Analysis of Glycerophospholipids in RAW264.7 Cell Inflammation Model Interfered by Diphenylheptane A with UPLC-Q/TOF MS

KeyWord:inflammation  glycerophospholipids  diphenylheptane A  ibuprofen  biomakers  ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF MS)

Abstract:

      The study aims to illustrate the effect of diphenylheptane A on the glycerophospholipids metabolism in the inflammatory process through analyzing glycerophospholipids components of RAW264.7 cells under different status(control£¬inflammation£¬administration) and screening potential pathological and pharmacological biomarkers.RAW264.7 cells were randomly divided into four groups£ºcontrol group(C)£¬inflammation group(L)£¬diphenylheptane A(D)£¬ibuprofen(B£¬positive control).C and L were treated with fresh medium while D and B were treated with medium containing 20¦Ìg/mL diphenylheptane A or 100¦Ìg/mL ibuprofen£¬respectively.1 h later£¬lipopolysaccharide(LPS) was added to L£¬D and B at a final concentration of 0.5¦Ìg/mL.24 h later£¬glycerophospholipids in RAW264.7 cells of four groups were extracted by modified Bligh-Dyer method and analyzed by ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF MS) in both positive and negative ion modes.Then£¬glycerophospholipids components were identified by combination of MS/MS fragment ions information£¬element composition in MassLynx 4.1 and the Lipid Maps database.Finally£¬potential pathological and pharmacological biomarkers were screened by unsupervised principal component analysis(PCA)£¬supervised partial least squares-discriminate analysis(PLS-DA)£¬supervised orthogonal partial least squares discriminate analysis(OPLS-DA)£¬and Student¡¯s t-test(P<0.05).The results showed that 27 potential pathological biomarkers were found by comparing C and L£¬23 potential pharmacological biomarkers were found by comparing D and L£¬and 17 potential pharmacological biomarkers were found by comparing B and L.The biomarkers mainly included phosphatidylcholine(PC)£¬lysophosphatidylcholine(lysoPC)£¬phosphatidylethanolamine(PE) and lysophosphatidylethanolamine(lysoPE).It was found that diphenylheptane A led to obvious changes of glycerophospholipids metabolism during the process of inflammation£¬which were closely related to the occurrence and development of inflammation.