Yangyin Qingre Formula Regulates Treg/Th17 Balance by Down-Regulating JAK/STAT Signaling Pathway

  OBJECTIVE  To investigate the effect and mechanism of Yangyin Qingre formula (YHF) on Treg/Th17 imbalance induced by lipopolysaccharide (LPS) in mice.

  METHODS  CD4⁺ T cells from mouse spleen were extracted by magnetic bead sorting method and CD4⁺ T cells differentiation was stimulated by LPS followed by intervention with 1, 2, 4 mg · mL^-1 YHF. Cells proliferation was detected by MTT method. The ratio of Treg/Th17 of CD4⁺ T cells subsets was detected by flow cytometry. The expression of cytokines (IL-10, TGF-β, IL-6, IL-12p70) in cells supernatant was measured by ELISA. The expression of Foxp3, RORγt, and the signalling molecules JAK/STAT in the cells were detected by Western blot.

  RESULTS  HPLC identified eight active ingredients of YHF, namely hydroxy saffron yellow pigment A, polydatin, rutin, 2, 3, 5, 4'-tetrahydroxystilbene glucoside, resveratrol, quercetin, emodin and curcumin. YHF dose-dependently inhibited the proliferation of LPS-stimulated mouse CD4⁺ T cells (P < 0.05), increased the proportion of Treg (P < 0.05) and decreased the proportion of Th17 (P < 0.01), promoted the secretion of Treg-related anti-inflammatory factors IL-10 and TGF-β in culture supernatant and inhibited the secretion of Th17-related pro-inflammatory factors IL-6 and IL-12p70 secretion. 1, 2, 4 mg · mL^-1 YHF increased the expression of Foxp3 protein (P < 0.05) and decreased the expression of RORγt protein (P < 0.01). 1, 2, 4 mg · mL^-1 YHF decreased the expression of JAK2, STAT3 and their phosphorylated proteins (P < 0.05). The combination of 4 mg · mL^-1 YHF with the JAK2-specific inhibitor AG490 increased culture supernatant IL-10 and TGF-β levels (P < 0.05), decreased IL-6 and IL-12p70 levels (P < 0.05) and more significantly decreased p-JAK2, p-STAT3 and RORγt protein expression (P < 0.01, P < 0.001) and increased Foxp3 protein expression (P < 0.01).

  CONCLUSION  YHF can effectively inhibit the proliferation of inflammatory LPS-induced CD4⁺ T cells by suppressing the expression of JAK2 and STAT3 signaling molecules, increase the expression of Foxp3 and the number of Treg, decrease the expression of RORγt and the number of Th17, increase the secretion of anti-inflammatory cytokines IL-10 and TGF-β and inhibit the secretion of pro-inflammatory cytokines IL-6 and IL-12p70.