Distinct inhibitory mechanisms of isoquercitrin gallate and its aglycone on zymosan-induced peroxynitrite production in macrophages

Nitric Oxide 17 (2007) 134–142 www.elsevier.com/locate/yniox

Distinct inhibitory mechanisms of isoquercitrin gallate and its aglycone on zymosan-induced peroxynitrite production in macrophages Byung-Hak Kim, In Jeong Lee, Hwa-Young Lee, Bang Yeon Hwang, Sang-Bae Han, Youngsoo Kim * College of Pharmacy & Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, Republic of Korea Received 17 April 2007; revised 10 June 2007 Available online 21 June 2007

Abstract Peroxynitrite, the coupling product of superoxide and nitric oxide (NO) radicals, plays as a pathogenic mediator in the oxidative stress-implicated diseases. Quercetin 3-O-b-(200 galloyl)-glucopyranoside (Q-32) is an isoquercitrin gallate. In this study, Q-32 was found to inhibit zymosan-induced production of protein-bound 3-nitrotyrosine, a stable metabolite of peroxynitrite, in macrophages RAW 264.7, and its inhibitory mechanism was also documented to be different from that of its aglycone, quercetin. Both Q-32 and quercetin inhibited not only zymosan- but also phorbol 12-myristate 13-acetate-induced superoxide productions in the macrophages. Q-32 did not affect NO production and inducible NO synthase (iNOS) expression in zymosan-stimulated macrophages RAW 2647. However, quercetin inhibited zymosan-induced NO production as well as down-regulated zymosan-induced iNOS expression at the transcription level. Further, quercetin inhibited zymosan-induced nuclear factor (NF)-jB transcriptional activity but also NF-jB-dependent iNOS promoter activity. Taken together, Q-32 and quercetin could provide invaluable tools to investigate zymosan-induced oxidative stress with distinct antioxidant mechanisms. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Isoquercitrin gallate; Quercetin; Protein-bound 3-nitrotyrosine; Zymosan; Macrophages

Peroxynitrite, the coupling product of superoxide and nitric oxide (NO) radicals, is considered as a possible beneficial molecule in the cellular immune response against invading microorganisms [1,2]. On the other hand, under oxidative stress, peroxynitrite has been evidenced to play as a pathogenic mediator in a number of disorders, including asthma, idiopathic pulmonary fibrosis and inflammatory bowel disease [3–5]. Therefore, inhibition of peroxynitrite formation, by preventing either superoxide or NO production, could be valuable to limit tissue damage in the inflammatory circumstances. The activated immune cells including macrophages can release excess amounts of peroxynitrite due to NADPH *

Corresponding author. Fax: +82 43 268 2732. E-mail address: [email protected] (Y. Kim).

1089-8603/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2007.06.002

oxidase- and inducible NO synthase (iNOS)-dependent generations of superoxide and NO, respectively [6,7]. The redox core of NADPH oxidase complex is membrane-associated cytochrome b558 [8]. The other p40phox, p47phox and p67phox components consist of cytoplasmic complex [9]. Upon activation of phagocytes, the cytoplasmic components are phosphorylated by protein kinases, primarily protein kinase C (PKC), and then translocated to the membrane for association with cytochrome b558 to be an active complex generating superoxide [10,11]. In addition, small GTP-binding proteins, Rac isoforms are essential for the activation of NADPH oxidase complex [12,13]. Free radical NO is synthesized from L-arginine by NO synthase (NOS). Endothelial and neuronal NOS isoforms are constitutive enzymes and produce moderate amounts of NO, regulating physiological processes such as relaxation

B.-H. Kim et al. / Nitric Oxide 17 (2007) 134–142

(St. Louis, MO, USA). A piNOS-luciferase (Luc) reporter construct was obtained from Dr. C.J. Lowenstein (Johns Hopkins University School of Medicine, Baltimore, MD, USA), pNF-jB-secretory alkaline phosphatase (SEAP)-NPT reporter construct from Dr. Y.S. Kim (Seoul National University, Seoul, Korea), and an expression vector of IKKb from Dr. J.H. Lee (KRIBB, Taejon, Korea).

