1258 Notes Biol. Pharm. Bull. 29(6) 1258—1261 (2006) Vol. 29, No.

Suppressive Effect of Punica granatum on the Production of Tumor

Necrosis Factor (Tnf) in BV2 Microglial Cells
Kyung Hee JUNG,a Mi-Ja KIM,b Eunyoung HA,c Yoon Kyung UHM,a Hye Kyung KIM,d
Joo-Ho CHUNG,a and Sung-Vin YIM*, a
a
Department of Pharmacology, College of Medicine, Kyung Hee University; Seoul 130–70, Korea: b Graduate School of
Obesity Science, Department of Obesity Management, Dongduk Women’s University; Seoul 136–714, Korea: c Department
of Biochemistry, College of Medicine, Kyung Hee University; Seoul 130–70, Korea: and d Department of Food and
Biotechnology, Hanseo University; Seosan 356–70, Korea. Received November 28, 2005; accepted February 23, 2006

While the anti-oxidant properties of Punica granatum methanol extract (PGMF) are well documented, little
is known concerning the anti-inflammatory effect of Punica granatum. PGMF was pretreated in BV2 microglial
cells and cells were stimulated to induce inflammation by lipopolysaccharide (LPS). The effect of PGME on the
production and expression of tumor necrosis factor a (Tnf, previously known as Tnf a ) was determined by en-
zyme-linked immunosorbent assay (ELISA), western blotting, and reverse transcription-polymerase chain reac-
tion (RT-PCR). In addition, the expression of nuclear factor kappa b (Nfk b) was measured using an elec-
trophoretic mobility shift assay (EMSA). By ELISA, PGME at the concentrations of 1, 5, 10, and 50 m g/ml inhib-
ited Tnf production in LPS-stimulated cells by 30.2, 42.3, 57.6, and 88.4%, respectively, compared to LPS-stimu-
lated cells. The LPS-stimulated Tnf production was reduced with a dose-dependent manner. Immuno blot and
RT-PCR analyses revealed that PGME of 5 and 50 m g/ml inhibited the expression of both protein and mRNA lev-
els of Tnf compared to LPS-stimulated cells. EMSA revealed that PGME of 5 and 50 m g/ml blocked the LPS-
stimulated activation of Nfk b. These data suggest that PGME may suppress LPS-stimulated Tnf production
through inhibition of Nfk b in BV2 microglia cells.
Key words Punica granatum; tumor necrosis factor (Tnf); nuclear factor kappa b (Nfk b); lipopolysaccharide (LPS); BV2 mi-
croglial cell

