Life Sciences 103 (2014) 59–67

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Pheophytin a and chlorophyll a suppress neuroinflammatory responses in lipopolysaccharide and interferon-γ-stimulated BV2 microglia Sunyoung Park a,1, Jeong June Choi b,1, Bo-Kyung Park a, Soo Jeong Yoon a, Jung Eun Choi a, Mirim Jin a,⁎ a b

Laboratory of Pathology, College of Korean Medicine, Daejeon University, Daejeon 300-716, Republic of Korea Natural Products Research Institute, Gyeonggi Institute of Science and Technology Promotion, Gyeonggi-do 443-270, Republic of Korea

a r t i c l e

i n f o

Article history: Received 31 December 2013 Accepted 31 March 2014 Available online 13 April 2014 Keywords: Pheophytin a Chlorophyll a Microglia BV2 cell Neuroinflammation

a b s t r a c t Aims: Microglia-mediated inflammation is associated with pathogenesis of various neuronal disorders. This study investigated inhibitory effects of pheophytin a (PP) and chlorophyll a (CP) on neuroinflammation and underlying cellular mechanisms in microglia cells. Main methods: BV2 murine microglia cells were stimulated by lipopolysaccharide (LPS, 100 ng/mL) and interferon (IFN)-γ (10 U/mL). The productions of nitric oxide (NO) and expressions of proinflammatory cytokines and chemokines were determined by ELISA and RT-PCR. Western blot and confocal microscopy were applied to analyze activation of transcription factors and mitogen activated protein kinase (MAPK). Key findings: PP and CP significantly reduced the levels of NO, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and chemokines including macrophage inhibitory protein (MIP)-1α, macrophage chemoattractant protein (MCP)-1 and IFN-γ inducible protein (IP)-10 in BV2 cells stimulated with LPS and IFN-γ (LI). The nuclear expression of p65 NF-κB was significantly suppressed, which was accompanied by reduced the levels of IFN-β, phospho-STAT-1, and interferon regulatory factor (IRF)-1. Activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but not p38 MAPK were prominently suppressed by PP and/or CP. Significance: PP and CP may suppress inflammatory responses by inhibiting NF-κB activation and type I IFN signaling pathway. These result suggested that PP and CP have potential as anti-inflammatory agents for microglia-mediated neuroinflammatory disorders. © 2014 Elsevier Inc. All rights reserved.

Introduction Bioactive phytochemicals rich in vegetables, fruits and medicinal herbs have been known to present beneficial and protective effects against neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS), which pathogenesis is implicated with inflammatory status (Sun et al., 2011; Essa et al., 2012). Microglia, representative of residence macrophage population in the CNS, has been proposed to play a role in host defense and tissue repair (Nimmerjahn et al., 2005). However, over-reactive microglia is thought to be responsible for inflammation-mediated brain tissue damage, and chronic activations of the cells seem to be involved in the development of various neurodegenerative diseases (Gonzalez-Scarano and Baltuch, 1999; Hirsch, 2000). Activated microglia are capable of producing a variety of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6

⁎ Corresponding author at: Laboratory of Pathology, College of Korean Medicine, Daejeon University, Daejeon 300-716, Republic of Korea. Tel.: + 82 42 280 2679; fax: +82 42 280 2624. E-mail address: [email protected] (M. Jin). 1 The authors equally contributed to this work.

http://dx.doi.org/10.1016/j.lfs.2014.04.003 0024-3205/© 2014 Elsevier Inc. All rights reserved.

(Dickson et al., 1993) and chemokines including MIP-1α, MCP-1 as well as IP-10, which are potentially involved in the pathogenesis of the disease through neuroinflammatory responses (Biber et al., 2008). NO, which is a neurotoxic chemical mediator generated by inducible nitric oxide synthase (iNOS), is rapidly transcribed and expressed in microglia after brain damage contributing to neuronal dysfunction (Chao et al., 1992; Liu et al., 2002). LPS and IFN-γ are potent inflammatory stimuli eliciting activation of microglia. Upon these stimuli, the expressions of iNOS, proinflammatory cytokines, and chemokines genes are rapidly activated under tight regulation at many levels, where transcription factors such as NF-κB and STAT-1 play pivotal roles for the gene expressions (Stark, 2007; Hayden and Ghosh, 2008). Toll-like receptor (TLR) activation by LPS stimulation transduces the cellular signals, resulting in activation of NF-κB and an interferon pathway mediator, IRF-3 (Toshchakov et al., 2002; Hu and Ivashkiv, 2009). In resting status, NF-κB consisting of Rel family p50 and p65 subunits reside in cytoplasm and form an inactive complex combined with inhibitory protein, IκB. However, various stimuli rapidly induce phosphorylation, ubiquitination, and degradation of IκB by 26S proteasome. The dissociation of IκB enables NF-κB free from the inhibitory proteins and translocate into nucleus, leading to binding to κB binding sites in the promoter of the various target genes (Hayden and Ghosh, 2008).

