Isoliensinine suppresses chondrocytes pyroptosis against osteoarthritis via the MAPK/NF-κB signaling pathway.

Background: Isoliensinine is an active compound derived from Nelumbo nucifera which has long been used for its anti-inflammatory properties. However, the mechanism of Isoliensinine in the treatment of osteoarthritis is poorly known.

Purpose: The present study aims to investigate whether Isoliensinine could alleviate osteoarthritis by regulating MAPK/NF-κB signaling pathway-mediated pyroptosis.

Methods: Network pharmacology and KEGG enrichment analysis were used to identify the therapeutic targets of Isoliensinine for OA. Molecular docking was used to confirm the binding ability of Isoliensinine and related proteins. In vitro, chondrocytes were stimulated with IL-1β to construct an inflammatory model and treated with Isoliensinine. The viability of the cells was assessed using the CCK-8 kit. The apoptosis rate of cells was measured using Annexin V-FITC/PI assay. And assessed the levels of ROS, lipid-ROS, and mitochondrial membrane potential. Corresponding assay kits were utilized to measure the levels of MDA and SOD. Subsequently, the anabolic and catabolic markers in chondrocytes, alongside inflammatory targets were measured by RT-PCR and Western blot. The expression level of pyroptosis and MAPK/NF-κB signaling pathway-related targets was examined. Furthermore, we constructed a rat osteoarthritis model using ACLT surgery. We then assessed the progression of osteoarthritis by Micro-CT, H&E staining, S&F staining and immunohistochemistry.

Results: Enrichment analysis showed that Isoliensinine treatment of osteoarthritis may be through the MAPK/NF-κB pathway, and molecular docking showed that Isoliensinine and MAPK/NF-κB pathway proteins had a good binding ability. Data showed that Isoliensinine could reduce ECM degradation and inflammation, and inhibit IL-1β-induced apoptosis. It also mitigated ROS and LPO activation, regulated mitochondrial dysfunction, and reduced intracellular oxidative stress levels. Furthermore, Western blot showed that Isoliensinine also inhibited the activation of the MAPK/NF-κB pathway, thereby inhibiting the pyroptosis of chondrocytes. In vivo, Micro-CT, H&E staining and S&F staining results showed that Isoliensinine could effectively improve joint damage caused by osteoarthritis. And IHC analyses indicated NLRP3, MMP3 protein expression were significantly diminished and Collagen II expression was increased in the Isoliensinine treatment groups.

Conclusion: In conclusion, our study suggested that Isoliensinine mitigates ECM degradation, oxidative stress, chondrocytes apoptosis, and pyroptosis through the inhibition of the MAPK and NF-κB pathways, thereby delaying the progression of osteoarthritis.