Quercetin pretreated umbilical cord mesenchymal stem cells alleviate inflammatory bowel disease via IL-10 /Janus kinase 2/STAT3 signaling

Figure 1 Isolation and identification of human umbilical cord-derived mesenchymal stem cells. A: Human umbilical cord-derived mesenchymal stem cell (hUCMSC) morphology. Scale bar = 100 μm; B: Detection of surface markers of hUCMSCs by flow cytometry; C: HUCMSCs differentiate into osteoblasts. Scale bar = 500 μm; D: HUCMSCs differentiate into adipocytes. Scale bar = 500 μm.

Figure 2 Quercetin pretreatment enhances human umbilical cord-derived mesenchymal stem cell viability without affecting their immunosuppressive ability. A: Pretreatment of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) with different concentrations of quercetin (QUR) for 24 hours; B: Pretreatment of hUCMSCs with different concentrations of QUR for 48 hours; C-H: QUR can decrease the mRNA expression of interleukin 1 beta (IL-1β), IL-6, colony-stimulating factor-6 (CSF-6), monocyte chemoattractant protein 1 (MCP-1), prostaglandin-endoperoxide synthase 2 (PTGS-2), and tumor necrosis factor-alpha-stimulated gene/protein-6 (TSG-6) in the untreated hUCMSC group. The data are expressed as the mean ± SEM, n = 3. ^aP < 0.05 vs untreated hUCMSC group, ^bP < 0.01 vs untreated hUCMC group.

Figure 3 Intraperitoneal injection of quercetin-human umbilical cord-derived mesenchymal stem cells can improve 2,4,6-trinitrobenzenesulfonic acid-induced colitis. A: Experimental flowchart; B: Weight changes, ^aP < 0.05; C: Macroscopic images of the colon; D: Colon length statistical chart, ^aP < 0.05, ^cP < 0.05; E: Survival rate; F: Disease Activity Index (DAI) score to assess the severity of the disease, ^aP < 0.05, 2,4,6-trinitrobenzenesulfonic acid (TNBS) vs TNBS + human umbilical cord-derived mesenchymal stem cells (hUCMSCs), ^cP < 0.05 TNBS vs TNBS + quercetin (QUR)-hUCMSCs; G: On the third day after TNBS induction, the percentage of mouse body weight; H: Colon hematoxylin and eosin staining pathological map. Scale bar = 100 μm; I: Histological Activity Index (HAI) score is used to evaluate the severity of tissue pathology, ^aP < 0.05, ^cP < 0.05; J: Myeloperoxidase (MPO) detection of neutrophil infiltration, ^aP < 0.05, ^cP < 0.05. The data are expressed as the mean ± SEM, n = 6. PBS: Phosphate-buffered saline.

Figure 4 Therapeutic effect of quercetin-human umbilical cord-derived mesenchymal stem cells on 2,4,6-trinitrobenzenesulfonic acid induced colitis in mice continues until the recovery period. A: Experimental flowchart; B: Survival rate; C: Weight changes, ^aP < 0.05, 2,4,6-trinitrobenzenesulfonic acid (TNBS) vs TNBS + human umbilical cord-derived mesenchymal stem cells (hUCMSCs), ^cP < 0.05 TNBS vs TNBS + quercetin (QUR)-hUCMSCs; D: Disease Activity Index (DAI) score to assess the severity of the disease, ^aP < 0.05, TNBS vs TNBS + hUCMSCs, ^cP < 0.05 TNBS vs TNBS + QUR-hUCMSCs; E: On the seventh day after TNBS induction, the percentage of mouse body weight, ^cP < 0.05; F: Macroscopic images of the colon; G: Colon length statistical chart, ^cP < 0.05; H: Colon hematoxylin and eosin staining pathological map. Scale bar = 100 μm; I: Histological Activity Index (HAI) score is used to evaluate the severity of tissue pathology, ^aP < 0.05, ^cP < 0.05; J: Myeloperoxidase (MPO) detection of neutrophil infiltration, ^cP < 0.05. The data are expressed as the mean ± SEM, n = 6. PBS: Phosphate-buffered saline.