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S. Park et al. / Life Sciences 103 (2014) 59–67

Upon IFN-γ stimulation, homodimers of phosphorylated STAT-1 translocate to the nucleus, bind to the GAS, and activate transcription of specific genes (Hu and Ivashkiv, 2009). The expression of IRF-1, a transcription factor that recognizes a sequence called the interferon stimulation response element (ISRE), is regulated by STAT-1 (Jaruga et al., 2004). Further, the MAPK family that consists of three main subfamily members including JNK, ERK, and p38 MAPK has been known to play important roles in the signaling by LPS and IFN-γ thereby leading to inflammatory activation of microglia (Kim et al., 2004). We investigated that PP and CP found in the most of green vegetables had suppressive effects on expression of proinflammatory mediators via NF-κB, STAT-1, IRF-1, and MAPKs in activated BV2 cells and suggested that these natural pigments may have potentials as antiinflammatory agents in neurodegenerative disorders mediated by microglia. Materials and methods

Table 1 Gene specific primer sequences for RT-PCR. Gene

Oligonucleotide sequence

Product size (bp)

iNOS

Sense; 5′- TGG TGG TGA CAA GCA CAT TT -3′ antisense; 5′- CTG AGT TCG TCC CCT TCT CTC C -3′ Sense; 5′- CTC CCA GGT TCT CTT CAA GG -3′ antisense; 5′- TGG AAG ACT CCT CCC AGG TA -3′ Sense; 5′- AAG CTC TCA CCT CAA TGG A -3′ antisense; 5′- TGC TTG AGA GGT GCT GAT GT -3′ Sense; 5′- CAT GTT CTC TGG GAA ATC GTG G -3′ antisense; 5′- AAC GCA CTA GGT TTG CCG AGT A -3′ Sense; 5′- TCT GCA ACC AAG TCT TCT CAG -3′ antisense; 5′- GAA GAG TCC CTC GAT GTG GGC TA -3′ Sense; 5′- CAG CAG GTG TCC CAA AGA -3′ antisense; 5′- CTT GAG GTG GTT GTG GAA A -3′ Sense; 5′- CCT ATC CTG CCC ACG TGT TG -3′ antisense; 5′- CGC ACC TCC ACA TAG CTT ACA -3′ Sense; 5′- TCC AAG AAA GGA CGA ACA TTC G -3′ antisense; 5′- TGA GGA CAT CTC CCA CGT CAA -3′ Sense; 5′- ACC GTG AAA AGA TGA CCC AG -3′ antisense; 5′- TCT CAG CTG TGG TGG TGA AG -3′

229

TNF-α IL-1β IL-6 MIP-1α MCP-1 IP-10 IFN-β β-Actin

Reagents and cell culture LPS and chlorophyll a were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pheophytin a was generated from chlorophyll a according to a previously reported method (You et al., 2011). IFN-γ was purchased from R&D Systems (Minneapolis, MN, USA). Murine BV2 microglial cells were obtained from Prof. HS Kim (Ewha Woman’s University) and cultured in DMEM medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (Lonza) and 100 μg/mL penicillin-streptomycin (Lonza) at 37 °C in a humidified atmosphere containing 5% CO2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay BV2 cells (5 × 105 cells/mL) were cultured in the presence of pheophytin a (0.5–2.5 μg/mL), chlorophyll a (1–5 μg/mL), or luteolin (20 μM) for 24 h and incubated with 10 μL (5 mg/mL) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) for 4 h at 37 °C. The formazan crystals were dissolved in 100 μL dimethyl sulfoxide. Absorbance was measured at 540 nm using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).

195 302 473 375 242 430 313 272

Western blot analysis Whole lysates were harvested using RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktail set III (Calbiochem, California, USA). Nuclear extracts were prepared using Nuclear Extract kit (Active Motif, Carlsbad, CA, USA.). Equal amounts (40 μg) of proteins were electrophoresed using 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA), which were blocked with 5% skim milk in TBS/T buffer (Tris buffered saline in 0.1% Tween-20) for 1 h. The membranes were incubated with antibodies specific to iNOS, NF-κB p65, phospho-STAT-1, STAT-1, IRF-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), phospho-p38 (Thr180/Tyr182), p38, phospho-ERK (Thr202/Tyr204), ERK, phosphoJNK (Thr183/Tyr185), and JNK (Cell Signaling Technology, Beverly, MA). Blots were incubated with HRP-conjugated secondary antibody. HRP was detected using a chemiluminescent detection reagent (Amersham Biosciences).

