Epigallocatechin-3-gallate inhibits angiotensin II

and interleukin-6-induced C-reactive protein

production in macrophages

Ming Li1,2, Jun-Tian Liu1, Xiao-Ming Pang1, Chun-Jie Han3, Jun-Jun Mao1

1Department of Pharmacology, Xi�an Jiaotong University School of Medicine, Xi�an, PR China2Department of Pharmacy, Lianyungang Hospital of Traditional Chinese Medicine, Lianyungang, PR China3Shaanxi Institute for Food and Drug Control, Xi�an, PR China

Correspondence: Jun-Tian Liu, e-mail: ljt@mail.xjtu.edu.cn

Abstract:

Background: Consumption of green tea has been associated with health benefits against multiple diseases including cardiovascular

diseases. However, the action mechanisms of green tea and its major ingredient epigallocatechin-3-gallate (EGCG) against cardio-

vascular diseases are still unclear. Emerging evidence has suggested a common role for C-reactive protein (CRP) in the pathogenesis

of inflammation and atherosclerosis. Therefore, the effect of EGCG on angiotensin II (Ang II)- and interleukin-6 (IL-6)-induced

CRP production in U937 macrophages and the possible mechanisms were observed.

Methods: U937 macrophages were cultured, and Ang II and IL-6 were used as stimulants for generation of CRP. U937 macrophages

were preincubated with EGCG at 1, 3, 10 µM for 1 h prior to the stimulation. mRNAexpression and protein level were determined by

RT-PCR and ELISA, respectively. ROS production was observed by a fluorescence microscope.

Results: Pretreatment of macrophages with EGCG prior to the stimulation concentration-dependently inhibited Ang II- and IL-6-

induced expression of CRP both in protein and mRNA levels. Meanwhile, EGCG reduced Ang II- and IL-6-stimulated generation of

ROS in macrophages.

Conclusion: EGCG is able to inhibit Ang II- and IL-6-stimulated CRP expression in macrophages to produce an anti-inflammation

by interfering with ROS generation. The finding is helpful to update understanding of anti-atherosclerotic effects of EGCG.

Key words:

epigallocatechin-3-gallate, angiotensin II, interleukin-6, C-reactive protein, macrophages

Introduction

Inflammation plays a pivotal role in all stages of athe-

rosclerosis. Numerous experimental studies and clinic

observations have confirmed that a variety of inflam-

matory cytokines, such as interleukin-6 (IL-6), inter-

leukin-1 (IL-1), tumor necrosis factor-a (TNF-a) and

C-reactive protein (CRP) directly participate in athe-

rosclerosis formation [10, 13]. CRP as an inflamma-

tory marker, not only stimulates the expressions of ad-

hesion molecules [15] and matrix metalloproteinase-1

[25], but also activates the complement system [14]

and impedes vasorelaxation [22]. Thus, CRP is in-

volved in all stages of atherosclerotic process.

Recent study showed that human macrophages are

able to generate CRP after stimulation with inflamma-

912 Pharmacological Reports, 2012, 64, 912�918

Pharmacological Reports2012, 64, 912�918ISSN 1734-1140

Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences

tory cytokines (IL-6, IL-1b and TNF-a) [4] and some

of vasoactive peptides, such as endothelin-1 (ET-1)

and angiotensin II (Ang II), which raises the possibil-

ity that human macrophages may be a source of lo-

cally produced CRP in the arterial wall [12, 24]. Our

previous study indicated that Ang II stimulates CRP

production in macrophages predominantly through

activating mitochondria-derived reactive oxygen spe-

cies (ROS) [12]. Thus, we presume that antioxidant

and anti-inflammatory agents may suppress CRP pro-

duction in macrophages so to relieve inflammation re-

action during atherosclerosis.

Numerous epidemiological and experimental stud-

ies have suggested that epigallocatechin-3-gallate

(EGCG) in red wine and green tea has a very strong

antioxidant activity, and possesses a significant role in

prevention of cancer and cardiovascular disease [27].

Although precise mechanisms of the cardiovascular

protection have not been completely defined, much

evidence suggests that the protection from EGCG

may involve anti-oxidation [27], anti-inflammation

[20, 26], anti-thrombosis [5] and anti-proliferation [1]

to relieve the risk of atherosclerosis [11]. An in vivo

study also finds that EGCG can significantly reduce

the formation of atherosclerosis plaque (55–77%),

and the degree of hyperlipidemia in Apo-E gene defi-

cient mice [15]. Despite these findings, the regulatory

effect of EGCG on Ang II- and IL-6-induced CRP

production in human macrophages is unknown.

