1546 Vol. 35, No. 9

© 2012 The Pharmaceutical Society of Japan

Biol. Pharm. Bull. 35(9) 1546–1552 (2012)

Ginsenoside Rg3 Attenuates Microglia Activation Following Systemic Lipopolysaccharide Treatment in MiceSun-Min Park, Moon-Suk Choi, Nak-Won Sohn, and Jung-Won Shin*Department of Oriental Medical Science, Graduate School of East-West Medical Science, Kyung Hee University; Yongin 446–701, South Korea. Received April 29, 2012; accepted June 28, 2012

Neuroinflammation, characterized by activation of microglia and expression of major inflammatory me-diators, contributes to neuronal damage in addition to acute and chronic central nervous system (CNS) dis-ease progression. The present study investigated the immune modulatory effects of ginsenoside Rg3, a prin-ciple active ingredient in Panax ginseng, on pro-inflammatory cytokines and microglia activation in brain tissue induced by systemic lipopolysaccharide (LPS) treatment in C57BL/6 mice. Systemic LPS treatment induces immediate microglia activation in the brain. Based on this information, ginsenoside Rg3 was treated orally with 10, 20, and 30 mg/kg 1 h prior to the LPS (3 mg/kg, intraperitoneally (i.p.)) injection. Ginsenoside Rg3 at 20 and 30 mg/kg oral doses significantly attenuated up-regulation of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and IL-6 mRNA in brain tissue at 4 h after LPS injection. Morphological activation of microglia and Iba1 protein expression by systemic LPS injection were reduced with ginsenoside Rg3 (30 mg/kg) treatment. In addition, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in brain tissue were also attenuated with oral treatment of ginsenoside Rg3 at 30 mg/kg. These results indicate that ginsenoside Rg3 plays a modulatory role in neuroinflammation. This study shows that ginsenoside Rg3 attenuates microglia activation using an in vivo animal model.

Key words ginsenoside Rg3; neuroinflammation; microglia; cytokine; lipopolysaccharide

Neuroinflammation which involves inflammation of the central nervous system (CNS) is now recognized to be a fea-ture of all neurological disorders. In a wide variety of acute and chronic CNS disorders, inflammatory processes contrib-ute to the damage of neurons and progression of disease.1) Neuroinflammation is considered a mediator for secondary damage and is characterized by activation of microglia, resident macrophages in the brain, and expression of major inflammatory mediators.2)

Cerebral ischemia leads to the activation of microglia and astrocytes and subsequent production of inflammatory media-tors.3) Once activated, inflammatory cells release a variety of cytotoxic agents including cytokines, matrix metalloprotein-ases (MMPs), nitric oxide (NO) and reactive oxygen species (ROS). These substances may induce more cell damage as well as cause disruption of the blood–brain barrier (BBB) and extracellular matrix.3,4) Furthermore, neurodegenerative conditions are characterized by chronic neuroinflammatory processes. The progressive deposition of amyloid beta-peptide (Aβ) in Alzheimer’s disease might provide a chronic stimulus to microglial cells.5) Parkinson’s disease is also characterized by chronic inflammation induced dopaminergic neuron degen-eration within the substantia nigra.6) Hence, determining the modulatory effects of various drugs on neuroinflammation is critical for developing therapeutic approaches for CNS disor-ders.

Ginseng (the root of Panax ginseng C.A. MEYER, Aralia-ceae) has been used for the treatment of neurological disorders and other diseases in traditional medicine. Ginsenosides, or ginseng saponins, are the principle active constituents in gin-seng.7) Ginsenoside Rg3 demonstrated neuroprotective effects against cerebral ischemia in rats through inhibition of mito-chondrial permeability transition pore and intracellular Ca2+ elevation by L-type Ca2+ channels or N-methyl-D-aspartate

(NMDA) receptors.8–11) Recently, ginsenoside Rg3 has also been known to play a role in the modulation of inflammatory processes.12) In BV-2 microglial cell culture studies, ginsen-oside Rg3 reduced cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), and pro-inflammatory cytokine ex-pressions, including tumor necrosis factor (TNF)-α and inter-leukin (IL)-1β, induced by lipopolysaccharide (LPS) or Aβ42 stimulation.13,14) These reports indicated that ginsenoside Rg3 might play a modulatory role on microglia in the brain.

