| Polyamines can protect mucous membrane barrier function by promoting gastrointestinal mucosa damage repair as well as regulating the formation and stabilization of tight junction and the adherin junction between cells.Gastrointestinal mucosal injury is a common pathological change in spleen deficiency syndrome.Sijunzi decoction is the representative of Yiqi Jianpi method,which is usually used to improve the symptoms of spleen deficiency syndrome of various gastrointestinal patients.The previous researchs of our group showed that,the aqueous extracts of Sijunzi decoction and the polysaccharides from Lanceolata,Astragalus,Atractylodes Macrocephala and Glycyrrhiza have protective effects on gastric mucosal injury in rats with stress ulcer.The mechanism of the effects is related to the increasing polyamine contents on gastric or intestinal mucosa.The researchs of migration of epithelial cells between damage repair of gastrointestinal mucosa show that,the extracts（polysaccharides,flavone,etc）of Atractylodes Macrocephala,Lanceolata,Astragalus,Glycyrrhiza and Sijunzi decoction can promote the migration of intestinal epithelial IEC-6 cells.The effect is connection with polyamine signal channel by a key point that is the regulation of Ca2+.Polysaccharides from Atractylodes Macrocephala can enhance the expression of the adherin junction protein E-cadherin,Beta-catenin,and Alpha-catenin in IEC-6 cells.However,the mechanism of gastrointestinal mucosa repair after injury by Yiqi Jianpi herbs from cell junction in vivo needs further study.ObjectiveTo observe the influences of Sijunzi Decoction（aqueous extract and polysaccharide）on gastric and intestinal mucosa in indometacin injury model rat.And explore the mechanism of the prevention and treatment of gastrointestinal mucosal injury by Sijunzi Decoction from the regulation of mucosal barrier and cell connective proteins by polyamine.Methods1 The extraction and purification of Sijunzi Dcoction aqueous extract and polysaccharide:Four herbs of Sijunzi Dcoction were extracted by water and analysed by the HPLC method;“Polysaccharide 1”and“Polysaccharide 2”from Sijunzi Dcoction were obtained through water extract-alcohol precipitation method and the sevag method.Phenol sulfuric acid method was used to detect polysaccharide contents of the two polysaccharides.HPLC method was used to analysis purity of“Polysaccharide 2”.2 The replication of the model of gastric and intestinal mucosal injury induced by indomethacin in rats:10mg/kg and 40mg/kg doses of indomethacin were injected subcutaneous for 7h,and the appropriate dose of gastric mucosal injury model was screened.4mg/kg and 8mg/kg doses of indomethacin were injected subcutaneous for 3d/7d to screen the appropriate dose of the small intestinal mucosal injury model.6mg/kg/d doses of indomethacin were injected subcutaneous for 3d/7d and 10g/kgdoses of Sijunzi Decoction aqueous extract were gavage for 3d/7d to screen the suitable time for the treatment of small intestinal mucosal injury.3 The prevention of Sijunzi Decoction to the gastric and intestinal mucosal injury in rats:（1）Study of gastric mucosal injury:Sijunzi Decoction aqueous extract（5g/kg,15g/kg）were gavage to the rats.And 40mg/kg doses of indomethacin were injected subcutaneous 1 hour later.The rats were executed at 7 hours.Collect the gastric mucosa and plasma.Record gastric mucosa injury score and analysis HE staining pathological grading;（2）Study of intestinal mucosal injury:6mg/kg/d doses of indomethacin were injected subcutaneous for6 days.And Sijunzi Decoction aqueous extract（5g/kg,15 g/kg）or polysaccharide（0.358 g/kg,1.074 g/kg）were gavage to the model rats between the 6 days.The rats were executed at 7 days.Collect the intestinal mucosa and plasma.Record the small intestinal mucosa injury score and analysis HE staining pathological grading.4 HPLC method was used to detect the polyamines content of mucosa in stomach and small intestine.5 D-lactic acid content in Plasma was detected by the kit.6 ELISA method was used to detect the total Ca2+volume of gastric and intestinal mucosa.7 Western blot and immunohistochemical methods were used to detect the tight junction proteins（Occludin,ZO-1,Claudin-3）and adherin junction Proteins（E-cadherin,Alpha-catenin,and Beta-catenin）in the gastric mucosa.The immunohistochemical method was used to detect the small intestinal mucosa tight junction proteins（ZO-1,Occludin,Claudin-3）and adherin junction proteins（Alpha-catenin,E-cadherin,and Beta-catenin）.Results1 The extraction and purification of Sijunzi Dcoction:The extraction rate of Sijunzi decoction aqueous extract is 35.42%.The ginseng saponin Rg1,Re,Rb1 and atractylodes lactone III,licorice glycosides can be detected by high performance liquid chromatography（HPLC）method.The extraction rate of Sijunzi decoction polysaccharide 1,2 are 8.77%and 7.16%respectively as well as the polysaccharide content are 92.34%and 81.59%respectively.According to previous results,polysaccharide 2 was elected as one of reagents because of a better efficacy.HPLC analysis shows that polysaccharides 2 has a concentrated molecular weight and is a high purity polysaccharide.But there are other smaller absorption peak,so it is not uniformity polysaccharide.2 The replication of the model of gastric and intestinal mucosal injury induced by indomethacin in rats:（1）Gastric mucosal injury model:indomethacin（40mg/kg）injected subcutaneous for 7h can cause acute gastric mucosal injury in rats,and it is selected as the model dosage of gastric mucosal injury model.