The Effection Of API6 On Cholesterol Trafficking In Lipid Loading And Inflammatory Macrophages

PurposeApi6(Apoptosis Inhibitor 6), also known as AIM or Spα,belongs to the macrophage scavenger receptor cysteine-rich domain super family. It has been implicated that Api6 played a very important role in immune regulation, tumor genesis, and in protecting macrophages from the apoptotic in different stages of atherosclerosis. However, as a scavenger receptor, its function in lipid metabolism is seldom studied. An over-expression system of Api6 in RAW264.7 cells was established in the present study using transient transfection technique, and the role of Api6 in cholesterol metabolism in lipid loading and inflammatory conditions were investigated.Material and method1. Api6 cDNA was inserted into PEGFP-N1 vector to form recombinant plasmid PEGFP-N1/Api6, and the recombinant vector was verified by restrictive end nuclease digestion analysis and DNA sequencing analysis.2. Mouse macrophages (RAW264.7) were transiently transfected with the PEGFP-N1/Api6 plasmid, cholesterol metabolism in lipid loading and inflammatory conditions were then investigated. Lipid Accumulations in macrophages were detected using Oil Red O staining and the expression of cholesterol trafficking related genes, such as SRA, CD36, LDLR, ABCA1, and ABCG1 were detected by real time quantitative polymerase chain reaction (RT-PCR).Result1. Plasmid PEGFP-N1/Api6 was constructed successfully. Western Blot test verified the increased expression of Api6 protein in PEGFP-N1/Api6 transfected RAW364.7 cells. RT-PCR results confirmed that compare to the empty vector transfected cells, the expression of Api6 in RAW264.7 cells transfected with PEGFP-N1/Api6 was increased 13 times.2. Oil red O experimental results showed that LDL loading with or without LPS treatment can lead to lipid accumulation in RAW264.7 cells. But the lipid accumulation is more obviously in PEGFP-N1/Api6 transfected RAW264.7 cells than in the empty vector transfected cells.3. Real-time PCR results showed that comparing to the group transfected with empty vector, over-expression of Api6 had no effects on the expression of SRA and ABCG1 genes in lipid loading and inflammatory conditions. However, it had influence in LDLR negative feedback regulation, increased scavenger receptor CD36 expression and decreased Cholesterol efflux related receptor ABCA1, thus led to the accumulation of intracellular cholesterol. ConclusionUnder conditions of cholesterol loading and inflammation, Api6 could increase the intracellular cholesterol accumulation, due to its possible role on disturbing LDLR negative feedback regulation, increasing scavenger receptor CD36 expression and decreasing Cholesterol efflux related receptor ABCA1.