Curcumin alleviates experimental colitis via a potential mechanism involving memory B cells and Bcl-6-Syk-BLNK signaling

Abstract

Background: Immune dysfunction is the crucial cause in the pathogenesis of inflammatory bowel disease (IBD), which is mainly related to lymphocytes (T or B cells, incl-uding memory B cells), mast cells, activated neutrophils, and macrophages. As the precursor of B cells, the activation of memory B cells can trigger and differentiate B cells to produce a giant variety of inducible B cells and tolerant B cells, whose dysfunction can easily lead to autoimmune diseases, including IBD.

Aim: To investigate whether or not curcumin (Cur) can alleviate experimental colitis by regulating memory B cells and Bcl-6-Syk-BLNK signaling.

Methods: Colitis was induced in mice with a dextran sulphate sodium (DSS) solution in drinking water. Colitis mice were given Cur (100 mg/kg/d) orally for 14 con-secutive days. The colonic weight, colonic length, intestinal weight index, occult blood scores, and histological scores of mice were examined to evaluate the curative effect. The levels of memory B cells in peripheral blood of mice were measured by flow cytometry, and IL-1β, IL-6, IL-10, IL-7A, and TNF-α expression in colonic tissue homogenates were analyzed by enzyme-linked immunosorbent assay. Western blot was used to measure the expression of Bcl-6, BLNK, Syk, and other signaling pathway related proteins.

Results: After Cur treatment for 14 d, the body weight, colonic weight, colonic length, colonic weight index, and colonic pathological injury of mice with colitis were ameliorated. The secretion of IL-1β, IL-6, TNF-α, and IL-7A was statistically decreased, while the IL-35 and IL-10 levels were considerably increased. Activation of memory B cell subsets in colitis mice was confirmed by a remarkable reduction in the expression of IgM, IgG, IgA, FCRL5, CD103, FasL, PD-1, CD38, and CXCR3 on the surface of CD19⁺ CD27⁺ B cells, while the number of CD19⁺ CD27⁺ IL-10⁺ and CD19⁺ CD27⁺ Tim-3⁺ B cells increased significantly. In addition, Cur significantly inhibited the protein levels of Syk, p-Syk, Bcl-6, and CIN85, and increased BLNK and p-BLNK expression in colitis mice.

Conclusion: Cur could effectively alleviate DSS-induced colitis in mice by regulating memory B cells and the Bcl-6-Syk-BLNK signaling pathway.

Keywords: BLNK; Bcl-6; Curcumin; Experimental colitis; Memory B cell.

Figure 1

Drugs and protocols used in the study. A: Molecular structural formula of curcumin (Cur); B: Cur used in the experiment; C: Colitis induction and Cur administration. The experiment lasted for 22 d, including 3 d of adaptive feeding, 0-7 d of treatment with 3% DSS treatment with drinking water, and 7 d of free drinking water. Animals were divided into four groups: Control (n = 10), DSS (n = 10), DSS + Cur (100 mg/kg/d, n = 10), and Ctrl + Cur (100 mg/kg/d, n = 10) groups.

Figure 2

Therapeutic evaluation of curcumin on DSS-induced experimental colitis. A: Body weight of mice from days 0-22; B: Weight change of rate in mice from days 1-22; C: OB scores of mice in the four groups from days 1-22; D: DAI scores of mice in the four groups from days 1-22; E: Gross changes in colonic length; F: Colonic length of mice on day 22 in the four groups; G: Colonic weight; H: Body weight; I: Colonic weight index; J: Colonic weight/colonic length; K: Spleen weight; L: Spleen weight/body weight; M: Histological appearance of colons from individual groups of mice (hematoxylin and eosin staining, magnification 50 ×, scale bar = 100 μm); N: Pathological injury score. Data are presented as the mean ± SE (n = 8-10). ^aP < 0.05, ^bP < 0.01 vs Control group; ^cP < 0.05, ^dP < 0.01 vs DSS group.

Figure 3

Curcumin effectively regulates inflammatory cytokine expression. A: Concentration of IL-10 in colonic tissue; B: Concentration of IL-35 in colonic tissue; C: Concentration of IL-6 in colonic tissue; D: Concentration of IL-17A in colonic tissue; E: Concentration of IL-1β in colonic tissue; F: Concentration of TNF-α in colonic tissue. Data are presented as the mean ± SE (n = 8-10). ^aP < 0.05, ^bP < 0.01 vs Control group; ^cP < 0.05, ^dP < 0.01 vs DSS group.

