In Vitro Study Of API6 Expression In Lipid Loading And Inflammatory Macrophages

ObjectiveApoptosis inhibitor 6 (Api6) , also known as AIM(Apoptosis Inhibitor expressed by Macrophage) or SPα(Soluble Protein alpha), is a newly defined member of the group B scavenger receptor cysteine-rich (SRCR) superfamily. Previous studies have suggested its important roles in immune system regulation, but the involvement of Api6 in lipid metabolism is seldomly studied. Two macrophages cell lines: mouse macrophages (RAW264.7) and human monocyte cell(THP-1)were choosed in this experiment to invertstigate :①lipid accumulation in lipid loading and inflammatory macrophages;②The affect of LPS and oxLDL loading on mRNA expression level of Api6,CD36 and SR-A in RAW264.7 and THP-1 marcrophages;③The relationship between Api6 expression level and the levels of LXRalpha and LXRbeta in lipid loading and inflammatory macrophagesMaterials and Methods1. The LDL was separated from fresh human blood and was oxdized to oxLDL. oxLDL and LPS treating were used to set up lipid loading condition and inflammatory condition. Lipid Accumulations in macrophages were detected using Oil Red O staining.2. mRNA was purifed from macrophages collected from peritoneal cavity of Balb/c mouse, Api6 cDNA was got from reverse transcription. Then it was inserted into pCDNA3.1 to form recombinant plasmid pCDNA3.1/Api6.3. mRNA level of Api6,CD36,SR-A,LXRalpha,LXRbeta,ABCA1,ABCG1 in RAW264.7 and THP-1 marcrophages with different treatments were detected by real time quantitative polymerase chain reaction (PCR).Results1. THP-1 cells and murine RAW264.7 macrophages were incubated in the presence of 100μg/ml oxLDL alone, LDL plus 200ng/ml LPS and LPS alone. Foam cell formation after treatment was evidenced by oil red O staining.2. The recombinant plasmid pCDNA3.1/Api6 was successfully cons- tructed and transfect it to RAW264.7 macrophages. The mRNA levels of Api6 increased 12500-fold after transfection.3. Real-time PCR results showed that after LPS or oxLDL treatment, the mRNA levels of Scavenger receptor SRA were increased 6-fold, 2.5-fold and 6-fold, 44-fold in Raw264.7 cells and THP-1 cellls respectively, but the expression of Api6 in Raw264.7 did not change. The mRNA levels of Api6 increased 2.7-fold after LPS and oxLDL treatment in THP-1 cells.4. ABCA1 and ABCG1 were target genes of marcrophages. T0901317 was agonist of LXR receptor. Real-time PCR results showed that after T0901317 treatment, the mRNA levels of ABCA1 and ABCG1 were increased 22-fold and 7-fold respectively in RAW264.7 cells and both increased 9-fold in THP-1 cells. But the expression of Api6 in RAW264.7 did not change by T0901317 treatment, while the mRNA levels of Api6 increased 2-fold in THP-1 marcrophages. The expression levels of Api6 in these macrophages were highly related to the presence of nuclear receptor liver X receptor(LXR) isoform LXRalpha.ConclusionsThe expression levels of Api6 in these macrophages were highly related to the presence of nuclear receptor liver X receptor(LXR) isoform LXRα. This study revealed that due to the lack of LXRαisoform, the basic expression level of Api6 was low in Raw264.7 macrophages, which makes it an ideal cell line for further study related to pathophysiological function of Api6 in lipid metabolism.