OH OH

Quercetin moiety 1 HO O

4’

1’

O

3

O OH

Galloyl moiety

OH

O

O 1”

2”

OH

O Glucopyranoside moiety

HO

135

OH OH

Cell culture Murine macrophages RAW 264.7 were cultured in DMEM medium (13.4 mg/ml Dulbecco’s modified Eagle’s medium, 24 mM NaHCO3, 10 mM Hepes, 143 U/ml benzylpenicillin potassium, 100 lg/ml streptomycin sulfate, pH 7.1) containing 10% FBS, and maintained at 37 °C with 5% CO2 atmosphere. The RAW 264.7 cells harboring pNF-jB-SEAP-NPT reporter construct were cultured in the same conditions except supplement of geneticin (500 lg/ml) to the media.

OH

Fig. 1. Chemical structures of Q-32 and its building moiety.

of blood vessels and neurotransmitter release in the brain [14,15]. Meanwhile, the immune cells such as macrophages also generate NO by iNOS, where it facilitates the killing of invading microorganisms [16]. However, dys-regulated overproduction of NO appears to contribute essentially to tissue injury [17]. Indeed, amounts of NO produced in the macrophages are related to the level of iNOS expression, which is dependent of several transcription factors, primarily nuclear factor (NF)-jB [18,19]. The NF-jB is functional as hetero- or homo-dimeric form of Rel family proteins and is usually sequestered in the cytoplasm by binding to inhibitory jB (IjB) proteins [20]. Upon activation of the macrophages, IjB kinase (IKK) complex can induce the phosphorylation of IjB proteins, which marks for ubiquitination followed by proteasome-mediated degradation [21–23]. NF-jB, free of IjB proteins, now moves into the nucleus and then binds to the promoter regions of inflammatory genes including iNOS for transcriptional activation [24]. Quercetin 3-O-b-(200 galloyl)-glucopyranoside (Q-32) is a naturally occurring isoquercitrin gallate (Fig. 1). Previously, we isolated Q-32 as one of antioxidant constituents from Persicaria lapathifolia (Polygonaceae) to protect superoxide production in human monocytes [25]. In the present study, Q-32 was found to inhibit zymosan-induced production of protein-bound 3-nitrotyrosine, a stable metabolite of peroxynitrite, in murine macrophages RAW 264.7, and its inhibitory mechanism was also documented to be distinct from that of its aglycone, quercetin. Experimental procedures

Enzyme-linked immunosorbent assay (ELISA) Macrophages RAW 264.7 were pre-treated with Q-32 or quercetin for 2 h and stimulated with zymosan (0.3 mg/ml) for 24 h. Amounts of protein-bound 3-nitrotyrosine in the cell lysates were quantified using an ELISA kit according to the supplier’s protocol (Upstate, Charlottesville, VA, USA). Protein was determined by the Bradford method using bovine serum albumin as a standard.

Superoxide quantification Macrophages RAW 264.7 were pre-treated with Q-32 or quercetin for 30 min, in the presence of lucigenin (25 lM), and stimulated with zymosan (0.3 mg/ml) or PMA (0.1 lg/ml). Immediately, chemiluminescence was measured as relative light unit (RLU) at 37 °C in the dark for 100 min period with 5-min intervals (zymosan challenge) or for 20-min period with 1-min intervals (PMA challenge). The chemiluminescence responses were also represented as integrated areas below the resulting chemiluminescence curves. In another experiment, phenazine methosulfate (PMS, 30 lM) and nitroblue tetrazolium (NBT, 100 lM) were mixed in 100 mM sodium phosphate buffer (pH 7.4), and treated with Q-32 or quercetin for 5 min. After incubating with NADH (150 lM) at room temperature for 2 min, absorbance values were measured at 560 nm.

Enzyme assay of cell-free PKC PKC activity was measured using a kit from Promega (Madison, WI, USA). Briefly, a purified PKCd (0.1 lg/ml) was activated with phosphatidylserine (200 lg/ml), in the presence or absence of either Q-32 or quercetin, and then incubated with a PKCd-specific peptide P-L-S-R-T-L-S-V-AA-K substrate at 30 °C for 30 min. The reaction mixture was resolved on agarose gel by electrophoresis and then photographed under UV transillumination.

NO quantification Macrophages RAW 264.7 were pre-treated with Q-32 or quercetin for 2 h and stimulated with zymosan (0.3 mg/ml) for 24 h. Amounts of NO in the culture media were measured using the Griess reaction [26]. Briefly, culture media (100 ll) were reacted with 1:1 mixture (100 ll) of 1% H3PO4 and 0.1% N-(1-naphthyl)ethylenediamine in water, and then measured the absorbance values at 540 nm.

