RÉSUMÉ
ObjectiveKey microRNAs （miRNAs） of colorectal adenoma （CRA） were identified and analyzed by bioinformatics methods， and differentially expressed genes （DEGs） were screened to construct regulatory relationships. The mechanism of Xiezhuo Jiedu recipe in preventing CRA was speculated and verified by animal experiments. MethodThe miRNAs dataset GSE50194 was obtained from the Gene Expression Omnibus （GEO） database of intestinal mucosal tissue of CRA patients， and the differentially expressed miRNAs were screened by GEO2R and Excel. TargetScan， miRTarbase， and miRDB databases were used to predict the target genes of the differentially expressed miRNAs， and an intersection was obtained. Key DEGs were screened through the STRING database and Cytoscape software， and the TRRUST database was used to predict downstream binding transcription factors （TFs）. The mRNA intersection was enriched by gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） in the Metascape database. DIANA TOOLS were applied to perform KEGG enrichment analysis of key miRNAs， and the key signaling pathways were selected for animal experiments. In animal experiments， the CRA mice model was established by using sodium glycan sulfate （DSS） drinking combined with intraperitoneal injection of azomethane oxide （AOM）， and Xiezhuo Jiedu recipe and aspirin were given by intragastric administration at the same time. The experiment lasted for nine weeks. The pathological changes in intestinal tissue were observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of miR-34a-5p in adenoma tissue. Protein expression levels of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， phosphoryl-PI3K （p-PI3K）， phosphoryl-Akt （p-Akt）， and B cell lymphoma （Bcl）-2 were detected by Western blot. The expression of Cyclin D1 （CCND1） was detected by immunohistochemistry （IHC）. In situ terminal transferase labeling （TUNEL） was used to detect apoptosis of adenoma tissue cells. ResultThe GEO database screened the GSE50194 dataset， and miR-34a-5p was selected as the research object from CRA and normal tissue. A total of 93 DEGs were selected. Among them， GO and KEGG enrichment analyses were closely related to biological processes such as transcriptional regulatory complex， RNA polymerase Ⅱ transcriptional regulatory complex， enzyme-linked receptor protein signaling pathway， and DNA-binding transcriptional activator activity， cancer pathway， PI3K/Akt pathway， etc. miR-34a-5p is mainly enriched in PI3K/Akt， cell cycle， and colorectal cancer pathways. Five key DEGs were screened out through the Matescape database， among which Bcl-2 and CCND1 were the key DEGs of miR-34a-5p. Further screening of the TFs of key DEGs revealed that E2F transcription factor 1 （E2F1） and tumor protein P53 （TP53） were the main TFs of Bcl-2 and CCND1. Animal experiments showed that Xiezhuo Jiedu recipe could effectively up-regulate mRNA level of miR-34a-5p， down-regulate the expression of PI3K， Akt， Bcl-2， p-PI3K， and p-Akt proteins in the intestinal tissue of CRA mice， down-regulate the positive expression rate of CCND1， and increase the apoptosis rate of intestinal epithelial cells. ConclusionIt is speculated that Xiezhuo Jiedu recipe may inhibit the abnormal proliferation and promote the apoptosis of intestinal epithelial cells in CRA mice by regulating the miR-34a-5p/PI3K/Akt signaling pathway， thus playing a role in the prevention of CRA.
