Mechanism Of NOX₂ Subunit P47^phox In Antiinflammatory Of Myricitrin

Objective:To establish an inflammatory model with LPS,to detect the anti-inflammatory effect of myricitrin,and to further explore its possible molecular mechanism,taking the mouse macrophage line RAW264.7 cells and the rat primary peritoneal macrophages as the research object.Methods:The cell inflammatory model was constructed by using 100 ng/ml LPS to stimulate the RAW264.7 cells of mouse macrophages and the primary peritoneal macrophages of rats,and the cell survival rate was detected by CCK-8 method,and the expression level of iNOS and COX-2 mRNA was detected by semi-quantitative RT-PCR method;using ELISA method to detect changes of inflammatory related cytokines TNF-?,IL-1?,IL-6 and Western blotting to detect iNOS and COX-2 protein expression levels.The activation of NF-?B signaling pathway and the protein expression level and membrane distribution of cytoplasmic subunits p47^phox and cytoplasmic subunits gp91^phoxhox in NOX₂,and the NF-?B-p65 nuclear translocation in cells were observed by laser confocal microscope,while in Western blotting in the experiment,the content of intracellular reactive oxygen ROS was detected by DCFH-DA fluorescence probe,and the effect of ROS as upstream signaling molecule on inflammatory response was verified by using ROS scavenger NAC.The pan-phosphorylation level of p47^phox and gp91^phoxhox was detected by immune co-precipitation method and laser confocal microscope,and p47^phox shRNA and gp91^phox shRNA interference plasmid were constructed respectively,p47^phox(S₃₀₄,S₃₂₁,S₃₂₉ and S₃₄₆)phosphorylated mutants and non-phosphorylated mutants,after instantaneous transfection of cells,the detection of related inflammatory indicators.Results:The vitality IC₅₀ value of RAW264.7 cells in the range of less than 200?g/ml concentration was 244.413?g/ml.In mouse macrophage line RAW264.7 cells and primary peritoneal macrophages in rats,myricitrin?50,100,200?g/ml?could effectively inhibit the expression level of iNOS protein and mRNA induced by LPS,and the expression of iNOS in 200?g/ml concentration group was restored to the background level.There was no significant effect on the expression of COX-2 protein and mRNA.In addition,myricitrin can significantly inhibit the release of inflammatory factors TNF-?,IL-1?,IL-6 and NO.LPS is able to activate the NF-?B signaling pathway,and the phosphorylation levels of p-IKK and p-I?B proteins peak at 30 minutes.The pretreatment cells of myricitrin can effectively inhibit the activation of p-IKK and p-I?B induced by LPS,and have dosage dependence.In addition,myricetrin can also prevent transcription factor NF-?B-p65 into the nucleus.myricitrin can inhibit the production of LPS induced endogenous ROS,and after NAC pretreatment cells,the related inflammatory reactions induced by LPS are effectively inhibited.myricitrin can effectively block the transposition of p47^phox subunits induced by LPS to cell membranes,and prevent p47^phoxhox and gp91^phox from forming complexes.In addition,the phosphorylation level of p47^phoxhox was significantly reduced after the pretreatment of the cells by myricitrin.After the instantaneous transfection of cells p47^phox shRNA and gp91^phox shRNA disturbed plasmid,the related inflammatory response and signal changes induced by LPS were significantly reduced,and the inhibition effect of the co-transfection group was better.However,there was no significant change in the phosphorylation levels of NF-?B pathway signaling molecules IKK and I?B after p47^phox(S₃₀₄,S₃₂₁,S₃₂₉ and S₃₄₆)phosphorylation mutants and non-phosphorylated mutants transfected cells.Conclusion:1.Myricitrin can effectively inhibit the expression of iNOS protein and mRNA levels in the inflammatory medium induced by LPS,but has no significant effect on the expression of COX-2.2.Myricitrin can reduce the release of inflammatory factor TNF-?,IL-1?,IL-6 and NO induced by LPS.3.Myricitrin can inhibit the activation of NF-?B signaling pathway induced by LPS.4.In LPS induced inflammatory response,ROS,as an upstream signaling molecule,mediated changes in downstream related inflammatory factors and signaling molecules,and myricitrin significantly inhibites the production of LPS induced endogenous ROS.5.LPS induces the translocation of NOX₂ cytoplasmic subunit p47^phox to cell membrane and the formation of gp91^phox complex of membrane subunit to produce ROS,but myricitrin inhibites the production of ROS and then inhibites the inflammatory response by reversing the process.6.Myricitrin can significantly inhibit the p47^phox of phosphorylation,but S₃₀₄,S₃₂₁,S₃₂₉and S₃₄₆ four serine sites may not participate in LPS induced p47^phox activation process.