The Preliminary Research Of FABP5 Participate In The Oxidation Of LDL In Raw264.7 Cells

Objective:Atherosclerosis is considered a chronic inflammatory response.Excessive-ly oxidized low-density lipoproteins are taken up by macrophages and become foam cells,causing a strong AS effect.In this study,the activity of phospholipase A2(PLA2)in cells was measured after using LDL to stimulate RAW264.7 cells,in order to exploring their transcription and translation expression levels,Real-time PCR and Western blot techniques were used to measured the expression levels of fatty acid-binding protein 5(FABP5)and peroxisome proliferating(PPARs).At the same time,the expression of FABP5 in RAW264.7 cells was measured after stimulated by unsaturated fatty acids(UFAs)to explore the interaction between UFAs and FABP5.The above experimental results were used to speculate interaction of phospholipase A2(PLA2),unsaturated fatty acids(UFAs),fatty acid-binding protein 5(FABP5),andperoxisomal proliferation-activated receptors(PPARs)are involved in the oxidation of low-density lipoprotein(LDL).what we do may be provide new ideas for LDL oxidation and provide guidance for the treatment of clinical diseases.Methods:Stimulated RAW264.7 cells with LDL for different time,and PLA2 total activity spectro ph-otometric assay kit was used to determine PLA2,activity;the expression levels of FABP5 genes and proteins were measured by Real-time PCR and western blot.Sucrose density gradient centrifugation was used to extracted the lipid rafts,using western blot technique to measure the content of FABP5 in lipid rafts and the part of except lipid rafts respectively.The RAW264.7 cells were cultured and stimulated with different concentrations of OA,ALA and AA.The expression of FABP5 was detected by Western blot;After LDL stimulated RAW264.7 cells for a long time,the expression levels of PPARs genes were measured by R western blot.Results:After short time stimulation to RAW264.7 cel,PLA2 activity increased;The expression of FABP5’s mRNA and protein were increased when using LDL stimulate RAW264.7 a long time;The expression of FABP5 increased after 12h of OA,s stimulation,and the expression of FABP5 both increased after AAs stimulation.The study of PPARs found no significant expression of PPARa after LDL stimulation,and the gene expression levels of PPARβ and PPARy increased.after separated lipid rafts,we found the levels of FABP5 in the lipid rafts is higher than control group,while the non-lipid rafts dispaly the opposite result.Conclusion:LDL stimulation can increase the activity of PLA2、the expression of FABP5 and translocation from non-lipid raft to lipid raft,indicating that FABP5 and PLA2 are very important factors in the oxidation of LDL.UFAs can change the expression of FABP5,indicating that UFAs may be downstream binding molecules of FABP5 in this process.After LDL stimulation,the expression of PPARs genes in cells was also increased.Combined with the relevant literature,we speculated that the LDL oxidation process:LDL stimulation will activate PLA2,while PLA2,hydrolyzes glycerophospholipids to produce UFAs(OA,ALA,AA,etc.)These UFAs recruit and bind FABP5 as a ligand and bond to PPARS,then combined with the PPRE,which is a reactive media-promoting factor response element of the target gene,then stimulate the oxidative stress pathway and promote the oxidation of LDL,and this process may be related to lipid rafts.