Shenjinhuoxue Mixture Attenuates Inflammation, Pain, and Cartilage Degeneration by Inhibiting TLR-4 and NF-κB Activation in Rats with Osteoarthritis: A Synergistic Combination of Multitarget Active Phytochemicals

Osteoarthritis (OA), a highly prevalent chronic joint disease, involves a complex network of inflammatory mediators that not only triggers pain and cartilage degeneration but also accelerates disease progression. Traditional Chinese medicinal shenjinhuoxue mixture (SHM) shows anti-inflammatory and analgesic effects against OA with remarkable clinical efficacy. This study explored the mechanism underlying anti-OA properties of SHM and evaluated its efficacy and safety via in vivo experiments. Through network pharmacology and published literature, we identified the key active phytochemicals in SHM, including β-sitosterol, oleanolic acid, licochalcone A, quercetin, isorhamnetin, kaempferol, morusin, lupeol, and pinocembrin; the pivotal targets of which are TLR-4 and NF-κB, eliciting anti-OA activity. These phytochemicals can enter the active pockets of TLR-4 and NF-κB with docking score≤−3.86 kcal/mol, as shown in molecular docking models. By using surface plasmon resonance assay, licochalcone A and oleanolic acid were found to have good TLR-4-binding affinity. In OA rats, oral SHM at mid and high doses (8.72 g/kg and 26.2 g/kg) over 6 weeks significantly alleviated mechanical and thermal hyperalgesia (P