Nitric oxide determination Immunocytochemistry BV2 cells (2 × 105 cells/mL) were activated with LPS (100 ng/mL) and IFN-γ (10 U/mL) (LI) for 24 h in the absence or presence of test reagents. The levels of nitric oxide in the culture supernatant were determined using a nitric oxide (NO) detection kit (iNtRON BioTechnology, Korea) according to manufacturer’s instruction. ELISA BV2 cells (2 × 105 cells/mL) were treated with LI for 16 h in the absence or presence of test reagents. The levels of TNF-α, IL-1β, IL-6, MIP-1α, MCP-1, and IP-10 in the culture supernatant were determined using a commercially available ELISA kit (BD Sciences, San Jose, CA, USA; eBioscience, San Diego, CA, USA; and R&D Systems) as manufacturer’s protocol.

BV2 cells (2 × 105 cells/mL) cultured on coverslips were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized by incubation for 5 min with 0.25% Triton X-100 in PBS. Nonspecific binding was blocked with 1% bovine serum albumin (Bovogen Biologicals Pty Ltd, Melbourne, Australia) in PBS. Then, the cells were incubated with anti-NF-κB p65 for 2 h, followed by Texas Red goat anti-rabbit IgG (Invitrogen, Oregon, USA) for 1 h. Cellular nuclei were stained with Hoechst33258 dye (Bis-benzimide, SigmaAldrich) for 10 min. Sections were mounted to slides with a gelatin medium (Sigma-Aldrich). Images were acquired using confocal laser scanning microscope (Carl Zeiss, Jena, Germany) with × 400 magnification. Statistical analysis

Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was isolated using Trizol® Reagent (Life Technologies, Carlsbad, CA, USA) and used as template for cDNA synthesis using a cDNA synthesis kit (iNtRON Biotechnology, Gyeonggi-do, Korea). The sequences for the RT-PCR primers were demonstrated in Table 1 (Ock et al., 2010). The PCR reaction was run for 35 cycles at 94 °C (20 s), 55 °C (30 s), and 72 °C (30 s).

All data are expressed as mean ± SD. The data presented are one representative experiment of three independent experiments. Oneway analysis of the variance (ANOVA) with Bonferroni multiple comparison test was used to analyze the differences between the control and experimental groups using SPSS statistical software (SPSS, Chicago, IL, USA). Student’s t-test was used to compare the mean values of the treatments. p b 0.05 was considered to be statistically significant.

S. Park et al. / Life Sciences 103 (2014) 59–67

Results

61

Table 2 Effects of pheophytin a and chlorophyll a on cell viability.

Pheophytin a and chlorophyll a suppressed LPS- and IFN-γ-induced NO formation and iNOS expression

Concentration (μg/mL)

In brain, NO is mainly produced by microglia and excessive production damages neurons (Chao et al., 1992). To test whether PP and CP have anti-inflammatory effect on microglia, we measured the level of NO production in the cell culture supernatant of BV2 cells. The production of NO was highly increased by co-treatment of LPS (100 ng/mL) plus IFN-γ (10 U/mL) (LI) up to 14-fold compared to that of normal group; however, treatment of PP and CP significantly blocked the formation of NO in a dose-dependent manner under the concentration of cytotoxicity (Fig. 1A, Table 2). Next, we investigated the effects of PP and CP on the expression of iNOS, which synthesizes NO in microglia. The protein level of iNOS was dramatically increased in activated BV2 cells, but the incubation of PP and CP greatly suppressed the levels to almost that of normal group (Fig. 1B). Further, the mRNA level of iNOS significantly increased by LI stimulation compared to that of normal cells; however, PP (2.5 μg/mL) and CP (5 μg/mL) greatly suppressed the gene expression to almost undetectable levels (Fig. 1C). Luteolin (20 μM), a positive control used in this study also, showed significant inhibitory effect on iNOS and NO production as previously reported (Kao et al., 2011). These data suggest that PP and CP might inhibit NO formation through down-regulation of iNOS expression.

0 0.25 0.5 1 2.5 5 7.5 10

100.00 119.27 97.35 92.99 91.04 85.00 80.95 82.24

100.00 99.28 94.04 96.24 95.48 105.33 95.88 89.48

± ± ± ± ± ± ± ±

1.46 11.85 4.66 0.89 4.92 8.94 8.83 7.19

± ± ± ± ± ± ± ±

1.46 14.30 5.33 5.38 5.54 28.40 0.36 7.22

which was greatly increased by the stimulants, was dramatically suppressed by PP and CP about 50% at the highest concentrations (Table 3). LI stimulation increased the level of IL-1β by approximately 44-fold; however, the pretreatment with PP and CP significantly inhibited the IL-1β production by 42% and 29% compared with control group, respectively (Table 3). The level of IL-6 was considerably suppressed by PP and CP at most 90%. Consistent with the protein levels, mRNA expressions of the cytokines were suppressed by treatments of PP and CP. The mRNA levels of the cytokines were determined 3 h after LI stimulation. In the presence of PP (2.5 μg/mL), the levels of TNF-α, IL-1β, and IL-6 were reduced to 31.4%, 18.9%, and 60.3% compared with control group, respectively (Fig. 2). CP (5 μg/mL) down-regulated the expressions of the cytokines about 17.4% (TNF-α), 30.3% (IL-1β), and 55.5% (IL-6) compared with control group. These

Next, the effects of PP and CP on productions of key proinflammatory cytokines, TNF-α, IL-1β, and IL-6 were examined. The level of TNF-α, ###

50 40

NO (µM)

Chlorophyll a

BV2 cells were treated with various concentrations of pheophytin a and chlorophyll a for 24 h, and then cell viability was measured by MTT assay. Values are expressed as mean ± SD from 3 independent experiments. Statistical significance was analyzed by one-way ANOVA test vs. 0 μg/mL treated group.