Materials Reverse transcription-polymerase chain reaction (RT-PCR) Isoquercitrin gallate Q-32 (>98% purity) was isolated from P. lapathifolia as described in our previous study [25]. Lipofectamine, fetal bovine serum (FBS) and other culture materials were purchased from Invitrogen (Carlsbad, CA, USA). The other chemicals, including quercetin, pyrrolidine dithiocarbamate (PDTC), zymosan A and phorbol 12-myristate 13-acetate (PMA), were otherwise purchased from Sigma–Aldrich

Macrophages RAW 264.7 were pre-treated with Q-32 or quercetin for 2 h and stimulated with zymosan (0.3 mg/ml) for 6 h. Total RNA of the cells was subjected to semi-quantitative RT-PCR using an RNA PCR kit (Bioneer, Taejon, Korea). Briefly, total RNA (1 lg) was reversely transcribed at 42 °C for 1 h and then subjected to 30 cycles of PCR consisting

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Table 1 Primer sequences for amplification and sizes of RT-PCR products iNOS

Sense Anti-sense

5 0 -GTCAACTGCAAGAGAACGGAGAA-3 0 5 0 -GAGCTCCTCCAGAGGGTAGGCTTG-3 0

457 bp

b-Actin

Sense Anti-sense

5 0 -CACCACACCTTCTACAATGAGCTGC-3 0 5 0 -GCTCAGGAGGAGCAATGATCTTGAT-3 0

745 bp

Luciferase assay Macrophages RAW 264.7 were transiently transfected with piNOSLuc reporter construct and pSV-b-galactosidase control vector (Promega, Madison, WI, USA) using Lipofectamine. The transfected cells were pretreated with Q-32 or quercetin for 2 h and stimulated with zymosan (0.3 mg/ml) for 16 h. Cell lysates were subjected to luciferase assay and b-galactosidase assay using corresponding kits from Promega (Madison, WI, USA). In another experiment, macrophages RAW 264.7 were transiently transfected with piNOS-Luc construct in combination with an expression vector of IKKb using Lipofectamine. The transfected cells were treated with Q-32 or quercetin for 16 h and then subjected to luciferase assay.

SEAP assay Macrophages RAW 264.7 harboring pNF-jB-SEAP-NPT reporter construct were pre-treated with Q-32 or quercetin for 2 h and stimulated with zymosan (0.3 mg/ml) for 16 h. Aliquots of the culture media were heated at 65 °C for 5 min and then reacted with a SEAP buffer (500 lM 4-methylumbelliferyl phosphate, 2 mM diethanolamine, 1 mM MgCl2) in the dark for 1 h. SEAP activity was measured as relative fluorescence units (RFU) with emission at wavelength 449 nm and excitation at wavelength 360 nm.

Cell viability assay Macrophages RAW 264.7 were incubated with various concentrations (0.3–100 lM) of Q-32 or quercetin for 24 h. The cells were treated with WST-1 solution (Dojindo Lab, Kumamoto, Japan) and then measured the absorbance values at wavelength 450 nm.

Statistical analysis Results are expressed as means ± SE. Data were analyzed by the ANOVA followed by the Dunnet test. Values of p < 0.01 were considered significant.

Results Effects of Q-32 and quercetin on peroxynitrite production Amounts of protein-bound 3-nitrotyrosine, a stable metabolite of peroxynitrite, were determined in cell lysates by ELISA. Upon exposure to zymosan alone for 24 h, macrophages RAW 264.7 resulted in a pronounced increase in tyrosine nitration from 20 ± 17 to 242 ± 27 pg of 3-nitrotyrosine per mg of cellular protein (Fig. 2). The Q-32 inhibited zymosan-induced production of protein-bound 3-nitrotyrosine in a dose-dependent manner, correspond-

300

NitroTyr (pg/mg)

of 30-s denaturation at 94 °C, 30-s annealing at 56 °C and 90-s extension at 72 °C [27]. The oligonucleotides used for amplification and sizes of the PCR products are described in Table 1. RT-PCR products were resolved on agarose gel by electrophoresis and stained with ethidium bromide.

