involved in the regulation of Ca²⁺homeostasis in the cell;Caveolin(Cav1)is the regulatory protein of calcium channel;TRPC1 and Cav1 regulate[Ca²⁺]cyt mainly by storing the stored operated Ca²⁺channels(SOC).Phospholipase C?-1(PLC?-1)is a membrane related phospholipase,which is involved in regulating Ca²⁺release of intracellular calcium pool[Ca²⁺]cyt can activate the GTP binding proteins Rho A and Rac1,and affect the cytoskeleton remodeling and promote cell migration.Protein complexes formed by the interaction of calcium regulatory proteins such as TRPC1/Rho A,TRPC1/Cav1,PLC?-1/Rac1 and play an important role in regulating the cell migration process.The experimental study of animal and cell in the early stage of the research group showed that the effect of Jianpi Yiqi prescription(Astragalus,Atractylodes,Licorice,Ginseng,Sijunzi Decoction,etc.)on the repair of gastrointestinal mucosa injury was related to the effect of polyamines and its regulatory mechanism;However,the precise regulation mechanism of the protective effect of the gastrointestinal mucosa of the traditional Chinese medicine of invigorating Spleen and Qi needs further study.Research objectiveThe previous study of the research group found that the effect of Astragalus Polysaccharide on the migration of intestinal epithelial cells(IEC-6)was related to its effect on the polyamine signaling pathway,and the regulation of Ca²⁺was the key index.Based on this,the effects of Astragalus Polysaccharide on Ca²⁺regulatory protein complex of the migration of polyamine signaling pathway in IEC-6 cells were further observed to explore the mechanism of Astragalus in promoting the repair of gastrointestinal mucosa injury.Research methods1.Preparation of Astragalus crude polysaccharide(Astragalus Polysaccharide 1)The powder of 200g Astragalus was soaked in 12 times volume pure water for 2 hours,decocted for 2 hours,filtered with gauze,then decocted for2 hours,after that collected twice water extracted.Astragalus water extraction was centrifuged(the centrifuge conditions were set at 12000rpm,20min).The supernatant is concentrated to 1/5 of the original volume.Add 95%ethanol 5times the volume to the concentrate,refrigerate the alcohol mixture at 4?for the night,and vacuum filter the sediment.The precipitate obtained from alcoholysis is continued to be alcohol precipitation,and the above steps are repeated 3 times.The final extract was frozen and dried.2.Preparation of Astragalus deprotein polysaccharide(Astragalus Polysaccharide 2)Sevag reagent is composed of chloroform and n-butanol(volume ratio:5:1).The crude Astragalus polysaccharide was dissolved in 400ml ultra pure water,and sevag reagent with a volume ratio of 1/5 was added.The mixture was shaken for 15 minutes in case of light ventilation and remained at rest for 3 hours;The lower layer of milk white protein was removed after the liquid was completely layered.Repeat the above steps 5 times until no opal protein layer appears and the reaction is terminated.The collecting water layer is frozen and dried.3.Preparation of purified polysaccharide from Astragalus(Astragalus Polysaccharide 3)Weigh 2g"Astragalus polysaccharide 2"and add 50ml of pure water to dissolve,add cellulose column in steps.After sample addition,let the solution flow into the bottom layer of column and close the drain switch,and the solution will stay overnight and fully adsorb.The next day,the sample was eluted with 600ml pure water,and the sample was connected by automatic sample receiving device with flow rate of 1.5m L/min.The content of polysaccharide in the eluate was determined by phenol sulfuric acid method,the absorbance value was combined,and the sample was freeze dried.4.Determination of"Astragalus polysaccharide 3"by phenol sulfuric acid methodThe content of Astragalus polysaccharide 3 was determined by phenol sulfuric acid method.UV spectrophotometer was used to detect the concentration of glucose concentration absorbance standard curve with wavelength of 490nm,horizontal coordinate(X)and absorbance value of Y.Sample content determination:take 2m L of Astragalus polysaccharide 3sample solution in a volumetric flask,and parallel 3 parts,and operate according to the blank tube method.The samples of the eluent collected by Astragalus polysaccharide 3 were taken as 200?l.Add pure water to 2m L in each tube,and follow the steps of handling glucose standard.5.Detection of"Astragalus polysaccharide 3"map by HPLCChromatographic conditions:column:ultrahydraulic TM liner(7.8×300mm)water soluble gel column;Mobile phase:super pure water;Flow rate:0.8ml/min;The column