Multi-omics perspective on Huoluo Xiaoling Pellet: key bioactive compounds and related mechanisms for the treatment of knee osteoarthritis

ETHNOPHARMACOLOGICAL RELEVANCE: Knee osteoarthritis (KOA) is a prevalent chronic knee joint disease, occurring mainly in the elderly and obese populations. In traditional Chinese medicine (TCM) theory, KOA is categorized as "knee bi" and "bi syndrome," primarily caused by external factors such as wind, cold, and dampness, as well as internal deficiencies. The treatment with Chinese herbal medicine focuses on dispelling wind, cold, and dampness, as well as promoting blood circulation and removing blood stasis. Huoluo Xiaoling Pellet (HXP), derived from Zhang Xichun's "The Records of Traditional Chinese and Western Medicine in Combination " during the Qing Dynasty, was known for its effects of activating blood circulation to remove stasis and alleviating pain by unblocking the collaterals, mainly used for treating conditions with stagnation of qi and blood. It has been included in the "Ancient Classic Prescriptions" by the National Administration of Traditional Chinese Medicine. However, the pharmacodynamic compounds, specific actions, and mechanisms of HXP on KOA have not yet been elucidated. AIM OF STUDY: Through qualitative and quantitative analysis, combined with multi-omics technology and network pharmacology, the key bioactive compounds and related mechanisms of HXP in improving knee osteoarthritis were explored. METHODS: UHPLC-Q-Orbitrap-MS and HPLC were used to analyze the chemical composition of HXP, chemometric analyses was used for quality control. The therapeutic effect of HXP was evaluated by Anterior Cruciate Ligament Transaction (ACLT)-induced KOA mouse model, and proteomics and lipid metabolomics were combined with network pharmacology and molecular docking techniques to explore for the key bioactive compounds and mechanisms. RESULTS: Identified 53 compounds in HXP, with Salvianolic acid B as a key quality marker and potential active compound for KOA. HXP reduced pain, protected chondrocytes, reduced oxidative stress, and improved bone parameters. The combined multi-omics analysis indicates that the protein targets of SHIP1, PTPRC, the cytochrome P450 enzyme family including Cyp1a2, Cyp2c29, Cyp2c50, Cyp2c544, as well as the JAK2/STAT3 pathway, play crucial roles in the improvement of KOA. CONCLUSION: Our results demonstrated that HXP was an effective drug to improve the development of KOA. Among them, Salvianolic acid B, the most abundant compound in HXP and the best computer simulation result, may be the key monomer compound of HXP for KOA treatment. The results of the visualisation analysis further contributed to our understanding of HXP treatment of KOA in terms of changes in SHIP1, PTPRC proteins and cytochrome P450 enzyme family. This is associated with the SHIP1/PI3K/AKT and JAK2/STAT3 pathways, as well as arachidonic acid metabolism and immune regulation.