Q-32 Quercetin PDTC

#

*

200

* * *

* * *

100

* * *

0

Sample

0

0

0.3

1

3

10 (μM)

Zymosan (0.3 mg/ml) Fig. 2. Zymosan-induced peroxynitrite production. Macrophages RAW 264.7 were pre-treated with Q-32, quercetin or PDTC for 2 h and stimulated with zymosan for 24 h. Amounts of protein-bound 3-nitrotyrosine (NitroTyr), a stable metabolite of peroxynitrite, were measured in the cell lysates by ELISA and are represented as NitroTyr (pg) per cellular protein (mg). Values are means ± SE of three separate experiments. #p < 0.01 vs. media alone-treated group. *p < 0.01 vs. zymosan alone-treated group.

ing to 25% inhibition at 0.3 lM, 36% at 1 lM, 63% at 3 lM and 99% at 10 lM, with an IC50 value of 2.1 lM (Fig. 2). Quercetin is an aglycone of Q-32 (Fig. 1) and inhibited the zymosan-induced tyrosine nitration in a dose-dependent manner, corresponding to 42% inhibition at 3 lM and 69% at 10 lM (Fig. 2). The antioxidant to suppress iNOS expression, PDTC also exhibited dosedependent inhibitory effect on zymosan-induced tyrosine nitration with an IC50 value of 0.9 lM (Fig. 2). Neither Q-32 nor quercetin at the effective concentrations affected cell viability of macrophages RAW 264.7 (data not shown). Effects of Q-32 and quercetin on superoxide production To investigate whether Q-32 or quercetin could affect NADPH oxidase-dependent superoxide production, lucigenin-enhanced chemiluminescence analysis was carried out. Upon exposure to zymosan alone, macrophages RAW 264.7 immediately increased the chemiluminescence to be maximal at 10 min, and its production gradually decreased to the basal level until 100 min (Fig. 3a). The zymosaninduced chemiluminescence was dose-dependently inhibited in the presence of superoxide dismutase (Table 2), indicating that the assay system is specific to superoxide generation. The Q-32 inhibited zymosan-induced superoxide production in a dose-dependent manner, corresponding

B.-H. Kim et al. / Nitric Oxide 17 (2007) 134–142

a

b Zym alone Zym+Q-32 (1 μM) Zym+Q-32 (3 μM) Zym+Q-32 (10 μM) Media alone

150

Q-32 Quercetin

100

Inhibition %

200

Superoxide (RLU)

137

100 50

**

75

*

50

*

25 0 1

0 0

25

50

75

100

3

10

Sample (μM)

Time (min) after Zym challenge

c

300

Q-32 Quercetin

100

Inhibition %

Superoxide (RLU)

d

PMA alone PMA+Q-32 (1 μM) PMA+Q-32 (3 μM) PMA+Q-32 (10 μM) Media alone

400

200 100

* *

75

*

50 25

*

**

0 1

0 0

5

10

15

20

3

10

Sample (μM)

Time (min) after PMA challenge Fig. 3. Zymosan or PMA-induced superoxide production. Macrophages RAW 264.7 were pre-treated with Q-32 or quercetin for 30 min and stimulated with zymosan (Zym, 0.3 mg/ml) (a and b) or PMA (0.1 lg/ml) (c and d) for the indicated times. Amounts of superoxide were measured as relative light units (RLU) using lucigenin-enhanced chemiluminescence (a and c). As the integrated areas below the resulting chemiluminescence curves, effect of Q-32 or quercetin on superoxide production is also represented as inhibition % (b and d). Values are means ± SE of three separate experiments. *p < 0.01 vs. zymosan or PMA alone-treated group.

Table 2 Effects of SOD and rottlerin on zymosan-induced superoxide production SOD (U/ml)

Superoxide (RLU)

Rottlerin (lM)

Superoxide (RLU)

0 102 103 104

1862 ± 155 1404 ± 173* 694 ± 89* 413 ± 102*

0 1 3 10

2011 ± 168 1937 ± 115* 931 ± 143* 678 ± 97*

Superoxide production in zymosan-stimulated macrophages RAW 264.7 was measured by lucigenin-enhanced chemiluminescence. Amounts of superoxide are relative light units (RLU) as integrated areas under the resulting chemiluminescence curves, until 100 min after zymosan challenge. Values are means ± SE of three separate experiments. * p < 0.01 vs. zymosan alone-treated group.

to 49% inhibition at 3 lM and 87% at 10 lM, with an IC50 value of 3.2 lM (Fig. 3a and b). Quercetin also inhibited zymosan-induced superoxide production with an IC50 value of 4.8 lM (Fig. 3b). Upon exposure to PMA alone, macrophages RAW 264.7 rapidly increased superoxide production to be maximal at 4 min and its production gradually decreased to the basal level until 20 min (Fig. 3c). Q-32 inhibited PMAinduced superoxide production in a dose-dependent manner, corresponding to 23% inhibition at 1 lM, 45% at

