Studies On The Effect Of Panax Ginseng Polysaccharide On Intestinal Mucosal Barrier From The Mechanism Of Ca²⁺ And HuR Mediated By Polyamine

ObjectiveTo observe the effects of panax ginseng polysaccharide on polyamine and Hu R-mediated epithelial barrier in small intestinal epithelial cells（IEC-6）,this study uses indicators such as intercellular permeability,expressions of adherens junctions proteins and cellular Ca²⁺level as the observation indexes;It also combines the study of the effects of Panax ginseng polysaccharide on dextran sodium sulfate（DSS）-induced intestinal mucosal injury in mice.To provide a reference for exploring the healing effect and mechanism of panax ginseng on intestinal mucosal injury in traditional Chinese medicine for strengthening qi and invigorating the spleen.Methods1.The effects of panax ginseng polysaccharide on epithelial barrier related indexes under different experimental conditions（normal culture,DFMO-loaded and/or calcium-free culture）:（1）Phenol red permeation assay by Transwell was used to detect intercellular permeability;（2）Western Blot was used to detect the expression of adherens junctions proteins（E-cadherin,α-catenin,β-catenin）;（3）Laser confocal microscopy was used to detect cellular Ca²⁺level.2.The effects of panax ginseng polysaccharide on related indexes mediated by polyamine and Hu R.（1）Phenol red permeation assay by Transwell was used to detect intercellular permeability under inhibition of Hu R（CMLD-2 loading）;（2）The nucleoplasmic expression of Hu R under inhibition of polyamine（DFMO loading）was detected by Western Blot after extraction of cytoplasmic/cytoplasmic protein.3.The effect of Panax ginseng polysaccharide on DSS induced intestinal mucosal injury in mice:Mice were free to drink 3%DSS for 6 consecutive days to establish the Model,while panax ginseng polysaccharide（0.35g/kg and 0.70g/kg）and Mesalazine（0.15g/kg）,control group and model group were given pure water.DAI score（body weight,fecal characteristics and occultic blood condition）was performed for each mouse on the 4th and 6th days.After the experiment,colon was taken and weighed and its length was measured.Spleen index was calculated by weighing.The colonic tissues were stained by HE and then pathological scores were performed under microscope.The expression of adhesion junctions（E-cadherin,α-catenin,β-catenin）in colonic tissues were detected by immunohistochemistry.Results1.Effects of panax ginseng polysaccharide on intercellular permeability of IEC-6:（1）Panax ginseng polysaccharide（80,160 mg/L）decreased intercellular permeability in normal calcium-containing culture（compared with the control group,P<0.01）;（2）Calcium-containing culture+DFMO loading increased intercellular permeability（compared with the control group,P<0.01）,that is,inhibition of polyamines can affect cellular barrier function.Panax ginseng polysaccharide（80,160 mg/L）interfered with the increase in intercellular permeability due to DFMO（compared with the model group,P<0.05 or P<0.01）;（3）Calcium-free culture resulted in increased intercellular permeability（compared with the control group,P<0.01）,that is,the lack of extracellular Ca²⁺could affect the cell barrier function,and panax ginseng polysaccharide（80 and 160 mg/L）interfered with the increased intercellular permeability due to calcium-free culture（compared with the calcium-free culture model group,P<0.01）;（4）Intercellular permeability was elevated under calcium-containing culture+CMLD-2 loading（compared with the control group,P<0.01）,indicating that the epithelial barrier was also disrupted when Hu R regulation was inhibited;Panax ginseng polysaccharide（80 mg/L）ameliorated the elevated intercellular permeability under CMLD-2 loading（compared with the CMLD-2 model group,P<0.05）.These results suggest that the enhancement of epithelial barrier function by panax ginseng polysaccharide is related to its influence on the polyamine-mediated Ca²⁺and Hu R regulatory mechanisms.2.Effects of panax ginseng polysaccharide on the expression of adhesion-linked proteins:（1）Panax ginseng polysaccharide（40 mg/L or 80 mg/L or 160 mg/L）increased the expression of E-cadherin,α-catenin andβ-catenin proteins in normal calcium-containing culture（compared with the control group,P<0.05 or P<0.01）;indicating that panax ginseng polysaccharide has the effect of increasing the expression of adherens junctions proteins E-cadherin,α-catenin andβ-catenin;（2）DFMO loading decreased the expression of adherens junctions proteins E-cadherin andα-catenin（compared with the control group,P<0.05）,but had no significant effect onβ-catenin protein expression（compared with the normal calcium-containing culture group,P>0.05）;Panax ginseng polysaccharide（80 mg/L