Figure 5 Colon tissue RNA sequencing: Transcriptomic analysis of colon tissue in an acute colitis model to investigate differential gene expression at transcriptional levels. A: Differentially gene expression (DEG) volcano map. 2,4,6-trinitrobenzenesulfonic acid (TNBS) vs quercetin (QUR)-human umbilical cord-derived mesenchymal stem cells (hUCMSCs); B: DEG volcano map. hUCMSCs vs QUR-hUCMSCs. The red dots represent upregulated genes, whereas the green dots represent downregulated genes; C: Hierarchical clustering analysis of DEGs; D: Gene Ontology enrichment analysis. The -log10(P-value) is used to measure the expression of DEGs; E: Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway. Point size indicates the number of genes in the pathway, and the point color corresponds to P-adjusted ranges; F: Protein network interaction analysis. Nodes represent proteins, and the connections between nodes represent the strength of protein-protein interactions. Target symbol size was related to the degree value; among them, the larger the target’s shape, the more critical the target in the network. BP: Biological processes; CC: Cellular component; JAK: Janus kinase; MF: Molecular function; STAT: Signal transducer and activator of transcription; Th17: T helper type 17.

Figure 6 Quercetin-human umbilical cord-derived mesenchymal stem cells treat inflammatory bowel disease mice through the interleukin 10 (IL-10), IL-6/Janus kinase/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 signaling pathway. A: Interleukin 10 (IL-10) mRNA expression in colon tissue through quantitative polymerase chain reaction (qPCR); B: Janus kinase 2 (JAK2) mRNA expression in colon tissue through qPCR, ^bP < 0.01, ^dP < 0.01; C: Signal transducer and activator of transcription 3 (STAT3) mRNA expression in colon tissue through qPCR, ^dP < 0.01; D: Detection of IL-10 and IL-6 protein expression through western blotting; E: Semi-quantitative expression analysis of IL-10 proteins in colon tissue, ^cP < 0.05; F: Semi-quantitative expression analysis of IL-6 proteins in colon tissue, ^cP < 0.05; G: Detection of JAK2, phosphorylated JAK2 (p-JAK2), STAT3, p-STAT3 and suppressor of cytokine signaling 3 (SOCS3) protein expression through western blotting; H: Semi-quantitative expression analysis of p-JAK2 proteins in colon tissue, ^cP < 0.05; I: Semi-quantitative expression analysis of p-STAT3 proteins in colon tissue, ^cP < 0.05; J: Semi-quantitative expression analysis of SOCS3 proteins in colon tissue, ^cP < 0.05. The data are expressed as the mean ± SEM, n = 6. hUCMSCs: Human umbilical cord-derived mesenchymal stem cells; MSCs: Mesenchymal stem cells; QUR: Quercetin; TNBS: 2,4,6-trinitrobenzenesulfonic acid.

Figure 7 Therapeutic effects of quercetin-human umbilical cord-derived mesenchymal stem cells on inflammatory bowel disease mice continues until the recovery period, also through the interleukin 10 (IL-10), IL-6/Janus kinase/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 signaling pathways. A: Interleukin 10 (IL-10) mRNA expression in colon tissue through quantitative polymerase chain reaction (qPCR), ^cP < 0.05; B: Janus kinase 2 (JAK2) mRNA expression in colon tissue through qPCR, ^cP < 0.05; C: Signal transducer and activator of transcription 3 (STAT3) mRNA expression in colon tissue through qPCR, ^cP < 0.05; D: Detection of IL-10 and IL-6 protein expression through western blotting; E: Detection of JAK2, phosphorylated JAK2 (p-JAK2), STAT3, p-STAT3, and suppressor of cytokine signaling 3 (SOCS3) protein expression through western blotting; F: Semi-quantitative expression analysis of IL-10 proteins in colon tissue, ^dP < 0.01; G: Semi-quantitative expression analysis of IL-6 proteins in colon tissue, ^cP < 0.05; H: Semi-quantitative expression analysis p-JAK2 proteins in colon tissue, ^aP < 0.05, ^dP < 0.01; I: Semi-quantitative expression analysis of p-STAT3 proteins in colon tissue, ^aP < 0.05, ^dP < 0.01; J: Semi-quantitative expression analysis of SOCS3 proteins in colon tissue. The data are expressed as the mean ± SEM, n = 6. hUCMSCs: Human umbilical cord-derived mesenchymal stem cells; MSCs: Mesenchymal stem cells; QUR: Quercetin; TNBS: 2,4,6-trinitrobenzenesulfonic acid.