3 lM and 67% at 10 lM, with an IC50 value of 4.7 lM (Fig. 3c and d). Quercetin also exhibited dose-dependent inhibitory effect on PMA-induced superoxide production with an IC50 value of 2.8 lM (Fig. 3d). To understand whether Q-32 or quercetin could directly scavenge reactive oxygen species, non-enzymatic production of superoxide and its analysis using NBT reduction were carried out. As shown in Fig. 4, significant amounts of superoxide were generated in the presence of PMS and NADH alone. A positive control, 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-one (PTIO) showed 91% scavenging effect against the reactive oxygen species. Q-32 at 1–10 lM did not scavenge the superoxide, significantly, but quercetin showed only 27% scavenging effect at the high concentration of 10 lM. PKCd has been evidenced to play an important role to phosphorylate and translocate p47phox, an essential component of NADPH oxidase-mediated superoxide production [10]. A positive control, rottlerin inhibited zymosaninduced superoxide production (Table 2), but also enzyme activity of PKCd (Fig. 5). However, neither Q-32 nor quercetin at concentrations of 3–10 lM exhibited significant inhibitory effects on phosphatidylserine-activated enzyme activity of cell-free PKCd (Fig. 5).

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B.-H. Kim et al. / Nitric Oxide 17 (2007) 134–142

Q-32 Quercetin PDTC

1.2

*

0.8

# 30

*

0.4

0.0 Sample

0

0

1

10

1

Q-32

10

NO (μM)

NBT (OD560)

#

10(μM)

QC

Fig. 4. Scavenging effect of Q-32 or quercetin against superoxide. Superoxide was non-enzymatically generated using phenazine methosulfate (PMS) and NADH, and its amounts were measured as optical density at 560 nm using nitroblue tetrazolium (NBT) reduction. Samples are Q-32 and quercetin (QC) and a positive control is 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (PTIO). Values are means ± SE of three separate experiments. #p < 0.01 vs. NBT alone-containing group. *p < 0.01 vs. PMS, NADH plus NBT alone-containing group.

Ratio %

*

* *

10

0 Sample

* *

PTIO

PMS + NADH

0

0

3

10

30

100 (μM)

Zymosan (0.3 mg/ml) Fig. 6. Zymosan-induced NO production. Macrophages RAW 264.7 were pre-treated with Q-32, quercetin or PDTC for 2 h and stimulated with zymosan for 24 h. Amounts of NO in the culture media were measured using the Griess reaction. Values are means ± SE of five separate experiments. #p < 0.01 vs. media alone-treated group. *p < 0.01 vs. zymosan alone-treated group.

at 100 lM, with an IC50 value of 28.3 lM (Fig. 6). The positive control, PDTC inhibited zymosan-induced NO production with an IC50 value of 9.6 lM (Fig. 6).

#

100

* 20

50

Effects of Q-32 and quercetin on iNOS expression

*

0

Substrate Product Sample

0

0

3

10

Q-32

3

10

QC

10(μM)

RO

PKCδ (0.1 μg/ml) Fig. 5. Effect of Q-32 or quercetin on enzyme activity of cell-free PKC. A purified PKCd was pre-incubated with Q-32, quercetin (QC) or rottlerin (RO) on ice for 1 h and then reacted with a PKCd-specific peptide substrate at 30 °C for 30 min. The reaction mixtures were resolved on agarose gel by electrophoresis. One of similar results is represented, and amounts of the product signal are also indicated as relative ratio %. Values are means ± SE of three separate experiments. #p < 0.01 vs. substrate alone-containing group. *p < 0.01 vs. substrate plus enzyme alonecontaining group.

Effects of Q-32 and quercetin on NO production To investigate whether Q-32 or quercetin could affect NO production, Griess analysis was carried out. Macrophages RAW 264.7 in the normal state produced 6.3 ± 2.3 lM of NO during incubation for 24 h, whereas the cells increased NO production, up to 28.2 ± 2.1 lM, upon exposure to zymosan alone (Fig. 6). The Q-32 at concentrations of 3–100 lM did not show significant inhibitory effects on zymosan-induced NO production (Fig. 6). On the other hand, quercetin inhibited zymosan-induced NO production in a dose-dependent manner, corresponding to 29% inhibition at 10 lM, 52% at 30 lM and 71%

