CP-25 inhibits the functions of activated human B cells through regulating BAFF-TRAF2-NF-κB and TNF-alpha-TRAF2-NF-κB signaling

摘要

OBJECTIVE This study was to investigate the effects of CP- 25 on the functions of activated human B cells through regulating BAFF and TNF-alpha signaling. METHODS B cells from peripheral blood mononuclear cells (PBMCs) of normal human were isolated using magnetic cell separation (MACS) by a positive selection. B cells (107 cells·mL-1) were stimulated by BAFF (100 ng·mL-1) or TNF-alpha (100 ng·mL-1) for two hours, and then were treated with CP-25 (10-5 mol·L-1) or Rituximab (5 μg·mL-1) or Etanercept (10 μg·mL-1). B cell proliferation was detected by CCK-8. B cell subsets and BAFF receptors (BAFFR, BCMA and TACI) were analyzed by flow cytometry. The expression of TNFR1 and TNFR2 on B cells was analyzed by flow cytometry. The expression of MKK3, MKK6, P-p38, P-p65, TRAF2 and p100/52 was analyzed by Western blotting. RESULTS CP-25 inhibited B cells proliferation stimulated by BAFF or TNF- alpha. CP- 25, Rituximab and Etanercept reduced the percentage and numbers of CD19+ B cells, CD19+CD20+ B cells, CD19+CD27+ B cells and CD19+CD20+CD27+ B cells induced by BAFF or TNF-alpha. CP-25 down-regulated the high expression of BAFFR, BCMA and TACI stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2 and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P- p65 in B cells stimulated by TNF-alpha. CONCLUSION CP- 25 regulated moderately activated B cells function by by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.