Yue Wang,1,2,* Jiadan Ren,1,* Huixin Chen,1 Xiaotian Chen,1 Bo Yan,1 Wenting Xu,2 Xiujuan Xiao,1 Qiang Yuan,1 Letian Shan,2,3 Li Zhou2 

1College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China; 2The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese Medicine), Hangzhou, 310053, People’s Republic of China; 3Fuyang Research Institute, Zhejiang Chinese Medical University, Hangzhou, 310053, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Qiang Yuan, Email yuanqiang0825@sina.com Li Zhou, Email zhouli@zcmu.edu.cn

Purpose: To assess the pharmacodynamic effects and therapeutic mechanisms of modified Fuzi decoction (MFZD) in osteoarthritis (OA), particularly OA-related inflammation.
Methods: The main components of MFZD were identified using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). An OA model was established in Sprague-Dawley rats via intra-articular injection of monoiodoacetate (MIA) to evaluate the anti-OA efficacy of MFZD via gavage. In vivo studies, including pain behavior evaluation, histopathological observation, immunohistochemical analysis, enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD), and Western blot (WB), were carried out to demonstrate the anti-inflammatory and anti-degenerative potency of MFZD against OA. Potential targets and pathways of MFZD were identified via network pharmacology analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, respectively. Subsequently, the tumor necrosis factor (TNF) signaling and related targets were validated in vitro by WB. For in vitro validations, primary synovial fibroblasts were isolated from rats and treated with MFZD-containing serum (MFCS) in the presence of TNF-α.
Results: UPLC-MS/MS analysis identified key compounds in MFZD, including trans-cinnamaldehyde, atractylenolide I, lobetyolin, paeoniflorin, pachymic acid, carmichaeline, talatisamine, fuziline, benzoylhypacoinine, benzoylmesaconine, benzoylaconine, hypaconitine, deoxyaconitine, mesaconitine, and aconitine. MFZD treatment improved the paw withdrawal threshold (PWT), alleviated histopathological damage, reduced TNF-α and monocyte chemotactic protein-1 (MCP-1) in the serum, and remedied the abnormal anabolism/catabolism of cartilage. TNF signaling was identified through network pharmacology analysis as the anti-inflammatory mechanism of MFZD, and validated by WB results showing that MFCS treatment reduced TNF-α-induced protein expression of p-MKK3/MKK6, p-p38, TRAF2, p-p65, and VCAM1.
Conclusion: This study demonstrated that MFZD exerts anti-degenerative and anti-inflammatory potency against OA and revealed the TNF-α/TRAF2/NF-κB signaling related anti-inflammatory mechanism of MFZD for the first time, offering mechanistic insights into its potential in OA therapy.

Keywords: modified Fuzi decoction, osteoarthritis, synovitis, network pharmacology, TNF-α/TRAF2/NF-κB signaling