RÉSUMÉ
ObjectiveThe bioinformatics method was used to screen ferroptosis differential genes （FRGs） closely related to ulcerative colitis （UC）， and animal experiments were conducted to verify whether the mechanism of Xiezhuo Jiedu recipe in treating UC is related to the regulation of ferroptosis. MethodThe differentially expressed genes （DEGs） of colonic mucosa tissue of UC patients were obtained from the GEO database， and the intersection of the genes with ferroptosis genes was used to obtain FRGs. The core FRGs were obtained by cluster analysis， minimum absolute contraction and selection operator （LASSO） regression， and receiver operating characteristic curve （ROC） curve analysis. In animal experiments， the UC mouse model was prepared by making the mouse freely drink 2.5% dextran sodium sulfate （DSS）. Xiezhuo Jiedu recipe and mesalazine were given by gavage for seven days， and the inflammatory infiltration of colonic mucosa was observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of E3 ubiquitin ligase （FBXW7）， zinc finger protein （ZFP36）， solute carrier family 7 member 11 （SLC7A11）， and Toll-like receptor 4 （TLR4） in colon tissue. The protein expression levels of FBXW7， ZFP36， SLC7A11， and TLR4 in colon tissue were detected by Western blot. ResultDataset GSE87466 was screened from the GEO database， and its intersections with the ferroptosis gene were analyzed to obtain 21 FRGs. After cluster analysis， LASSO regression， and ROC analysis， core FRGs （FBXW7， ZFP36， SLC7A11， and TLR4） were obtained. Immunoinfiltration analysis showed significant differences in the expression of initial B cells， M1 macrophages， plasma cells， and M2 macrophages in the colonic mucosa tissue of UC mice， and there was a significant correlation between core FRGs and these immune cells. Further animal experiments showed that the colonic mucosa tissue of mice in the model group was disorganized and infiltrated by a large number of inflammatory cells. The inflammation of the colonic mucosa tissue of mice in each group was relieved to varying degrees after treatment with Xiezhuo Jiedu recipe and mesalazine， while the colonic mucosa tissue of mice in the high-dose group of Xiezhuo Jiedu recipe showed almost no inflammatory changes. Compared with the normal group， the protein and mRNA expressions of FBXW7， ZFP36， SLC7A11， and TLR4 in the model group were significantly increased， and the expression of core FRGs in colonic mucosa tissue of mice in all groups was significantly down-regulated after treatment with Xiezhuo Jiedu recipe and mesalazine. ConclusionFBXW7， ZFP36， SLC7A11， and TLR4 are ferroptosis genes closely related to the pathogenesis of UC， and Xiezhuo Jiedu recipe can significantly alleviate colonic mucosa inflammation in mice by down-regulating core ferroptosis genes.
RÉSUMÉ
ObjectiveThe differential expression of microRNAs （miRNAs） between the active stage and the remission stage of ulcerative colitis （UC） was analyzed by bioinformatics method， and the regulatory relationship was constructed by screening the differentially expressed genes （DEGs）. The mechanism of Xizhuo Jiedu recipe in the treatment of UC was speculated and verified by animal experiments. MethodThe miRNAs data set of colonic mucosa tissue of UC patients was obtained from the gene expression database （GEO）， and the most differentially expressed miRNAs were screened by GEO2R， Excel， and other tools as research objects. TargetScan， miRTarbase， miRDB， STRING， TRRUST， and Matescape databases were used to screen key DEGs， predict downstream transcription factors （TFs）， gene ontology （GO）， and conduct Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. The key signaling pathways were selected for animal experiments. In animal experiments， the UC mouse model was prepared by making the mouse freely drink 2.5% dextran sodium sulfate （DSS）. Xiezhu Jiedu recipe and mesalazine were given by gavage for seven days， and the inflammatory infiltration of colonic mucosa was observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of miR-155-5p in colon tissue. Immunohistochemistry and Western blot were used to detect the protein expression levels of cytokine signal transduction inhibitor （SOCS1）， phosphorylated transcriptional signal transductor and activator 3 （p-STAT3）， phosphorylated Janus kinase 2 （p-JAK2）， and retinoic acid-associated orphan receptor-γt （ROR-γt）. The expression levels of transforming growth factor-β （TGF-β）， interleukin-17 （IL-17）， interleukin-6 （IL-6）， and interleukin-10 （IL-10） in serum were detected by enzyme linked immunosorbent assay （ELISA）. ResultThe GSE48957 dataset was screened from the GEO database， and miR-155-5p was selected as the research object from the samples in the active and remission stages. 131 DEGs were screened. The GO/KEGG enrichment analysis was closely related to biological processes such as positive regulation of miRNA transcription and protein phosphorylation， as well as signaling pathways such as stem cell signaling pathway， IL-17 signaling pathway， and helper T cell 17 （Th17） cell differentiation. The Matescape database was used to screen out 10 key DEGs， among which SOCS1 was one of the key DEGs of miR-155-5p. Further screening of the TFS of key DEGs revealed that STAT3 was one of the main TFs of SOCS1. The results of animal experiments showed that Xiezhu Jiedu Recipe could effectively down-regulate the mRNA expression of miR-155-5p and protein expression of p-STAT3， p-JAK2， and ROR-γt in colon tissue of UC mice and the expression of IL-17 and IL-6 in serum of UC mice， up-regulate the protein expression of SOCS1 and the expression of TGF-β and IL-10， increase the level of anti-inflammatory factors， and reduce inflammatory cell infiltration. ConclusionIt is speculated that Xizhuo Jiedu recipe may interfere with SOCS1 by regulating the expression of miR-155-5p in UC mice， inhibit the phosphorylation of STAT3， inhibit the differentiation of CD4+ T cells into Th17 cells， reduce the levels of pro-inflammatory factors （IL-17 and IL-6）， and increase the levels of anti-inflammatory factors （TGF-β and IL-10）. As a result， the inflammation of colon mucosa in UC mice was alleviated.