Study On Mechanism Of Guchang Zhixie Pill Acting On Ulcerative Colitis With "Liver Depression And Spleen Deficiency Syndrome Type"

Objective: To uncover mechanisms and molecular targets of Guchang Zhixie Pill(GCZXP)acting on UC with TCM syndrome of liver depression and spleen deficiency in silico and vivoMethods: The chemical components and related targets of six traditional Chinese medicines were extracted from the Traditional Chinese Medicine Datasets.Differential genes were acquired after R packages processed UC datasets,and the disease targets were obtained from Genecards database.The intersection target of the above three was regarded as a potential therapeutic targets.PPI network of therapeutic targets was constructed via String database,and then key genes and hub genes were obtained via Mcode and cyto Hubba apps in the Cytoscape.We used Cytoscape to build a multi-dimensional network of "TCMs-active ingredients-core targets-disease".We calculated the degree value in the network to get the top three compounds as the core components of GCZXP acting on UC.Enrichment analysis and the key transcription factors were carried out.The expression profiles of UC dataset were obtained by R packages,and the proportion of immune cells in normal group and UC group was calculated by CIBERSORT.T test(P <0.05),LASSO regression and Random Forest claasification were used to screen characteristic immune cells.The correlation between hub genes and immune cells was evaluated by Pearson correlation.We verifed the significant difference between normal and UC groups in each dataset.We built a variety of machine learning diagnosis models and screened the best models through R package "caret",compared with logistic regression model.We drew diagnostic nomogram and clinical decision curve.Auto dock Vina were used to molecule docking.The UC rats with liver depression and spleen deficiency was established by restraint stress,4%DSS solution free drinking and fasting every other day.Open field test and sugar water preference test were carried out on day 0,21 and 33 of treatment.DAI index of rats in each group was evaluated at the end of treatment.He staining was used to evaluate the colon injury in rats;HE staining was used to evaluate the colon injury in rats;Inflammatory factors were detected in colonic tissue and serum by Elisa.The expression levels of NF-κB p65,IKBα and Bcl2 were tested by Western Blot.Result: In this study,there were 51 active components of GCZXP against UC,including 3core components such as quercetin,kaempferol and berberine,and regulatimg 13 core targets including IL1β,CXCL-8 and PTGS2.GO enrichment showed that GCZXP involved the reaction to lipopolysaccharide,the reaction to inflammation,the interaction of chemokines,etc.KEGG enrichment analysis showed that it was mainly through NF-κ B,Toll-like and NOD-like signal pathways.The prediction of key transcription factors is closely related to NF-κ B p65 and IKBA.Immune infiltration analysis predicted that there were significant differences in 15 kinds of immune cells between the two groups by T test,18 kinds of immune cells were obtained by LASSO regression as well as 11 kinds of them in RF modle.The three methods mentioned above intersected.The result showed that a total of 8 kinds of immune cells were involved in M1 macrophages,M2 macrophages,neutrophils,etc.The correlation study between the three hub genes and eight immune cells showed that they were positively correlated with M1 macrophages,neutrophils and activated memory CD4 cells,and negatively correlated with plasma cells..The best diagnostic model obtained through package R “caret” screening was SVM model.In the training set and verification set,the AUC of SVM model was higher than that of logistic model,and the clinical decision curve showed a well diagnostic effect,which indicates that the three hub genes have good diagnostic and predictive potential.The results of molecular docking showed that the binding energies of quercetin,kaempferol and berberine with hub genes as well as key transcripts in GCZXP were all less than-5 kcal/mol,which was higher than that of mesalazine.Animal studies predicted that GCZXP coould significantly reduce the DAI in UC rats,increase the body weight,and allevate intestinal bleeding.The results of Two-Bottle choice and open field proved that GCZXP could increase the intake rate of sugar water,the number of upright and horizontal crossing,as well as relieve the symptoms of liver depression and spleen deficiency.HE staining proved that both high-dose GCZXP group and positive drug mesalazine groups could significantly improve intestinal cell injury,restore intestinal crypt structure and villus length,and reduce intestinal inflammation in UC rats.The results of ELISA predicted that GCZXP could significantly reduce the concentrations of IL-1 β and CXCL-8 in colon tissue and serum,reduce the concentration of COX-2 in colon tissue,increase the content of SOD in colon tissue,and improve inflammatory injury.Western-blot test showed that GCZXP could act on NF-κ B signaling pathway,inhibit the activity of NF-κ B p65,and increase the expression of IKBa and anti-apoptotic protein Bcl-2.Conclusion: GCZXP reduced the release of cytokines and regulated Bcl-2 in the treatment of UC by blocking NF-κB signaling pathway to prevent cell necrosis,promote ulcer healing,and improve the symptoms of liver depression and spleen deficiency.