Polyunsaturated fatty acid relatively decreases cholesterol content in THP-1 macrophage-derived foam cell: partly correlates with expression profile of CIDE and PAT members
Yue Song
0
Li-Jun Zhang
Hang Li
0
Yu Gu
0
Fan-Fan Li
0
Li-Na Jiang
0
Fang Liu
0
Jing Ye
0
Qing Li
0
0
State Key Laboratory of Cancer Biology and Department of Pathology, Xijing Hospital, Fourth Military Medical University
,
Xi'an
,
China
Background: Polyunsaturated fatty acids (PUFAs) have positive effect on the regulation of plasma lipids. But the mechanism for them to modulate lipid homeostasis in macrophage is still unclear. In this study, we employed PUFA to pretreat macrophages and evaluated the variations of lipid droplet (LD) content, lipid composition, and expressions of LD-associated genes in macrophage-derived foam cells. Method: THP-1-derived macrophages or human peripheral blood monocyte-derived macrophages were pre-treated with four non-esterified fatty acids (NEFAs) separately: saturated fatty acid (SFA)-palmitic acid (PA), monounsaturated fatty acids (MUFAs)-oleic acid (OA), PUFAs-linoleic acid (LA) and eicosapentaenoic acid (EPA). Intracellular lipid content and cholesterol efflux were analyzed in THP-1 macrophage-derived foam cells. Related gene expressions were detected by quantitative real-time PCR. Results: PUFA pre-treatment reduced cholesterol content in foam cells and increased cholesterol efflux to lipid-free apoAI in conditioned medium compared with PA or OA group. Cell death-inducing DFF45 like effector (CIDE) and Perilipin-Adipophilin-TIP47 (PAT) family members, as LD-associated proteins, showed specific gene expression profiles after PUFA pre-treatment. These results may help to explain the process of lipid metabolism within foam cells. Conclusion: PUFA (LA or EPA) had a potential protective effect against cholesterol accumulation. The specific expressions of CIDE and PAT genes may provide clues to explore the protective mechanism of PUFA in foam cells.
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Background
PUFAs are essential components in lipid metabolism. It
has been reported that PUFAs can affect lipid storage
and reduce the risk factors of cardiovascular diseases [1].
However, the potential role of PUFA in the formation of
fatty streak and atherosclerotic plaque is not yet clear.
The molecular mechanism for PUFA-induced lipid
influx and efflux in foam cells is inconclusive.
Abnormal lipid deposition in the intima of artery wall
leads to the formation of fatty streak. Macrophages
takeup the oxidized LDL (ox-LDL), stores a large portion of
lipids in their cytoplasm, and turn into foam cells [2,3].
The formation, morphology and lipolysis of intracellular
LDs are regulated by a number of LD-associated
proteins [4]. These proteins are located on the surface of
LDs and play active roles in the regulation of
intracellular lipid storage, nascent LD biogenesis and
transportation [5-8]. CIDE and PAT proteins are by far the most
specific LD-associated proteins being found. Proteomic
analyses also confirmed that the most abundant proteins
in LDs were the CIDE and PAT family members [9]. The
peroxisome proliferator-activated receptor (PPAR) is
considered as an important transcription factor involved
in the regulation of gene expression of most of
LDassociated proteins [10-12]. Microarray analysis in our
previous study [8] showed that CIDE and PAT members,
together with PPAR regulated proteins, had meaningful
expression changes in the process of THP-1
macrophagederived foam cells formation. These data suggested that
CIDE or PAT proteins were closely related to intracellular
LD formation. But different NEFA-induced protein
expressions are still not clear.
In this study, THP-1-derived macrophages and
peripheral blood monocyte-derived macrophages were
pretreated separately with PA, OA, LA and EPA (difference
in saturation). We intend to simulate a microenvironment
of monocyte-derived macrophages in artery wall. The lipid
composition of intracellular LDs was identified by
quantitative analysis in two cell groups separately. By analyzing
cholesterol efflux and mRNA expression profiles of CIDE,
PAT and PPAR transcriptional regulatory proteins in
THP-1 macrophage-derived foam cells, we try to present a
potential relationship among PUFA pre-treatment, lipid
loading and LD-associated gene expression.
Results
Identification of the optimum incubation condition
Before the experiment, we examined the cytotoxicity of
employed fatty acids on THP-1 macrophages with MTT
assay. NEFA pre-treatment increased cell death rate in a
time- and dose-dependent manner (Figure 1). Compared
with the control group, 48 hours incubation by each
NEFA had no effect on macrophage viability. While after
72 hours incubation, there were at least 75% cells
survived in each group, which is sufficient for the following
experiments. Concentration of NEFAs can also affect
macrophage viability. Cell survival rate decreased slightly
Figure 1 Cell viability of THP-1 macrophage after pre-treatment
with different NEFAs at different concentrations for 72 hours.
The THP-1-derived macrophages were pretreated by PA, OA, LA and
EPA separately with time and dose gradien (...truncated)