To understand whether Q-32 or quercetin could affect iNOS expression, semi-quantitative RT-PCR was carried out. iNOS transcript was hardly detectable in the normal state of macrophages RAW 264.7, but markedly increased when the cells were stimulated with zymosan alone (Fig. 7a and b). The Q-32 at concentrations of 3–100 lM did not inhibit zymosan-induced synthesis of iNOS transcript, significantly (Fig. 7a). However, quercetin inhibited zymosaninduced synthesis of iNOS transcript in a dose-dependent manner, corresponding to 65% inhibition at 30 lM and 94% at 100 lM (Fig. 7b). Transcriptional regulation of iNOS expression by quercetin was further documented using macrophages RAW 264.7 transfected transiently with piNOS-Luc construct encoding iNOS promoter (1592/+183) fused to luciferase gene as a reporter [18]. Upon exposure to zymosan alone, the transfected cells increased luciferase expression, up to 8-fold over the basal level (Fig. 7c). Quercetin inhibited zymosan-induced luciferase expression in a dose-dependent manner, corresponding to 48% inhibition at 10 lM, 72% at 30 lM and 90% at 100 lM (Fig. 7c). However, Q-32 at concentrations of 3–100 lM did not exhibit significant inhibitory effect on the zymosan-induced luciferase expression (Fig. 7c). A redox-sensitive transcription factor NF-jB has been evidenced to play a critical role in the expression of zymosan-inducible iNOS [18,19]. To clarify the distinct mechanisms of Q-32 and quercetin on iNOS expression, NF-jB transcriptional activity was monitored using macrophages RAW 264.7 harboring pNF-jB-SEAP-NPT construct

B.-H. Kim et al. / Nitric Oxide 17 (2007) 134–142

139

Q-32

a #

Ratio %

100

a

Quercetin

#

50

600

* β-actin iNOS

Q-32

0

0

3

10

30

100 (μM)

Zymosan (0.3 mg/ml)

SEAP (RFU)

0

*

400

* 200

b

#

Ratio %

100

50

0 Sample

*

0

0

3

10

30

100 (μM)

Zymosan (0.3 mg/ml)

* 0

β-actin

b

Q-32 Quercetin

iNOS Quercetin

0

0

3

10

30 100 (μM)

c

Luciferase (fold)

9

Luciferase (fold)

Q-32 Quercetin

#

#

9

Zymosan (0.3 mg/ml)

6

*

3

*

6

*

* *

3

*

0 Sample

0

0

3

10

30

100 (μM)

IKKβ vector (0.3 pg/cell) 0 Sample

0

0

3

10

30 100 (μM)

Zymosan (0.3 mg/ml) Fig. 7. Zymosan-induced iNOS expression. Macrophages RAW 264.7 were pre-treated with Q-32 (a) or quercetin (b) for 2 h and stimulated with zymosan for 6 h. Total RNA of the cells was subjected to semi-quantitative RT-PCR. One of similar results is represented and relative ratio % is also shown, where iNOS signal was normalized to b-actin signal. (c) The RAW 264.7 cells transfected transiently with both piNOS-Luc reporter construct and pSV-b-galactosidase control vector were pre-treated with Q-32 or quercetin for 2 h and stimulated with zymosan for 16 h. Cell lysates were subjected to luciferase and b-galactosidase assays. Luciferase expression as a reporter for iNOS promoter activity is represented as relative fold, where luciferase activity was normalized to b-galactosidase activity. Values are means ± SE of three separate experiments. #p < 0.01 vs. media alonetreated group. *p < 0.01 vs. zymosan alone-treated group.

Fig. 8. Zymosan-induced NF-jB transcriptional activity and expression vector IKKb-elicited iNOS promoter activity. (a) Macrophages RAW 264.7 harboring pNF-jB-SEAP-NPT reporter construct were pre-treated with Q-32 or quercetin for 2 h and stimulated with zymosan for 16 h. SEAP activity as a reporter for NF-jB transcriptional activity was measured as relative fluorescence units (RFU). (b) Macrophages RAW 264.7 were transiently transfected with piNOS-Luc reporter construct in combination with an expression vector of IKKb. The transfected cells were treated with Q-32 or quercetin for 16 h. Luciferase expression as a reporter for iNOS promoter activity was measured with cell lysates, and is represented as relative fold. Values are means ± SE of three separate experiments. #p < 0.01 vs. media alone-treated group (a) or reporter construct alone-transfected group (b). *p < 0.01 vs. zymosan alone-treated group (a) or reporter construct plus expression vector alone-transfected group (b).

