Er Miao San and its main components phellodendrine and atractylenolide-I exert anti-rheumatoid arthritis effects by inhibiting PAD4 and thereby reducing the formation of NETs

Background: Er Miao San (EMS) is an important Chinese herbal formula for the treatment of damp-heat underflow syndrome, and has been clinically proven to be effective in the treatment of rheumatoid arthritis (RA). Previous studies have shown that EMS can regulate the function of T cells and dendritic cells, affect the polarisation of macrophages, and inhibit the abnormal activation and angiogenesis of fibroblast-like synoviocytes (FLSs). However, it is unclear whether the inhibitory effect of EMS on RA is related to neutrophil dysfunction.

Purpose: In this study, we investigated whether EMS and its main active substances, phellodendrine (PHE) and atractylenolide-I (ATL-I), could treat RA by acting on neutrophils and their potential mechanisms.

Methods: The effects of EMS on collagen-induced arthritis (CIA) in mice were detected by colour Doppler scanning, hematoxylin-eosin (H&E) staining, immunohistochemistry and ELISA. Immunofluorescence and scanning electron microscopy were used to detect the effects of EMS and the main active ingredients PHE and ATL-I on neutrophils. Mouse peripheral blood neutrophils were extracted and Phorbol 12-myristate 13-acetate (PMA) stimulated the formation of neutrophils form neutrophil extracellular traps (NETs). The effects of EMS and its main active components PHE and ATL-I on the formation of NETs were assessed by molecular docking, immunofluorescence, western blotting, and ELISA.

Results: The results showed that EMS reduced inflammation scores, pro-inflammatory cytokine levels and neutrophil infiltration in CIA mice. In in vitro experiments, EMS maintained neutrophil morphology, inhibited PMA-induced NETs formation, and suppressed CitH3 and PAD4 expression in neutrophils. Moreover, PHE and ATL-I exerted effects similar to those of EMS.

Conclusion: The present study demonstrated that EMS can inhibit the formation of NETs, and the mechanism of action may be related to the reduction of histone citrullination by inhibition of PAD4, and that PHE and ATL-I have the same effect. Therefore, whether a new dosage form containing PHE and ATL-I can be investigated as an alternative to EMS for the treatment of RA deserves further study.

Atractylenolide-I; Er Miao san; Neutrophil extracellular traps; Phellodendrine; Rheumatoid arthritis.