GC-MS Analysis Of Fatty Acid Metabolomics In RAW264.7 Cell Inflammatory Model Interfered With Nsaids

Objective: The research was to study the fatty acid metabolomics ofRAW264.7 cell under the condition of inflammation based on GC-MS technology.The biological technique was applied to explore the biological information on related biomarkers.Methods: The inflammation model of RAW264.7 macrophage was induced by LPS and interfered with aspirin,ibuprofen and meloxicam.With the GC-MS technology,fatty acid components in RAW264.7 cell were extracted by the method of two-step extraction-methylation and determined after the key parameters of GC-MS were optimized,such as column selection,temperature programming,injection mode,flow rate and so on.Finally,fatty acid constituents were identified by referenced to the fatty acid standard products information and the NIST library(National Institute of Standards and Technology).The obtained data was imported into Simca-p 14.0 soft,and the learning methods of Principal Component Analysis(PCA),Partial least squares discrimination analysis(PLS-DA)and Orthogonal Partial Least Squares Discriminant Analysis(OPLS-DA)were established.With the ANOVA analysis,fatty acid components with VIP>1 and P<0.05 were selected as the significant difference biomarkers.At last,the biological significance of fatty acid biomarkers was explored in combination with biological methods(PCR,WB).Results: 21 fatty acids in RAW264.7 cells were successfully identified by GC-MS.The normal group,model group and medication administration group were successfully grouped with the learning method established by chemometrics.6 significant differential biomarkers were screened in the normal vs inflammatory group;seven significant differential biomarkers were screened in the inflammatory group vs aspirin group;nine significant differential biomarkers were screened in the inflammatory group vs ibuprofen group;three significant differential biomarkers were screened in the inflammatory group vs meloxicam group.Finally,with the ELISA,PCR and WB biological technology,the result revealed that mRNA and protein expression levels of IL-1?,NLRP3,GPR120 and ?-Arrestin-2 were up-regulated in RAW264.7 cells induced by LPS and Nigericin.After drug intervention in the inflammatory model,their expressions were statistically decreased.The result preliminarily indicated that the signaling pathway centered on the NLRP3 inflammasome is involved in the anti-inflammatory process of ibuprofen.Conclusion: Based on GC-MS combined with chemometrics,fatty acid biomarkers of different groups could be rapidly screened out for related metabolomics studies.It is indicated that the fatty acid metabolomics under the inflammatory model could be effectively studied with GC-MS combined with chemometrics.On the research,GC-MS combined with chemometrics is firstly applied to the metabolomics study of fatty acids in the inflammatory model of RAW264.7 macrophage.Application with biological means,the anti-inflammatory mechanism of ibuprofen was firstly elucidated from the perspective of NLRP3 inflammasome,the anti-inflammatory signaling pathway of ibuprofen was enriched.