Journal of Ethnopharmacology 105 (2006) 251254

Effect of curcumin on the expression of LDL

receptor in mouse macrophages
Chunlei Fan , Xingde Wo, Ying Qian, Jin Yin, Liping Gao
Institute of Molecular Medicine, Zhejiang College of Traditional Chinese Medicine, Hangzhou 310053, China
Received 20 July 2004; received in revised form 1 October 2005; accepted 14 November 2005
Available online 6 January 2006

Abstract
To investigate the molecular mechanisms of lipid-lowering drug, Rhizoma Curcumae Longae, we treated the mouse macrophages with curcumin,
which was purified from the ethanol extraction of Rhizoma Curcumae Longae. The LDL receptors expressed in the macrophages were determined
by ELISA, FLISA and assay of LDL uptake. Here for the first time, we found that curcumin obviously up-regulated the expression of LDL receptor
in mouse macrophages, and the doseeffect relationship was demonstrated.
2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Curcumin; Low density lipoprotein receptor; Gene expression; Macrophage

1. Introduction
Elevated level of low density lipoprotein-cholesterol (LDLC) in plasma is one of major causes of atherosclerosis and
coronary heart disease. HMG-CoA reductase inhibitors, such
as statins, already in clinical use to reduce cholesterol levels, are
effective in atherosclerosis patients. They show a good safety
profile in patients with high cholesterol levels and/or cardiovascular disease, however, statins may be potentially associated
with development of hepatic damage, myalgias, or polyneuropathy (Gaist et al., 2002; Guis et al., 2003). Approximately
two-thirds of LDL clearance is normally mediated by the LDL
receptor (Brown and Goldstein, 1986). In most cases, high level
of plasma LDL-C is due to mutations of LDL receptor, such
as familial hypercholesterolemia (FH) or suppression of LDL
receptors. This makes us to propose that the expression of LDL
receptor meditated by drugs would be an effective way to control
LDL-C levels in plasma.

Abbreviations: LDL, Low density lipoprotein; ELISA, enzyme-linked

immunosorbent assay; FLISA, fluorescein-linked immunosorbent assay; FH,
familial hypercholesterolemia; LDL-C, low density lipoprotein-cholesterol;
SREBP, sterol regulatory element-binding protein; SCAP, SREBP cleavageactivating protein; FBS, fetal bovine serum; PBS, phosphate-buffered saline
Corresponding author. Tel.: +86 571 86613598; fax: +86 571 86613598.
E-mail addresses: taijispear@126.com, snow@zjtcm.net (C. Fan).
0378-8741/$ see front matter 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2005.11.009

In recent years, studies show that curcumin [1,7-bis(4hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, structure displayed in Fig. 1], an ethanol extracted compound of
the traditional Chinese medicine-Rhizoma Curcumae Longae,
plays essential pharmacological roles, such as antioxidant, aniinfammatory agent, anti-thromobotic agent, hepatoprotector
(Naika et al., 2004), etc. More and more evidences come to
prove that curcumin is an active lipid-lowering compound that
can significantly decrease the level of serum lipid peroxides,
increase HDL-C, and decrease total serum cholesterol (Soni
and Kuttan, 1992; Soudamini et al., 1992; Babu and Srinivasan,
1997). But, all these studies are at the level of plasma lipid (Sree
and Rao, 1994), the evidences of molecular mechanisms of this
lipid-lowering drug are still poor. We report here that curcumin
obviously up-regulated the expression of LDL receptor in
mouse macrophages. And we proved that one of the lipidlowering mechanisms of the Traditional Chinese Medicine,
Rhizoma Curcumae Longae was by the effect of curcumin on
the up-regulation of the expression of LDL receptor.

2. Materials and methods

2.1. Materials
Curcumin (99.0% pure, HPLC) was obtained from National
Institute for the Control of Pharmaceutical and Biological Prod-

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C. Fan et al. / Journal of Ethnopharmacology 105 (2006) 251254

Fig. 1. Chemical structure of curcumin.

