The Protective Effect And Mechanism Of Glycyrrhiza Polysaccharide On Intestinal Mucosal Barrier

Radix Glycyrrhizae is a kind of herb in the genus Glycyrrhiza,which has the pharmacological properties of clearing away heat and detoxification,moistening lung and relieving cough,and enhancing qi.Glycyrrhiza Polysacchiade is a macromolecular carbohydrate extracted from licorice by traditional methods of water extraction,alcohol precipitation,enzyme promotion and microwave.It is also one of the main active components in licorice.Studies have shown that glycyrrhiza polysaccharide has a variety of biological effects,such as improving the body’s ability of immune regulation,anti-inflammation and sterilization,anti-oxidation,antivirus and so on.Glycyrrhiza polysaccharides should play a role in the gastrointestinal tract,because of the highmolecular weight of glycyrrhiza polysaccharides,it is difficult to be absorbed directly in the body to play an effect.The gastrointestinal tract is an important digestive organ and immune organ of the human body.It contains the mechanical barrier,immune barrier,biological barrier of intestinal mucosal immunity and the complex and varied intestinal flora which affects the function of the intestinal tract through multiple ways.Traditional pharmacological and pharmacodynamic studies of glycyrrhiza polysaccharide are difficult to elucidate the mechanism of action due to the particularity of the way glycyrrhiza polysaccharide exerts its biological activity.Based on the above conditions,this study aims to study the effects of glycyrrhiza polysaccharide on intestinal permeability and the improvement of related inflammatory response in mice,and verify its protective effect on mucosal barrier function in an in vitro cell model.The results are as follows:In this study,Xinjiang Glycyrrhiza was used as raw material to obtain crude licorice polysaccharide by traditional water extraction and alcohol precipitation method.After protein removal by Sevage method and dialysis method to remove small molecular impurities,glycyrrhiza polysaccharide（GPS）were obtained.The total sugar content in GPS measured by phenol-sulfuric acid method is 44.5%,and the protein content in GPS measured by Coomassie brilliant blue method is 4.5%.The content of reducing sugar in GPS measured by DNS method is 4.75%,and the molecular weight of GPS measured by Ubbelohde’s viscosity method is 1.27×105 Da.Acute ulcerative colitis（UC）mice induced by sodium dextran sulfate（DSS）were used as the animal model.The mice were treated with different doses of glycyrrhiza polysaccharide by gavage,and sulfasalazine（SASP）was used as the positive control drug.The intestinal permeability and inflammation indexes were detected.To analyze the effect of GPS on intestinal permeability and the therapeutic effect of inflammation in mice.①After GPS treatment,the clinical symptoms such as weight loss,colon shortening,spleen enlargement,diarrhea and stool bleeding in UC mice induced by DSS could be alleviated.②It also inhibits the release of pro-inflammatory cytokines IL-1,IL-6,and TNF-alpha,inhibiting the production of anti-inflammatory cytokines IL-10 and inhibiting the release of inflammatory cytokines.③GPS can improve the intestinal permeability of UC-induced acute ulcerative colitis mice.After GPS intervention,the contents of serum FITC-Dextran and D-lactic acid in mice were significantly reduced,and intestinal permeability was decreased,suggesting that the mucosal barrier may be repaired.According to the data of multiple indicators observed in this study,the therapeutic effect of GPS on DSS-induced UC mice was better than that of the positive drug SASP.Monolayer Caco2 cell model was used as the experimental object in vitro,and a monolayer cell barrier model was established on the Transwell compartment.CCK-8 assay was used to detect the effects of LPS and GPS on the viability and virulence of Caco2 cells.The passage volume of FITC in the monolayer barrier of Caco2 cells in Transwell compartment was detected.To investigate the protective effect of GPS on the monolayer barrier function of Caco2 cells induced by LPS.①On the first day of cell culture,the cells adhered to the wall and were in a transparent state.At the 7th day of culture,the monolayer of cells was basically fused and the intercellular junctions were close.After 10 days of culture,the cells differentiated obviously and differentiated into the brush border of small intestine epithelium.②Caco2 cells were used to establish a monolayer model of intestinal mucosal barrier.After LPS stimulation,the tight junction structure of epithelial cells could be destroyed,leading to the increase of intestinal mucosal permeability and the formation of intestinal barrier damage.CCK-8 method was used to detect the effects of LPS and GPS on cell proliferation and cell viability.The results showed that the concentration of LPS in this experiment could not inhibit the proliferation of Caco2cells,and had no effect on cell viability.Compared with model group,there was no difference in cell proliferation after LPS induction after different doses of GPS treatment（P>0.05）.There was no difference in cell proliferation in GPS alone treatment group（P>0.05）.The results showed that LPS and GPS at the concentration of this experiment did not affect the growth state of Caco2 cells and had little damage to cell viability.On this basis,we could carry out subsequent experiments.③Compared with the normal group,after LPS treatment,the content of FITC in BL side of Transwell compartment was significantly increased in model group,which proved that the permeability of monolayer barrier was significantly increased（P<0.01）.Compared with the model group,after GPS intervention,the content of FITC in BL side of Transwell compartment was significantly decreased（P<0.01）,indicating that GPS intervention could significantly improve the increased permeability of monolayer barrier caused by LPS.Compared with the normal group,without LPS-induced administration of GPS alone,the content of FITC in the BL side of Transwell compartment still decreased（P<0.01）,indicating that GPS can enhance the barrier function of monolayer cells.Conclusion:GPS can reduce the clinical symptoms of DSS-induced UC mice,reduce the intestinal permeability of UC mice,and regulate the level of inflammatory factors,thereby achieving the effect of inhibiting the inflammatory response.GPS has a protective effect on the mechanical barrier of intestinal mucosa,can reduce the permeability of intestinal epithelial cells,and relieve the damage of intestinal mucosa.In an in vitro cell model,it was verified that GPS can reduce the increase in the permeability of the monolayer cell barrier induced by LPS.This study provides an experimental basis for the treatment of UC and related gastrointestinal inflammatory diseases by GPS.The mechanism may be achieved through the effect of GPS on intestinal permeability and increased expression of tight junction proteins in epithelial cells to enhance the mechanical barrier function of the intestinal mucosa.The results of this study provide new experimental evidence for the mucosal repair and anti-inflammatory and anti-ulcer related effects of licorice.