Pheophytin a and chlorophyll a suppressed LPS- and IFN-γ-induced proinflammatory cytokines expressions

A

Cell viability (%) Pheophytin a

***

30

*** ***

***

20

***

***

***

10 0

Normal

2.5

5

PP

CP

0

LUT

0.5

1

2.5

1

2.5

PP

5

(µg/mL)

CP

LPS+IFN-γ

B (µg/mL) 0 LPS+IFN-γ

–

PP

CP

2.5

5

0

PP

–

–

+

LUT 0.5

+

+

CP

1

2.5

1

2.5

5

+

+

+

+

+

iNOS β-actin

C

PP (µg/mL)

LPS+IFN-γ

CP

0

0

0.5

2.5

1

5

LUT

–

+

+

+

+

+

+

iNOS β-actin Fig. 1. Effects of PP and CP on NO production and iNOS expression in BV2 cells. (A) BV2 cells were treated with various concentrations of PP and CP for 1 h followed by stimulation with LI for 24 h. The levels of NO in the cell culture supernatant were measured by NO detection Kit. The data represent the mean ± SD of three independent experiments. (B) The cells were incubated with PP and CP in the presence or absence of LI for 16 h. The level of iNOS was determined by Western blot analysis. (C) The cells were incubated with PP and CP in the presence or absence of LI for 6 h. Total RNAs were isolated and used for semi-quantitative RT-PCR. β-Actin was used as a loading control. ***p b 0.001 vs. control group, ###p b 0.001 vs. normal group (one-way ANOVA test). PP, pheophytin a; CP, chlorophyll a; LUT, luteolin.

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Table 3 Effects of pheophytin a and chlorophyll a on various cytokines production in activated BV2 cells. Group

Concentration (μg/mL)

ELISA (pg/mL) TNF-α

Normal Control Pheophytin a

Chlorophyll a

Luteolin

LPS + IFN-γ 0.5 1 2.5 1 2.5 5 20 μM

IL-1β

57.17 4688.42 2737.22 2508.81 2265.74 2983.43 2514.0.5 2343.69 2048.93

± ± ± ± ± ± ± ± ±

5.94 143.53††† 144.39*** 54.38*** 92.11*** 173.64*** 138.72*** 82.71*** 108.24***

21.44 935.52 699.05 577.01 545.34 729.14 705.43 671.83 524.46

IL-6 ± ± ± ± ± ± ± ± ±

2.65 53.59††† 57.73* 79.50*** 22.38*** 114.16 102.23* 88.71** 19.26***

0.37 7178.92 1967.69 856.48 216.90 3818.74 1963.55 911.64 1823.75

± ± ± ± ± ± ± ± ±

0.24 447.17††† 228.50*** 48.86*** 15.41*** 533.22*** 49.68*** 52.99*** 23.44***

BV2 cells were treated with various concentrations pheophytin a and chlorophyll a for 1 h followed by stimulation with LI for 16 h. The levels of TNF-α, IL-1β, and IL-6 in the cell culture supernatant were measured by ELISA. The data represent the mean ± SD of three independent experiments. *p b 0.05, **p b 0.01, ***p b 0.001 vs. control group. †††p b 0.001 vs. normal group (one-way ANOVA test).

as same manner as the proinflammatory cytokines. The levels of MIP1α MCP-1, and IP-10 were increased by LI approximately 9-, 14-, and 81-fold, respectively (Table 4). PP (2.5 μg/mL) lowered the levels of MIP-1α and MCP-1 approximately 27% and 17% compared with control group, respectively. There were approximately 23% and 32% inhibition in the levels of MIP-1α and MCP-1, respectively, in the cells treated with CP (5 μg/mL). Treatment of PP (2.5 μg/mL) and CP (5 μg/mL) led to approximately 50% inhibition of IP-10. The expression patterns of mRNA were very similar to those shown in the protein levels. PP (2.5 μg/mL) decreased the levels of MIP-1α, MCP-1, and IP-10 about 35.6%, 23.7%, and 20.7%, respectively, compared with control group (Fig. 3). In the presence of CP (5 μg/mL), the levels of the chemokines

data suggested that PP and CP might suppress expressions of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in activated microglia. Pheophytin a and chlorophyll a suppressed LPS- and IFN-γ-induced chemokines expressions The activated microglia produces high levels of chemokines such as MIP-1α, MCP-1, and IP-10 (Lee et al., 2002). Therefore, we confirmed the basal levels of the chemokines in BV2 cells as shown in Table 4. BV2 cells produced relatively higher levels of the chemokines than proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 under no stimulation. LI stimulation induced the expressions of the chemokines