Figure 4

Curcumin regulates differentiation of memory B cells in DSS-induced ulcerative colitis mice. A: Representative flow cytometry profile of peripheral blood mononuclear cells (PBMC); B: Representative flow cytometry profile of CD19⁺ cells in peripheral blood; C: Representative flow cytometry profile of CD19⁺ CD27⁺ cells in peripheral blood; D: Bar chart of CD19⁺ CD2 ⁺ memory B cells. Data are presented as the mean ± SE (n = 8-10). ^aP < 0.05, ^bP < 0.01 vs Control group; ^cP < 0.05, ^dP < 0.01 vs DSS group.

Figure 5

Curcumin regulates differentiation of memory B cells in DSS-induced ulcerative colitis mice. A: Representative flow cytometry profile of CD19⁺ CD27⁺ IgG⁺ cells in peripheral blood; B: Bar chart of CD19⁺ CD27⁺ IgG⁺ cells; C: Representative flow cytometry profile of CD19⁺ CD27⁺ IgM⁺ cells in peripheral blood; D: Bar chart of CD19⁺ CD27⁺ IgM⁺ cells; E: Representative flow cytometry profile of CD19⁺ CD27⁺ IgA⁺ cells in peripheral blood; F: Bar chart of CD19⁺ CD27⁺ IgA⁺ cells. Data are presented as the mean ± SE (n = 8-10). ^aP < 0.05, ^bP < 0.01 vs Control group; ^cP < 0.05, ^dP < 0.01 vs DSS group.

Figure 6

Curcumin regulates differentiation of memory B cells in DSS-induced ulcerative colitis mice. A: Representative flow cytometry profile of CD19⁺ CD27⁺ FCRL5⁺ cells in peripheral blood; B: Bar chart of CD19⁺ CD27⁺ FCRL5⁺ cells; C: Representative flow cytometry profile of CD19⁺ CD27⁺ CD103⁺ cells in peripheral blood; D: Bar chart of CD19⁺ CD27⁺ CD103⁺ cell; E: Representative flow cytometry profile of CD19⁺ CD27⁺ IL-10⁺ cells in peripheral blood; F: Bar chart of CD19⁺ CD27⁺ IL-10⁺ cells; G: Representative flow cytometry profile of CD19⁺ CD27⁺ FasL⁺ cells in peripheral blood; H: Bar chart of CD19⁺ CD27⁺ FasL⁺ cells. Data are presented as the mean ± SE (n = 8-10). ^aP < 0.05, ^bP < 0.01 vs Control group; ^cP < 0.05, ^dP < 0.01 vs DSS group.

Figure 7

Curcumin regulates differentiation of memory B cells in DSS-induced ulcerative colitis mice. A: Representative flow cytometry profile of CD19⁺ CD27⁺ PD-1⁺ cells in peripheral blood; B: Bar chart of CD19⁺ CD27⁺ PD-1⁺ cells; C: Representative flow cytometry profile of CD19⁺ CD27⁺ CD38⁺ cells in peripheral blood; D: Bar chart of CD19⁺ CD27⁺ CD38⁺ cells; E: Representative flow cytometry profile of CD19⁺ CD27⁺ CXCR3⁺ cells in peripheral blood; F: Bar chart of CD19⁺ CD27⁺ CXCR3⁺ cells; G: Representative flow cytometry profile of CD19⁺ CD27⁺ Tim-3⁺ cells in peripheral blood; H: Bar chart of CD19⁺ CD27⁺ Tim-3⁺ cells. Data are presented as the mean ± SE (n = 8-10). ^aP < 0.05, ^bP < 0.01 vs Control group; ^cP < 0.05, ^dP < 0.01 vs DSS group.

Figure 8

Curcumin regulates expression of memory B cell-related proteins in DSS-induced ulcerative colitis mice. A: Expression of Bcl-6, BLNK, p-BLNK, CIN85, Syk, and p-Syk in colon tissues as revealed by Western blot analysis. GAPDH served as the internal reference; B-G: Quantitative evaluation of (B) Bcl-6, (C) BLNK, (D) p-BLNK, (E) CIN85, (F) Syk, and (G) p-Syk. Data are presented as the mean ± SE (n = 8-10). ^aP < 0.05, ^bP < 0.01 vs Control group; ^cP < 0.05, ^dP < 0.01 vs DSS group.