| Objective:By employing a comprehensive modeling approach,we established an experimental model of ulcerative colitis(UC)characterized by spleen and kidney yang deficiency concomitant with liver depression.This model was utilized to investigate the pharmacodynamics of Sishen pills combined with Tongxie Yaofang in treating UC of this particular subtype.Additionally,we employed network pharmacology to explore potential targets of Sishen pills combined with Tongxie Yaofang in treating UC with spleen and kidney yang deficiency concomitant with liver depression.By leveraging network pharmacology to predict targets,we aimed to elucidate the underlying mechanisms of action of Sishen pills combined with Tongxie Yaofang in UC rats with spleen and kidney yang deficiency concomitant with liver depression.Methods:1.We obtained datasets with the accession numbers GSE36807,GSE87466,and GSE53306 from the GEO database to analyze the differential genes between ulcerative colitis and healthy individuals.These differentially expressed genes were then intersected and de-duplicated with ulcerative colitis gene targets collected from databases such as GeneCard,OMIM,DrugBank,and DisGeNET to identify disease-specific targets for ulcerative colitis.To identify the active ingredients of Sishen pills combined with Tongxie Yaofang,we searched databases such as TCMSP and BATMAN-TCM.Subsequently,we constructed a protein-protein interaction(PPI)network of the UC targets for the treatment of ulcerative colitis with Sishen pills combined with Tongxie Yaofang.From this network,we screened for the core targets of Sishen pills combined with Tongxie Yaofang in the treatment of UC.To gain insights into the potential pathways involved,we performed enrichment analysis using KEGG and GO databases,thereby preliminary screening the possible pathways through which Sishen pills combined with Tongxie Yaofang may exert their therapeutic effects in ulcerative colitis.2.The rats were randomly divided into six groups:the normal group,the model group,the positive drug group(mesalazine,0.4 g/kg),and the high,medium,and low dose groups of Sishen pills combined with Tongxie Yaofang(26.2 g/kg,13.1 g/kg,6.55 g/kg).After one week of adaptation,except for the normal group,the remaining groups underwent ice-cold senna infusion,rectal distention,and intramuscular injection of hydrocortisone for four weeks.Starting from the 15th day,each treatment group received the corresponding medication,while the model group and normal group were administered an equal volume of sterile water for two consecutive weeks.On the 21st day of the experiment,ulcerative colitis characterized by spleen and kidney Yang deficiency concomitant with liver depression was induced by colonic instillation of TNBS mixed with ethanol,while the normal group received a saline solution.Two days before tissue collection,a sucrose preference test was conducted,and on the day before tissue collection,all groups were orally administered D-xylose.One hour after the last administration,blood was collected from the orbit,and serum was separated and stored.At the same time,hippocampus and colon tissues were collected.A portion of the colon tissue was fixed with 4%paraformaldehyde,and the pathological changes of the colon tissue were observed under a microscope after HE staining in all groups of rats.3.Serum samples were collected from the rats,and enzyme-linked immunosorbent assay(ELISA)was performed to measure the levels of tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6),gastrin(Gas),motilin(MTL),cortisol(Cort),and serotonin(5-HT)in the serum.Additionally,visible spectrophotometry was used to determine the concentration of Dxylose in the rat serum.These analyses aimed to validate the successful establishment of the ulcerative colitis model characterized by spleen and kidney Yang deficiency concomitant with liver depression and evaluate the therapeutic efficacy of Sishen pills combined with Tongxie Yaofang.4.Real-time polymerase chain reaction(PCR)was used to detect the expressions of phosphatidylinositol-3-kinase(PI3K),protein kinase B(AKT),brain-derived neurotrophic factor(BDNF),and tyrosine kinase receptor B(TrkB)mRNA in the colon and hippocampus.5.The protein expression levels of PI3K,AKT,BDNF,and TrkB in the colon and hippocampus of each group were detected using Western Blot.Results:1.By integrating network pharmacology and bioinformatics,a preliminary exploration of the therapeutic mechanisms of Sishen pills combined with Tongxie Yaofang in the treatment of ulcerative colitis was conducted.In the analysis of protein-protein interaction(PPI)network,the most connected target was AKT.The involved gene functions primarily include response to exogenous substances,organic acid catabolic processes,intracellular and extracellular substance transport,DNA binding transcription factor activity,ligand-activated transcription factor activity,and other related functions.The pathways implicated in this study mainly include lipid and atherosclerosis pathways,cancer pathways,PI3K-AKT signaling pathway,human cytomegalovirus infection,and other related pathways.2.After a certain period of modeling,rats in the model group showed reduced activity,lethargy,slowed weight gain,irritability,and noticeable fecal staining around the anus,indicating varying degrees of liver depression,spleen deficiency,and kidney yang deficiency.The administration of Sishen pills combined with Tongxie Yaofang at different doses generally improved the