A

PP

CP

(µg/mL) 0

0

0.5

2.5

1

5

LUT

LPS+IFN-γ –

+

+

+

+

+

+

TNF-α IL-1β IL-6 β-actin

B Sample

Control

Concentration (µg/mL)

RT-PCR TNF-α

IL-1β

IL-6

LPS + IFN-γ

100

100

100

0.5

76.61 ± 22.15

89.99 ± 18.31

72.64 ± 7.89

2.5

68.55 ± 4.64*

82.09 ± 7.84*

39.66 ± 17.39***

1

77.16 ± 17.36

95.75 ± 18.41

73.46 ± 16.37*

5

82.56 ± 20.20

69.69 ± 6.21*

44.46 ± 5.34***

20 µM

48.89 ± 26.69

94.44 ± 6.50

28.06 ± 13.09*

PP

CP Luteolin

Fig. 2. Effects of PP and CP on mRNA expressions of cytokines in BV2 cells. (A) BV2 cells were pretreated with PP and CP for 1 h followed by LI stimulation for 6 h. Levels of TNF-α, IL-1β, and IL-6 mRNA were determined by RT-PCR. β-Actin was used as a loading control. The data presented are representative of three independent experiments. (B) The mRNA expression levels of the cytokines were determined by measuring the band intensities. The relative expression of each gene was calculated by comparing with control group. *p b 0.05, ***p b 0.001 vs. control group (Student’s t-test). PP, pheophytin a; CP, chlorophyll a; LUT, luteolin.

S. Park et al. / Life Sciences 103 (2014) 59–67

63

Table 4 Effects of pheophytin a and chlorophyll a on various chemokines production in activated BV2 cells. Group

Concentration (μg/mL)

ELISA (ng/mL) MIP-1α

Normal Control Pheophytin a

LPS + IFN-γ 0.5 1 2.5 1 2.5 5 20 μM

Chlorophyll a

Luteolin

17.85 156.74 145.81 139.78 115.35 155.89 149.18 120.81 116.01

MCP-1

± ± ± ± ± ± ± ± ±

1.64 6.20††† 6.36 1.21 6.05*** 12.50 7.53 4.77*** 7.26***

1.11 15.36 15.81 15.32 12.80 16.43 15.99 10.46 10.08

± ± ± ± ± ± ± ± ±

IP-10 0.10 0.84††† 0.65 0.87 0.11 1.40 0.74 0.98*** 1.00***

0.08 6.50 5.16 5.10 3.49 4.80 4.48 3.17 5.04

± ± ± ± ± ± ± ± ±

0.01 0.08††† 0.05 0.07 0.09*** 0.07* 0.03*** 0.04*** 0.05

BV2 cells were treated with various concentrations of pheophytin a and chlorophyll a for 1 h followed by stimulation with LI for 16 h. The levels of MIP-1α, MCP-1, and IP-10 in the cell culture supernatant were measured by ELISA. The data represent the mean ± SD of three independent experiments. *p b 0.05, ***p b 0.001 vs. control group. †††p b 0.001 vs. normal group (one-way ANOVA test).

1995; Cheng et al., 1998; Zhou et al., 1998; Lim and Garzino-Demo, 2000; Fernandez et al., 2002; Choi et al., 2006; Zhao et al., 2008; Lu et al., 2011). Therefore, we first investigated effects of PP and CP on the NF-κB activated by LI stimulation in BV2 cells. The Western blot analysis using nuclear extract indicated that the nuclear expressions of p65, which were significantly increased by LI stimulation, were significantly inhibited by pretreatment with PP and CP (Fig. 5A). In addition, data from confocal microscopy indicated the nuclear translocation of NF-κB is significantly blocked in the cells treated with PP and CP (Fig. 5B), while p65 mostly translocated into nucleus in LI only treated cells. Second, we investigated the effects of PP and CP on activations of transcription factors mediating IFN signaling. We examined the

were reduced to 31.6%, 28.3%, and 8.2%, respectively. These data suggest that PP and CP might suppress chemokines expressions such as MIP-1α, MCP-1, and IP-10 in activated microglia. Pheophytin a and chlorophyll a suppressed activation of NF-κB and STAT-1 pathway As shown in Fig. 4, NF-κB and STAT-1 are known to control the expression of iNOS, proinflammatory cytokines, and chemokine genes examined in this study by acting on their promoters (Shimizu et al., 1990; Grove and Plumb, 1993; Lowenstein et al., 1993; Ohmori and Hamilton, 1993; Xie et al., 1993; Martin et al., 1994; Sanceau et al.,

