基于PI3K/AKT/HIF-1α通路探讨泄浊解毒方调控糖酵解改善肠黏膜屏障治疗溃疡性结肠炎的机制研究OA

Objective:To investigate the mechanism of Xiezhuo Jiedu Formula(泄浊解毒方)in treating ulcerative colitis(UC)by regulating glycolysis to improve the intestinal mucosal barrier through bioinformatics,machine learning,and animal experiments.Methods:UC gene microarray data were extracted from the Gene Expression Omnibus(GEO)database to identify differentially expressed genes(DEGs).Glycolysis-related gene sets were retrieved from the Gene-Cards database.Intersecting genes(Gly-DEGs)were identified using a Venn diagram.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed using the Metascape data-base.Core Gly-DEGs with diagnostic and therapeutic potential were screened through machine learning algorithms,inclu-ding Support Vector Machine(SVM),Random Forest(RF),and Receiver Operating Characteristic(ROC)analysis.Immune infiltration analysis was conducted using the CIBERSORT deconvolution method.A UC rat model was induced using 2,4,6-trinitrobenzenesulfonic acid(TNBS)-absolute ethanol.Blood and colon tissue samples were collected from the rats.Histopathological morphology of the colon was observed by hematoxylin-eosin(HE)staining.Serum levels of the pro-inflammatory cytokines interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α),and interleukin-10(IL-10)were measured by enzyme-linked immunosorbent assay(ELISA).The expression levels of phosphoenolpyruvate car-boxykinase 1(PCK1),platelet endothelial cell adhesion molecule-1(PECAM1),matrix metalloproteinase-1(MMP1),lactate dehydrogenase A(LDHA),and hexokinase 2(HK2)in colon tissue were detected by immunohistochemistry(IHC).The expression levels of occludin,mucin 2(MUC2),and junctional adhesion molecule A(JAM-A)in colon tis-sue were detected by immunofluorescence(IF)assay.The mRNA expression levels of Pck1,Pecam1,Mmp1,Ldha,and Hk2 in colon tissue were quantified by real-time quantitative polymerase chain reaction(Real-time PCR).The protein expression levels of PCK1,PECAM1,MMP1,PI3K,phosphorylated PI3K(p-PI3K),AKT,phosphorylated AKT(p-AKT),and hypoxia-inducible factor-1α(HIF-1α)in colon tissue were determined by Western blot.Results:Through the GEO database,the datasets GSE206285 and GSE87466 were retrieved,yielding 1510 and 957 DEGs,respectively.A total of 4682 glycolysis-related genes were obtained from the GeneCards database,and by intersecting these,144 Gly-DEGs were identified.KEGG pathway enrichment analysis revealed that these Gly-DEGs were mainly enriched in the IL-17,PI3K-Akt,PPAR,and HIF-1 signaling pathways.Using SVM,RF,and ROC models,three core Gly-DEGs(PCK1,PECAM1,MMP1)were ultimately identified.Immune infiltration analysis demonstrated significant differences in memory B cells,plasma cells,CD8+T cells,natural killer cells,M0 macrophages,M1 macrophages,and mast cells(P<0.01).The expression of the core Gly-DEGs showed significant correlations with T cells,macrophages,dendritic cells,and neu-trophils.Correlation analysis between the core Gly-DEGs and intestinal mucosal barrier markers indicated significant cor-relations with occludin,MUC2,and F11R(JAM-A)(P<0.05).Animal experiment results indicated that compared with the normal control group,the model control group showed significant pathological changes in the colonic mucosa,inclu-ding disrupted epithelial integrity,ulcer formation,disorganized,reduced,or absent glandular structures,and inflammatory cell infiltration in the mucosal layer and lamina propria.Serum levels of the inflammatory factors IL-1β and TNF-α sig-nificantly increased,while the IL-10 level significantly decreased.The positive expression areas of MMP1,PECAM1,LDHA,and HK2 in colon tissue significantly increased,while protein and mRNA expressions were significantly upregulat-ed(P<0.01).The immunofluorescence intensity of occludin,MUC2,and F11R(JAM-A)significantly decreased(P<0.01),and the protein expression levels of PI3K,p-PI3K,AKT,p-AKT,and HIF-1α were significantly upregulated(P<0.01).PCK1 expression was negative or weakly positive,while its protein and mRNA expression were significantly downregulated(P<0.01).Compared with the model control group,the Xiezhuo Jiedu Formula groups showed largely in-tact epithelial structures,basically regular gland arrangement,and only a small amount of inflammatory cell infiltration.The serum IL-1β and TNF-α levels significantly decreased,while the IL-10 level significantly increased.The positive ex-pression areas of MMP1,PECAM1,LDHA,and HK2 in colon tissue were significantly reduced,with their protein and mRNA expression levels significantly downregulated(P<0.01).The immunofluorescence intensity of occludin,MUC2,and F11R(JAM-A)significantly increased(P<0.01).The protein expression levels of PI3K,p-PI3K,AKT,p-AKT,and HIF-1α were significantly downregulated(P<0.01).PCK1 expression was positive and significantly increased,and its protein and mRNA expression levels were significantly upregulated(P<0.01).Conclusion:PCK1,PECAM1,and MMP1 are core Gly-DEGs markers with diagnostic and therapeutic potential,mediating metabolic pathways involving vari-ous immune cells,such as T cells,macrophages,dendritic cells,neutrophils,as well as intestinal epithelial cells.Xiezhuo Jiedu Formula can treat UC by targeting the PI3K-AKT-HIF-1α-glycolysis-intestinal mucosal barrier axis.