Embelin protects against apoptosis and inflammation by regulating PI3K/Akt signaling in IL-1β-stimulated human nucleus pulposus cells

Abstract

Embelin is a natural benzoquinone compound that displays a beneficial effect in various inflammatory-related diseases. However, the effect of embelin on degeneration of intervertebral disc (IDD), a chronic inflammatory disorder, has not been reported. This study was attempted to explore the therapeutic action of embelin on IDD in vitro. Network pharmacology analysis was performed for evaluating the link between embelin and IDD. The human nucleus pulposus cells (NPCs) were stimulated with IL-1β to induce inflammation. Cell viability of NPCs was assessed by CCK-8 assay. Western blotting was conducted to detect the expression levels of PI3K, p-PI3K, Akt, p-Akt, cleaved caspase-3, caspase-3, Bax, Bcl-2, p65 and p-p65. Apoptotic deaths of NPCs were examined by TUNEL assay. The production of COX-2, IL-6, IL-8, and TNF-α was examined by ELISA. It can be seen that 16 overlapping genes were selected from 109 possible targets of embelin and 342 possible targets of IDD. KEGG pathway enrichment analysis showed that the PI3K/Akt signaling pathway was a close link between embelin and IDD. We found that embelin dose-dependently improved the cell viability in IL-1β-stimulated NPCs. Embelin elevated the relative levels of p-PI3K/PI3K and p-Akt/Akt in IL-1β-stimulated NPCs. IL-1β induced a significant increase in apoptotic deaths of NPCs, which was attenuated by embelin treatment. IL-1β-induced alternations in expression levels of apoptotic-related proteins including cleaved caspase-3, Bax and Bcl-2 were prevented by embelin treatment. Pretreatment with LY294002 (an inhibitor of PI3K) reversed the inhibitory effect of embelin on IL-1β-induced apoptosis in NPCs. Embelin treatment caused inhibitory effects on the IL-1β-stimulated production of COX-2, IL-6, IL-8, and TNF-α, which were abolished by LY294002 treatment. Furthermore, embelin treatment prevented IL-1β-induced phosphorylation of p65 in NPCs, while LY294002 elevated the embelin-caused decrease in p-p65/p65 level. Overall, embelin protected human NPCs against IL-1β-stimulated apoptosis and inflammation by regulating the PI3K/Akt signaling pathway. These findings provided new ideas for the clinical usage of embelin in the prevention and treatment of IDD.

Keywords: Degeneration of intervertebral disc; Embelin; Inflammation; Nucleus pulposus cells; PI3K/Akt pathway.