Elucidation of the key therapeutic targets and potential mechanisms of Andrographolide multi-targets against osteoarthritis via network pharmacological analysis and experimental validation

Objective: Our purpose is to unveil Andrographolide's potential multi-target and multi-mechanism therapeutic effects in treating OA via systematic network pharmacological analysis and cell experimental validation.

Materials and methods: Initially, we gathered data from Andrographolide and OA-related databases to obtain information on Andrographolide's biological properties and the targets linked with OA. We developed a bioinformatic network about Andrographolide and OA, whereby we analyzed the network to identify potential therapeutic targets and mechanisms of action of Andrographolide. Subsequently, we used molecular docking to analyze the binding sites of Andrographolide to the target proteins. At the same time, SDF-1 was used to construct an OA cell model to verify the therapeutic effect of Andrographolide on OA and its effect on target proteins.

Results: Our experimental results show that Andrographolide has excellent pharmaceutical properties, by Lipinski's rules for drugs, suggesting that this compound can be considered to have a high therapeutic potential in drug development. 233 targets were preliminarily investigated, the mechanisms through which Andrographolide targets OA primarily involve the TNF signaling pathway, PI3K-AKT signaling pathway, IL-17 signaling pathway, and TLR signaling pathway. These mechanisms target OA by influencing immune and inflammatory responses in the joints, regulating Apoptosis to prevent chondrocyte death. Finally, TNF-α, STAT3, TP53, IL-6, JUN, IL-1β, HIF-1α, TGF-β1, and Akt1 were identified as 9 key targets of Andrographolide anti-OA. In addition, our molecular docking analyzes with cell experimental validation further confirm the network pharmacology results. According to our molecular docking results, Andrographolide can bind to all the hub target proteins and has a good binding ability (binding energy < -5 kcal/mol), with the strongest binding affinity to Akt1 of -9.2 kcal/ mol. The results of cell experiments showed that Andrographolide treatment significantly increased the cell viability and the expression of COL2A1 and ACAN proteins. Moreover, 30 μM Andrographolide significantly reversed SDF-1-induced increases in the protein expression of TNF-α, STAT3, TP53, IL-6, JUN, IL-1β, HIF-1α, and TGF-β1, and decreases in the protein expression of Akt1.

Conclusion: This study provides a comprehensive understanding of the potential therapeutic targets and mechanisms of action of Andrographolide in OA treatment. Our findings suggest that Andrographolide is a promising candidate for drug development in the management of OA.

Andrographolide; Multi-target therapy; Network pharmacology; Osteoarthritis.