A

PP

CP

(µg/mL)

0

0

0.5

2.5

1

5

LUT

LPS+IFN-γ

–

+

+

+

+

+

+

MIP-1α MCP-1 IP-10 β ti β-actin

B Sample

Control

Concentration (µg/mL)

RT-PCR MIP-1α

MCP-1

IP-10

LPS + IFN-γ

100

100

100

0.5

83.81 ± 0.34***

82.23 ± 1.24*

94.98 ± 6.26

2.5

64.43 ± 3.28***

76.28 ± 11.80

79.26 ± 15.76

1

73.84 ± 5.59**

66.27 ± 5.89*

86.19 ± 3.24*

5

68.40 ± 6.05*

71.72 ± 6.52*

91.79 ± 0.78*

20 µM

77.86 ± 38.47

79.44 ± 7.38

120.53 ± 3.35*

PP

CP Luteolin

Fig. 3. Effects of PP and CP on mRNA expressions of chemokines in BV2 cells. (A) BV2 cells were pretreated with PP and CP for 1 h followed by LI stimulation for 6 h. Levels of MIP-1α, MCP1, and IP-10 mRNA were determined by RT-PCR. β-Actin was used as a loading control. The data presented are representative of three independent experiments. (B) The mRNA expression levels of the chemokines were determined by measuring the band intensities. The relative expression of each gene was calculated by comparing with control group. *p b 0.05, **p b 0.01, ***p b 0.001 vs. control group (Student’s t-test). PP, pheophytin a; CP, chlorophyll a; LUT, luteolin.

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S. Park et al. / Life Sciences 103 (2014) 59–67

Promoter

References Xie et al. , 1993 Lowenstein et al. , 1993 Martin et al. , 1994

Choi et al. , 2006 Zhao et al. , 2008

Choi et al. , 2006

Shimizu et al. , 1990 Sanceau et al. , 1995

Fernandez et al. , 2002 Grove and Plumb, 1993

Lim and Garzino-Demo, 2000 Zhou et al. , 1998

Lu et al. , 2011 Ohmori and Hamilton, 1993

Fig. 4. Schematic diagram of transcription factor binding sites locating in the promoter region of inflammatory genes investigated in this study. The scale does not reflect relative distance of each cis-element.

expression of a type I IFN, IFN-β. As shown in Fig. 6A LI stimulation induced the expression of IFN-β; however, PP and CP significantly reduced mRNA level of the type I IFN (Fig. 6A). Also the levels of phospho-STAT-1 and IRF-1 were highly increased in activated BV2 cells. However, the treatments of PP and CP significantly suppressed the increased phospho-STAT-1 and IRF-1 (Fig. 6B and C). The treatment of LUT (20 μM) significantly inhibited LI induced activation of NF-κB and STAT-1 as previously reported (Kao et al., 2011), whereas IFN-β expression levels were not affected. Taken together, these data suggested that inhibitory effects of PP and CP on inflammatory responses might be mediated by suppression of NF-κB activation and type I IFN signaling mediators such as STAT-1 and IRF-1 in LI-stimulated BV2 microglia. Pheophytin a and chlorophyll a suppressed activation of MAPKs In an effort to understand possible signaling pathway, we investigated the effects of PP and CP on MAPKs such as JNK, ERK, and p38. The levels of phosphorylated MAPKs were dramatically increased at 10 min following LI treatment (data not shown). The levels of phosphorylated JNK, ERK, and p38, which were hardly detectable in resting BV2 cells, were dramatically increased by LI treatment. The incubation with PP (2.5 μg/mL) or CP (5 μg/mL) greatly lowered the phosphorylation of JNK in BV2 cells (Fig. 7A). CP significantly decreased the level of

phosphorylated ERK, but not PP (Fig. 7B). However, the level of phosphorylated p38 was not changed by PP and CP treatments (Fig. 7C). In contrast, LUT did not show any inhibitory effects on MAPKs at this time point. These data suggested that PP and CP might inhibit neuronal inflammatory responses via suppressing the activation of MAPKs such as JNK and ERK.

Discussion CP is a photosynthetic pigment present in almost green vegetables. Structurally, it consists of a central magnesium ion encased in a fourion nitrogen ring as well as side chains and a hydrocarbon tail. CP can be converted to PP by heat or under mildly acidic conditions, which removes the central magnesium ion (Subramoniam et al., 2012; Islam et al., 2013). As a result, CP and PP are mainly consumed in the human diet as raw and cooked green vegetables, respectively. Importantly, we found that PP and CP have comparable anti-inflammatory effects. Thus, although the cooking process alters the chemical composition of CP, it may be expected that raw and cooked vegetables provide similar levels of anti-inflammatory effects. This study suggested that both phytochemicals are effective on microglia-induced neuroinflammatory responses.

