Direct TNF-α targeting by Periploca forrestii-derived compounds confers anti-rheumatoid arthritis activity

Periploca forrestii Schltr. (PF), traditionally used by the Miao ethnic group in China, has been reported to exert a therapeutic effect on rheumatoid arthritis (RA), potentially through regulating of the TNF-α signaling pathway. This study aimed to identify PF-derived TNF-α-targeting compounds and elucidate their anti-RA mechanisms.

TNF-α-binding constituents in PF were screened using affinity ultrafiltration plus liquid chromatography-mass spectrometry (AU-LC/MS). Initial screening for TNF-α antagonistic activity was performed in L929 cells. Binding interactions were verified by biolayer interferometry (BLI), surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS). LPS-induced RAW264.7 cells were used to evaluate anti-inflammatory activity, while MH7A cells were employed to assess the effects of active compounds on inflammation and TNF-α-mediated NF-κB signaling. The underlying mechanisms were further investigated using molecular docking and competitive binding assays. The anti-RA efficacy of selected compounds was evaluated in a collagen-induced arthritis (CIA) rat model.

Nine TNF-α-binding compounds were identified, among which isochlorogenic acid B, daucosterol, and vitamin E exhibited strong TNF-α binding affinity and effectively protected L929 cells from TNF-α-induced cytotoxicity. These compounds directly interacted with TNF-α as confirmed by BLI, SPR, CETSA, and DARTS, and interfered with TNF-α/TNFR1 interaction. Consequently, they suppressed NF-κB activation, decreased phosphorylation of IκBα and p65, and reduced the levels of NO and proinflammatory cytokine (such as TNF-α, IL-6, and IL-1β). Further analyses, including molecular dynamics simulations and molecular docking, demonstrated stable binding of these compounds to TNF-α, consistent with experimental findings. In CIA rats, all three compounds markedly alleviated joint swelling, histopathological damage, and inflammatory cytokine levels.

Isochlorogenic acid B, daucosterol, and vitamin E are key TNF-α-targeting constituents of Periploca forrestii that exert anti-RA effects by blocking TNF-α/TNFR1 interaction and inhibiting NF-κB-mediated inflammatory responses. These findings suggest that PF may serve as a source of natural small-molecule TNF-α inhibitors for RA therapy.