The Research Of Inflammatory Cytokines Inhibiting Macrophage Cholesterol Efflux Via MiR-33a-5P

Aims: Atherosclerosis leads to serious health problems, includingcoronary atherosclerotic heart disease, stroke and peripheral vascular disease,etc. The inflammation and excessive cholesterol accumulation in vessel wallare two key factors of the development of atherosclerosis. Inflammation，even in the absence of traditional risk factors (dyslipidemia, hypertension,etc.)，can facilitate arterial hyperplasia and lipid accumulation, promote theformation of atherosclerotic plaque. Macrophage is the main source of foamcells in atherosclerotic plaque. Cholesterol efflux impairment inmacrophages will affect the progress of atherosclerosis. ABCA1andABCG1are responsible for the major part of macrophage cholesterol efflux.ABCA1mediates the efflux of cholesterol and phospholipid frommacrophages to apolipoprotein with poor lipid molecule like apolipoproteinA-I (apoA-I). ABCG1mediates cholesterol efflux from macrophages toapolipoprotein with abundant lipid molecule like HDL.miR-33a, which islocated in intron16of the SREBP-2gene (on chromosome22) involved incholesterol biosynthesis and cholesterol uptake，is co-transcribed with SREBP-2. miR-33a-5P binds to the3’UTR of ABCA1/ABCG1mRNA toinhibit ABCA1/ABCG1translation and regulating cholesterol efflux.We have previously demonstrated that inflammation increasescholesterol accumulation by disrupting SREBP2-LDLR negative feedbackregulation in THP-1macrophages.We also demonstrated that inflammationcan inhibit ABCA1/ABCG1mediated cholesterol efflux. However, themechanism by which inflammation inhibits cholesterol efflux remainsunclear.In this study, the experiments were undertaken to investigatewhether inflammation activates miR-33a-5P expression, inhibitingABCA1/ABCG1expression and cholesterol efflux in human THP-1macrophages.Methods: We used interleukin-6(IL-6), tumor necrosis factor-alpha(TNF-α)，in the presence or absence of low density lipoprotein (LDL)，to stimulate THP-1macrophages. THP-1macrophages were infected byeither control lentivirus vectors (Con-miR or Con-Anti-miR) or lentivirusencoding miR-33a-5P or Anti-miR-33a-5P. The effects of inflammatorycytokines， over expression of miR-33a-5P and Anti-miR-33a-5P onintracellular lipids accumulation and cholesterol contents were assessed byoil red O staining and quantitative intracellular cholesterol assay（enzymicmethod）. We examined apoA-I-mediated cholesterol efflux using afluorescent sterol (BODIPY-cholesterol). The gene and protein expressionsof the relevant molecules （miR-33a-5P，SREBP-2，ABCA1and ABCG1） involved in cholesterol trafficking were examined using quantitativereal-time polymerase chain reaction and Western blotting.Results:1.Our data demonstrated that inflammatory cytokine IL-6or TNF-α，in the absence or presence of LDL，promoted lipids accumulation，increasedTC and CE contents in THP-1macrophages. Inflammatory cytokinesdecreased apoA-I-mediated cholesterol efflux，increased the expression ofmiR-33a-5P and mRNA and protein expression of SREBP2, and decreasedmRNA and protein expression of ABCA1and ABCG1.2.In our study, we demonstrated that，in the absence or presence ofLDL,inflammation or over-expression of miR-33a-5P caused lipidsaccumulation and decreased ABCA1/ABCG1mediated cholesterol efflux.However, Anti-miR33a-5P overrode the inhibitory effects of cholesterolefflux induced by inflammatory stress. Over expression of Anti-miR-33a-5Pdecreased lipids accumulation and cholesterol contents in THP-1macrophages, decreased ApoA-I-mediated cholesterol efflux, decreasedmiR-33a-5P expression, increased mRNA and protein expression of ABCA1and ABCG1.Conclusions: In summary, inflammatory cytokines via activatingmiR-33a-5P expression, inhibit ABCA1/ABCG1expression and ABCA1/ABCG1mediated cholesterol efflux in human THP-1macrophages. Theseresults suggest that miR-33a-5P plays important roles in inflammatory responses and cholesterol homeostasis and indicate that Anti-miR-33a-5Pmay help to prevent inflammatory cytokine-associated macrophage foamcell formation.