Research On The Mechanism Of Tanshinone ⅡA In The Treatment Of Inflammatory Bowel Disease Based On Bioinformatics And Metabonomics

Objective:First,through animal experiments to study the therapeutic effect of TanshinoneⅡA(Tanshinone ⅡA,Tan ⅡA)on inflammatory bowel disease(IBD),and then use bioinformatics to explore the possible targets and signal pathways of Tan ⅡA’s therapeutic effect on IBD,UPLC-Q-Orbitrap-MS technology detects changes in mouse colon tissue metabolites,finds out potential biomarkers,and analyzes related metabolic pathways.The linoleic acid pathway(CLA)shared by them was selected for in vivo and in vitro mechanism pathway verification experiments,which proved that Tan ⅡA has therapeutic effects on mouse IBD and can alleviate inflammation in mice by regulating the CLA pathway.Methods:4%sodium dextran sulfate(DSS)was used to induce mouse model of inflammatory bowel disease,and disease activity index(DAI)score was made by combining body weight,fecal shape and hematochezia of mice.The length and weight of the colonic tissue of mice were measured,and the histological score of the colonic histological section was made.The expression of inflammatory cytokines such as TNF-α,IL-1β,IL-6 and IL-10 in serum was detected by enzyme-linked immunosorbent assay(ELISA),indicating the efficacy of Tan ⅡA in the treatment of IBD.Based on Gene Expression Omnibus(GEO)database,the active differential genes of IBD were obtained.Gene functional annotation analysis(GO)and signaling pathway enrichment analysis(KEGG)were performed using David database to research the interaction between differential genes and signaling pathways,and finally to explore the molecular mechanism of the efficacy of Tan ⅡA.Metabolomics uses UPLC-Q-Orbitrap-MS to detect changes in serum metabolites in mice,find potential biomarkers through multivariate statistical analysis,and analyze related metabolic pathways.In vivo pathway verification,we used immunohistochemical staining and Western blot to detect protein expression levels related to the linoleic acid pathway in mouse colon tissue,such as:△9dehydrogenase,PPARγ,NF-κB,phosphorylation of IκBα,COX-2,iNOS etc.In vitro pathway verification,we selected the THP-1 acute myeloid leukemia cell line,PMA induced it to become a macrophage,and then used lipopolysaccharide(LPS)to stimulate it to produce an inflammatory response.By using the CCK-8 kit method,Western blot and qRT-PCR method,the cell activity and the linoleic acid the expression levels of a protein involved in signaling pathways were dectected respectively,such as△9dehydrogenase,PPARγ,NF-κB,IκBa protein expression and mRNA level.Through the above detection at the cellular level combined with animal experiments to verify the conjecture and hypothesis of drug action pathways.Results:4%DSS was administered continuously for 7 days,which successfully induced a mouse colitis model.Tan IIA significantly improved the weight of mice and reduced the DAI score.Tan IIA can significantly reduce the activity of MPO,a related inflammation marker in colitis tissue,and decrease intestinal permeability.The results of HE staining showed that compared with the model,the pathological changes of the mouse colon tissue in the middle and high dose group were significantly reduced.1.ELISA method to detect the levels of inflammatory factors(TNF-α,IL-1β,IL-6,IL-10)in mouse tissues,and the pro-inflammatory factors TNF-α,IL-1β,IL-6 after administration of Tan IIA There is a degree of decrease,and the anti-inflammatory factor IL-10 has a certain degree of increase.Among them,the medium and high dose group of Tan IIA has a more obvious effect.By selecting GSE95095 data set for difference analysis,with |log2FC| greater than 1 and FDR less than 0.05 as the thresholds,a total of 170 differential genes were screened out.To obtain the related items of cell composition(CC),molecular function(MF)and biological process(BP),different gene function annotation analysis was carried out.And the enrichment analysis of KEGG signaling pathway showed that,most genes were enriched in protein digestion and absorption pathway,retinol pathway,PI3K-AKT pathway,post-adolescent diabetes and linoleic acid pathway.UPLC-Q-Orbitrap-MS was adopted to detect the changes of serum metabolites in mice.Through multivariate data analysis and processing the metabolic profile data obtained,20 kinds of endogenous components were identified and screened out,such as taurine and 1-gluten.Aminoamide,betaine,hydroxypyruvate,linoleic etc.,can become potential biomarkers of Tan IIA treatment effect.According to the system network analysis of the corresponding pathways,the main pathways that Tan IIA affects DSS-induced colitis include taurine and hypotaurine metabolism,glyoxylic acid and dicarboxylic acid metabolism,alanine,aspartic acid and glutaric acid,linoleic acid metabolism,the metabolism of acid,the metabolism of glycine,serine and threonine,and the metabolism of arachidonic acid.After the administering of Tan IIA,the levels of inflammatory factors decreased respectively,especially for the Tan IIA mediumium with high dose.The immunohistochemical staining method showed that compared with the model,Tan IIA can significantly increase PPARy and reduce the expression of NF-κB p65 in the nucleus.The results of Western blot method showed that Tan IIA can cause the increase of △9 dehydrogenase and PPARy in mouse colon tissue,and inhibit the increase of phosphorylated IκBα,COX-2,iNOS,etc.To detect cell proliferation,the CCK-8 method was used and the resulted data demonstrated that,Tan IIA has no obvious toxic effect on THP-1 cells.Western blot and qRT-PCR methods were used to detect the protein expression and mRNA levels of△9dehydrogenase,PPARγ,NF-κB and p-κBα in the differentiated THP-1 cells stimulated by LPS,and it was found that Tan IIA can increase the expression andmRNA of △9dehydrogenase and PPARy Level,inhibit the expression of NF-κB and the phosphorylation level of p-IκBα.Conclusions:This study confirmed through experiments that Tan IIA does have a good therapeutic effect on DSS-induced IBD,and it can regulate downstream NF-κB signaling pathways to reduce inflammation in mice through the mediation of CLA and PPARy.Lay a theoretical foundation for the elucidation of the pharmacological mechanism of Tan IIA,and provide ideas and guidance for the study of molecular mechanisms in the treatment of inflammatory bowel disease.