Honglin Zhang,1,* Haixu Jiang,2,* Qiuzhu Wei,1,* Jie Xu,1 Zihan Zhao,1 Yuhe Sun,1 Qingyi Lu1 

1School of Life Sciences, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 2School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Qingyi Lu, School of Life Sciences, Beijing University of Chinese Medicine, Beijing, 102488, People’s Republic of China, Email lu_qingyi@126.com

Objective: Si Miao San is effective in ameliorating rheumatoid arthritis (RA) both clinically and experimentally. NETs play a fundamental role in the onset and progression of RA. The goal of this study was to explore the therapeutic effects of Si Miao San (SMS) on adjuvant-induced arthritis in mice and the regulatory mechanisms of NETs both in vivo and in vitro.
Methods: SMS decoctions were identified using LC-MS/MS analysis and TCMSP. In the adjuvant induced RA murine model, SMS decoction and methotrexate were administered orally. Disease progression was analysed by assessing arthritic scores and joint diameter, H&E staining, safranin O-fast green staining, toluidine blue staining and micro-CT analysis. The expression of NE, MPO, PAD4, LC3B, CitH3, p-AKT and p-PI3K and the production of ROS were detected using IHC, WB and IF analyses. Cytokines in the sera of the mice were detected using cytometric bead arrays. After the in vitro culture of neutrophils, NE, MPO, PAD4, LC3B, CitH3, ROS, p-PI3K and p-AKT were measured using IF and WB analyses. Autophagy was further observed with TEM.
Results: SMS decoction compounds were first identified. Compared with the model group, SMS significantly inhibited joint swelling, inflammation progression and bone destruction. The levels of NE, MPO, PAD4, CitH3, LC3B, ROS production and relative expression of p-AKT and p-PI3K in joint tissues were significantly reduced in the SMS group compared to the model group (P < 0.05). In vitro culture, SMS-containing serum significantly reduced the LC3B-II/LC3B-I ratio and the relative expression levels of p-AKT and p-PI3K, as well as the levels of ROS, NE, MPO, PAD4, and CitH3 compared with those in the PMA-treated group (P < 0.05), which was abolished by the treatment with the AKT activator SC79.
Conclusion: The SMS-induced suppression of inflammation in experimental RA occurred through the modulation of the AKT/ROS/autophagy axis.

Keywords: rheumatoid arthritis, neutrophil extracellular traps, reactive oxygen species, autophagy, protein kinase B