Journal of Ethnopharmacology 105 (2006) 251–254

Effect of curcumin on the expression of LDLreceptor in mouse macrophages

Chunlei Fan ∗, Xingde Wo, Ying Qian, Jin Yin, Liping GaoInstitute 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 2005Available 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 determinedby ELISA, FLISA and assay of LDL uptake. Here for the first time, we found that curcumin obviously up-regulated the expression of LDL receptorin mouse macrophages, and the dose–effect relationship was demonstrated.© 2005 Elsevier Ireland Ltd. All rights reserved.

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eywords: Curcumin; Low density lipoprotein receptor; Gene expression; Macrophage

. Introduction

Elevated level of low density lipoprotein-cholesterol (LDL-) in plasma is one of major causes of atherosclerosis andoronary heart disease. HMG-CoA reductase inhibitors, suchs statins, already in clinical use to reduce cholesterol levels, areffective in atherosclerosis patients. They show a good safetyrofile in patients with high cholesterol levels and/or cardiovas-ular disease, however, statins may be potentially associatedith development of hepatic damage, myalgias, or polyneu-

opathy (Gaist et al., 2002; Guis et al., 2003). Approximatelywo-thirds of LDL clearance is normally mediated by the LDLeceptor (Brown and Goldstein, 1986). In most cases, high levelf plasma LDL-C is due to mutations of LDL receptor, suchs familial hypercholesterolemia (FH) or suppression of LDLeceptors. This makes us to propose that the expression of LDLeceptor meditated by drugs would be an effective way to controlDL-C levels in plasma.

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

Abbreviations: LDL, Low density lipoprotein; ELISA, enzyme-linkedmmunosorbent assay; FLISA, fluorescein-linked immunosorbent assay; FH,amilial hypercholesterolemia; LDL-C, low density lipoprotein-cholesterol;REBP, sterol regulatory element-binding protein; SCAP, SREBP cleavage-ctivating protein; FBS, fetal bovine serum; PBS, phosphate-buffered saline∗ Corresponding author. Tel.: +86 571 86613598; fax: +86 571 86613598.

2. Materials and methods

2.1. Materials

Curcumin (99.0% pure, HPLC) was obtained from NationalI

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E-mail addresses: taijispear@126.com, snow@zjtcm.net (C. Fan).

378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reservedoi:10.1016/j.jep.2005.11.009

nstitute for the Control of Pharmaceutical and Biological Prod-

252 C. Fan et al. / Journal of Ethnopharmacology 105 (2006) 251–254

Fig. 1. Chemical structure of curcumin.

ucts. ANA-1 cell lines (mouse macrophages) were provided byShanghai Cell Bank, Chinese Academy of Science. Cell culturemedium was from Gibco. Rabbit anti-bovine LDL receptor wasprovided by Graz University, Austria. Goat anti-rabbit IgG-HRPwas obtained from KPL. DiI was from Biotium Inc. LDL waspurified from human plasma by our own laboratory. All otherreagents are purchased from Shanghai Sangon.

2.2. Cell culture and determination of number of LDLrceptors

ELISA: ANA-1 cell lines were cultured in two 24-well platesin RPMI1640 medium containing 10% bovine calf serum withsix groups by seven repeated wells (each well has a total volumeof 2.5 ml, density 1.0 × 106 cells ml−1). 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 wastreated with the medium containing no curcumin. After 24 h,1.5 ml of the cell solution was transferred from each of all thewells to each of 1.5 ml Eppendorf tubes (which were treatedwith 1% FBS-PBS, pH 7.4 to block non-specific antibody bind-ing before use) respectively and the tubes were centrifuged at900 × g for 5 min. The supernatant was discarded, and 1 ml of4% formaldehyde was added to each of the tubes to fix thecells for 10 min. Then all the tubes were centrifuged at 900 × gfor 5 min. The supernatant was discarded. PBS (pH 7.4) wasacfrPtfsowctrTw9ocTsadafM

FLISA: The procedure was the same as that of ELISA until“. . .0.5 ml of diluted secondary antibody was added to eachof the tubes”. But here, goat anti-rabbit IgG-FITC, 1:1000 inPBS, were used as the secondary antibody and 6 groups with-out repeated well were set. After incubated with the secondaryantibody and washed by PBS, 10,000 cells of each group weremeasured by FACSan (BD FACSort).

