Jiawei Wutou decoction attenuates knee osteoarthritis by modulating mitochondrial oxidative stress via the JAK2/STAT3 pathway: Insights from network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicine formula effectively used for the clinical treatment of Knee Osteoarthritis (KOA), the mechanism of action of Jiawei Wutou Decoction (JWD) remains unclear.

AIM OF THE STUDY: This study aims to preliminarily explore the potential mechanism of JWD in regulating mitochondrial oxidative stress for the treatment of KOA through the integration of UHPLC-MS/MS, network pharmacology, molecular docking technology and experimental validation.

MATERIALS AND METHODS: UHPLC-MS/MS was used to identify the main components of the water extract of JWD. HPLC was employed to determine the contents of Salidroside, Paeoniflorin, Calycosin- 7-O-β-D-glucopyranoside, Benzoylmesaconitine, Benzoylhypaconitine, Icariin and Glycyrrhizic Acid, which are the signature components of JWD. A DMM-induced KOA rat model was established. The pharmacodynamics, morphology and histopathological analysis were applied to preliminary determination of the preventive and therapeutic effects of JWD on KOA. A network of key compounds, core targets and key signaling pathways of JWD was constructed by combining network pharmacology and the binding activity between key components and core targets was verified by molecular docking techniques. Western Blot (WB), immunohistochemistry (IHC), CCK-8 assay and immunofluorescence techniques were employed to validate the therapeutic mechanism of JWD on KOA in both in vitro and in vivo experiments.

RESULTS: A total of 55 compounds were identified in JWD by UHPLC-MS/MS. Animal experiments showed JWD had a protective effect on DMM-induced KOA rats model, mainly in the amelioration of sclerosis, cartilage damage, increasing the pain threshold, and inhibition of oxidative stress. The potential targets of 55 compounds from JWD were predicted using online compound target databases. Protein-Protein Interaction (PPI) network analysis revealed that key targets such as AKT1, IL6, TNF, TP53, BCL2 and STAT3 are potential therapeutic targets of JWD for KOA treatment via regulating mitochondrial oxidative stress. Functional enrichment analysis indicated that apoptosis, cellular senescence, and the JAK/STAT signaling pathway were significantly enriched. Molecular docking results demonstrated that 18-α glycyrrhetinic acid, icariin, benzoylmesaconine, glabrol, rhodiosin, and benzoylpaeoniflorin in JWD exhibited high binding activity to JAK2 and STAT3. Therefore, the JAK2/STAT3 signaling pathway was identified as the potential target for the experimental validation study. JWD could upregulate the expression of Collagen II, Aggrecan and Bcl-2, downregulate the expression of Bax, Caspase-3, IL-6 and JAK2/STAT3 signaling related proteins in KOA rats. Besides, JWD improved mitochondrial morphology, reduced ROS generation and decreased MMP levels, which was attributed to the activation of JAK/STAT signaling molecules in TBHP-induced chondrocytes.

CONCLUSIONS: This study demonstrates that JWD exhibits potential therapeutic effects on KOA, which may be associated with the suppression of mitochondrial oxidative stress via the JAK/STAT signaling pathway.