or 160mg/L）had an intervention effect on the reduced expression of E-cadherin andα-catenin due to DFMO loading（compared with the model group,P<0.05）,but no significant effect onβ-catenin protein expression（compared with the model group,P>0.05）.It was suggested that panax ginseng polysaccharide could improve the decrease of E-cadherin andα-catenin expression of adhesion-linked proteins caused by DFMO loading;（3）Calcium-free culture could cause the decrease of E-cadherin,α-catenin andβ-catenin expression（compared with the calcium-containing culture control group,P<0.01）;Panax ginseng polysaccharide（40mg/L or 80 mg/L）interfered with the decrease in E-cadherin andα-catenin protein expression due to DFMO（compared with the calcium-free model group,P<0.05 or P<0.01）,but had no significant effect onβ-catenin protein expression（compared with the calcium-free model group,P>0.05）.It was suggested that the lack of extracellular Ca²⁺could decrease the expression of adherens junctions proteins（E-cadherin andα-catenin）,and panax ginseng polysaccharide had a certain improvement on the decreased expression of E-cadherin andα-catenin proteins caused by calcium-free culture.The above results suggest that panax ginseng polysaccharide promote the expression of IEC-6 adherens junctions proteins（E-cadherin,α-catenin andβ-catenin）in relation to their effects on polyamine and Ca²⁺regulatory mechanisms.3.Effect of panax ginseng polysaccharide on cellular Ca²⁺levels:（1）Panax ginseng polysaccharide（40,80,160 mg/L）increased cellular Ca²⁺levels without loading（compared with the control group,P<0.05）;（2）DFMO loading caused a decrease in cellular Ca²⁺levels（compared with the control group,P<0.01）,and panax ginseng polysaccharide（40,80,160 mg/L）had an ameliorating effect on DFMO-induced panax ginseng polysaccharide（40,80,160 mg/L）had an ameliorating effect on the decrease of Ca²⁺level caused by DFMO（compared with the model group,P<0.01）.It is suggested that the effect of panax ginseng polysaccharide on increasing cellular Ca²⁺levels is one of the mechanisms by which it increases the expression of adherens junctions proteins and thus affects intercellular permeability.4.Effects of panax ginseng polysaccharide on the nucleoplasmic distribution of Hu R:（1）DFMO loading reduced the expression level of Hu R in the nucleus and increased the abundance in the cytoplasm（compared with the control group,both P<0.01）,suggesting that polyamine depletion could change the nucleoplasmic distribution of Hu R;（2）Panax ginseng polysaccharide could increase the expression of Hu R in the nucleus and decrease the content of Hu R in the cytoplasm under DFMO loading（compared with the model group,P<0.05 or P<0.01）,suggesting that panax ginseng polysaccharide could antagonize the increase of Hu R at cytoplasmic level and decrease of cytoplasmic level caused by DFMO loading,and its effect was similar to the addition of exogenous putrescine;（3）The content of total Hu R protein in cells was not significantly changed（compared with the control group,P>0.05）.5.Effects of panax ginseng polysaccharide（0.35g/kg or 0.70g/kg）on intestinal mucosal injury in mice caused by DSS.（1）Decrease the DAI score of model mice（compared with the model group,P<0.05）and improve the symptoms of intestinal injury;（2）Prolong the colon length and spleen index of model mice（compared with the model group,P<0.05 or P<0.01）;（3）Decrease the histopathological score of colon mucosa of model mice（compared with the model group,P<0.05）;（4）Increase the adherens junctions of colon tissue of model mice E-cadherin,α-catenin,β-catenin protein expression pathological histological scores in model mice（compared with the model group,P<0.01）.It is suggested that the effect of panax ginseng polysaccharide in preventing and treating DSS-induced intestinal mucosal barrier damage in mice is related to its effect on the expression of adherens junctions,which is mutually supported by the cell experiment results.ConclusionPanax ginseng polysaccharide could enhance epithelial barrier function through polyamine-mediated Ca²⁺and Hu R regulation by a mechanism related to its effects on intercellular permeability,promotion of the expression of adherens junctions proteins（E-cadherin,α-catenin andβ-catenin）,regulation of cellular Ca²⁺level and Hu R subcellular distribution;The effect of panax ginseng polysaccharide on colon mucosal injury induced by DSS in mice was related to its effect on the expression of E-cadheren,α-catenin andβ-catenin in the adherens junctions of colon tissue.The results of the study provide a reference to explore the protective effect of panax ginseng on intestinal mucosa in the Chinese medicine of benefitting the qi and strengthening the spleen.