Regulation Effect And Mechanism Of Danggui Buxue Decoction On Intestinal Mucosal Barrier In Immunosuppression Mice

Danggui Buxue decoction(DBD)is a classic prescription with hematopoietic functions,immunomodulatory and anti-tumor.It is composed of Astragali Radix and Angelica sinensis Radix.Modern studies have proved that DBD has the pharmacological effects of promoting hematopoietic function,regulating immunity and anti-tumor.At present,the immunomodulatory effects of DBD and its mechanism have not been thoroughly studied.In particular,the mechanism of immune protection in IS state is not clear.Therefore,this project takes mice as the research object.A mouse model of IS induced by cyclophosphamide(CY).Pathological examination,ELISA and molecular biology techniques were used to investigate the immunomodulatory effects of DBD.On this basis,the possible mechanism of action was explained through network pharmacology and experimental verification.The main results are as follows:1.In order to explore the regulation effect of DBD on mice body weight and spleen lymphocyte immunity.Forty SPF KM mice were randomly divided into four groups according to body weight,which were control group(saline),model group(CY+normal),positive group(CY+levamisole hydrochloride)and DBD group(CY+DBD).The results showed that DBD could alleviate CY induced decreases in body weight,thymus index and CD4+/CD8+ in lymphocyte subsets of spleen in mice.And the pathological enlargement of spleen caused by CY was significantly improved(P<0.05).The results suggest that DBD can alleviate CY induced immune damage,protect the immune organs of mice,and significantly enhance cellular immune function.2.By observing the intestinal morphology of mice and detecting the expression levels of intestinal mucosal barrier related genes and proteins.The effects of DBD on the intestinal mucosal barrier of IS mice were explored.The results showed that DBD could significantly increase the length of intestinal villi The results showed that DBD could significantly increase the length of intestinal villi and decrease the depth of crypt,and the ratio of villi length to crypt depth was significantly increased(P<0.05).At the same time,the level of SIg A secreted by intestinal tract increased significantly after DBD administration.And DBD up-regulated m RNA expression of ZO-1,MUC-2,Ig A and protein expression of ZO-1 in ileum tissue.The results suggest that DBD can slow down CY-induced intestinal damage and enhance intestinal mucosal immune function through physical and immune barriers.3.In order to further elucidate the role of DBD in intestinal mucosal immunity,from the perspective of biological barrier,16 S r RNA sequencing was performed on cecum contents of mice by 16 S sequencing technology.The results showed that compared with the model group,DBD could increase the relative abundance and species diversity of intestinal flora.And can increase Shannon,Chao1 and Simpson indexes.The ratio of Firmicutes to Bacteroidetes at the level of intestinal flora was significantly reduced.The results suggest that DBD can improve the imbalance of intestinal flora caused by CY,thus maintaining the stability of biological barrier in mice.4.In order to explore the active ingredients of DBD and its possible mechanism of action.This study used network pharmacological methods to predict the potential active ingredients,targets and signaling pathways of DBD in the treatment of IS diseases.And then verified by molecular docking technology.The results showed that the key effective ingredients of DBD included quercetin,kaempferol and formononetin.The core targets of DBD involved TP53,RELA,TNF,AKT1 and IL-6.According to KEGG analysis,PI3K-AKT signaling pathway may play a key role in DBD treatment of IS diseases.Molecular docking verified that each core target had good binding activity with corresponding ingredients.5.To verify the predicted results of network pharmacology,the active ingredients of DBD were identified by HPLC-MS and animal tests were conducted.On this basis,animal tests were carried out.The results showed that quercetin,kaempferol and formononetin were detected in DBD by HPLC-MS.The m RNA and protein expression levels of serum RELA,TNF and IL-6 were significantly down-regulated after DBD administration(P<0.05).Meanwhile,the m RNA expression level of PI3 K and AKT(P<0.05)and the PI3 K protein expression level of ileum were down-regulated(P<0.01).It is suggested that DBD plays an immunomodulatory role by regulating PI3K-AKT signaling pathway.In conclusion,DBD can improve the immunity of IS mice,reduce damage to the intestinal physical barrier and enhance immune barrier function,positive regulation of intestinal flora.On this basis,network pharmacology predicted that its mechanism of action might be related to the regulation of PI3K-AKT signaling pathway.