However, little information is available about the effects of orally administered ginsenoside Rg3 on neuroinflammation using an in vivo animal study. Therefore, to better under-standing the neuroprotective and anti-inflammatory effects of ginsenoside Rg3, the present study investigated its effects on the early stage of neuroinflammation induced by systemic LPS treatment in mice.

MATERIALS AND METHODS

Animals Male C57BL/6 mice (25–28 g, Nara Biotechnol-ogy, Korea) were used for this study. All animal protocols were approved by the Ethics Committee for the Care and Use of Laboratory Animals at Kyung Hee University. The animals were housed in plastic cages at a constant temperature (22± 2°C) and humidity (55± 10%) with 12 h–12 h light–dark condi-tions. The animals were allowed free access to food and water before the experiment.

Reagents 20(S)-Ginsenoside Rg3 (C42H72O13; formula weight, 785.01) was purchased from LKT Laboratories (Saint Paul, MN, U.S.A.). Lipopolysaccharide (LPS; from Esch-erichia coli O55:B5) and 3,3-diaminobenzidine (DAB) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Rab-bit anti-ionized calcium binding adaptor molecule 1 (Iba1) antibodies (#016-20001, #019-19741) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Mouse anti-actin antibody was purchased from Chemicon International

Regular Article

* To whom correspondence should be addressed. e-mail: shinarago@khu.ac.kr

The authors declare no conflict of interest.

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(Temecula, CA, U.S.A.). Mouse anti-iNOS antibody was pur-chased from BD Biosciences (Laguna Hills, CA, U.S.A.). Rabbit anti-COX-2 antibody was purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). Goat anti-rabbit and goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP) conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Cy2-conjugated donkey anti-mouse or donkey anti-rabbit IgG, and Cy3-conjugated donkey anti-mouse IgG were purchased from Jackson ImmunoReseach Laboratories (West Grove, PA, U.S.A.). The other chemicals and reagents used were of high quality and obtained from various commercial sources.

Experimental Groups Mice were randomly divided into five groups. The normal group (Normal) was allowed free access to food and water without any treatment. The control group (LPS) was intraperitoneally (i.p.) injected with a single dose of LPS (3 mg/kg) and received vehicle (normal saline) orally 1 h before the LPS injection. The ginsenoside Rg3 treat-ment groups [LPS+ Rg3(10), LPS+ Rg3(20), LPS+ Rg3(30)] were administered 3 oral doses of ginsenoside Rg3 (10, 20, or 30 mg/kg, dissolved in normal saline) once 1 h prior to LPS injection. A total of 48 mice were used in this study.

Real-Time Polymerase Chain Reaction (PCR) Measure-ment Pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) mRNA expression in the brain tissue was measured by the quantitative real-time PCR method. At 4 h after the LPS injec-tion, mice were sacrificed by decapitation and brain tissue was rapidly dissected. Total RNA was extracted from the samples with Trizol (Qiagen, Germany) according to the manufac-turer’s protocol. One microgram of total RNA was transcribed into DNA by using iScript cDNA synthesis Kit (Bio-Rad, U.S.A.). After reverse transcription, quantitative real-time PCR was performed using preoptimized primer/probe mixture with iQ SYBR Green Supermix kit (Bio-Rad, U.S.A.) and the CFX 96 REAL-TIME PCR Detection System (Bio-Rad, U.S.A.). Primer sequences for the analyzed genes are listed in Table 1. The relative difference in expression between samples was represented using cycle time values normalized by mea-surement of the housekeeping gene β-actin as a reference. The sample values represent x-fold differences from a normal sample (given as a designated value of 1) within the same experiment.

