Effect Of Polysaccharide From Sijunzi Decotion On Ca²⁺ Related Regulators During Intestinal Epithelial Cell Migration

BackgroudYiqi jianpi herbs has the function of protecting gastrointestinal mucosa,which can promote the repair of gastrointestinal mucosal damage.Epithelial cell migration is an important step in the repair of gastrointestinal mucosal damage.It is found that yiqi jianpi prescription has a good therapeutic effect on the treatment of gastrointestinal mucosal damage,Sijunzi Decoction is the representative of yiqi jianpi prescription,Previous studies in our research group found that sijunzi Decoction can prevent gastric mucosal damage induced by stress ulcer in rats,which is related to the increase of gastric and small intestinal mucosal polyamine（spermidine）;Sijunzi Decoction can promote the migration of IEC-6 cells in the intestinal epithelium.The mechanism is related to the influence of polyamine signaling pathway,and calcium ion（Ca2+）is one of the key regulatory indicators.In the process of intestinal epithelial cell migration,the intracellular flow（SOCE）is one of the main methods of Ca²⁺internal flow.After exhaustion of polyamines,the intracellular free calcium ion([Ca²⁺]cyt)is reduced and the calcium pool in the endoplasmic reticulum is released,which can cause the transient increase of intracellular Ca²⁺,and the Ca²⁺in the cells was depleted;When calcium depletion,calcium store depletion induced influx of Ca²⁺that capacitive Ca²⁺flow（capacitative calcium entry,CCE）is influenced by many factors,TRPC1 as the SOC channel protein is important in the regulation of CCE,STIM1 as a transmembrane protein in the regulation of Ca²⁺has a significant effect in enhancing CCE,When Ca²⁺depleted in cells,STIM1 migrated from endoplasmic reticulum to cell membrane and formed STIM1/TRPC1 complex with TRPC1,which increased intracellular calcium flow and promoted cell migration.STIM2 is also one of the regulatory proteins of SOC,but with the opposite role of STIM1,it competes with TRPC1 for STIM1,forming STIM1/STIM2 complex,reducing TRPC1/STIM1 complex formation,regulating TRPC1 mediated Ca²⁺influx and regulating cell migration.Objective:Surrounding the effect of sijunzi Decoction on polyamine signaling pathway during intestinal epithelial cell migration to further observe its effects on Ca2+regulates related indicators,to interpret the mechanism of yiqi jianpi herbs sijunzi Decoction on the action of gastric mucosal damage repair.Method1、Sijunzi Decoction polysaccharides extraction,separation and purification:Sijunzi Decoction polysaccharides pieces through the water extraction and alcohol precipitation,and then by Sevag method to remove protein,reoccupy DEAE-52 on polysaccharide cellulose column chromatography,the three-step operation sijunzi decoction can be obtained respectively polysaccharide samples 1,2,3,sugar content of the polysaccharides by phenol sulfuric acid method;A high performance liquid chromatography（HPLC）method was used to detect the purity of sijunzi decoction.The choice of"Sijunzi Decoction polysaccharide 3"as a follow-up experiment（hereinafter referred to as"four gentlemen’s soup polysaccharide"）.2、The cell migration model was established to verify the efficacy of sijunzi decoction:In this paper,iec-6 cell migration pharmacological experiment model was established to observe the effect of sijunzi Decoction on cell migration,and the optimal dose of sijunzi Decoction was selected to promote cell migration.3、Western Blot were used to detect STIM1 and STIM2 protein expression.4、Immunofluorescence method was used to observe the expression of STIM1 protein and its migration to the cell membrane.5、The expression of STIM1/TRPC1 and STIM1/STIM2 complex protein was detected by immunoprecipitation.Results:1、Sijunzi Decoction polysaccharide on the effects of the IEC-6cell migration:sijunzi Decoction polysaccharide（40,80,160 mg/L）can promote the efficacy of cell migration（compared with the blank control group P<0.01）,and improve and reverse the phenomenon of cell migration speed slow caused by DFMO（polyamine synthesis inhibitors）(compared with DFMO load group（P<0.01）.2、The effect of Sijunzi Decoction polysaccharide on the expression of STIM1 and STIM2:the expression of STIM1 can be improved by Sijunzi Decoction（20,40,80,160 mg/L）,and the expression of STIM2（P<0.05）is reduced.The expression inhibition of STIM1 protein caused by DFMO was reversed,and the expression of STIM2 protein induced by DFMO was decreased.（the DFMO model group compared with the normal group P<0.05,and the comparison between Sijunzi Decoction group and DFMO model group was P<0.05）.3、The effect of SJZDP on the shift of STIM1 protein:SJZDP（80 mg/L）can promote the migration of STIM1 to the cell membrane and improve the delay and decrease of the displacement of STIM1 to the cell membrane caused by DFMO.4、The effect of SJZDP on the expression of protein complex TRPC1/STIM1 and STIM1/STIM2:Treatment with SJZDP could increase STIM1/TRPC1 protein complex expression and reduces STIM1/STIM2 protein complex expression,the inhibitory expression of STIM1/TRPC1 protein complex induced by DFMO was reversed,the elevation expression of STIM1/STIM2 protein complex induced by DFMO was reducted（compared with the model group P<0.05）.ConclusionSJZDP has the effect of promotes cell migration,The mechanism is related to its difference regulate Ca2+receptor positive and negative regulatory proteins（STIM1,STIM2）and protein complexes（STIM1/TRPC1,STIM1/STIM2）expression,and increases the Ca2+flowing mediated by TRPC1,so as to promote cell migration.