Paeoniflorin attenuates experimental autoimmune encephalomyelitis in mice in association with enhanced Treg differentiation and activation of Raf/MEK1/2/ERK1/2/Egr-1 signaling

White peony root ( Paeonia lactiflora Pall.) is the dried root of peony. White peony root has a long history in Traditional Chinese Medicine (TCM) and is primarily used to treat various conditions, including pain, inflammation, and immune system disorders. Paeoniflorin (PF) is one of the main active components of white peony root, and it has multiple functions such as anti-inflammation, immune regulation, and neuroprotection. However, the impact of PF on multiple sclerosis (MS) remains to be fully elucidated. In this study, we observed that PF treatment markedly attenuated neurological symptoms in experimental autoimmune encephalomyelitis (EAE) mice, reduced disease severity, and alleviated spinal cord inflammation and demyelination, indicating a significant neuroprotective effect. PF administration was accompanied by reduced CD4 + T cell infiltration, decreased Th17 cell proportions, and increased Treg cell frequency in vivo. It also balanced Th17/Treg-associated cytokine secretion by suppressing IL-17 and promoting IL-10 expression. In addition, PF modulated the expression of differentiation-related proteins in lymphoid tissues and inhibited inflammatory gene and protein levels. In primary murine CD4 + T cells, PF enhanced Treg differentiation under Treg-polarizing conditions. In Jurkat cells, PF induced phosphorylation of Raf, MEK 1/2 , and ERK 1/2 , together with increased expression of Egr-1 and Foxp3. These findings indicate that PF treatment is associated with modulation of the Treg/Th17 balance in EAE mice and with changes in Raf/MEK 1/2 /ERK 1/2 /Egr-1 signaling in Jurkat cells. In summary, PF treatment ameliorated EAE manifestations, together with restoration of Treg/Th17 homeostasis and activation of the Raf/MEK 1/2 /ERK 1/2 /Egr-1 pathway. However, whether the Raf/MEK 1/2 /ERK 1/2 /Egr-1 signaling axis directly contributes to PF-induced Treg differentiation in primary T cells or to the therapeutic effects of PF in vivo remains to be determined. Graphical abstract PF treatment ameliorated EAE manifestations and was associated with reduced CD4 + T-cell infiltration, restoration of the Th17/Treg balance, and attenuation of CNS inflammation and demyelination in EAE mice. PF also enhanced Treg differentiation in primary murine CD4 + T cells and was associated with the activation of the Raf/MEK 1/2 /ERK 1/2 /Egr-1 signaling axis in Jurkat cells. The causal contribution of this signaling axis to Treg differentiation and the therapeutic effects of PF remains to be determined.