Therefore, the present study observed the inhibitory

effect of EGCG on Ang II- and IL-6-induced CRP ex-

pression in macrophages so as to provide further evi-

dence for its anti-inflammatory and anti-athero-

sclerotic effects.

Materials and Methods

Reagents

RPMI-1640, penicillin and streptomycin were pro-

duced by Gibco BRL (Grand Island, NY, USA). Fetal

bovine serum (FBS) was from HyClone (Logan, UT,

USA). Ang II was obtained from Sigma-Aldrich (St.

Louis, MO, USA). IL-6 was provided by R&D Sys-

tems (USA). EGCG was purchased from Alexis Bio-

chemicals (San Diego, CA, USA). Human CRP

ELISA kit was bought from Alpha Diagnostics (San

Antonio, TX, USA). ROS assay kits were ordered

from the Beyotime Institute of Biotechnology (Jiangsu,

China). BCA protein assay kit was supplied by Pierce

(Rockford, IL, USA).

Cell culture

U937 histiocyte (American Type Culture Collection)

is also called resident macrophage [21]. U937 macro-

phages were cultured in a 5% CO2 atmosphere in

RPMI-1640 supplemented with 10% FBS, 2 mM glu-

tamine, 25 mM HEPES, penicillin (100 U/ml) and

streptomycin (100 µg/ml). The medium was changed

every 2 or 3 days. When the cells were seeded into

a 6-well plate at 1×106 cells/ml, the medium was

changed to the serum free medium for an additional

24 h incubation to render the cells quiescent before

adding the stimuli. In EGCG experiments, U937

macrophages were exposed to Ang II (1 µM) or IL-6

(0.1 µM) for 12 h after pretreated with EGCG for 1 h.

All experimental procedures were performed in ac-

cordance with the international, national and institu-

tional rules, and approved by the Institutional Animal

Care Committee of Xi’an Jiaotong University.

ELISA

U937 macrophages were pretreated with the different

concentrations of EGCG (1, 3, 10 µM) for 1 h and

stimulated by IL-6 (0.1 µM) or Ang II (1 µM) for

12 h. Then, the cells were washed with PBS, lysed

and scraped with 200 µl of ice-cold RIPA lysis buffer.

After that, the cell lysates were centrifuged at 12,000

rpm for 20 min at 4°C, and the supernatant containing

the protein fraction was extracted. Protein concentra-

tion was quantified with the BCA protein assay kit

[24]. Finally, equal amount of protein extract was as-

sayed for CRP by ELISA kit specific for human CRP

according to the manufacturer’s instruction.

RT-PCR

Total RNA was isolated from U937 macrophages

after the treatment using Trizol (Invitrogen, NY,

USA) according to manufacturer’s instruction. The

total cellular RNA was quantified by measuring the

absorbance at 260 and 280 nm, and RNA purity

(A260/A280) was between 1.6 and 1.9. One micro-

gram of isolated RNA from each sample was tran-

scribed into complementary (cDNA) using oligo

(dT)18 Primer and Revert AidTM M-MuLV Reverse

Pharmacological Reports, 2012, 64, 912�918 913

EGCG inhibits Ang II- and IL-6-induced CRP in macrophagesMing Li et al.

Transcriptase (Fermentas, St. Leon-Rot, Germany).

The cDNA was amplified using 1.5 µl of each of the

CRP forward primer (5’-TCGTATGCCACCAAG-

AGACAAGACA-3’) and reverse primer (5’-AAC-

ACTTCGCCTTGCACTTCATACT-3’). This resulted

in a 440 bp PCR product (35 amplification cycles of

94°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for

45 s, and 72°C for 5 min). The housekeeping gene

(GAPDH) was amplified as an internal control for

normalization using the forward primer (5’-GGA-

TTTGGTCGTATTGGG-3’) and the reverse primer

(5’-GGAAGATGGTGATGGGATT-3’). This resulted

in a 205 bp product (35 amplification cycles of 94°C

for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 45 s,

and 72°C for 5 min). In all the experiments, control

reactions were performed by substituting sterile

nuclease-free water for the RNA template in the reac-

tion. PCR products were separated on 2% agarose

gels and the relative amount of each mRNA was nor-

malized to the housekeeping gene (GAPDH) mRNA.

Measurement of ROS

To evaluate inhibition of ROS generation, U937

macrophages were pretreated with the different con-

centrations of EGCG (1, 3, 10 µM) for 1 h before ex-

posed to Ang II (1 µM) or IL-6 (0.1 µM) for 12 h.