Western Blotting The brain tissue was homogenized and sonicated on ice in lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediamine-tetraacetic acid (EDTA), 1% protease inhibitor cocktail; Sig-ma). After centrifugation, the supernatant was collected and assayed for protein concentration by using the Bradford meth-od. The lysates containing 50 µg proteins were fractionated

by SDS–10% polyacrylamide gel electrophoresis, and then subjected to Western blot analysis. The primary antibodies used in this study were rabbit anti-Iba1 antibody (#016-20001, Wako, Japan) and mouse anti-β-actin antibody (Chemicon, U.S.A.).

Immunohistochemistry Microglia activation in the brain tissue was observed with immunohistochemistry. At 4 h after the LPS injection, the mice were deeply anesthetized and perfused transcardially with 0.05 M phosphate-buffered saline (PBS) containing 4% paraformaldehyde. The brain was re-moved and was postfixed in the same perfusing solution over-night at 4°C. Coronal sections of 30 µm thickness were made using a freezing microtome (Leica, 2800N, Germany). The brain sections were stained by a free-floating DAB reaction. The sections were rinsed with 0.05 M PBS and then incubated for 15 min in 1% hydrogen peroxide PBS at room temperature. The sections were incubated overnight at 4°C with primary antibodies against Iba1 (1 : 500, #019-19741, Wako, Japan), as a microglia marker. The sections were then incubated with bio-tinylated anti-mouse secondary antibody (1 : 200, Vector Labo-ratories, U.S.A.) for 1 h at room temperature, after which the avidin–biotin complex (Vector Laboratories, U.S.A.) method was carried out with peroxidase coupling in a mixture con-taining 0.05% DAB (Sigma-Aldrich, U.S.A.) and 0.03% H2O2 for 2–5 min. For observation of iNOS and COX-2 expression in the brain tissue, immunofluorescent labeling was performed using primary antibodies against iNOS (1 : 200, #610329, BD, U.S.A.) and COX-2 (1 : 200, #160106, Cayman, U.S.A.). The anti-rabbit or anti-mouse Cy2 (Jackson ImmunoResearch, U.S.A.) was used as a secondary antibody. Images of the DAB-colorized brain sections were captured using a light microscope (BX51, Olympus, Japan) equipped CCD camera (DP70, Olympus, Japan) and the fluorescence-labeled images were captured using confocal laser-scanning microscopy (Carl Zeiss, LSM 510 META, Germany).

Count of Iba1 Immuno-positive Cells Immunohisto-chemistry stained sections were used for analysis of the num-ber of the immuno-positive cells. The count of Iba1 immuno-positive microglia was analyzed by using ImageJ software (Ver. 1.44p, NIH, U.S.A.). Four sections and four fields per section were chosen for analysis in each rat. Data were nor-malized with the same area (105 µm2) and the mean values for the four sections in each rat were used for statistical analysis.

Area Percentage Measurement of iNOS and COX-2 Ex-pression The images captured using confocal laser-scanning microscopy and ImageJ software were used for analysis of the area % of iNOS and COX-2 expression. In brief, image con-trast was adjusted adequately (step-1), then the image inverted to black–white binary image (step-2). Threshold gray value was determined and pixels which gray value was greater than

Table 1. Primer Sequences

TNF-α Forward 5′-TGA GAA GTT CCC AAA TGG C-3′Reverse 5′-GCT ACA GGC TTG TCA CTC-3′

IL-1β Forward 5′-TGA GCA CCT TCT TTT CCT TCA-3′Reverse 5′-TTG TCT AAT GGG AAC GTC ACA C-3′

IL-6 Forward 5′-AGA CTT CAC AGA GGA TAC CA-3′Reverse 5′-GCA TCA TCG TTG TTC ATA CA-3′

β-Actin Forward 5′-TTT CCA GCC TTC CTT GGG TAT G-3′Reverse 5′-CAC TGT GTT GGC ATA GAG GTC TTT AC-3′

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the threshold were defined, refer to original image (step-3). All pixels in the same area (52714 µm2) were measured and calcu-lated to area percentage (step-4). Four sections and four fields per section were chosen for analysis in each rat. The mean values for the four sections in each rat were used for statistical analysis.