S. Park et al. / Life Sciences 103 (2014) 59–67

A

PP (µ g/mL)

LPS+IFN-γ

0

0

–

+

0. 5 2. 5 +

+

65

CP 1

5

LUT

+

+

+

NF- κB p65 YY-1

B

LPS + IFN-γ Normal

Control

PP

CP

LUT

NF-κB p65

Hoechst

Merge

Fig. 5. Effects of PP and CP on LI-induced activation of NF-κB in BV2 cells. BV2 cells were pretreated with PP (2.5 μg/mL) and CP (5 μg/mL) for 1 h followed by stimulation with LI for 1 h. (A) The level of NF-κB p65 in the nuclear extract was determined by Western blot analysis. YY-1 was used as a loading control of nuclear extracts. (B) Translocation of p65 into nucleus was visualized by immunocytochemistry. Images were acquired using confocal laser scanning microscopy (×400). PP, pheophytin a; CP, chlorophyll a; LUT, luteolin.

Findings of human epidemiological studies and in vitro and in vivo experimental data have suggested that vegetables, fruits, grains, seeds, as well as medicinal herbs and their active constituents protect against neurodegenerative diseases through regulation of inflammation (Gonzalez-Gallego et al., 2010; Essa et al., 2012). For example, high consumption of fruits and vegetables, in particular, green and yellow vegetables, is associated with a statistically significant reduction in the risk of ALS, a progressive neurodegenerative disease with impaired spinal cord motor neurons resulting in atrophy of skeletal muscles and paralysis (Okamoto et al., 2009). Further, tea consumption is inversely correlated with the incidence of dementia, AD, and PD (Mandel et al., 2008). Epigallocatechin-3-gallate (EGCG), a component of tea, has been suggested as an active compound that can prevent AD (Mandel et al., 2008), and amyloid beta (Aβ) peptide-induced inflammation has been implicated in the pathogenesis of this disease (Kim et al., 2009). EGCG pretreatment effectively decreased Aβ-induced iNOS and NO production and cytotoxicity in activated microglia (Kim et al., 2009). Resveratrol, a polyphenolic compound rich in purple grape, was reported not only to decrease neurodegeneration in the hippocampus and prevent learning impairment in AD animal models but also to suppress Aβ-induced neuroinflammation and attenuate NF-κBmediated expression of iNOS and cyclooxygenase-2 (COX-2) (Bi et al., 2005). Furthermore, the symptoms of Parkinson’s disease (PD), a chronic and progressive degenerative disease associated with impaired motor control and cognitive dysfunction, are thought to be caused by loss of dopaminergic neurons in the substantia nigra (Gonzalez-Hernandez et al., 1996). Oxidative events are considered the causes underlying the damage to the dopaminergic neurons, and NO released by inflammation-induced microglia may also play a role in pathogenesis of PD (Gonzalez-Hernandez et al., 1996). Curcumin, a component of the medicinal plant Curcuma longa Linn, is known to exert neuroprotective effects and to ameliorate PD symptoms; this protective effect is

largely due to its ability to block induction of microglial cells and antioxidant properties (Lee et al., 2007), which are mediated by targeting multiple components in cells, including transcription factors and signaling pathways (Goel et al., 2008) In this study, we explored the potential preventive effects of PP and CP on neurodegenerative disorders, which are mediated by activation of inflammatory responses in microglia. One of the main functions of microglia in the pathologic condition is that the cells act as sources of free radicals, proinflammatory cytokines, and chemokines, which initiate neuroinflammation leading to neuronal toxicity and degeneration (Gonzalez-Scarano and Baltuch, 1999). In the present investigation PP and CP showed strong inhibition of microglia-mediated inflammation. PP and CP inhibited NO production by suppressing iNOS mRNA and protein expression. Also, our data clearly showed that the treatment of PP and CP significantly reduced the productions of MIP-1α, MCP-1, and IP-10 as well as proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in activated microglia. Chemokines present in the brain increase infiltration of inflammatory immune cells. MIP-1α and MCP1 are well known to recruit monocytes, T cells, and dendritic cells, whereas IP-10 recruits monocytes/macrophages, T cells, NK cells, and dendritic cells, (Man et al., 2007). Such recruitment may lead to neurodegeneration such as ischemia and ALS (Biber et al., 2008) and contribute to neuropathic pain caused by nerve injury (Kiguchi et al., 2012). Taken together, our data demonstrate that phytochemicals have the potential to treat inflammation-mediated neuronal disease and suggest that increased intake of green vegetables would be beneficial. Upon LPS stimulus, TLR4/MyD88 signaling pathway is predominantly used to induce the expression of iNOS, proinflammatory cytokines and chemokines. Activation of MyD88 triggers activations of both NF-κB and MAPKs signaling molecules. TLR also activate MyD88 independent pathway leading to phosphorylation and nuclear translocation of IRF-3 and induction of IFN-β (Doyle et al., 2002), which in turn initiate the

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A

PP

CP

A

(µg/mL)

