Effect of Chang’an decoction (肠安方) on ulcerative colitis by regulating T helper 17 cells and regulatory T cells via Rab27 in the p53/high mobility group box 1 pathway

doi:10.19852/j.cnki.jtcm.2025.05.007

Abstract

Abstract:

OBJECTIVE: To explore the effect of Chang’an decoction (肠安方, CAD) of ameliorating the immune imbalances in ulcerative colitis (UC) by regulating Rab27 in the P53/high mobility group box 1 pathway.

METHODS: The functions and important signaling pathways of the Rab27- and UC-related genes were analyzed viathe use of microarray data from the gene expression omnibus database, gene ontology database, Kyoto encyclopedia of genes and genomes database and gene set enrichment analysis. Dextran sulfate sodium salt-induced colitis mouse model was used to verify the bioinformatics results. Colon length, body weight, and disease activity index were measured. Hematoxylin and eosin staining was applied to validate the histopathology. Tight junction proteins were detected by immunohistochemistry. The proportions of T helper 17 cells (Th17) and regulatory T cells (Treg) in mesenteric lymph nodes were measured viaflow cytometry. Proinflammatory cytokines like interleukin (IL) 17 (IL-17), IL-21 and IL-22 and anti-inflammatory cytokines like transforming growth factor β and IL-10 in the serum and colon of mice were detected by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction, respectively. The expression levels of high mobility group box 1 (HMGB1), P53 and phospho- P53 (P-P53) in colonic tissues were detected by immunofluorescence and Western blotting.

RESULTS: Bioinformatics analysis revealed that compared with normal tissues, the expression of Rab27 was significantly increased in UC tissues. Receiver operating characteristic curve showed that Rab27 has the potential to be used as a biomarker for the diagnosis of disease activity. Enrichment analysis showed that UC and Rab27 were mainly associated with small molecule transport, nutrient metabolism, transmembrane transport and the downstream pathway of P53. According to animal experiments, the expression of Rab27 was increased in UC tissues, which aggravated the colonic pathological damage, activated the expression of HMGB1, and also leaded to the imbalance of Th17 and Treg cells. After CAD intervention, Rab27 overexpression, weight loss, colon shortening, and pathological damage were substantial reduced, the expression of tight junction proteins, zona occludens 1 and Occludin were increased. The effect of CAD at high-dose was more obvious. In addition, CAD upgraded the number of Treg cells and the production of TGF-β and IL-10, while decreasing the number of Th17 cells and the expression of inflammatory cytokines (IL-17, IL-21, and IL-22). Moreover, colon inflammation was alleviated by CAD, as indicated by the regulation of HMGB1 and P-P53 expression.

CONCLUSION: The expression of Rab27, HMGB1 and P-P53 could be decreased by CAD, and the balance of Th17 and Treg cells as well as their related cytokines could be regulated by CAD.

Key words: ulcerative colitis, Rab27, high mobility group box 1, T helper 17 cells, regulatory T cells, bioinformatics, Chang’an decoction

ZHENG Li, JIN Ting, WANG Xiaojing, WANG Yingqi, LIU Fengbin, MI Hong. Effect of Chang’an decoction (肠安方) on ulcerative colitis by regulating T helper 17 cells and regulatory T cells via Rab27 in the p53/high mobility group box 1 pathway[J]. Journal of Traditional Chinese Medicine, 2025, 45(5): 998-1008.

Figures/Tables 4

Figure 1 The intestinal inflammation of DSS-induced mice could be alleviated by Rab27 knockout A: mRNA expression (n = 3); A1: Rab27A; A2: Rab27B; B: weight change (n = 6); C: DAI scores (n = 6); D: colon length (n = 6); D1: measurement of the colon; D2: statistical graph of colon length; E: HE staining (scale bar: 100 μm × 200); E1: Water; E2: DSS; E3: Rab27-/-+Water; E4: Rab27-/-+DSS; Water: control group; DSS: ulcerative colitis group; Rab27-/-+Water: Rab27 knock out mice with normal drinking water; Rab27-/-+DSS: Rab27 knock out mice with ulcerative colitis. DAI: disease activity index; HE: hematoxylin and eosin. One-way analysis of variance was utilized. Results are presented as mean ± standard deviation. aP < 0.01, cP < 0.05 vs water; bP < 0.001, dP < 0.05 vs DSS.