2.3. LDL uptake assays

The cell culture was similar as above, but no repeat wellsand one more groups were set for negative control. Thus, fivegroups were treated with the medium containing 10, 20, 30, 40and 50 �M of curcumin, respectively. The control group wastreated with the medium containing no curcumin. The neg-ative control group was treated with the medium containing30 �M of curcumin. After 24 h, 1.5 ml of the cell solution fromeach of the groups was transferred to each of 1.5 ml Eppen-dorf tubes, respectively, and they were centrifuged at 900 × gfor 5 min, and the supernatant was discarded. About 1 ml ofserum-free RPMI1640 medium was added to each tube towash the cells for 2 min, and then the tubes were centrifugedat 900 × g for 5 min. The supernatant was discarded. For thenegative control group, 1 ml of diluted LDL receptor antibody(1:1000 in serum-free RPMI1640 medium) was added and thecells were incubated at 37 ◦C for 1.5 h to block the functionoosaD1octiaftTPLS

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dded to the tubes to regulate the cell concentration to 1.0 × 106

ells ml−1of each tube. The tubes were centrifuged at 900 × gor 5 min. The supernatant was discarded. 0.5 ml of diluted LDLeceptor antibody (rabbit anti-bovine LDL receptor, 1:1000 inBS) was added to each of the tubes. The tubes were shaken

o re-suspend the cells and the cells were incubated at 37 ◦Cor 1.5 h. The tubes were centrifuged at 900 × g for 5 min. Theupernatant was discarded. About 1 ml PBS was added to eachf the tubes to wash the cells for 2 min, and then the tubesere centrifuged at 900 × g for 5 min. The supernatant was dis-

arded. The wash and centrifugation were repeated for threeimes. About 0.5 ml of diluted secondary antibody (goat anti-abbit IgG-HRP, 1:1000 in PBS) was added to each of the tubes.he tubes were shaken to re-suspend the cells and the cellsere incubated at 37 ◦C for 1.5 h. The tubes were centrifuged at00 × g for 5 min. The supernatant was discarded. About 1 mlf PBS was added to wash the cells for 2 min. The tubes wereentrifuged at 900 × g for 5min. The supernatant was discarded.he wash and centrifugation were repeated for three times. Theupernatant was discarded. About 0.1 ml of OPD reagent wasdded into each of the tubes and incubated at RT for 15 min in theark. The reaction was stopped by adding 0.1 ml of 20% sulfuriccid in each of the tubes. 0.1 ml of reaction mixture was takenrom each of the tubes and measured at 492 nm by Multiscan

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f LDL receptor. The other groups were treated with 1 mlf diluted mouse IgG (non-LDL receptor antibody, 1:1000 inerum-free RPMI1640 medium). The tubes were centrifugednd the supernatant was discarded. About 1 ml (15 �g ml−1) ofiI-LDL [labeled as the method of Barak (Barak and Webb,981)] in serum-free RPMI1640 medium was added to eachf the tubes. The medium in the negative control group stillontained the receptor antibody (1:1000 diluted) for blockinghe interaction between the receptor and DiI-LDL, and thatn the other groups contained mouse IgG (non-LDL receptorntibody, 1:1000 diluted). The cells were incubated at 37 ◦Cor 4.5 h and then at 4 ◦C for 0.5 h. The tubes were cen-rifuged at 900 × g for 5 min. The supernatant was discarded.he cells were fixed by 4% formaldehyde, and then washed byBS for three times. The uptake of DiI-LDL was detected bySCM (Zeiss LSM 510) and measured by FACSan (BD FAC-ort).

. Results

.1. Curcumin increases the number of LDL receptors

To study effect of curumin on the expression of LDL receptor,e chose a stable mouse macrophage cell line, ANA-1. Quan-

itative analysis of LDL receptor numbers was performed byLISA and FLISA.

In Tables 1 and 2, we found that the expressed LDL receptorsn ANA-1 were remarkably increased by curcumin. Comparingo the control, it increased them to 107% (by ELISA) or 121%by FLISA) at 10 �M, 186% (by ELISA) or 204% (by FLISA) at0 �M, 284% (by ELISA) or 292% (by FLISA) at 30 �M, 227%

C. Fan et al. / Journal of Ethnopharmacology 105 (2006) 251–254 253

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

Control Curcumin (�M)

10 20 30 40 50

0.6435 ± 0.0107 0.6899 ± 0.0315** 1.1949 ± 0.0244*** 1.8293 ± 0.0284*** 1.4609 ± 0.0336*** 0.9411 ± 0.0493***

Percentage of control 107 186 284 227 146

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 2Effect of curcumin on the numbers of LDL receptors expressed in mousemacrophages (FLISA)

Control Curcumin (�M)

10 20 30 40 50

5.68 6.88 11.58 16.61 14.82 10.95Percentage of control 121 204 292 261 193

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

(by ELISA) or 261% (by FLISA) at 40 �M, 146% (by ELISA) or193% (by FLISA) at 50 �M. Obviously, there was a dose–effectrelationship at the range from 10 to 30 �M. Curcumin at 40and 50 �M still had a significant effect, but 30 �M of curcuminfunctioned best.