Increasing evidence indicates that fruit and natural extracts MATERIALS AND METHODS
consumption is associated with reduced risk of diseases, in-
cluding cardiovascular disease, stroke, cancer and inflamma- Preparation of Extract Punica granatum was pur-
tion related diseases.1,2) Punica granatum, commonly known chased from Kyungdong market (Seoul, Korea) and was au-
as pomegranate, belongs to the Punicaceae family and con- thenticated by College of Oriental Medicine, Semyung Uni-
sumed around the world as edible fruit.3) Punica granatum versity. Methanol extract of Punica granatum (yield; 19.7%
(Pomegranate) showed various biological effects including of dry weight) was obtained by 48 h maceration at room tem-
cardiovascular protection and anti-cancer.4,5) Also, it was re- perature and was filtered through a 0.45 m M filter (Osmonics,
ported that pomegranate fruit extract possess effect of anti- Minnetonka, MN, U.S.A.), lysophilized, and kept at 4 °C.
carcinogenesis6,7) and skin tumorigenesis8) and pomegranate Cell Culture and Treatment The BV2 microglial cells
wine also inhibited production of Tnf-a .9) In several study, were grown and maintained in Dulbecco’s modified Eagle’s
Pomegranate fruit extract were shown to possess antioxidant medium (DMEM) supplemented with 10% fetal bovine
activity, its antioxidant activity was higher than well-known serum, 2 mM L-glutamine, streptomycin and penicillin
natural antioxidant such as vitamin E (a -tocopherol) and (Gibco, Grand Island, NY, U.S.A.). Cells were treated with
flovonoid.10—13) PGME for the indicated time and harvested for further analy-
Microglia is the immunocompetent, macrophage-like cells sis.
that reside in the central nervous system.14) However, the Enzyme-Linked Immunosorbent Assay (ELISA) For
over-activation of microglial cells and their release of neuro- the analysis of Tnf, the BV2 microglial cells were plated
toxic inflammatory mediators can also contribute to neuronal onto 24 well culture plates. Cells were (1
105 cell/ml)
cell death in many neurodegenerative diseases.15) Therefore, serum-starvated for 2 h and were pretreated with 1, 5, 10 and
controlling microglial activation may have therapeutic bene- 50 m g/ml of PGME for 2 h and then treated with lipopolysac-
fit. Upon their activation, microglial cells release a number of caride (LPS, 1 m g/ml). After incubation with LPS for 24 h,
pro-inflammatory cytokines including tumor necrosis factor supernatants were collected and immediately frozen at
(Tnf), that have been suggested to induce tissue damage and 70 °C. Harvested supernatants were tested for Tnf by
is considered to be important mediators of the inflammatory ELISA. The plates were coated overnight with 2 m g/ml anti-
response.16,17) The expression of these cytokines depends on Tnf capture monoclonal antibody (R&D systems, Minneapo-
the activation of the transcriptional factor, nuclear factor lis, MN, U.S.A.) in 0.1 M Na2HPO4 pH 9 buffer and blocked
kappa b (Nfk b) which is a critical intracelluar mediator of with phosphate buffered saline (PBS)-Tween 20. A biotin-la-
the inflammatory reaction.18,19) beled 1 m g/ml anti-Tnf detecting antibody (R&D systems,
This study was to investigate effects of PGME on the pro- Minneapolis, MN, U.S.A.) was used. The plates were devel-
duction Tnf in LPL-stimulated BV2 microglia cells. oped using streptavidin-horseradish peroxidase (Vector,
Burlingame, CA, U.S.A.) and 2,2-azino-bis substrate (Sigma,
St. Louis, MO, U.S.A.).
∗ To whom correspondence should be addressed. e-mail: 94princess@hanmail.net; ysvin@khu.ac.kr © 2006 Pharmaceutical Society of Japan
June 2006 1259