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encoding four copies of the jB sequence fused to SEAP gene as a reporter [28]. Upon exposure to zymosan alone, the transfected cells increased SEAP expression, up to 3-fold over the basal level, indicating that cellular NF-jB is transcriptionally functional (Fig. 8a). Q-32 at concentrations of 3–100 lM did not inhibit zymosan-induced SEAP expression, significantly (Fig. 8a). Quercetin inhibited zymosan-induced SEAP expression in a dose-dependent manner, corresponding to 32% inhibition at 10 lM, 53% at 30 lM and 77% at 100 lM (Fig. 8a). To further document whether quercetin could inhibit NF-jB-dependent iNOS expression, macrophages RAW 264.7 were transiently transfected with piNOS-Luc construct in combination with an expression vector encoding IKKb. Luciferase expression as a reporter for iNOS promoter activity was efficiently increased by transfection of expression vector IKKb (Fig. 8b). Quercetin inhibited expression vector IKKb-elicited SEAP expression in a dose-dependent manner, corresponding to 46% inhibition at 10 lM, 77% at 30 lM and 91% at 100 lM (Fig. 8b). However, the expression vector IKKb-elicited SEAP expression was not impaired in the presence of Q-32 (Fig. 8b). Discussion Q-32 is a naturally occurring isoquercitrin gallate and its aglycone is quercetin (Fig. 1). In the present study, Q-32 was found to inhibit tyrosine nitration due to peroxynitrite production in zymosan-stimulated macrophages RAW 264.7 (Fig. 2). Quercetin also inhibited the zymosaninduced tyrosine nitration in a dose-dependent manner (Fig. 2). Peroxynitrite production is dependent upon the generations of superoxide and NO radicals in zymosan-stimulated macrophages RAW 264.7. Both Q-32 and quercetin inhibited not only zymosan- but also PMA-induced superoxide productions in dose-dependent manners (Fig. 3). In another experiment, Q-32 could not directly scavenge the superoxide but quercetin showed a weak scavenging effect at the high concentration (Fig. 4). Therefore, Q-32 and quercetin preferentially inhibited NADPH oxidase-dependent superoxide production instead of scavenging the reactive oxygen species in zymosan-stimulated macrophages. In order to generate superoxide by NADPH oxidase complex, PKC should be activated to phosphorylate the p47phox and then the cytoplasmic components are translocated for the assembly with membrane-associated cytochrome b558 [10]. Zymosan activates PKC via a receptormediated signaling pathway and PMA is a direct activator of PKC [29,30]. In this study, both Q-32 and quercetin at the effective concentrations on superoxide production did not show significant inhibitory effects on the enzyme activity of cell-free PKCd (Fig. 5). Therefore, these findings suggest that Q-32 or quercetin could possibly prevent the membrane-associated assembly of NADPH oxidase complex, an event downstream PKC-mediated phosphorylation of cytoplasmic p47phox component, which is similar

to the NADPH oxidase-inhibitory mechanism shown by apocynin, a polyphenolic compound [31]. We next determined that Q-32 or quercetin could affect NO production. Q-32 at concentrations of 3–100 lM did not show significant inhibitory effect on zymosan-induced NO production but quercetin dose-dependently inhibited (Fig. 6). However, quercetin showed much less effectiveness on NO production than superoxide production in zymosan-stimulated macrophages, in view of IC50 values (Figs. 3b and 6). Therefore, inhibitory levels of Q-32 or quercetin on 3-nitrotyrosine production seem to be correlated to those, IC50 values on superoxide production in zymosanstimulated macrophages (Figs. 2 and 3b). To understand whether differential effects of Q-32 or quercetin on NO production could be dependent upon iNOS expression, RT-PCR and promoter activity were further analyzed. Consistently, Q-32 did not affect zymosaninduced iNOS transcript synthesis and iNOS promoter activity (Fig. 7a and c), but quercetin attenuated zymosan-induced iNOS transcript synthesis but also iNOS promoter activity, in parallel (Fig. 7b and c), indicating that quercetin could down-regulate zymosan-induced iNOS expression at the transcription level. The iNOS promoter behaves as a sophisticated biosensor with multiple responsive elements, including NF-jB binding site, activator protein-1 (AP-1) site, CCAAT/ enhancer-binding protein (C/EBP) site, interferon-a-stimulated response element (ISRE) and tumor necrosis factor responsive element (TNF-RE) [18,32]. Among these elements, a redox-sensitive NF-jB transcription factor has been evidenced to play a major mechanism on the expression of zymosan-inducible iNOS [18,19]. Quercetin inhibited zymosan-induced NF-jB transcriptional activity in a dose-dependent manner (Fig. 8a). As well as, expression vector IKKb-elicited iNOS promoter activity was also impaired in the presence of quercetin (Fig. 8b). These findings indicate that inhibitory effect of quercetin on zymosaninduced NO production is attributable to its down-regulatory mechanism on NF-jB-dependent iNOS expression. However, Q-32 did not affect zymosan-induced NF-jB transcriptional activity (Fig. 8a) and expression vector IKKb-elicited iNOS promoter activity (Fig. 8b). We reported previously that quercetin 3-O-b-(200 galloyl)rhamnopyranoside, a structural analog of Q-32, could suppress iNOS expression and NF-jB activation in LPS-stimulated macrophages [33]. This unexpected result could be speculated due to either distinct signaling pathways derived from zymosan and LPS [34] or importance of subtle structural difference in the sugar moiety. More recently, zymosan-recognizing receptors such as toll-like receptor (TLR)-2 and dectin-1 have been characterized in the immune cells including macrophages [35,36]. Specific binding of zymosan to TLR-2 can trigger NF-jB activation that up-regulates the expression of proinflammatory proteins, including iNOS and certain cytokines [35,37]. On the other hand, non-opsonic recognition of zymosan by dectin-1 can stimulate phagocytosis and