ucts. ANA-1 cell lines (mouse macrophages) were provided by

Shanghai Cell Bank, Chinese Academy of Science. Cell culture
medium was from Gibco. Rabbit anti-bovine LDL receptor was
provided by Graz University, Austria. Goat anti-rabbit IgG-HRP
was obtained from KPL. DiI was from Biotium Inc. LDL was
purified from human plasma by our own laboratory. All other
reagents are purchased from Shanghai Sangon.
2.2. Cell culture and determination of number of LDL
rceptors
ELISA: ANA-1 cell lines were cultured in two 24-well plates
in RPMI1640 medium containing 10% bovine calf serum with
six groups by seven repeated wells (each well has a total volume
of 2.5 ml, density 1.0 106 cells ml1 ). Of the all six groups,
five groups were treated with the medium containing 10, 20, 30,
40 and 50 M of curcumin, respectively. The control group was
treated with the medium containing no curcumin. After 24 h,
1.5 ml of the cell solution was transferred from each of all the
wells to each of 1.5 ml Eppendorf tubes (which were treated
with 1% FBS-PBS, pH 7.4 to block non-specific antibody binding before use) respectively and the tubes were centrifuged at
900 g for 5 min. The supernatant was discarded, and 1 ml of
4% formaldehyde was added to each of the tubes to fix the
cells for 10 min. Then all the tubes were centrifuged at 900 g
for 5 min. The supernatant was discarded. PBS (pH 7.4) was
added to the tubes to regulate the cell concentration to 1.0 106
cells ml1 of each tube. The tubes were centrifuged at 900 g
for 5 min. The supernatant was discarded. 0.5 ml of diluted LDL
receptor antibody (rabbit anti-bovine LDL receptor, 1:1000 in
PBS) was added to each of the tubes. The tubes were shaken
to re-suspend the cells and the cells were incubated at 37 C
for 1.5 h. The tubes were centrifuged at 900 g for 5 min. The
supernatant was discarded. About 1 ml PBS was added to each
of the tubes to wash the cells for 2 min, and then the tubes
were centrifuged at 900 g for 5 min. The supernatant was discarded. The wash and centrifugation were repeated for three
times. About 0.5 ml of diluted secondary antibody (goat antirabbit IgG-HRP, 1:1000 in PBS) was added to each of the tubes.
The tubes were shaken to re-suspend the cells and the cells
were incubated at 37 C for 1.5 h. The tubes were centrifuged at
900 g for 5 min. The supernatant was discarded. About 1 ml
of PBS was added to wash the cells for 2 min. The tubes were
centrifuged at 900 g for 5min. The supernatant was discarded.
The wash and centrifugation were repeated for three times. The
supernatant was discarded. About 0.1 ml of OPD reagent was
added into each of the tubes and incubated at RT for 15 min in the
dark. The reaction was stopped by adding 0.1 ml of 20% sulfuric
acid in each of the tubes. 0.1 ml of reaction mixture was taken
from each of the tubes and measured at 492 nm by Multiscan
MK3.

FLISA: The procedure was the same as that of ELISA until

. . .0.5 ml of diluted secondary antibody was added to each
of the tubes. But here, goat anti-rabbit IgG-FITC, 1:1000 in
PBS, were used as the secondary antibody and 6 groups without repeated well were set. After incubated with the secondary
antibody and washed by PBS, 10,000 cells of each group were
measured by FACSan (BD FACSort).
2.3. LDL uptake assays
The cell culture was similar as above, but no repeat wells
and one more groups were set for negative control. Thus, five
groups were treated with the medium containing 10, 20, 30, 40
and 50 M of curcumin, respectively. The control group was
treated with the medium containing no curcumin. The negative control group was treated with the medium containing
30 M of curcumin. After 24 h, 1.5 ml of the cell solution from
each of the groups was transferred to each of 1.5 ml Eppendorf tubes, respectively, and they were centrifuged at 900 g
for 5 min, and the supernatant was discarded. About 1 ml of
serum-free RPMI1640 medium was added to each tube to
wash the cells for 2 min, and then the tubes were centrifuged
at 900 g for 5 min. The supernatant was discarded. For the
negative control group, 1 ml of diluted LDL receptor antibody
(1:1000 in serum-free RPMI1640 medium) was added and the
cells were incubated at 37 C for 1.5 h to block the function
of LDL receptor. The other groups were treated with 1 ml
of diluted mouse IgG (non-LDL receptor antibody, 1:1000 in
serum-free RPMI1640 medium). The tubes were centrifuged
and the supernatant was discarded. About 1 ml (15 g ml1 ) of
DiI-LDL [labeled as the method of Barak (Barak and Webb,
1981)] in serum-free RPMI1640 medium was added to each
of the tubes. The medium in the negative control group still
contained the receptor antibody (1:1000 diluted) for blocking
the interaction between the receptor and DiI-LDL, and that
in the other groups contained mouse IgG (non-LDL receptor
antibody, 1:1000 diluted). The cells were incubated at 37 C
for 4.5 h and then at 4 C for 0.5 h. The tubes were centrifuged at 900 g for 5 min. The supernatant was discarded.
The cells were fixed by 4% formaldehyde, and then washed by
PBS for three times. The uptake of DiI-LDL was detected by
LSCM (Zeiss LSM 510) and measured by FACSan (BD FACSort).
3. Results
3.1. Curcumin increases the number of LDL receptors
To study effect of curumin on the expression of LDL receptor,
we chose a stable mouse macrophage cell line, ANA-1. Quantitative analysis of LDL receptor numbers was performed by
ELISA and FLISA.
In Tables 1 and 2, we found that the expressed LDL receptors
in ANA-1 were remarkably increased by curcumin. Comparing
to the control, it increased them to 107% (by ELISA) or 121%
(by FLISA) at 10 M, 186% (by ELISA) or 204% (by FLISA) at
20 M, 284% (by ELISA) or 292% (by FLISA) at 30 M, 227%

C. Fan et al. / Journal of Ethnopharmacology 105 (2006) 251254

253

Table 1
Effect of curcumin on the numbers of LDL receptors expressed in mouse macrophages (ELISA)
Control

Curcumin (M)

0.6435 0.0107
Percentage of control

10

20

30

40

50

0.6899 0.0315**

1.1949 0.0244***

1.8293 0.0284***

1.4609 0.0336***

107

186

284

227

0.9411 0.0493***
146

Note: Data were collected at the mean S.D. of seven cell wells, 1.0 106 cells each well, measured by enzyme-linked immunosorbent assay (ELISA).
** P < 0.01.
*** P < 0.001.