Statistical Analysis All data in this study are presented as means± standard errors. Differences between groups were evaluated using Student’s t-test and one way analysis of vari-ance (ANOVA). A probability value of less than 0.05 was used to indicate a significant difference.

RESULTS

Ginsenoside Rg3 Attenuates Pro-inflammatory Cy-tokines mRNA Expression in the Brain Tissue of LPS-Treated Mice To determine the effect of ginsenoside Rg3 on TNF-α, IL-1β, and IL-6 mRNA expression in the brain tissue following systemic LPS treatment, LPS (3 mg/kg) was treated with i.p. injection and 3 doses of ginsenoside Rg3 (10, 20, or 30 mg/kg) were treated orally 1 h before the LPS injection into male C57BL/6 mice. Quantitative real-time PCR measurement of brain TNF-α, IL-1β, and IL-6 mRNA was performed at 4 h after the LPS injection. LPS increased the brain TNF-α mRNA ∼80-fold (80.3± 6.2 fold), while ginsenoside Rg3 treatment attenuated TNF-α mRNA of the brain tissue sig-nificantly at both 20 and 30 mg/kg (59.8± 6.4 fold, p<0.05;

51.1± 6.6 fold, p<0.01) compared to the LPS group. Inhibitory effect of ginsenoside Rg3 on brain TNF-α mRNA expression was showed a dose dependent pattern (Fig. 2A). LPS also increased brain IL-1β mRNA ∼110-fold (109.4± 14.1 fold), while ginsenoside Rg3 treatment attenuated IL-1β mRNA in brain tissue significantly at doses of 20 and 30 mg/kg (71.1± 8.1 fold, 73.2± 6.2 fold, p<0.05, respectively), as compared to the LPS group (Fig. 2B). Similarly, brain IL-6 mRNA was increased ca. 210-fold (207.9± 31.1 fold) by the LPS injection and ginsenoside Rg3 treatment attenuated IL-1β mRNA in brain tissue significantly at both 20 and 30 mg/kg (127.6± 12.5 fold, 133.4± 11.2 fold, p<0.05, respectively) compared to the LPS group (Fig. 2C). These results support that ginsenoside Rg3 attenuated the over-expression of cytokines in the brain induced by systemic LPS injection.

Fig. 1. Chemical Structure of 20(S)-Ginsenoside Rg3

Fig. 2. Effects of Ginsenoside Rg3 on TNF-α, IL-1β, and IL-6 mRNA in Brain TissueLPS increases brain inflammatory cytokines mRNA, while ginsenoside Rg3 treatment attenuates brain TNF-α (A), IL-1β (Β), and IL-6 (C) mRNA at 20 and 30 mg/

kg administration. Data are represented by mean±S.E.M. (n=6 in each group). Statistical significances are based on comparison to the LPS group (* p<0.05; ** p<0.01).

Fig. 3. Effect of Ginsenoside Rg3 on Iba1 Expression in the BrainRepresentative Western blots illustrate differences in the bands of Iba1 (A).

LPS up-regulates Iba1 expression in brain tissue, while ginsenoside Rg3 treatment attenuates up-regulation of Iba1 expression at doses of 20 and 30 mg/kg adminis-tration (B). Data are represented by mean±S.E.M. (n=6 in each group). Statistical significances are based on comparison to the LPS group (* p<0.05; *** p<0.001).