（2）Model of small intestinal mucosal injury:Indomethacin（6mg/kg/d）injected subcutaneously for 6 days can injury intestinal mucosa in rats;Sijunzi decoction aqueous extracts（10g/kg）have prevention and treatment effectson the gastric mucosal injury in the model rats;Indomethacin（6mg/kg/d）injected subcutaneously for 6 days,and execute at the 7 day is selected as the model dosage of intestinal mucosal injury model.3 The influence of Sijunzi Decoction to the gastrointestinal mucosal injury in rats:（1）The Sijunzi decoction aqueous extracts（5 g/kg,15 g/kg）have prevention and treatment effects on the gastric mucosal injury in the model rats.（2）The Sijunzi Decoction aqueous extract（15 g/kg）and polysaccharide（0.358 g/kg,1.074 g/kg）have prevention and treatment effects on the intestinal mucosal injury in the model rats.Macroscopicscore and pathological scorew which compared with the model group were lower（P<0.05 or P<0.01）.At a equivalent dose of herbs,the effect of Sijunzi Decoction polysaccharide was better than that of aqueous extract.4 Effects of Sijunzi Decoction extract on gastrointestinal mucosa of polyamine content:（1）Sijunzi Decoction aqueous extract（5 g/kg,15 g/kg）can improve the gastric mucosa in rat models of polyamine（spermidine）content.（2）Sijunzi Decoction aqueous extract（15 g/kg）and polysaccharide（0.358 g/kg,1.074 g/kg）can improve the small intestinal mucosa in rat models of polyamine（spermidine）content.These imply that the prevention and treatment effects on the gastrointestinal mucosal injury of Sijunzi Decoction might be related to the improvement of polyamines content.5 Effects of Sijunzi Decoction extracts on the intestinal permeability（D-lactate in plasma）of model rats:（1）The study of gastric mucosal injury model by indometacin shows that the D-lactate in plasma of model group was increased significantly（compared with control group P<0.01）.This implys that indometacin can increase the intestinal permeability of rat.But Sijunzi Decoction aqueous extracts（5 g/kg,15g/kg）have no influence to the increasing D-lactate in plasma of model of rats.（2）The study of intestinal mucosal injury model by indometacin shows that the D-lactate in plasma of model group was increased significantly（compared with control group P<0.01）.And the D-lactate in plasma of the intestinal injury model rat was significantly reduced by the Sijunzi decoction aqueous extract（15 g/kg）and polysaccharide（0.358,1.074 g/kg）（compared with model group P<0.05 or P<0.01）for 6 days.These imply that Sijunzi Decoction can improve the increasing intestinal permeability that is cause by the intestinal permeability.6 Effects of Sijunzi Decoction extract on the total Ca2+volume of gastric and intestinal mucosa:（1）The study of gastric mucosal injury model by indometacin shows that the total Ca2+volume of gastric mucosa of the model group was decreased（compared with control group P<0.05）,which imply that gastric mucosal injury can cause a decrease of the total Ca2+volume.Sijunzi Decoction aqueous extract（5 g/kg,15 g/kg）can slightly increase the total Ca2+volume of gastric mucosa in gastric mucosa injury model of rat（P>0.05）at 8 hours.（2）The study of intestinal mucosal injury model by indometacin shows that the total Ca2+volume of intestinal mucosa of the model group was decreased slightly compared with control group（P>0.05）.And the total Ca2+volume of intestinal mucosa of the intestinal injury model rat was significantly increased by the Sijunzi decoction aqueous extract（15 g/kg）and polysaccharide（1.074 g/kg）（compared with model group P<0.05）.These imply that Sijunzi Decoction can improve the total Ca2+volume of intestinal mucosa of the model rat.7 Effects of Sijunzi Decoction extract on the tight junction and adherin junction proteins of gastric and intestinal mucosa of model rat:（1）The study of gastric mucosal injury model by indometacin shows that Sijunzi Decoction aqueous extract（5 g/kg,15 g/kg）can increase tight junction proteins Occludin,ZO-1,Claudin-3 and adherin junction proteins E-cadherin,Alpha-catenin of gastric mucosa in model rat（compared with model group P<0.05or P<0.01）,but not the Beta-catenin.（2）The study of intestinal mucosal injury model by indometacin shows that Sijunzi Decoction aqueous extract（15 g/kg）and polysaccharide（0.358 g/kg,1.074 g/kg）can increase tight junction proteins Occludin,ZO-1,Claudin-3 and adherin junction proteins E-cadherin,Alpha-catenin of intestinal mucosa in model rat（compared with model group P<0.05 or P<0.01）.And Sijunzi Decoction aqueous extract（15 g/kg）and polysaccharide（1.074 g/kg）can also increase the Beta-catenin（compared with model group P<0.05）.ConclusionSijunzi Decoction（aqueous extract and polysaccharide）have prevention and treatment effects on the gastric and intestinal mucosal injury by indomethacin.They can increase polyamine（spermidine）content of the gastric and intestinal mucosa in model rat,improve the expression of tight junction and adherin junction proteins in gastric and intestinal mucosa.They also have a improvement to the increasing intestinal permeability and the reduction of the total Ca2+volume of mucosa.These imply that the prevention and treatment effects on the gastric and intestinal mucosal injury by Sijunzi Decoction might relative to the influence in polyamines and mucosal barrier.The polysaccharide was one of the main effective components of Sijunzi Decoction in gastrointestinal mucosal protection.The results provide a reference for the study in mechanism of gastrointestinal mucosal protection of Yiqi Jianpi prescriptions. |
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