Table 2
Effect of curcumin on the numbers of LDL receptors expressed in mouse
macrophages (FLISA)
Control

5.68
Percentage of control

Curcumin (M)
10

20

30

40

50

6.88
121

11.58
204

16.61
292

14.82
261

10.95
193

Note: Data are geo mean of fluorescence value of 104 cells measured by
fluorescein-linked immunosorbent assay (FLISA).

(by ELISA) or 261% (by FLISA) at 40 M, 146% (by ELISA) or

193% (by FLISA) at 50 M. Obviously, there was a doseeffect
relationship at the range from 10 to 30 M. Curcumin at 40
and 50 M still had a significant effect, but 30 M of curcumin
functioned best.
3.2. The expression of a functional LDL receptor was
induced by curcumin
The expression of a functional LDL receptor was taken by
LDL-uptake assay. ANA-1 cells were treated with different concentrations of curcumin for 24 h, then incubated in serum-free
medium with 15 g/ml of DiI-LDL for 4.5 h at 37 C and 0.5 h at
4 C. The functional LDL receptors induced by curcumin could
be observed clearly using LSCM (Fig. 2). About 1050 M of
curcumin remarkably increased the activity of LDL receptors
(Fig. 2cg). The quantitative analysis of functional LDL receptors in ANA-1 cells was performed by FACSan analysis. About
1050 M of curcumin significantly increased the geo mean fluorescent value in ANA-1 cells (Table 3), which proved that the
uptake of DiI-LDL was increased.
4. Discussion and conclusions
As seeing in Tables 1 and 2, curcumin increased the numbers of LDL receptors on the cell surface. In Fig. 2 and Table 3,
curcumin significantly activated the uptake of LDL. The best
concentration of curcumin was at 30 M, and its action of LDL
uptake could be blocked by the antibodies of LDL receptor,
proving that curcumin was exactly effect on this receptor. The
doseeffect relationship from 0 to 30 M was demonstrated.
But the effect decreased at high concentration as over 40 M of
curcumin. We know that curcumin is a small-molecular-weight
lipid-soluble compound, which can easily pass by or stay on the

Fig. 2. The functional LDL receptors expressed in macrophages were induced

by curcumin. (af) The uptake of DiI-LDL were detected by LSCM. (a) Blank
control, the cells were treated without curcumin. The expressed LDL receptors were very low. (b) Negative control, the cells were treated with 30 M of
curcumin, but also treated with LDL receptor antibodies before and during the
incubation of DiI-LDL to block the interaction between the receptors and DiILDL. (cg) The cells were treated with 10, 20, 30, 40 and 50 M of curcumin,
respectively.

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C. Fan et al. / Journal of Ethnopharmacology 105 (2006) 251254

Table 3
Effect of curcumin on the functional LDL receptor expression in mouse macrophages
Normal control

4.05
Percentage of normal control

Negative control

2.66
65.78

Curcumin (M)
10

20

30

40

50

12.47
308

20.66
510

55.58
1372

50.19
1239

42.04
1038

Note: Data are geo mean of fluorescence value of 104 cells. Negative control: 30 M curcumin + LDL receptor antibodies.

lipid bilayer of the plasma membrane. A functional LDL receptor can recycle by the receptor-mediated endocytosis (Brown
and Goldstein, 1986). Curcumin is also a weak acidic polyphenic
compound and too high concentration of curcumin in the cells
or on the lipid bilayer may reduce the pH value of the cellular
extra- or intra-environment. The conformation of LDL receptor in acidic pH will suppress the LDL binding to its receptor
(Rudenko et al., 2002).
To increase the activity of LDL receptor is valuable for a
lipid-lowering drug. According to LDL receptor-mediated pathway, one functional LDL receptor could take up to several
hundred LDL particles into the cell. This can explain why curcumin increased uptake of LDL up to 1372% of the control,
much higher than the numbers increased [284% (by ELISA) or
292% (by FLISA) of the control]. The action of curcumin is
just like a signal that suddenly opened the expression system
of LDL receptor. The expression of LDL receptor is regulated
by cellular sterols through the SCAP/SREBP pathway (Brown
and Goldstein, 1997). It would be very interesting to further
research on curcumin to see if curcumin would be effect on the
SCAP/SREBP pathway.
As a nature compound extracted from Rhizoma Curcumae
Longae, curcumin shows a significant effect on increasing the
expression of LDL receptor. It must be a new potential drug
in hypercholesterolemia or atherosclerosis therapy by lowering
cholesterol level in plasma with low or no side-actions.
Acknowledgments
We thank Prof. Hong Xingqiu for extraction, purification and
determination of curcumin; Dr. Yang yu (Zhejiang University)
for LSCM and FACSan technical assistance. We also thank Ms.

Qingyan Hu (University of Toronto, Canada) for reading the

manuscript.
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