Study On Mechanism Of ’Tonifying Fire To Supplement Earth And Promote Tissue Regeneration’ Method To Repair Intestinal Mucosal Mechanical Barrier In Ulcerative Colitis With Spleen Kidney Yang Deficiency

ObjectiveBy establishing an experimental model of ulcerative colitis（UC）rats with spleen and kidney yang deficiency,observe the effects of Qifulizhong Enema Decoction on the pathological changes of colon tissue,intestinal mucosal permeability and intestinal epithelial tight junction related proteins Occludin and JAM-A in UC rats,and explore possible mechanism to provide new strategy and theoretical basis for the treatment of UC.Methods70 male SD rats were randomly divided into a normal control group,model group,Qifulizhong Enema Decoction high,medium and low dose group(3,1.5,0.75 g·mL^-1),mesalazine group(0.03 g·mL^-1).Except the normal control group,the UC rat model of spleen-kidney yang deficiency was established by the ways that‘Rhubarb free Decoction was given by gavage+trinitrobenzene sulfonic acid（TNBS）-55%ethanol enema’.The rats treated by enema for 2 weeks.At the end of the experiment,blood was collected and colon tissue was taken.The UC rats were evaluated and scored.Use hematoxylin-eosin（HE）staining observing the pathological changes of colonic tissue,transmission electron microscope（TEM）observing the ultrastructure of colonic epithelial cells,and the enzyme-linked immunosorbent assay（ELISA）detecting D-lactic acid（D-LA）and diamine oxidase（DAO）content of Serum.Use immunohistochemistry（IHC）and Western blot methods detecting the expression of tight junction proteins in colon tissue for example Occludin and JAM-A.Results1.The effect on the general condition of ratsThe model group rats have unformed stools,decreased weight,lethargy,hunched back,and significantly reduced food intake.After treatment with Qifulizhong Enema Decoction,the symptoms of rats in each dose group alleviated to varying degrees,and the diet increased.Stool gradually formed,the number of stools decreased,and the activity level improved.The DAI score was significantly lower than the model group（P<0.05,P<0.01）.2.Effect on HE staining of colon tissueHistopathology of the colon of rats in the model group showed obvious shedding and necrosis of mucosal epithelial cells,changes in crypt structure and a decrease in number,and a large number of inflammatory cells infiltrated.After treatment with Qifulizhong Enema Decoction,the structure of the colon mucosal epithelium of rats in each dose group was relatively complete,and the lamina propria glands were arranged more neatly which were improved compared with the model group,and the infiltration of lymphocytes,neutrophils and other inflammatory cells was reduced.The ulcer surface is reduced and more limited.The overall score of TDI decreased compared with the model group（P<0.05,P<0.01）.3.Influence on the ultrastructure of intestinal epithelial cellsThe space between the intestinal epithelial cells of the model group was significantly widened,the tight junction structure was loose and fuzzy,the cell junction structure was destroyed,and the cell free surface was sparsely microvilli.After treatment with Qifulizhong Enema Decoction,The density of tight junctions between the intestinal epithelial cells of rats in each dose group increased,the structure was clear,the intercellular space was narrowed,and the microvilli on the free surface of the cells were arranged more neatly.4.Influence on serum D-LA and DAO contentThe levels of serum D-LA and DAO in the model group were higher than normal control group（P<0.01）,suggesting the mucosal permeability of UC rats increased.After treatment with Qifulizhong Enema Decoction,the D-LA and DAO levels of rats in each dose group decreased significantly（P<0.05,P<0.01）.It is suggested that the therapeutic effect of Qifulizhong Enema Decoction on UC model rats may be achieved by improving the permeability of the intestinal mucosa and repairing the intestinal mucosal barrier.5.Effect on the expression of TJ protein Occludin and JAM-A in colon tissueThe expression of Occludin and JAM-A protein in the model group colon tissue was significantly lower than normal control group（P<0.01）.After treatment with Qifulizhong Enema Decoction,the expression of Occludin and JAM-A protein of Qifulizhong enema Decoction high and medium dose groups could significantly up-regulate（P<0.05,P<0.01）,suggesting that Qifulizhong Enema Decoction,which has the effect of tonifying fire to supplement earth and promote tissue regeneration,can play a protective effect on the mechanical barrier of colonic mucosa by regulating the tight junction Occludin and JAM-A protein.Conclusions1.Using‘Rhubarb free Decoction+TNBS-55%ethanol’to replicate the UC rat model of spleen and kidney yang deficiency,the rats appeared typical symptoms of UC such as diarrhea and mucus pus and blood in the stool after modeling,as well as symptoms of spleen and kidney yang deficiency such as mental atrophy,liking to get together and crouching.Consistent with the clinical features of UC prolonged and repeatedly difficult to heal,it shows that the UC rat model of spleen and kidney yang deficiency was successfully replicated.2.Qifulizhong Enema Decoction can effectively relieve stool symptoms of UC rats,increase diet and activity,reduce intestinal inflammation and infiltration,and have a good therapeutic effect on UC of spleen and kidney yang deficiency.3.Qifulizhong Enema Decoction,which has the effect of tonifying fire to supplement earth and promote tissue regeneration,can effectively improve the permeability of the intestinal mucosa,increase the expression of tight junction proteins for Occludin and JAM-A.Repair the mechanical barrier of the intestinal mucosa and protect the integrity of the intestinal mucosa.