Effects And Mechanism Of FABP3 Gene On LPS-induced Inflammation Response In Bovine Mammary Epithelial Cells

The mastitis has made terrible damages for dairy health and industry around the world.As the increasing demonds of quantity and quality,it is crucial to establish effective technology of to prevent and treat mastitis.With the development of molecular biology techniques,marker-assisted selection(MSA)recently has become an important approach for genetic improvement and breeding in cow industry.While MAS depended on the most critical basis that mastitis related genes or molecular markers were known.Genome-wide association study(GWAS)GWAS was an efficient approach to find the significant SNPs associated with dairy cow mastitis.Fatty acid binding protein(FABP)family genes located with 1Mb around the significant SNPs were suggested as the candidate genes.this SNP within 1MB scope.FABP3 is a important fatty acid transporters in cells,and active in multiple tissues such as breast and myocardial.FABP3 can involve in various life activities by mediatng fatty acid transporting,such as lipid metabolism,energy metabolism,immune regulation and so on.In this study,we built LPS-induced cellular inflammation model to research function and mechanism of FABP3 gene on LPS-induced inflammation response in bovine mammary epithelial cells.Experiment results are as follows:(1)The FABPs gene expression level in bovin peripheral blood leucocyte(PBL)and mammary epithelial cells(bMECs):In bovin peripheral blood leucocyte,FABP3 and FABP5 expressed efficiently,but not FABP4,FABP8 or FABP9;in bovine mammary epithelial cells,the expression level of FABP4 and FABP5 is high,but FABP3?FABP8 and FABP9 is low;(2)Built LPS-induced mammary epithelial cells(bMECs)inflammation model:we treated cells with LPS concentration of 0.5ug/ml for 24 h to induce inflammation response.Then the cell viability was measured by flow cytemetry.The result demonstrated LPS failed to influence cell viability,which suggested the model was built successfully;(3)The influence on FABP3 mRNA expression of LPS stimulating in PBL and bMECs: Compared with the control group,LPS increased significantly the mRNA expression of FABP3 at 3-12h(P<0.01),with maximal level at 24 h in PBL.InbMECs,FABP3 mRNA expression with highest level at 24 h was also increased significantly(P<0.01),compared with the control group at 12-24h;(4)The function of FABP3 gene on LPS-induced inflammation response in bMECs: FABP3 gene silencing by siRNA inhibited LPS-induced inflammation factor(IL-6 and IL-8)mRNA expression(P<0.001),and FABP3 overexpression using the FABP3 overexpression plasmid increased IL-6,IL-8 mRNA expression(P<0.001).Therefore we found FABP3 regulated LPS-stimulated crucial inflammation NF-?B passway activation by mediating PPAR?(peroxisome proliferator activated receptors)expression;(5)The function of long-chain fatty acid on FABP3 mRNA expression and inflammation response LPS-induced:palmitic acid(saturated fatty acid)can significantly promote the expression of FABP3(P<0.001),and increased the mRNA expression of IL-6 and IL-8 induced by LPS in bMECs(P<0.01);but stearic acid(saturated fatty acid)failed to influence.It is interesting that Oleic acid(unsaturated fatty acid)significantly inhibited FABP3 expression,and expression of the inflammation factor(IL-6and IL-8)(P<0.01).In summary,FABP3 gene significantly expressed in PBL and bMECs,and LPS significantly induced FABP3 mRNA expression.FABP3 gene silencing efficiently inhibited activation of NF-?B passway by increasing PPAR? expression,which regulated pro-inflammation factor IL-6 and IL-8 expression to prevent inflammation response.As FABP3 transporting long-chain fatty acid,different long-chain fatty acid influenced the FABP3 mRNA expression and inflammation response in LPS-induced bMECs.The results of our research provided new experimental basis and theory reference for researching on immune mechanism related to mastitis and controling dairy cow mastitis efficiently.