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Ginsenoside Rg3 Attenuates Microglia Activation in the Brain Tissue of LPS-Treated Mice Studies indicated that systemic LPS treatment induced activation of microglia in the brain immediately.15) Iba1 protein is specifically expressed in microglia and is upregulated during the activation of these cells.16) The effects of ginsenoside Rg3 on microglia activation following systemic LPS injection were tested by quantifica-tion of Iba1 protein with Western blotting and immunohis-tochemistry against Iba1 antibody in the brain tissue at 4 h after LPS injection (Fig. 3A). LPS increased the % increase of Iba1 expression in brain tissue (188.7± 5.5%). Ginsenoside Rg3 treatment attenuated, significantly and dose dependently, the % increase of Iba1 expression in the brain tissue at doses of 20 and 30 mg/kg (159.7± 7.9%, p<0.05; 142.9± 7.2%, p<0.001; respectively) as compared to the LPS group (Fig. 3-B).

Immunohistochemistry against Iba1 antibody was processed with 30 mg/kg dose of ginsenoside Rg3 administration because 30 mg/kg ginsenoside Rg3 was most effective on inhibition of cytokines and Iba1 expression. The LPS injection activated microglia, as displayed by an increase in cell size, irregular shape, thickened and shortened processes and intensified Iba1 immuno-staining density in the cerebral cortex, dentate gyrus (DG) of hippocampus and hypothalamic region of the brain (Fig. 4A). However, ginsenoside Rg3 treatment lessened the morphological changes to activated-form of microglia in all brain regions compared with the LPS group (Fig. 4A). To de-termine the effect of ginsenoside Rg3 on microglia activation, the number of Iba1 immuno-stained microlgia was counted and normalized with the same area (105 µm2). Moreover, gin-senoside Rg3 treatment reduced numbers of Iba1-expressed microglia in the corresponding area significantly, compared to that of LPS group (Fig. 4B). In the cerebral cortex, LPS group was 59.0± 1.8 cells/105 µm2 and LPS+ Rg3 group was 53.7±

1.4 cells/105 µm2 (p<0.05). In the hypothalalmic region, LPS group was 47.7± 2.0 cells/105 µm2 and LPS+ Rg3(30) group was 41.3± 1.8 cells/105 µm2 (p<0.05). The number Iba1-expressed microglia in the DG was not counted due to the heterogeneous tissue properties. These results indicate that ginsenoside Rg3 attenuated microglia activation in the brain induced by sys-temic LPS injection.

Ginsenoside Rg3 Attenuates iNOS and COX-2 Expres-sion in the Brain Tissue of LPS-Treated Mice In order to better understand the effect of ginsenoside Rg3 on inflam-matory responses in the brain, immunofluorescent labeling on iNOS and COX-2 expression in the brain tissue was per-formed with 30 mg/kg dose of ginsenoside Rg3 administration. Effect of ginsenoside Rg3 was evaluated by the area % of iNOS or COX-2 expression in the brain tissue. Ginsenoside Rg3 treatment significantly reduced the area % of iNOS ex-pression (1.72± 0.11% vs. 1.31± 0.14%, p<0.05) with respect to the LPS group (Figs. 5A, B). The area % of COX-2 expression in the cerebral cortex was also reduced significantly (4.94± 0.59% vs. 2.66± 0.60%, p<0.05) with respect to the LPS group (Figs. 5C, D). This result also indicates that ginsenoside Rg3 attenuated the inflammatoy response in the brain induced by systemic LPS injection.

