| BackgroundGastrointestinal mucosal injury is one of the pathogenetic basis of digestive disease.The healing of gastrointestinal mucosal injury includes early restitution and following-up repair,the cell migration runs through these two stages.Study on IEC-6(Intestinal epithelial cell)found that polyamines are necessary for cell migration,and[Ca2+]cyt is the key factor during polyamines-mediated cell migration.The increased[Ca2+]cyt would promote IEC-6 cell migration,in contrast,down-regulation of[Ca2+]cyt could inhibit cell migration.[Ca2+]cyt depends on the release of intracellular Ca2+ from endoplasmic reticulum,or the influx of extracellular Ca2+which is the critical mechanism involved in maintaining suitable[Ca2+]cyt.Phospholipase C-yl(PLC-yl)hydrolyses Phosphatidylinositol(4,5)bisphosphate(PIP2)into diglyceride and inositol(1,4,5)-tresphospate(IP3)which could combine with the IP3 receptor on endoplasmic reticulum,then implicates in the regulation of[Ca2+]cyt through modulating both Ca2+store release and Ca2+ influx.Besides,Racl could directly interacts with PLC-yl to form the Racl/PLC-yl complex which participates in the regulation of capacitative Ca2+entry(CCE)and[Ca2+]cyt.Canonical transient receptor potential-1(TRPC1)functions as store-operated Ca2+channels(SOC)and plays an important role in IEC-6 cell migration by controlling CCE and[ca2+]cyt.Furthermore,RhoA and Cav-1(caveolin-1)could respectively interact with TRPC1,and form the RhoA/TRPC1 or Cav-1/TRPC1 complexes,then accelerate the Ca2+influx mediated by TRPC1.The histopathological features of gastrointestinal mucosal injury are found in patients with spleen-deficiency syndrome for which the herbs of reinforcing Qi strengthening spleen could effectively reduce gastrointestinal mucosal injury.Our previous works showed that polysaccharide from sijunzi decoction could prevent indomethacin-induced intestinal mucosal injury in rats,and evaluated the content of both polyamines and Ca2+ in intestinal mucosa,suggesting that the mechanism was related to its effect on polyamines and Ca2+content.Polysaccharides from Sijunzi decoction(its sovereign drug is Panax ginseng).Astragalus membranaceus and Atractylodes macrocephala could promote cell migration in vitro,of which the mechanism was associative with polyamines signaling pathway including the regulation of Ca2+.Polysaccharide is one of the active ingredient in herbs of reinforcing Qi strengthening spleen.ObjectiveTo observe preventive and therapeutic effect of Panax ginseng polysaccharide on indomethacin-induced intestinal mucosal injury,and to investigate its the potential effect on Ca2+ related regulators during IEC-6 cell migration.Thus,further explore the pharmacodynamic mechanism of the herbs of reinforcing Qi strengthening spleen in promoting the healing of gastrointestinal mucosal injury,in order to provide a reference for its clinical efficacy in treating gastrointestinal mucosal injury.Methods1.Extraction,isolation and purification of Panax ginseng polysaccharide:samples of Panax ginseng polysaccharide NO.1-NO.3 were obtained from Panax ginseng by water extraction and alcohol precipitation,Sevag method of de-protein and DEAR-52 cellulose column.Phenol-sulfuric acid assay was carried out to determine the total carbohydrate of 3 samples.Lowery's method was used to detect the protein content of Panax ginseng polysaccharide 2.High-performance gel permeation chromatograph was conducted to analyzethe the purity of Panax ginseng polysaccharide 3.2.Cell migration assays and tested drug selection:IEC-6 cell scratching migration model were conducted to exam the effects of all three samples of Panax ginseng polysaccharide on cell migration,so as to select the polysaccharide with the well pharmacological activity for the following experiments.3.Effect of Panax ginseng polysaccharide on indomethacin-induced intestinal mucosal injury:indomethacin(5mg/kg,subcutaneous injection)to induce intestinal mucosal