0

0

0.5 2.5

1

5

LUT

LPS+IFN-γ

–

+

+

+

+

+

+

PP (µg/mL)

LPS+IFN-γ

IFN-β

0

0

–

+

CP

0. 5 2. 5 +

+

1

5

LUT

+

+

+ 54 kDa 46 kDa

p-JNK

β-actin

54 kDa JNK

PP

B

CP

β-actin

(µg/mL)

0

0

0.5 2.5

1

5

LUT

LPS+IFN-γ

–

+

+

+

+

+

+

46 kDa

B

p-STAT-1 p STAT 1

42 kDa PP

(µg/mL)

LPS+IFN-γ

0

0

–

+

0. 5 2. 5 +

+

CP 1

5

LUT

+

+

+

STAT-1

p-ERK

44 kDa 42 kDa

β-actin

ERK

44 kDa 42 kDa

β-actin

42 kDa

PP

C

CP

(µg/mL)

0

0

0.5 2.5

1

5

LUT

LPS+IFN-γ

–

+

+

+

+

+

+

IRF-1 β-actin Fig. 6. Effects of PP and CP on signal molecules in IFN signaling pathways in BV2 cells. BV2 cells were pretreated with PP and CP for 1 h. (A) The cells were stimulated by LI for 6 h. Total RNAs were isolated and used for RT-PCR using IFN-β specific primers. β-Actin was used as a loading control. The cells were stimulated with LI for 1 h to determine the levels of (B) phospho-STAT-1, STAT-1, (C) and IRF-1 from total cell lysates. The data presented are representative of three independent experiments. PP, pheophytin a; CP, chlorophyll a.

STAT-1 activation. Our study found that PP and CP suppressed the LIinduced activation of both NF-κB and IFN-β. These results suggest that the herbal pigments might regulate activity of TLR pathways at the upstream of MyD88. Also, MAPKs might be the target signaling pathway, which plays a role in LPS-induced neuroinflammatory and neurodegenerative disease mediated by microglia. It has been known that LPS increases the total amount of nuclear JNK and induces TNF-α, IL-6, and MCP-1 expressions in microglia through activation of JNK (Waetzig et al., 2005). ERK and p38 are also suggested that the signaling pathways activated by neuropathic substances including LPS and amyloid beta, leading to NO and TNF-α productions in microglia (Pyo et al., 1998; Bae et al., 2006). In accordance with these reports, inhibition of JNK, ERK, and p38 decreased the production of iNOS, TNF-α, and IL-1β in LPS stimulated microglia (Han et al., 2002; Kim et al., 2004; Uesugi et al., 2006). The expression of neuropathological chemokine, IP-10, is also under regulation of MAPK in cooperation with NF-κB in BV2 microglia (Shen et al., 2006). Our present study indicated that PP and CP suppressed the phosphorylation of JNK and ERK in BV2 microglia. Those suppressive effects of PP and CP on specific MAPKs might lead to diminish the levels of neuropathological factors such as NO, neuroinflammatory cytokines, and chemokines in BV2 microglia. Also, IFN-γ is a potent inducer of inflammatory responses. IFN-γ is considered to enhance TLR signaling for the efficient induction of inflammatory mediators. Recently, it has been suggested that the synergy between IFN-γ and TLR is mediated by STAT-1 (Hu and Ivashkiv, 2009). In an effort to understand underlying molecular mechanisms for the

PP

C (µg/mL) LPS+IFN-γ

0

0

–

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0. 5 2. 5 +

+

CP 1

5

LUT

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+

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p-p38

43 kDa

p38

43 kDa

β-actin

42 kDa

Fig. 7. Effects of PP and CP on phosphorylation of MAPKs in BV2 cells. BV2 cells were pretreated with PP and CP for 1 h followed by incubation with LI for 10 min. The total cell lysates were electrophoresed and analyzed by Western blot using (A) phospho-JNK, JNK, (B) phospho-ERK, ERK, (C) phospho-p38, and p38 specific antibodies. The data presented are representative of three independent experiments. PP, pheophytin a; CP, chlorophyll a.

suppressive effects of PP and CP on inflammatory responses, we investigated the effects of these small natural molecules on transcription factors inducing inflammatory responses in microglia. Our data indicated that both NF-κB and STAT-1 pathways are seemed to be the targets of PP and CP in the down-regulation of the inflammatory mediators in microglia. This study suggests that chlorophyll could act as a neuroprotective agent by inhibiting microglia-mediated inflammation. However, further investigations are set forth to elucidate the efficacy of chlorophyll as an active component of green vegetables that protect from inflammationassociated neurodegenerative disorders. Conflict of Interest statement The authors declare that there are no conflicts of interest.

Acknowledgment This work was supported by “Food Functionality Evaluation program” under the Ministry of Agriculture, Food and Rural Affairs, and by The Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (grant numbers HI12C1954 and HI13C0493).

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