Then, the cells were loaded with 10 µM 2’,7’-

dichlorodihydrofluorescein diacetate (H2DCF-DA)

for 30 min at 37°C, and washed twice with PBS. Fi-

nally, the cells were imaged with fluorescence micro-

scope (Olympus BX51, Japan) in a dark environment

every 2 min for 10 min after the specimen was

mounted on the object stage. Fluorescence images

were acquired at the excitation and emission wave-

lengths of 488 and 525 nm, respectively. The fluores-

cence intensity of an experimental field comprising

20–25 cells of each group was measured and analyzed

with Image-pro plus software. The relative fluores-

cence intensity was taken as the average of values

from three repeated experiments.

Statistical analysis

All values were shown as the mean ± SEM. Statistical

significance between groups was assessed by one-

way ANOVA, followed by Tukey’s multiple compari-

son with the SPSS 16.0 program. A value of p < 0.05

was considered statistically significant.

Results

Effect of EGCG on Ang II- and IL-6-induced pro-

tein production of CRP in macrophages

The results from Figures 1 and 2 indicated that the un-

stimulated macrophages exhibited a low basal CRP

914 Pharmacological Reports, 2012, 64, 912�918

Fig. 1. Effect of EGCG on Ang II-induced CRP production in macro-phages. U937 macrophages were preincubated with the differentconcentrations of EGCG for 1 h before stimulated with 1 µM Ang II for12 h. Then, protein extracts were collected from the cell lysates, andassayed for CRP concentration by ELISA. Data from three independ-ent experiments are expressed as the means ± SEM. ** p < 0.01 and*** p < 0.001 vs. Ang II alone

Fig. 2. Effect of EGCG on IL-6 -induced CRP production in macro-phages. U937 macrophages were pretreated with the different con-centrations of EGCG for 1 h before exposure to 0.1 µM IL-6 for 12 h.Then, protein extracts were collected from the cell lysates, and as-sayed for CRP concentration by ELISA. Data from three independentexperiments are expressed as the means ± SEM. ** p < 0.01 and*** p < 0.001 vs. IL-6 alone

release of 2.65–3.50 ng/ml, and CRP production was

increased to 3.47 or 3.33 times over control after

macrophages were stimulated with 1 µM Ang II or

0.1 µM IL-6 for 12 h (p < 0.001). However, pretreat-

ment of the cells with the different concentrations of

EGCG for 1 h prior to Ang II or IL-6 stimulation sig-

nificantly inhibited Ang II- and IL-6-induced CRP

production in a concentration-dependent manner (p <

0.01 or p < 0.001 vs. Ang II or IL-6 alone).

Effect of EGCG on Ang II- and IL-6 -induced

mRNA expression of CRP in macrophages

As shown in Figures 3 and 4, Ang II and IL-6 at con-

centrations used both up-regulated CRP mRNA ex-

pression in macrophages with 2.75 or 3.59 times over

control (p < 0.01 or p < 0.001). But, preincubation of

the cells with the same concentrations of EGCG also

reduced Ang II- and IL-6-induced CRP mRNA accu-

mulation in macrophages (p < 0.05, p < 0.01 or p <

0.001 vs. Ang II or IL-6 alone).

Effect of EGCG on Ang II- and IL-6 -stimulated

ROS generation in macrophages

As seen in Figures 5 and 6, there was a minor DCF

fluorescence in control macrophages, which repre-

sented ROS generation. Exposure of macrophages to

Ang II or IL-6 resulted in a noticeable raise of DCF

fluorescence with 5.47 or 6.90 times over control (p <

0.001). However, pretreatment of the cells with EGCG

obviously depressed ROS generation in a concentra-

tion-dependent manner (p < 0.05 or p < 0.001 vs.

Ang II or IL-6 alone).

Discussion

Inflammation is an important event in the develop-

ment of cardiovascular diseases such as atherosclero-

sis, restenosis and hypertension. CRP may contribute

to development of atherosclerosis and the subsequent

Pharmacological Reports, 2012, 64, 912�918 915

EGCG inhibits Ang II- and IL-6-induced CRP in macrophagesMing Li et al.

