Mechanism Of Blumea Balsamifera Oil On Lipopolysaccharides-induced Secretion Of Inflammatory Cytokines In Macrophages

Inflammation is a defense and protective response to multiple harmful stimuli.Over and uncontrolled inflammation can lead to local tissues or even systemic damages and injuries,and it is the fundament of the pathogenesis of a variety of inflammatory diseases.Our previous study found that Blumea balsamifera oil has good anti-inflammatory activity,however,its potential mechanism of action is not well understood.The purpose of this study was to explore the effect of Blumea balsamifera oil on LPS-induced secretion of inflammatory factors by macrophage and its potential mechanism.Firstly,using the GC-MS method to analyze the five Blumea balsamifera oil from Guizhou,determine and compare the main components,and calculate the relative percentage by the peak area normalization method;The model of mouse ear swelling induced by xylene and the inhibition test of allergic slow-reacting substances（SRS-A）were used to screen Blumea balsamifera oil with relatively good anti-inflammatory activity in five oils,which laid the material foundation for the subsequent research.Secondly,the toxicity of the best source of Blumea balsamifera oil to macrophages was detected by MTT assay;and the secretion of tumor necrosis factor（TNF-α）,interleukin-1β（IL-1β）,interleukin-6（IL-6）,prostaglandin E₂（PGE₂）and cyclooxygenase-2（COX-2）by LPS-induced macrophages was detected by ELISA;and the effects of nitric oxide（NO）and nitric oxide synthase（iNOS）on LPS-inducedmouseperitonealmacrophageswasdetectbyUltraviolet spectrophotometry.Finally,the effect of Blumea balsamifera oil on the relative mRNA expression of CD14,MD-2,TLR4,MyD88,IRAK-1,TAK-1,NF-κBp65,iNOS and COX-2 in LPS-indeced RAW264.7 cells by qPCR;and the relative protein expression of CD14,MD-2,TLR4,MyD88,IRAK-1,TAK-1,NF-κBp65 and nlrp3 in LPS-indeced RAW264.7 cells was detected by Western blot.The experimental results showedthatthecontentof（-）-Borneol,trans-Caryophyllene,Camphor,Caryophyllene-oxide,Alloaromadendrene,Xanthoxylin,Linalool is relatively high in Blumea balsamifera oil of different origins,and it has 21 common ingredients;The four oils can significantly reduce the swelling of xylene-induced ear swelling in mice（P﹤0.05,P﹤0.01）,and the five oils can significantly reduce SRS-A production（P﹤0.01）,obtaining the best actived Blumea balsamifera oil,and the IC₅₀0 value of the oil to macrophages is 254.4μg·mL^-1.And the oil can significantly inhibit LPS-mediated secretion of TNF-α,IL-1β,IL-6,PGE₂ and COX-2,NO and iNOS activity（P﹤0.05,P﹤0.01）,also inhibited the relative mRNA expression of CD14,MD-2,TLR4,MyD88,IRAK-1,TAK-1,NF-κBp65,iNOS,COX-2 and inhibit the relative protein expression of CD14,MD-2,TLR4,MyD88,IRAK-1,TAK-1,NF-κBp65,nlrp3 in macrophages（P﹤0.05,P﹤0.01）.The results suggest that the five Blumea balsamifera oil from Guizhouhavedifferentdegreesofanti-inflammatoryeffects,andthe anti-inflammatory mechanism may inhibit the phosphorylation of NF-κBp65 into the nucleus through the LPS-TLR4 signaling pathway,reduce the production of PGE₂ and inhibit the activity of iNOS,thereby reducing the production and expression of NO and TNF-α,IL-1β,IL-6;it is also possible to inhibit the occurrence of inflammation by inhibiting the activation of the nlrp3 inflammasome pathway in macrophages by LPS.The results of this experiment will provide theoretical data for further research on Blumea balsamifera oil from Guizhou.