Systematic insight into the dual COX-2/5-LOX inhibitory mechanism of Duhuo Jisheng decoction for treatment of osteoarthritis based on in silico and bioassay

Abstract

Ethnopharmacological relevance: Traditional Chinese medicine (TCM) is frequently used to treat osteoarthritis (OA). Duhuo Jisheng decoction (DHJSD), a Chinese patent medicine, was commonly used Chinese herbal formula for the treatment of OA. In Western medicine, dual inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzyme has been proved to be a promising strategy to treat inflammatory diseases with reduced side effects.

Aim of the study: To elucidate the dual action mechanism of DHJSD targeting COX-2 and 5-LOX against OA.

Materials and methods: DHJSD, containing 1495 compounds was screened using a virtual screening approach based on molecular docking. The inhibitory effect of hit compounds against COX-2 and 5-LOX was validated using enzyme-based assays. In vitro, rat chondrocytes were treated with IL-1β (10 ng/mL) for 24 h to induce OA model in vitro. The chondrocyte viability was evaluated using an CCK-8 assay. ELISA was used to detect inflammatory factors expression. Immunofluorescence was used to assess the expression level of collagen II and MMP-13. In addition, a rat cartilage explants culture model was established, and safranin O and HE staining analysis were carried to assess cartilage matrix degradation and cartilage damage, respectively. In vivo, carrageenan-induced paw edema assay was used to examine anti-inflammatory activity, and the gastric ulcerogenic effect was further detected. Finally, Molecular dynamics simulations and binding free energy analysis were carried to explore the binding mechanism.

Results: 13 compounds from DHJSD were identified as promising candidates by a virtual screening approach. Among these candidates, three hits 7,4'-dimethoxyisoflavone, genistein, and fraxetin displayed dual inhibition of COX-2 and 5-LOX. Further in vitro assay indicated that 7,4'-dimethoxyisoflavone, genistein, and fraxetin could inhibit PGE2, LTB4, TNF-α, IL-6, or NO production in IL-1β-induced chondrocytes. In addition, the three compounds reduced IL-1β-induced degradation of collagen II and expression of MMP-13 in rat chondrocytes. The results of anti-inflammatory activity of the three compounds in vivo showed that the highest anti-inflammatory activity with edema inhibition percentages of 50.00%, 56.00%, and 51.00% after 3 h, respectively. Moreover, it was found that 7,4'-dimethoxyisoflavone, genistein, and fraxetin have a superior gastric safety profile comparable to indomethacin. Finally, molecular dynamics simulations, binding free energy analysis, and detailed interaction mode demonstrated that 7,4'-dimethoxyisoflavone, genistein, and fraxetin interacted well with both COX-2 and 5-LOX.

Conclusions: 7,4'-dimethoxyisoflavone, genistein, and fraxetin from DHJSD with excellent anti-inflammatory effects and no gastric ulceration effects, which helps to explain the dual action mechanism and potential material basis of DHJSD in treating OA and provide evidence to support DHJSD's clinical use.

Keywords: 5-LOX; COX-2; Duhuo Jisheng decoction; Molecular dynamics simulation; Osteoarthritis; Virtual screening.