Astilbin combined with lipopolysaccharide induces IL-10-producing regulatory B cells via the STAT3 signalling pathway

Astilbin exerts immunoregulatory activities and plays anti-inflammatory effects in inflammation-associated diseases. IL-10-producing B cells are the major subset of regulatory B cells (Bregs) and inhibit inflammation and autoimmune diseases. This study aimed to analyse the inducing effect of astilbin on Bregs and investigate the involved molecular mechanisms. The frequencies and activities of IL-10-producing Bregs were observed using the co-treatment of astilbin and lipopolysaccharide (LPS) ex vivo. The protective effect of astilbin/LPS-induced Bregs on dextran sulphate sodium (DSS)-induced colitis was confirmed in vivo. The molecular signalling events of Breg induction were checked via Western blot. CD40−/− and toll-like receptor (TLR) 4−/− B cells were treated with astilbin/LPS to determine the modulatory role of CD40 or TLR4 on astilbin/LPS-induced Bregs. Although astilbin alone could not affect Bregs, the co-treatment of astilbin and LPS remarkably induced CD19+ CD1dhi and CD19+ TIM-1+ cells which produced IL-10 ex vivo. Colonic CD19+ CD1dhi and CD19+ TIM-1+ cells were also increased in astilbin-treated mice with DSS-induced colitis. The adoptive transfer of CD19+ TIM-1+ cells pre-induced by astilbin/LPS directly suppressed the progression of DSS-induced colitis. Combined astilbin and LPS stimulated the STAT3 activation of CD19+ TIM-1+ cells but had no effects on SOCS3, AKT, NF-κB, Erk, JNK nor P38. Inhibiting the STAT3 phosphorylation of CD19+ TIM-1+ cells abolished Breg induction by astilbin/LPS. Furthermore, Breg induction was weakened in CD40−/− B cells with the decrease in STAT3 activation, but had disappeared in TLR4−/− B cells with no STAT3 activation, thereby confirming the indispensable role of TLR4 signalling in the induction of IL-10-producing Bregs. This study reports the new immunoregulatory role of astilbin for promoting IL-10-producing B cells and suggests the possible use of astilbin in the therapy of inflammatory diseases.