NOX2 Mediated Myricitrin Inhibition On LPS-induced Inflammatory Response

Objectives: In the present study,We clarified the effect of myricitrin on NOX2-drived ROS-mediated JAKs/STAT1-dependent inflammatory response in LPS-induced mouse monocytes / macrophages RAW264.7 cells in vitro.Meantime,we assessed the effect of myricitrin on LPS-induced mouse acute lung injury in vivo.Thereby,we clarified the pharmacological mechanism of myricitrin on the inflammatory response induced by LPS.Methods: CCK-8 assay was used to detect the RAW264.7 cytotoxicity administrated by myricitrin.Western blotting and RT-PCR were used to detect the effect of myricitrin on i NOS,COX-2 protein and m RNA levels in RAW264.7 macrophage induced by LPS,and confirmed the effective dose range of myricitrin.ELISA assay was used to detect the effect of myricitrin on pro-inflammatory cytokines and inflammatory mediator TNF-α,IL-6,MCP-1,NO and PGE2 expression.Western blotting was used to detect the phosphorylation time point of MAPKs and JAK-STAT signalling transduction pathway induced by LPS,and furthermore assessed the effect of myricitrin.The effect of myricitrin on nuclear translocation and transcriptional activity of STAT1 transcription factor were observed by laser scanning confocal microscope and EMSA.The effect of myricitrin on ROS accumulation was observed by ROS probe DCFH-DA.The distribution changes of NOX2 subunits gp91 ^phox and p47 ^phox in cytoplasm and plasma membrane were observed by western blotting,laser scanning confocal microscope.NOX2 inhibitor apocynin,ROS scavenger NAC,JAK inhibitor ruxolitinib were used to confirm the relationship between NOX2,ROS,JAKs/STAT1 signalling and the downstream pro-inflammatory cytokines as well as inflammatory mediator expression.NOX2 subunits gp91 ^phox and p47 ^phox sh RNA plasmid vector were constructed and its interference effect is validated.After knocking down the gp91 ^phox and p47 ^phox,the ROS production,JAK1,JAK2,STAT1 activation and i NOS,COX-2 expression were detected.The effect of myricitrin on acute lung injury in mice stimulated by LPS was observed by HE staining assay.Results: Non-cytotoxic does of myricitrin markedly inhibited iNOS protein and mRNA levels induced by LPS in RAW264.7 macrophage,and ensure the appropriate effects of high,medium,and low therapeutic doses of myricitrin（100,200,and 400 μg/ml）.Myricitrin significantly inhibited the release of tumour necrosis factor（TNF）-α,interleukin-6（IL-6）,and MCP-1,as well as the production of nitric oxide and the expression of i NOS.In addition,myricitrin had no effect on COX-2 and PGE2 in LPSinduced RAW264.7 macrophage.Myricitrin significantly relieved the acute lung injury in mice stimulated by LPS.The activation of ERK,JNK and P38 achieved the peak at30 min.The phosphorylation of JAK1 and JAK2 reached the highest level at 10 min.Meanwhile,LPS induced the STAT1 and STAT3 maximum activation at 4 h.Myricitrin could significantly down-regulated the LPS-induced phosphorylation of JAK1,JAK2 and STAT1 in a dose-dependent manner,but had no effect on the ERK,JNK,P38 and STAT3 activation.Myricitrin inhibited the LPS-induced nuclear translocation and transcriptional activity of STAT1 in RAW264.7 macrophage.Myricitrin effectively eliminated the LPS-induced ROS accumulation in RAW264.7 macrophage.Myricitrin could block the aggregation of NOX2 subunits gp91 ^phox and p47 ^phox in plasma membrane induced by LPS.Myricitrin down-regulated LPS-induced JAKs/STAT1-dependent inflammatory response through down-regulating the ROS level generated from NOX2.NOX2 subunits gp91 ^phox and p47 ^phox sh RNA plasmid vector markedly inhibits LPS-induced ROS production,JAK1,JAK2,STAT1 activation and i NOS,COX-2 expression in RAW264.7 macrophage.Conclusion:1)Non-cytotoxic levels of myricitrin alleviated inflammatory response in RAW264.7macrophage and acute lung injury.2)Myricitrin down-regulated LPS-induced pro-inflammatory cytokines and inflammatory mediator i NOS,NO,IL-6,TNF-α,MCP-1 and had no effect on COX-2and PGE2.3)Myricitrin inhibited LPS-induced inflammatory response by blocking JAKs/STAT1 rather than MAPKs signaling pathway.4)Myricitrin down-regulated LPS-induced JAKs/STAT1-dependent inflammatory response through down-regulating the ROS level generated from NOX2.5)The anti-inflammatory effect of myricitrin were through blocking NOX2 subunits p47 ^phox and gp91 ^phox polymerization in LPS-induced RAW264.7 macrophage.