Western Blotting Forty micrograms of proteins were

separated by SDS-PAGE gel electrophoresis and transferred
to nitrocellulose membrane (Schleicher & Schuell, Middle-
sex, U.K.). The membrane was blocked with 5% skin milk in
10 mM Tris–HCl containing 150 mM NaCl and 0.5% Tween
20 (TBS-T). After brief washing with TBS-T the membrane
was then incubated with primary antibody (1 : 1000) that rec-
ognizes Tnf protein (Cell Signaling, Beverly, MA, U.S.A.).
After thorough washing with TBS-T, horseradish peroxidase-
conjugated secondary antibody (New England Biolabs, Bev- Fig. 1. The Production of Tnf on Lipopolysaccharide (LPS)-Stimulated in
erly, MA, U.S.A., 1 : 2000 dilution in TBS-T) was applied to BV2 Microglial Cells at Various Times
the membrane and the blot was developed using an enhanced The BV2 microglial cells were plated on 24 well plates. BV2 microglial cells
(1
105 cm/ml) were treated with LPS (1 m g/ml). The media were harvested for 2, 6, 12
chemiluminescence detection kit (Amersham, Piscataway, and 24 h after LPS treatment and then measured by ELISA for Tnf production
NJ, U.S.A.). ∗ p0.05 vs. control, ∗∗ p0.01 vs. control.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR) The cells were cultured with or without PGME
and/or LPS (1 m g/ml) for 8 h. Total cellular RNA was iso-
lated by RNA isolation kit (Zymo Research, Orange, CA,
U.S.A.). The levels of Tnf mRNA were determined with Re-
verse Transcription System (Promega, Madison, WI, U.S.A.)
in a 25 m l reaction volume using 1 m g total RNA and 10 units
of reverse transcriptase. Samples were incubated at 42 °C for
60 min, and the reaction was terminated by heating to 95 °C
for 5 min. PCR reactions included 5 min at 94 °C for denatu-
ration and 35 cycles (30 s at 94 °C, 30 s at 60 °C, and 30 s at
72 °C), followed by incubation for 10 min at 72 °C for final
extension before holding at 4 °C. Five microliters aliquot of Fig. 2. The Effect of PGME on Tnf Production
each sample was analyzed by electrophoresis in 1.5% BV2 microglial cells (1
105 cm/ml) were pretreated with 1, 5, 10 and 50 m g/ml of
PGME for 2 h and then treated with lipopolysaccharide (1 m g/ml). The media were har-
agarose gel in the presence of 5 ng/ml ethidium bromide and vested 24 h later and assayed for Tnf production. LPS, lipopolysaccharide; PGME,
visualized under ultraviolet light. Punica granatum. # p0.01 vs. control, ∗ p0.01 vs. LPS.
Nuclear Extract Preparation and Electrophoretic Mo-
bility Shift Assay (EMSA) Nuclear extracts from mi- LPS was set to 24 h.
croglia cells were prepared as follows; cells (2
107 cells on a Production of LPS-Stimulated Tnf Was Decreased by
100 mm dish) were treated with 1 ml of lysis buffer (10 mM PGME To investigate anti-inflammatory effect of PGME,
Tris–HCl, pH 7.9, 10 mM NaCl, 3 mM MgCl2, 1% NP-40) on we determined the production of Tnf, a potent inflammatory
ice for 4 min. After 10 min of centrifugation at 1500 g, the mediator, by ELISA (Fig. 2). The cells were pre-incubated
pellet was resuspended in 50 m l of extraction buffer (20 mM with 1, 5, 10, or 50 m g/ml of PGME for 2 h and then with
HEPES, pH 7.9, 20% glycerol, 1.5 mM MgCl2, 0.2 mM ethyl- LPS (1.0 m g/ml)20) for 24 h. As shown in Fig. 2, Tnf produc-
enediaminetetraacetic acid (EDTA), 1 mM deloitte touch tion by PGME was significantly inhibited about 50% com-
tohmatsu (DTT), 1 mM phenylmethyl sulfonyl fluoride pared to LPS-stimulated Tnf production (100%, p0.01).
(PMSF) and incubated on ice for 30 min. After centrifugation The inhibitory effect of PGME on Tnf production was dose-
at 12000 g for 5 min, the supernatant was harvested as the dependent through the concentration range from 1 to
nuclear protein extract and stored at 70 °C. Protein concen- 50 m g/ml. At the concentrations of 1, 5, 10, and 50 m g/ml,
tration was determined with a Lowry protein assay reagent Tnf productions by PGME were 1.310.13, 1.110.08,
from Bio-Rad (Hercules, CA, U.S.A.). Ten micrograms of 0.810.19, and 0.220.04 ng/ml, respectively, compared to
the nuclear proteins were incubated with 32P-labeled Nfk b control. The most potent inhibitory effect of Tnf production
probe on ice for 30 min and resolved on 5% acrylamide gel. by PGME was observed at 50 m g/ml.
Statistical Analysis Statistical analysis was performed Tnf Protein Expressions Were Decreased by PGME
using Student’s t-test and one way analysis of variance (one Western blot analysis was executed to determine protein level
way-ANOVA). The accepted level of significance was preset of Tnf in BV2 microglial cells (Fig. 3). The result was that
as p value 0.05. Data are represented as meansS.E.M. Tnf protein expressions by PGME were decreased at both 5
and 50 m g/ml. The expression level of Tnf by PGME at
RESULTS 50 m g/ml was similar to that of control. This result showed
the inhibitory effect of PGME on the LPS-stimulated Tnf
The Production of Tnf Was Increased by LPS As production.
shown in Fig. 1, the amount of Tnf secreted into extracellular Tnf mRNA Expression Was Decreased by PGME To
space was gradually increased at 2, 6, and 12 h incubations. elucidate the mechanism responsible for the inhibitory effect
After 24 h incubation with LPS the secreted concentration of of PGME on Tnf production, we determined the level of Tnf
Tnf was markedly increased almost to 2 ng/ml. Since the pro- mRNA by RT-PCR analysis. When the cells were pretreated
duction of LPS-stimulated Tnf was greatest at 24 h incuba- with PGME for 8 h, the Tnf mRNA level induced by LPS
tion, throughout the experiment, the incubation time with (1 m g/ml) was greatly decreased (Fig. 4). Pretreatment of
1260 Vol. 29, No. 6

respectively.