DISCUSSION

Ginsenoside Rg3 is an effective neuroprotective ingredi-ent in Panax ginseng.8–11) Recently, it has been reported that ginsenoside Rg3 plays an important role in modulating the inflammatory processes with in vitro studies.13,14) Modula-tory effect of ginsenoside Rg3 on microglia activation could be presupposed depending on its neuroprotective and anti-inflammatory effects reported previously through in vivo and

Fig. 4. Effect of Ginsenoside Rg3 on Microglia Activation in the BrainRepresentative photographs show Iba1 immuno-stained microglia of the mouse brain (A). LPS group shows mostly an activated-form of microglia which display an in-

creased cell size, irregular shape, thickened and shortened processes and intensified Iba1 immuno-staining density, in the cerebral cortex (Cortex), dentate gyrus of hippo-campus (DG) and hypothalamic region (Hypothalamus) compared to the normal group. Moreover, ginsenoside Rg3 treated group (LPS+Rg3, 30 mg/kg) shows a decrease of morphological activation of microglia in all brain regions with respect to LPS group. Scale bar is 100 µm, applicable to all photographs. The number of microglia was counted and normalized in the corresponding same area (B). LPS increases the number of Iba1-expressed microglia in the brain. Ginsenoside Rg3 treatment significantly reduces the number of Iba1-expressed microglia both in the cerebral cortex and hypothalamus. Data are represented by mean±S.E.M. (n=6 in each group). Statistical sig-nificances are based on comparison to the LPS group (* p<0.05).

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in vitro studies. Ginsenosides (protopanaxadiols; Rb1, Rb2, Rd, Rh2, etc.) which has a similar chemical structure to Rg3 may also affect microglia activation and neuroinflammation. Ginsenoside Rb1 depressed the activation of microglia in the penumbra around a cerebral ischemic insult in rats.17) Ginsen-oside Rh2 showed anti-inflammatory effect against LPS and interferon-gamma-stimulated BV-2 microglial cells.13) The present study further extends these observations by showing that ginsenoside Rg3 exhibits suppressive effects against neu-roinflammation induced by systemic LPS treatment in vivo. As a result, ginsenoside Rg3 significantly attenuated microglia activation, pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), iNOS and COX-2 expressions in the brain tissue in-duced by systemic LPS injection.

Systemic LPS injection (intraperitoneal) activates the inflammatory response in the brain through the toll-like receptors (TLRs) located on glia.18) This in turn promotes inflammation through activation of microgia and overproduc-tion of inflammatory cytokines.19) Moreover, concerning with neurodegenerative disease’s study models, it was reported that systemic LPS treatment increased expression of amyloid precursor protein (APP) and the Aβ generation and also in-duced delayed and progressive loss of dopaminergic neurons in the substantia nigra.19,20) In an acute systemic LPS treat-ment study, inflammatory responses in the brain, including morphological activation of microglia, neutrophil infiltration, and mRNA/protein expression of inflammatory mediators, ap-peared within 4–8 h and subsided within 1–3 d.15) In the pres-ent study, there were robust increases of TNF-α, IL-1β, IL-6 mRNA, microglia activation, iNOS and COX-2 expression in the brain tissue at 4 h after the single LPS (3 mg/kg) i.p. injec-tion. These parameters show the features of acute inflamma-tion in the brain by LPS treatment.

There is abundant evidence suggesting that pro-inflam-matory cytokine production and signaling results in neuronal cell death and is closely related to neurodegeneration.21) In previous studies, ginsenoside Rg3 effectively suppressed pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in LPS-stimulated BV-2 microglial cells,13) in Aβ-treated BV-2 microglial cells,14) and in oxazolone applied anaphylactic mice.22) These suppressive effects against pro-inflammatory cytokines suggested that ginsenoside Rg3 might be associated with intracellular NF-κB which is a major transcription factor that regulates genes responsible for both the innate and adap-tive immune response.23) This study shows that ginsenoside Rg3 significantly reduced TNF-α, IL-1β, and IL-6 mRNA in the brain tissue following systemic LPS treatment. This result may expand our understandings of the immune modulatory effects of ginsenoside Rg3 on neuroinflammation from in vitro to in vivo.