temperature was 30?;Sample quantity 10ml.Standard curve preparation:take the dextran reference solution,inject the sample separately,record the peak time,take the retention time(RT)of the chromatographic peak as the horizontal coordinate,and the reference molecular weight pair value(Ig M)as the vertical coordinate,and carry out regression analysis.Determination of molecular weight:take astragalus polysaccharide 3 to prepare 1.5 mg/ml solution.The relative molecular weight of Astragalus polysaccharide 3 can be obtained by the regression equation made by standard curve.6.Cell migration experimentTaking"Astragalus polysaccharide 3"as the test drug,hereinafter referred to as Astragalus polysaccharide.Cells with 4×10⁵/ml density was inoculated on the six hole plate,2.5ml per hole;Culture in incubator;When the cell is full,use 200?l.The suction head of 200?Lpiper scratches along the direction of45 degrees vertically in the center of 6 hole plate;PBS flushing;Add 2.5ml of DMEM medium containing 2%serum.The effective concentration range of Astragalus polysaccharide promoting cell migration was divided into the following groups:normal group,Astragalus polysaccharide 20mg/L,Astragalus polysaccharide 40mg/L,Astragalus polysaccharide 80mg/L,Astragalus polysaccharide 160mg/L.The effective concentration of Astragalus polysaccharide was divided into the following groups:Normal group,DFMO+Astragalus polysaccharide 40 mg/L,DFMO+Astragalus polysaccharide 80 mg/L,DFMO+SPD 5?mol/L.The effects of Astragalus polysaccharide on the migration of si-Cav1 treated cells were observed as follows:Normal group,si-Cav1+Astragalus polysaccharide 40 mg/L,si-Cav1+Astragalus polysaccharide 80 mg/L,si-Cav1+SPD 5?mol/L;The effects of Astragalus polysaccharide on the migration of NSC 23766 treated cells were divided into the following groups:Normal group,NSC23766+Astragalus polysaccharide 40 mg/L,NSC 23766+Astragalus polysaccharide 80 mg/L,NSC 23766+SPD 5?mol/L;The effects of Astragalus polysaccharide on the migration of Rhosin treated cells by Rho A inhibitor were divided into the following groups:Normal group,Rhosin+Astragaluspolysaccharide40mg/L,Rhosin+Astragalus polysaccharide 80mg/L,Rhosin+SPD 5?mol/L.Each group was observed and photographed at 0h and 12h.Five fields of vision were collected for each hole,and the area before and after scratch was calculated by imag J.Relative migration area rate=[(0h area-12h area)/0h average area]×100%.7.Western blot method for detection of Rho A,Rac1,Cav1,TRPC1,PLC?-1 protein expressionThe effects of Astragalus psaccharide on the signal pathway of polyamine calcium ion in cell migration were divided into the following groups:Normal group,stragalus polysaccharide 40mg/l,astragalus polysaccharide 80mg/L,SPD 5?mol/L;The effects of Astragalus polysaccharide on the signal pathway of polyamine calcium ion in cell migration were studied as follows:normal group,DFMO+Astragalus polysaccharide 40 mg/L,DFMO+Astragalus polysaccharide 80 mg/L DFMO+SPD 5?mol/L.The lysate of each group was mixed with SDS,and the boiling denaturation was 5min.The protein mixture was added with 10%SDS PAGE electrophoresis;After electrophoresis,the protein gel was transferred to the PVDF membrane.Tbst strip with 5%skimmed milk powder.With TRPC1 and PLC?-1.The TBST4?of antibody of Cav1,Rac1 and Rho A were incubated overnight.The two groups were incubated with the strip for 1 hour at room temperature.The immune complex reacted in ECL chemiluminescence substratefor 5 minutes,and the protein bands were displayed by chemiluminescence imaging.8.Detection of TRPC1/Rho A,TRPC1/Cav1 and PLC?-1/Rac1 by immunoprecipitation expression of complexThe effects of Astragalus Polysaccharide on the protein complex of cellularmigration polyamine calcium signaling pathway were divided into the following groups:Normal group,Astragalus polysaccharide 40mg/L,Astragalus polysaccharide 80mg/L,SPD 5?mol/L.The effects of Astragalus Polysaccharide on the protein complex of polyamine calcium signaling pathway in the cell migration were investigated as follows:in the normal group,DFMO+Astragalus polysaccharide 40 mg/L,DFMO+Astragalus polysaccharide 80 mg/L,DFMO+SPD 5?mol/L.The cells were divided and centrifuged to get the supernatant.A small amount of cracking liquid was taken for Western blot analysis,and 1 was added to the remaining cracking liquid 1?g TRPC1 antibody(PLC?