Fig. 3. Effect of EGCG on Ang II-induced CRP mRNA expression inmacrophages. After preincubation with the different concentrationsof EGCG for 1 h, U937 macrophages were exposed to 1 µM Ang II for12 h. Then, the total RNA of the cells was extracted with the Trizolagent, followed by reverse transcription to obtain cDNA. GAPDH wasused as an internal control. Results from three independent experi-ments are expressed as themeans ± SEM. ** p < 0.01 and *** p < 0.001vs. Ang II alone

Fig. 4. Effect of EGCG on IL-6 -induced CRP mRNA expression inmacrophages. After preincubation with the different concentrationsof EGCG for 1 h, U937 macrophages were exposed to 0.1 µM IL-6 for12 h. Then, the total RNA of the cells was extracted with the Trizolagent, followed by reverse transcription to obtain cDNA. GAPDH wasused as an internal control. Results from three independent experi-ments are expressed as the means ± SEM. * p < 0.05, ** p < 0.01 and*** p < 0.001 vs. IL-6 alone

cardiovascular events by inducing inflammatory re-

sponses [23]. On the basis of its pleiotropic roles in

cardiovascular diseases, CRP provides a pivotal link

between inflammation and atherogenesis.

Growing evidence confirms that green tea and its

major ingredient EGCG exhibit the wide pharmacol-

ogical activities, such as antioxidation, decreasing

blood lipids and serum CRP level in rats, relieving

atherosclerotic lesions in various animal models of

hyperlipidemia [3], which suggests that consumption

of green tea can provide many beneficial effects for

prevention and treatment of multiple diseases includ-

ing, atherosclerosis [6, 7]. In the present study, we

found that EGCG inhibited Ang II- and IL-6-induced

protein and mRNA expression of CRP in macro-

phages in a concentration-dependent manner. Moreo-

ver, the suppression of CRP generation occurred at the

transcriptional level, as shown by the RT-PCR study.

The results are also consistent with the data from

Wang et al. [24] and Peng et al. [16] in vascular

smooth muscle cells (VSMCs). Since CRP is an im-

portant inflammatory molecule, it is suggested that

EGCG relieves inflammatory response involved in

atherosclerotic process through inhibiting CRP pro-

duction in macrophages.

ROS participate in cellular signaling transduction

and regulate a number of important cellular events

[17]. ROS generated in oxidative stress may up-

regulate production of proinflammatory cytokines

such as CRP, IL-1b, IL-6 and TNF-a through activat-

ing the transcription nuclear factor kB (NF-kB) to

participate in atherogenesis [2, 9, 18]. Our previous

experiment demonstrated that Ang II can induce CRP

production through activating ROS in VSMCs [24],

916 Pharmacological Reports, 2012, 64, 912�918

Fig. 5. Effect of EGCG on Ang II-stimulated ROS generation in macro-phages. U937 macrophages were pretreated with the different con-centrations of EGCG for 1 h before stimulated with 1 µM Ang II for 12 h.Then, the cells were incubated for 30 min with 10 µM 2�,7�-dichlorodi-hydrofluorescein diacetate (H2DCF-DA) in serum-free RPMI-1640.Finally, the fluorescent intensity wasmeasured by a fluorescencemicro-scope. Data from three independent experiments are expressed as themeans ± SEM. * p < 0.05 and *** p < 0.001 vs. Ang II alone

Fig. 6. Effect of EGCG on IL-6 -induced ROS generation in macro-phages. U937 macrophages were pretreated with the different con-centrations of EGCG for 1 h before stimulated with 0.1 µM IL-6 for 12 h.Then, the cells were incubated for 30 min with 10 µM 2�,7�-dichloro-dihydrofluorescein diacetate (H2DCF-DA) in serum-free RPMI-1640.Finally, the fluorescent intensity was measured by a fluorescence mi-croscope. Data from three independent experiments are expressedas the means ± SEM. *** p < 0.001 vs. IL-6 alone

macrophages [12] and human aortic endothelial cells

[8]. Schaefer et al. also find that IL-6-induced ROS

generation depends on NAD(P)H oxidase [19]. The

present result showed that Ang II and IL-6 stimulated

ROS production in macrophages while EGCG pre-

treatment significantly reduced ROS formation. Con-

sidering antioxidative property of EGCG and our pre-

vious reports, it is inferred that the inhibitory effect of

EGCG on CRP production in macrophages is related,

at least in part, to decreasing ROS.

Taken together, our data suggest that EGCG is able

to inhibit Ang II- and IL-6-stimulated CRP expression

in macrophages to produce an anti-inflammation by

interfering with ROS generation. The finding is help-

ful to update understanding of anti-atherosclerotic ef-

fects of EGCG, but further investigations are needed

to elucidate anti-inflammatory mechanisms of EGCG

and to establish the feasibility and efficacy of using

EGCG in a clinical setting.

Acknowledgment:

This work was supported by the Doctoral Fund of the Ministry ofEducation of China (No. 20100201110053).

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Received: December 25, 2011; in the revised form: March 26, 2012;accepted: April 23, 2012.

918 Pharmacological Reports, 2012, 64, 912�918