Chrysin Inhibits LPS-induced Inflammatory Via ROS Mediated Multiple Signaling Pathways

Objective: To investigate the molecular mechanism of chrysin in inhibiting LPS-induced inflammation.Methods: RAW264.7 cells were pretreated using different concentration of chrysin for 24 h,and then treated with LPS.Cell viability was measured using CCK-8 assay for screening the safe cellular dose of chrysin.Meanwhile,the levels of i NOS and COX-2 expressions were also detected by Western bloting and RT-PCR methods for determining the effectively anti-inflammatory dose of chrysin.The important signaling pathways were also determined using Antibody Array Kit.The levels of anti-inflammatory cytokines and mediators,including IL-6,TNF-?,NO and MCP-1 were also detected using ELISA method.The relevant signaling pathways including PI3K-AKT-m TOR,JAK-STAT and MAPK were performed using Western bloting.The nuclear entrance of transcript factors,STAT1 and STAT3 was also observed under laser scanning confocal microscope,as well as the production of reactive oxygen species(ROS)labelled with ROS probe.The effect of ROS on AKT-m TOR,JAK-STAT and MAPK signaling pathways was also detected in the RAW264.7 cells treated with ROS inhibitor N-acetyl-L-cysteine(NAC).Results: The cellular viability of RAW264.7 cell line was not affected under the treatment of less than 60 ?g/ml of chrysin.Chrysin treatment decreased the expression levels of i NOS and COX2 in RAW264.7 cells treated with LPS.So three concentration of 10,30 and 60 ?g / ml of chrysin were used as low,median,and high dose in the following experiments.The results showed that chrysin can effectively inhibit the released levels of TNF-?,IL-6,MCP-1 and NO in RAW264.7 cell treated by LPS.Different signaling pathways were activated by LPS.The maximum activated time of AKT,p-PRAS40,p-m TOR,p-70S6 K,and p-PS6 proteins in PI3K-AKT-m TOR signaling pathway was about 1 h,1.5 h,2.5 h,3 h,and 4 h,respectively.Furthermore,the maximum activated time of JAK and STAT proteins in JAK-STAT signaling pathway was 10 min and 4 h,respectively;as well as MAPK signaling pathway about 30 min.The nuclear translocation of STAT-1 and STAT-3 was also inhibited by chrysin.The production of ROS in RAW264.7 cells was also decreased under chrysin treatment.The phosphorylation of PI3K-AKT-m TOR,JAK-STAT and MAPK signaling pathway proteins were inhibited by NAC.Conclusion: 1.Chrysin can effectively inhibit inflammatory response of RAW264.7 cells induced by LPS.2.Chrysin attenuated the release of pro-inflammatory cytokines and inflammatory mediator in the i NOS,MCP-1,COX-2,TNF-?,NO,and IL-6 in RAW264.7 cells induced by LPS.3.The anti-inflammatory effects of chrysin was through blocking ROS-mediated PI3K-AKT-m TOR,JAK-STAT and MAPK signaling pathways in RAW264.7 cells.