PNS Alleviates Rheumatoid Arthritis by Regulating HIF-1α/PKM2-mediated Glycolysis to Inhibit Th17 Differentiation

Purpose To assess the therapeutic potential of PNS against rheumatoid arthritis (RA) and its modulation of Th17 differentiation, specifically investigating in vitro the mechanism by which PNS inhibits glycolysis in Th17 cells through the HIF-1α signaling pathway. Methods CIA model were established and allocated into control, model, and PNS (100 mg/kg) groups to assess anti-arthritic efficacy and effects on Th17 differentiation. Network pharmacology identified PNS components and RA-related targets across databases. Protein-protein interaction (PPI) network construction and pathway enrichment analysis predicted core targets and pathways. Naive CD4⁺ T cells were differentiated into Th17 cells using IL-6, IL-23, and TGF-β. Cells (excluding the control group) were treated with PNS (5, 10, 20 µg/mL). Th17 cell differentiation was assessed by flow cytometry, and IL-17A secretion was quantified by ELISA. Expression of pathway-related genes and proteins was determined via RT-qPCR and immunoblotting, respectively. The PKM2-specific inhibitor Compound 3K and HIF-1α activator DFOM were employed to validate the mechanistic pathway. Results PNS attenuated the severity of arthritis in CIA mice, which was evidenced by the swelling of the hind paws, arthritis scores, body weight and pathological changes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that Th17 cell differentiation, PKM2 and HIF-1α signaling pathways may be related to the anti-RA effect of PNS. PNS effectively reduced the ratio of Th17 cells in vitro and in vivo. PNS inhibited the expression of PKM2 and HIF-1α in synovial cells and spleen T cells of CIA mice. PNS treatment significantly inhibited PKM2-mediated glycolysis. Furthermore, hyperactivation of the HIF-1α pathway in Th17 cells was reversed by PNS treatment. Conclusions PNS relieved the severity of CIA mice by inhibiting Th17 differentiation. Mechanistically, PNS suppressed HIF-1α-mediated PKM2 expression, inhibiting glycolysis-dependent Th17 differentiation., thereby alleviating rheumatoid arthritis.