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induce superoxide production through NADPH oxidase complex [36]. The dectin-1-dependent signaling pathway has been evidenced to be independent but amplify TLR2-mediated inflammatory responsiveness [38]. In conclusion, Q-32 could inhibit zymosan-induced tyrosine nitration due to peroxynitrite production, which is attributable to its preventive mechanism on superoxide production without affecting NO production. Quercetin also inhibits zymosan-induced tyrosine nitration, in which the compound prevented superoxide production but also NO production. Finally, Q-32 and quercetin could provide invaluable tools to investigate zymosan-induced oxidative stress with distinct antioxidant mechanisms. Acknowledgments This work was financially supported by the research grant from Chungbuk National University in 2006. We appreciated Drs. C.J. Lowenstein, Y.S. Kim, and J.H. Lee for their kind supply of piNOS-Luc construct, pNFjB-SEAP-NPT construct, and expression vector IKKb, respectively. References [1] A. Vazquez-Torres, J. Jones-Carson, P. Mastroeni, H. Ischiropoulos, F.C. Fang, Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental Salmonellosis: effects on microbial killing by activated peritoneal macrophages in vitro, J. Exp. Med. 192 (2000) 227–236. [2] J.M. Hickman-Davis, P. O’Reilly, I.C. Davis, J. Peti-Peterdi, G. Davis, K.R. Young, R.B. Devlin, S. Matalon, Killing of Klebsiella pneumoniae by human alveolar macrophages, Am. J. Physiol. Lung Cell Mol. Physiol. 282 (2002) L944–L956. [3] D. Saleh, P. Ernst, S. Lim, P.J. Barnes, A. Giaid, Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid, FASEB J. 12 (1998) 929–937. [4] D. Saleh, P.J. Barnes, A. Giaid, Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis, Am. J. Respir. Crit. Care Med. 155 (1997) 1763–1769. [5] I.I. Singer, D.W. Kawka, S. Scott, J.R. Weidner, R.A. Mumford, T.E. Riehl, W.F. Stenson, Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease, Gastroenterology 111 (1996) 871–885. [6] B.M. Babior, NADPH oxidase, Curr. Opin. Immunol. 16 (2004) 42– 47. [7] J.B. Hibbs, Infection and nitric oxide, J. Infect. Dis. 185 (2002) S9– S17. [8] D. Rotrosen, C.L. Yeung, J.P. Katkin, Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins, J. Biol. Chem. 268 (1993) 14256–14260. [9] F.B. Wientjes, G. Panayotou, E. Reeves, A.W. Segal, Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox, Biochem. J. 317 (1996) 919– 924. [10] E.A. Bey, B. Xu, A. Bhattacharjee, C.M. Oldfield, X. Zhao, Q. Li, V. Subbulakshmi, G.M. Feldman, F.B. Wientjes, M.K. Cathcart, Protein kinase Cd is required for p47phox phosphorylation and translocation in activated human monocytes, J. Immunol. 173 (2004) 5730–5738.

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