Mechanism of PI3K/AKT/mTOR signaling pathway for mediating anti-inflammatory and anti-oxidant effects of chrysin: a protein microarray-based study

Abstract

To investigate the mechanism of PI3K/AKT/mTOR signaling pathway for mediating the anti-inflammatory and anti-oxidant effects of chrysin. RAW264.7 cells were treated with different concentrations of chrysin for 24 h, and the changes in cell viability were detected using CCK-8 method. The cells with or without chrysin pretreatment for 2 h were stimulated with lipopolysaccharide (LPS) for different lengths of time, and the related signal molecules were screened using protein chip technique. In cells pretreated with chrysin for 2 h followed by LPS stimulation for 18 h, the release of IL-6, MCP-1 and TNF-α by the cells was detected with ELISA, and NO production was examined using Griess method, and ROS level was determined using DCFH-DA. The effects of chrysin, LPS, and their combination on the mRNA expressions of iNOS and COX-2 were detected using RT-PCR; Western blotting was performed to examine the changes in cellular expressions of p-AKT, p-PRAS40, p-mTOR, mTOR, p-P70S6k, p-S6RP and S6RP following the treatments with LPS, N-Acetyl-L-cysteine, and chrysin, alone or in combinations. Chrysin below 60 μg/mL did not significantly affect the viability of RAW264.7 cells (P>0.05). Chrysin treatment significantly reduced the release of IL-6, MCP-1, and TNF-α and the level of NO (P < 0.01), and inhibited the mRNA and protein expressions of iNOS and COX-2 (P < 0.01) in the cells. The results of protein chip screening suggested that LPS could activate the AKT/mTOR pathway, which was significantly inhibited by chrysin pretreatment, and the results were verified by Western blotting (P < 0.01). Chrysin treatment significantly reduced the generation of endogenous ROS, and treatment with N-Acetyl-L-cysteine to eliminate intracellular ROS obviously reduced the expressions of iNOS and COX-2 (P < 0.05) and blocked the AKT/mTOR pathway (P < 0.05). Chrysin can inhibit the synthesis of the upstream signaling molecule ROS to inhibit the activation of AKT/mTOR signaling pathway, regulate the translation process of ribosomes, down-regulate the synthesis and release of pro-inflammatory cytokines and inflammatory mediators, and thus produce anti-inflammatory effects.