-1 antibody or Ig G of the same genus)added to the lysate,4?shake and incubate overnight.Take 10?L protein A+G Agarose beads were added to the lysate of the cells incubated overnight with the antibody.The antibody was incubated for 2-4 hours at 4?slowly,so that the antibody was connected with the protein A+G agarose beads.After immunoprecipitation,centrifugation was performed at 3000rpm for 3min,and supernatant was carefully removed.Agarose beads were washed with 1ml lysate buffer for 3-4 times.Join 15?L 2×SDS sample buffer solution,boiling water for 5min.The western-blotting analysis was used later.9.Detection of Ca²⁺level of cells by laser confocal microscopeThe calcium ion was detected by Fluo-4AM.After 12 hours of culture,the treated cells were added 1?l.The excitation wavelength and the emission wavelength of 516nm were detected by Fluo 4 AM under the Leika laser confocal microscope.Adjust the parameters to the same,take photos and record the calcium ion Fluorescence pictures,and take 5 photos in each group.The Fluorescence values were recorded using Image J analysis.10.Small RNA interference Cav-1 experiment(si-Cav1)Si-cav1 and Lipofectamine 2000 are used as ratio 100pmol:5?mol.The compound was mixed,incubated at room temperature for 20 minutes,and then added to the six hole plate containing 2.5ml of Opti-MEM medium(Invitrogen).After 48 hours of culture,the cells were harvested for subsequent analysis.Experimental results1.The results of the test of Astragalus Polysaccharide samples:the yield of Astragalus Polysaccharide 1,2 and 3 were 5.82%,3.38%,1.25%,32.20%,52.40%and 74.97%respectively;The content of Astragalus Polysaccharide 3was the highest,and then the related tests were carried out.The results of HPLC showed that Astragalus Polysaccharide 3 was heteropolysaccharide,and the peak proportion was;The relative molecular weight was 6130Da;The monosaccharide content of Astragalus Polysaccharide 3 was 47.13%,Arabinose 33.40%,Mannose 9.95%,Ribose 7%,Galactose 2.88%,respectively;The results showed that there would be different absorption peaks in different time periods.2.Dose screening of Astragalus polysaccharide samples on cell migration:in this study,"Astragalus polysaccharide 3"(hereinafter referred to as"Astragalus polysaccharide")was selected as the test drug for cell experiment.The results showed that Astragalus polysaccharide promoted the migration of IEC-6 cells(cell mobility of Astragalus polysaccharide 20mg/L compared with the blank group P<0.05,Astragalus polysaccharide 40,80,160mg/L compared with the blank group P<0.01),and the effective dose was20mg/L-160mg/L;The results showed that the best effect was40mg/L,80mg/L,so the following experimental dose was set as 40mg/L and80mg/L.3.The effect of Astragalus polysaccharide on cell migration:Astragalus polysaccharide(40mg/L,80mg/L)can promote cell migration and reverse the inhibition of DFMO(polyamine synthesis inhibitor,2.5mmol/L)on cell migration(P<0.05).It is suggested that the effect of Astragalus polysaccharide on cell migration is related to its effect on polyamines.4.The effect of Astragalus Polysaccharide on Ca²⁺regulatory protein and its protein complex of polyamine signaling pathway:(1)Astragalus polysaccharide(40mg/l,80mg/l)can improve Rho A,Rac1,Cav1,TRPC1,PLC?-1 expression(P<0.05 compared with normal group).The inhibition of DFMO on the above protein expression was reversed(P<0.05 compared with DFMO model group);(2)It can improve TRPC1/Rho A,TRPC1/Cav1 and PLC?-1/Rac1 expression and antagonizes DFMO on TRPC1/Rho A,TRPC1/Cav1,PLC?-1/Rac1 expression.It is suggested that the effect of Astragalus polysaccharide on cell migration is related to the effect of calcium library release and TRPC1 mediated Ca²⁺influx.5.The effect of Astragalus polysaccharide on the related indexes under specific loading:(1)It can reverse the decrease of expression of Cav1,decrease of Ca²⁺level and inhibition of cell migration caused by si-Cav1(P<0.05 compared with si-Cav1 model group);(2)The decrease of Ca²⁺and the inhibition of cell migration caused by Rhosin were reversed(P<0.05);(3)The decrease of Ca²⁺and the inhibition of cell migration caused by NSC23766were reversed(P<0.05).It suggested that Astragalus polysaccharide could improve the decrease of Ca²⁺level and cell migration inhibition caused by specific loading.Research conclusionAstragalus polysaccharide is one of the effective components of Astragalus to promote the migration of IEC-6 cells.The mechanism of action is related to the effect of the expression of Ca²⁺regulatory protein and its complex in the signal pathway of polyamine migration,thus promoting the TRPC1 mediated Ca²⁺flow.The results provide a reference for the protection of gastrointestinal mucosa of Astragalus,a Traditional Chinese Medicine,which can help to invigorate Qi and invigorate Spleen.