Figure 2 Th17/Treg cell balance could be regulated by CAD A: number of Th17 and Treg cells in mesenteric lymph nodes (n = 3); A1:Th17 cells in the Water group; A2: Th17 cells in the DSS group; A3: Th17 cells in the DSS + CAD group; A4: Treg cells in the Water group; A5: Treg cells in the DSS group; A6: Treg cells in the DSS + CAD group; A7: statistical graph of Th17 cells; A8: statistical graph of Treg cells; B: protein expressions of RORγt and Foxp3 in colon tissues (n = 3); B1: protein band; B2: statistical graph of RORγt; B3: statistical graph of Foxp3; C: immunofluorescence results of RORγt and Foxp3 in colon tissues (scale bars: 100 μm × 200); C1: RORγt in the Water group; C2: RORγt in the DSS group; C3: RORγt in the DSS + CAD group; C4: Foxp3 in the Water group; C5: Foxp3 in the DSS group; C6: Foxp3 in the DSS + CAD group; CAD-H: high-dose Chang’an decoction (26 g·kg-1·d-1); Water: control group; DSS: ulcerative colitis group; DSS + CAD: ulcerative colitis mice with high-dose Chang’an decoction (26 g·kg-1·d-1); CAD: Chang’an decoction. The drug was administered for 6 d. Th17: T helper 17 cells; Treg: regulatory T cells; UC: ulcerative colitis; RORγt: RAR-related orphan receptor γt; Foxp3: forkhead box protein 3; MLNs: mesenteric lymph nodes. One-way analysis of variance was utilized. Results are presented as mean ± standard deviation. aP < 0.01, cP < 0.001 vs water; bP < 0.01, dP < 0.05, eP < 0.001 vs DSS.

Figure 3 The inflammation of DSS-induced mice could be alleviated by CAD via affecting Rab27 A: mRNA expression (n = 3); A1: Rab27A; A2: Rab27B; B: weight change (n = 6); C: DAI scores (n = 6); D: colon length (n = 6); D1: measurement of colon; D2: statistical graph of colon length; E: HE staining (scale bar: 100 μm × 200); E1: DSS; E2: Rab27-/- + DSS; E3: DSS + CAD; E4: Rab27-/- + DSS + CAD; DSS: ulcerative colitis group; Rab27-/- + DSS: Rab27 knock out mice with ulcerative colitis; DSS + CAD: ulcerative colitis mice with high-dose Chang’an decoction (26 g·kg-1·d-1); Rab27-/- + DSS + CAD: Rab27 knock out mice with ulcerative colitis were given high-dose Chang’an decoction (26 g·kg-1·d-1); CAD: Chang’an decoction. The drug was administered for 6 d. DAI: disease activity index; HE: hematoxylin and eosin; PCR: polymerase chain reaction. One-way analysis of variance was utilized. Results are presented as mean ± standard deviation. aP < 0.01 vs water, bP < 0.05 vs DSS, cP < 0.01 vs DSS, dP < 0.001 vs DSS.

Figure 4 Th17/Treg balance could be regulated by CAD via effecting Rab27 in P53/HMGB1 pathway A: protein expression levels of P-P53, P53 and HMGB1 in colon tissues (n = 3); A1: protein band; A2: statistical graph of HMGB1; A3: statistical graph of P-P53; B: number of Th17 and Treg cells in mesenteric lymph nodes of each group (n = 3). B1:Th17 cells in the DSS group; B2: Th17 cells in the Rab27-/- + DSS group; B3: Th17 cells in the DSS + CAD group; B4: Th17 cells in the Rab27-/- + DSS + CAD group; B5: Treg cells in the DSS group; B6: Treg cells in the Rab27-/- + DSS group; B7: Treg cells in the DSS + CAD group; B8: Treg cells in the Rab27-/- + DSS + CAD group; B9: statistical graph of Th17 cells; B10: statistical graph of Treg cells; DSS: ulcerative colitis group; Rab27-/- + DSS: Rab27 knock out mice with ulcerative colitis; DSS + CAD: ulcerative colitis mice with high-dose Chang’an decoction (26 g·kg-1·d-1); Rab27-/- + DSS + CAD: Rab27 knock out mice with ulcerative colitis were given high-dose Chang’an decoction (26 g·kg-1·d-1); CAD: Chang’an decoction (26 g·kg-1·d-1). The drug was administered for 6 d. HMGB1: high mobility group box 1; P-P53: phospho- P53; IL-17: interleukin 17; IL-21: interleukin 21; IL-22: interleukin 22; TGF-β: transforming growth factor β; IL-10: interleukin 10; PCR: polymerase chain reaction; MLNs: mesenteric lymph nodes; UC: ulcerative colitis; Th17: T helper 17 cells; Treg: regulatory T cells; NF-κB: nuclear factor kappa-B. One-way analysis of variance was utilized. Results are presented as mean ± standard deviation. aP < 0.05, bP < 0.01, cP < 0.001 vs DSS.

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