DISCUSSION

The proinflammatory and anti-inflammatory immune re-

sponses of the healthy body are maintained in a carefully
controlled dynamic equilibrium. Tnf, a kind of cytokines
Fig. 3. Effect of PGME on Lipopolysaccharide (LPS)-Stimulated Expres- which mediate inflammation pathway, is major target of ther-
sion of Tnf Protein
apeutic strategy on in many chronic inflammatory con-
BV2 microglial cells were pretreated with PGME (5, 50 m g/ml) and treated with LPS
(1 m g/ml). After 24 h of incubation, the cell lysates (40 m g/ml) were separated by SDS- ditions.21,22) The induction of inflammatory mediators is reg-
PAGE, transferred to a nitrocellulose membrane and then developed using the chemilu- ulated by transcription factors, such as NFk b.23) Systemic in-
minescence kit. Con, control; LPS, lipopolysaccharide; PG, Punica granatum. Tnf,
tumor necrosis factor a ; Actb, beta actin. jection of a sublethal LPS dose induces acute inflammation
in susceptible strains of rats24) and mice.25) LPS triggers the
generation of reactive oxygen intermediates as well as the se-
cretion of Tnf,26) which activates the DNA binding ability of
NFk b. It is known that Tnf production, induced by LPS,
plays an important role in inflammatory conditions.27)
In this study, PGME decreased the production of Tnf dose-
dependently at 1, 5, 10, and 50 m g/ml in LPS-stimulated mi-
croglial cells (Fig. 2). By western blot analysis, the LPS-sim-
ulated protein expression of Tnf was decreased by PGME
Fig. 4. Effect of PGME on LPS-Stimulated Expression of Tnf mRNA
BV2 microglia cells were pretreated with PGME (5, 50 m g/ml) and treated with LPS
(Fig. 3) and the present study also showed that PGME in-
(1 m g/ml). After 8 h of incubation, total RNA was prepared and Reverse transcription- hibits the expression of Tnf mRNA in LPS-stimulated BV2
polymerase chain reaction (RT-PCR) was performed. The PCR products were separated microglial cells (Fig. 4). These results suggest that PGME
on an 1.5% agarose gel and stained with ethidium bromide. Con, control; LPS,
lipopolysaccharide; PGME, Punica granatum; Tnf, tumor necrosis factor a ; Gapdh, suppresses Tnf mRNA expression, and thus contributes to
glyceraldehyhe-3-phosphate dehydrogenase. Gapdh was used as an internal control. decreasing the production of Tnf.
Antioxidant properties of PGME have been proposed to
influence beneficially on local inflammatory and tissue dam-
aging, which were triggered by oxidative stress.8)
Ginko and grape seed extracts, which has high antioxidant
ability, also show inflammatory inhibition effect.28,29) Ilieva et
al.28) reported that ginko exerted an anti-inflammatory effect
on inflammatory cells by suppressing the production of ac-
tive oxygen. Vitseva et al.29) suggested that grape seed ex-
tracts inhibited inflammation by decreasing release of reac-
tive oxygen intermediates.
Immue and inflammatory reactions are regulated by NFk b
through the NFk b binding sites in their promoter regions.30)
We examined whether increased Tnf production is mediated
via Nfk b mediated pathway by EMSA (Fig. 5) and EMSA
Fig. 5. Effect of PGME on Nuclear Factor k b (Nfk b) DNA Binding Ac- analysis revealed that the DNA binding ability of Nfk b was
tivity
inhibited by PGME. In conclusion, LPS-stimulated PGME
BV2 microglial cells were pretreated with PGME (5, 50 m g/ml) and treated with LPS
(1 m g/ml) for 8 h. Nuclear extracts were isolated and used in an electrophoretic mobility supresses Tnf in BV2 microglial cells, the mechanism of
shift assay with 32P-labeled Nfk b oligonucleotide as a probe. The arrow indicates the which, at least in part, might involve the inhibition of Nfk b.
p65 Nfk b binding complex. Con, control; LPS, lipopolysacchaide; PGME, Punica
granatum; nuclear factor k b, Nfk b.
REFERENCES

