Tang Ran, Qin Zhi-Fang, Yin Jia-Hua, Wang Jia-Yu, Su Wen-Rui, Xuan Zi-Hua, Chen Bin, Jia Xiao-Yi
School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China; Anhui Province Key Laboratory of Bioactive Natural Products, Hefei 230012, China.
School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China; Anhui Province Key Laboratory of Bioactive Natural Products, Hefei 230012, China.
Fitoterapia. 2025 Jul 28;185:106771. doi: 10.1016/j.fitote.2025.106771.
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.
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.
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.
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.
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.
二妙散（EMS）是治疗湿热下注证的重要中药方剂，临床已证实其对类风湿关节炎（RA）有效。既往研究表明，EMS可调节T细胞和树突状细胞功能，影响巨噬细胞极化，并抑制成纤维样滑膜细胞（FLS）的异常活化和血管生成。然而，EMS对RA的抑制作用是否与中性粒细胞功能障碍有关尚不清楚。
本研究探讨EMS及其主要活性成分黄柏碱（PHE）和白术内酯-I（ATL-I）是否通过作用于中性粒细胞治疗RA及其潜在机制。
采用彩色多普勒扫描、苏木精-伊红（H&E）染色、免疫组化和酶联免疫吸附测定（ELISA）检测EMS对小鼠胶原诱导性关节炎（CIA）的影响。采用免疫荧光和扫描电子显微镜检测EMS及其主要活性成分PHE和ATL-I对中性粒细胞的影响。提取小鼠外周血中性粒细胞，用佛波酯（PMA）刺激中性粒细胞胞外诱捕网（NETs）形成。通过分子对接、免疫荧光、蛋白质印迹法和ELISA评估EMS及其主要活性成分PHE和ATL-I对NETs形成的影响。
结果显示，EMS降低了CIA小鼠的炎症评分、促炎细胞因子水平和中性粒细胞浸润。在体外实验中，EMS维持中性粒细胞形态，抑制PMA诱导的NETs形成，并抑制中性粒细胞中瓜氨酸化组蛋白H3（CitH3）和肽基精氨酸脱亚氨酶4（PAD4）表达。此外，PHE和ATL-I发挥了与EMS相似的作用。
本研究表明，EMS可抑制NETs形成，其作用机制可能与通过抑制PAD4减少组蛋白瓜氨酸化有关，且PHE和ATL-I具有相同作用。因此，是否可研究含PHE和ATL-I的新剂型作为EMS治疗RA的替代方案值得进一步研究。