injury model in rats daily for 4 days.1 hour before subcutaneous injection,treatment of Panax ginseng polysaccharide(5g/kg or 15g/kg,dissolve in pure water)was administered via gavage daily for 4 days.At the fifth day,animals were sacrificed by anesthesia,the intestinal mucosal ulcer damage was recorded,and the mucosa was scraped for the detection of Ca2+content.4.Colorimetric determination of Ca2+ content of intestinal mucosa in rats.5.Detection of Ca2+ related regulator:?Western-blot was used to determine the expression of RhoA,Cav-1,TRPC1,Racl and PLC-?1 proteins.?Co-immunoprecipitation was carried out to detect the expression of RhoA/TRPCl,Cav-1/TRPC1 and Racl/PLC-yl complexes.?Observe the effect of NSC-23766(the GTP-Rac-1 inhibitor)on IEC-6 cell migration,and detect the expression of Racl,PLC-?l and Racl/PLC-yl complexes in cells treated with NSC-23766 by western-blot or co-immunoprecipitation.Results1.Extraction,isolation and purification of Panax ginseng polysaccharide:?The extraction yields of Panax ginseng polysaccharide NO.1-NO.3 were 6.50%,5.06%and 2.70%.?The total carbohydrate content in Panax ginseng polysaccharide NO.1-NO.3 were 65.25%,77.38%and 97.21%,in which the Panax ginseng polysaccharide 3 had the highest total carbohydrate content.2.Cell migration assays and tested drug selection:all the 3 samples of Panax ginseng polysaccharide could promote IEC-6 cell migration,Panax ginseng polysaccharide 2 was selected as the tested drug in vivo with two dose groups of 5g/kg and 15g/kg(according to the amount of crude herbs);Panax ginseng polysaccharide 3 was selected as the tested drug in vitro,four dose groups of 20,40,80 and 160mg/L were set up,and some experiments were set up with two dose groups of 80 and 160mg/L.3.Effect of Panax ginseng polysaccharide on indomethacin-induced intestinal mucosal injury:?Panax ginseng polysaccharide alleviated the indomethacin-induced intestinal mucosal ulcer damage in rats;? increased the Ca2+ content of intestinal mucosa in rats,indicating that the preventive effect of Panax ginseng polysaccharide on intestinal mucosal injury may be associated with the Ca2+content in mucosa,which also provided a research basis for the investigation of the effect of Panax ginseng polysaccharide on gastrointestinal mucosal protection in vitro.4.Effect of Panax ginseng polysaccharide on Ca2+related regulators during IEC-6 cell migration:?Panax ginseng polysaccharide promoted the expression of RhoA and TRPC1 proteins,and enhanced the level of RhoA/TRPC1 complex during cell migration.?Panax ginseng polysaccharide increased the expression of Cav-1 protein,and caused up-regualtion of RhoA/TRPC1 complex during cell migration.?Panax ginseng polysaccharide enhanced the expression of Rac1 and PLC-?1 protein,and elevated the level of Racl/PLC-?1 complex during cell migration.?NSC-23766 could significantly inhibit IEC-6 cell migration,and down-regulated the level of Rac1/PLC-yl complex.However,concomitant treatment with Panax ginseng polysaccharide could enhanced the expression of Racl,PLC-yl protein as well as Racl/PLC-yl complex,and reverse the effect of NSC-23766 on cell migration.These results suggest that the herbs of reinforcing Qi strengthening spleen may affect the Ca2+ influx by up-regulating the level of Ca2+related regulators during cell migration.ConclusionPanax ginseng polysaccharide could promote IEC-6 cell migration,of which the mechanism is related to its influence on polyamines pathway Ca2+related regulators;Panax ginseng polysaccharide also could alleviate the indomethacin-induced intestinal mucosal injury,and increased the Ca2+ content of intestinal mucosa in rats,suggesting that polysaccharide is one of the effective components of Panax ginseng.These findings provide the reference for the herbs of reinforcing Qi strengthening spleen for treating enteropathy characterized by mucosal damage in clinical practice. |
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