3.2. The expression of a functional LDL receptor wasinduced by curcumin

The expression of a functional LDL receptor was taken byLDL-uptake assay. ANA-1 cells were treated with different con-centrations of curcumin for 24 h, then incubated in serum-freemedium with 15 �g/ml of DiI-LDL for 4.5 h at 37 ◦C and 0.5 h at4 ◦C. The functional LDL receptors induced by curcumin couldbe observed clearly using LSCM (Fig. 2). About 10–50 �M ofcurcumin remarkably increased the activity of LDL receptors(Fig. 2c–g). The quantitative analysis of functional LDL recep-tors in ANA-1 cells was performed by FACSan analysis. About10–50 �M of curcumin significantly increased the geo mean flu-orescent value in ANA-1 cells (Table 3), which proved that theuptake of DiI-LDL was increased.

4. Discussion and conclusions

As seeing in Tables 1 and 2, curcumin increased the num-bers of LDL receptors on the cell surface. In Fig. 2 and Table 3,ccupdBcl

Fig. 2. The functional LDL receptors expressed in macrophages were inducedby curcumin. (a–f) The uptake of DiI-LDL were detected by LSCM. (a) Blankcontrol, the cells were treated without curcumin. The expressed LDL recep-tors were very low. (b) Negative control, the cells were treated with 30 �M ofcurcumin, but also treated with LDL receptor antibodies before and during theincubation of DiI-LDL to block the interaction between the receptors and DiI-LDL. (c–g) The cells were treated with 10, 20, 30, 40 and 50 �M of curcumin,respectively.

urcumin significantly activated the uptake of LDL. The bestoncentration of curcumin was at 30 �M, and its action of LDLptake could be blocked by the antibodies of LDL receptor,roving that curcumin was exactly effect on this receptor. Theose–effect relationship from 0 to 30 �M was demonstrated.ut the effect decreased at high concentration as over 40 �M ofurcumin. We know that curcumin is a small-molecular-weightipid-soluble compound, which can easily pass by or stay on the

254 C. Fan et al. / Journal of Ethnopharmacology 105 (2006) 251–254

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

Normal control Negative control Curcumin (�M)

10 20 30 40 50

4.05 2.66 12.47 20.66 55.58 50.19 42.04Percentage of normal control 65.78 308 510 1372 1239 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 recep-tor can recycle by the receptor-mediated endocytosis (Brownand Goldstein, 1986). Curcumin is also a weak acidic polypheniccompound and too high concentration of curcumin in the cellsor on the lipid bilayer may reduce the pH value of the cellularextra- or intra-environment. The conformation of LDL recep-tor 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 alipid-lowering drug. According to LDL receptor-mediated path-way, one functional LDL receptor could take up to severalhundred LDL particles into the cell. This can explain why cur-cumin increased uptake of LDL up to 1372% of the control,much higher than the numbers increased [284% (by ELISA) or292% (by FLISA) of the control]. The action of curcumin isjust like a signal that suddenly opened the expression systemof LDL receptor. The expression of LDL receptor is regulatedby cellular sterols through the SCAP/SREBP pathway (Brownand Goldstein, 1997). It would be very interesting to furtherresearch on curcumin to see if curcumin would be effect on theSCAP/SREBP pathway.

As a nature compound extracted from Rhizoma CurcumaeLongae, curcumin shows a significant effect on increasing theexpression of LDL receptor. It must be a new potential drugin hypercholesterolemia or atherosclerosis therapy by loweringcholesterol level in plasma with low or no side-actions.

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Qingyan Hu (University of Toronto, Canada) for reading themanuscript.

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cknowledgments

We thank Prof. Hong Xingqiu for extraction, purification andetermination of curcumin; Dr. Yang yu (Zhejiang University)or LSCM and FACSan technical assistance. We also thank Ms.

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