overall condition of the rats.Following the administration,the body weight of rats in each treatment group showed a significant increase(P<0.05).3.Under microscopic observation of colon samples stained with hematoxylin and eosin(HE),rats in the model group exhibited varying degrees of ulcers,inflammatory cell infiltration,and disrupted or even lost intestinal villi structure.In comparison,the positive drug(mesalazine)group and the high and medium dose groups of Sishen pills combined with Tongxie Yaofang showed a significant reduction in inflammatory cells and less severe damage to the intestinal villi structure.The ulceration was noticeably alleviated.Although the low dose group of Sishen pills combined with Tongxie Yaofang did not exhibit as significant improvements as the other treatment groups,there was still a certain degree of symptom relief observed compared to the model group.4.Compared to the normal group,the sucrose preference of rats in the model group significantly decreased during the sucrose preference test(P<0.01).However,when comparing with the model group,the high,medium,and low dose groups of Sishen pills combined with Tongxie Yaofang showed a significant increase in sucrose preference(P<0.01).In comparison to the normal group,the levels of TNF-α,IL-6,Gas,MTL,and 5-HT in the serum of the model group were significantly elevated(P<0.01),while the levels of Dxylose and Cort were significantly reduced(P<0.01).However,when comparing with the model group,the high,medium,and low dose groups of Sishen pills combined with Tongxie Yaofang exhibited a significant decrease in the levels of TNF-α,IL-6,Gas,MTL,and 5-HT in the serum(P<0.01).The medium dose group showed a decrease in 5-HT,and the low dose group showed a decrease in TNF-α,although the differences were not statistically significant.Additionally,when comparing with the model group,the serum levels of Dxylose and Cort in the high,medium,and low dose groups of Sishen pills combined with Tongxie Yaofang increased,and the differences between the high and medium dose groups were statistically significant(P<0.01).Although the low dose group showed an increasing trend in the levels of D-xylose and Cort,the differences were not significant.5.The real-time PCR results demonstrated that compared to the normal group,the mRNA levels of PI3K,AKT,BDNF,and TrkB in the colon tissue of rats in the model group were significantly elevated(P<0.01).However,when comparing with the model group,the high,medium,and low dose groups of Sishen pills combined with Tongxie Yaofang were able to significantly reduce the mRNA levels of PI3K,AKT,BDNF,and TrkB in the colon tissue(P<0.01).It should be noted that the decrease in BDNF in the low dose group was not statistically significant.In comparison to the normal group,the mRNA levels of PI3K,AKT,BDNF,and TrkB in the hippocampal tissue of rats in the model group were decreased(P<0.01).However,when comparing with the model group,the high,medium,and low dose groups of Sishen pills combined with Tongxie Yaofang were able to significantly increase the mRNA levels of PI3K,AKT,BDNF,and TrkB in the hippocampal tissue(P<0.01).It is worth noting that the increase in AKT and BDNF in the low dose group was not statistically significant.6.The Western blot results revealed that compared to the normal group,the expression levels of PI3K,AKT,BDNF,and TrkB in the colon tissue of rats in the model group were significantly increased(P<0.01).However,when comparing with the model group,the high,medium,and low dose groups of Sishen pills combined with Tongxie Yaofang were able to significantly reduce the expression levels of PI3K,AKT,BDNF,and TrkB in the colon tissue(P<0.01).It should be noted that the decrease in PI3K in the low dose group was not statistically significant.In comparison to the normal group,the expression levels of PI3K,AKT,BDNF,and TrkB in the hippocampal tissue of rats in the model group were significantly decreased(P<0.01).However,when comparing with the model group,the high,medium,and low dose groups of Sishen pills combined with Tongxie Yaofang were able to increase the expression levels of PI3K,AKT,BDNF,and TrkB in the hippocampal tissue(P<0.01).However,the increase in TrkB in the low dose group was not statistically significant.Conclusion:1.The combined modeling approach involving rectal instillation of Aloe vera gel,rectal irritation by clamping the tail,intramuscular injection of dexamethasone,and colonic instillation of 2,4,6-trinitrobenzene sulfonic acid(TNBS)mixed with ethanol successfully replicates a model of ulcerative colitis characterized by spleen-kidney yang deficiency and liver depression,which closely resembles the clinical condition.2.The combined use of Sishen Pill and Tongxie Yaofang exerts its therapeutic effect by regulating gastrointestinal motility,restoring adrenal cortex function,improving intestinal mucosal permeability,promoting small intestine absorption function,and reducing 5-HT secretion.This explains the scientific connotation of traditional Chinese medicine in treating ulcerative colitis characterized by spleen and kidney Yang deficiency in conjunction with liver depression,which involves tonifying the spleen and soothing the liver,warming the kidney,and strengthening the intestines.3.Sishen pills combined with Tongxie Yaofang may treat ulcerative colitis with spleen-kidney Yang deficiency and liver depression by regulating the PI3K、AKT signaling pathway. |
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