PGME (5, 50 m g/ml) decreased Tnf mRNA expressions to 1) Black G., Dietrich M., Norkus E., Morrow J., Hudes M., Caan B.,
Packer L., Am. J. Epidemiol., 156, 274—285 (2002).
near basal level. 2) Barzi F., Woodward M., Marfisi R. M., Tavazzi L., Valagussa F., Mar-
Nfk b Binding Activities Were Decreased by PGME chioli R., Eur. J. Clin., 57, 604—611 (2003).
We next examined the influence of PGME on the Nfk b DNA 3) Schubert S. Y., Lansky E. P., Neeman I., J. Ethnopharmacol., 66, 11—
binding activity by EMSA (Fig. 5). Nfk b is a well known 17 (1999).
transcription factor to transduce extracellular signals to the 4) Aviram M., Dornfield L., Coleman R., Drugs Exp. Clin. Res., 28, 49—
62 (2002).
increased expression of inflammatory cytokines including 5) Malik A., Afaq F., Sarfaraz S., Adhami V. M., Syed D. N., Mukhtar H.,
Tnf.18) As shown in Fig. 5, the incubation of cells with LPS Proc. Natl. Acad. Sci. U.S.A., 102, 14813—14818 (2005).
(1 m g/ml) increased the Nfk b binding activity and pretreat- 6) Afaq F., Malik A., Syed D., Maes D., Matsui M. S., Mukhtar H., Pho-
ment of BV2 microglia cells with PGME decreased the LPS- tochem. Photobiol., 81, 38—45 (2005).
stimulated DNA binding activity of Nfk b at both 5 and 7) Kawaii S., Lansky E. P., J. Med. Food, 7, 13—18 (2004).
8) Farrukh A., Mohammad S., Christian G. K., Jess D. R., Hasan M., Int.
50 m g/ml. Densitometer analysis revealed that PGME at con- J. Cancer, 113, 423—433 (2005).
centrations of 5 and 50 m g/ml, decreased the levels of LPS- 9) Schubert S. Y., Neeman I., Resnick N., FASEB J., 14, 1931—1933
stimulated Nfk b DNA binding activity to 60.8 and 89.9%, (2002).
June 2006 1261

10) Ajaikumar K. B., Asheef M., Babu B. H., Padikkala J., J. Ethnophar- K. T., Biol. Pharm. Bull., 25, 472—476 (2002).
macol., 96, 171—176 (2005). 21) Bolton A. E., Am. J. Cardiol., 95, 24—29 (2005).
11) Seeram N. P., Adams L. S., Henning S. M., Niu Y., Zhang Y., Nair M. 22) Brennan F. M., Maini R. N., Feldmann M., Br. Med. Bull., 51, 368—
G., Heber D., J. Nutr. Biochem., 6, 360—367 (2005). 384 (1995).
12) Chidambara Murthy K. N., Jayaprakasha G. K., Singh R. P., J. Agric. 23) Rahman I., Macnee W., Thorax., 53, 601—612 (1998).
Food Chem., 17, 4791—4795 (2002). 24) Rosenbaum J. T., Mcdevitt H. O., Guss R. B., Egbert P. R., Nature
13) Youdim K. A., Mcdonald J., Kalt W., Joseph J. A., J. Nutr. Biochem., (London), 286, 611—613 (1980).
13, 282—288 (2002). 25) Li Q., Peng B., Whitcup S. M., Jang S. U., Chan C. C., Exp. Eye Res.,
14) Barron K. D., J. Neurol. Sci., 134, 57—68 (1995). 61, 629—632 (1995).
15) Gonzalez-Scarano F., Baltuch G., Annu. Rev. Neurosci., 22, 219—240 26) Tracey K. J., Cerami A., Ann. Rev. Med., 45, 491—503 (1994).
(1999). 27) White J. E., Tsan M. F., Am. J. Respir. Cell Mol. Biol., 24, 164—169
16) Perry V. H., Brain Behav. Immun., 18, 407—413 (2004). (2001).
17) Mcdonald D. R., Brunden K. P., Landreth C. E., J. Neurosci., 17, 28) Ilieva I., Ohgami K., Shiratori K., Koyama Y., Yoshida K., Kase S., Ki-
2284—2294 (1997). tamei H., Takemoto Y., Yazawa K., Ohno S., Exp. Eye Res., 79, 181—
18) Siebenlist U., Franzoso G., Brown K., Annu. Rev. Cell Biol., 10, 405— 187 (2004).
455 (1994). 29) Vitseva O., Varghese S., Chakrabarti S., Folts J. D., Freedman J. E., J.
19) Kuprash D. V., Udalova I. A., Turetskaya R. L., Nedospasov S. A., Cardiovasc. Pharmacol., 46, 445—451 (2005).
Rice N. R., Oncogene, 11, 97—106 (1995). 30) Gil M. I., Tomas-Barbern F. A., Hess-Pierce B., Holcroft D. M., Kedar
20) Kim Y. K., Kim R. G., Park S. J., Ha J. H., Choi J. W., Park H. J., Lee A. A., J. Agric. Food Chem., 10, 4581—4589 (2000).