Microglia, the resident immune cells in the CNS, are key cellular elements of the acute neuroinflammatory response and is the primary source for pro-inflammatory cytokines detected in the brain. Microglia are activated in response to brain injuries and immunological stimuli to undergo dramatic alterations in morphology, changing from resting, ramified mi-croglia into activated, amoeboid microglia, which is believed to favor phagocytosis and mobility.24) Microglia are readily activated by an extensive list of pro-inflammatory stimuli, such as LPS and Aβ.25,26) Despite major roles of microglia on inflammatory responses in the brain, there were no reports to demonstrate effects of ginsenoside Rg3 on microglia activation in vivo. This effect of ginsenoside Rg3 was investigated only with in vitro BV-2 microglial cell cultures.13,14) In the present study, ginsenoside Rg3 lessened the microglia morphological changes to an activated-form which can manifest as increased

Fig. 5. Effects of Ginsenoside Rg3 on iNOS and COX-2 Expression in the BrainGinsenoside Rg3 treated group (LPS+Rg3, 30 mg/kg) shows a decrease in fluorescence density against iNOS (A) and COX-2 (C) compared to the LPS group. Scale

bars are 100 µm. Ginsenoside Rg3 treatment reduces the area % of iNOS (B) and COX-2 (D) expression significantly. Data are represented by mean±S.E.M. (n=6 in each group). Statistical significances are based on comparison to the LPS group (* p<0.05).

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cell size, irregular shape, thickened and shortened processes in all brain regions. Moreover, ginsenoside Rg3 significantly reduced Iba1 protein expression in the brain compared to that of LPS group. Iba1, which is highly and specifically ex-pressed in microglia and macrophage,16) and is involved in RacGTPase-dependent membrane ruffling and phagocytosis.27) By immunohistochemical analyses, anti-Iba1 antibody was found to specifically recognize ramified microglia in normal rat brain, and Iba1 protein was strongly upregulated in acti-vated microglia.28) Therefore, Iba1 may play significant roles in the regulation of some immunological and pathophysiologi-cal functions of microglia, and serve as a novel marker for detecting the activation of microglia. However, this study is the first to show that ginsenoside Rg3 plays a modulatory role in microglia activation using an in vivo animal study.

Activated microglia are responsible for the induction of iNOS and COX-2 in the brain by pro-inflammatory cytokines or LPS stimulation.29) Nitric oxide (NO) and prostaglandins are major pleiotrophic mediators produced by iNOS, COX-1 and COX-2.29,30) Microglia can generate superoxide, by using the constitutively expressed NADPH oxidase, which reacts with NO to form the powerful oxidant peroxynitrite.31) Cy-clooxygenase plays a central role in the inflammatory cascade by converting arachidonic acid into bioactive prostanoids.32) Therefore, induction of iNOS and COX-2 leads microglia-mediated neurotoxicity. On the other hand, genetic deletion of microglial iNOS, pharmacological suppression of COX-2 activity, or the addition of exogenous superoxide dismutase (SOD) reduces microglia-mediated neurotoxicity.33) In previ-ous reports, ginsenoside Rg3 attenuated iNOS and COX-2 ex-pressions in the liver and kidney of the LPS-treated rats12) and in LPS/interferon-gamma-stimulated BV-2 microglial cells.13) Based on our study findings, ginsenoside Rg3 significantly at-tenuated both iNOS and COX-2 over-expressions in the brain induced by systemic LPS treatment. These results suggest that ginsenoside Rg3 may play a role in the microglia-mediated neurotoxicity as well as in the modulation of inflammation.

The findings from this study indicate that ginsenoside Rg3 can effectively attenuate pro-inflammatory cytokines and mi-croglia activation in the brain induced by systemic LPS treat-ment and may potentially protect against microglia-mediated neurodegenerative diseases. Further in-depth and long-term in vivo research is required to confirm these findings.

Acknowledgement This work was supported partly by a Grant (KHU-20110208) from the Kyung Hee University in 2011.

REFERENCES

1) Wee Yong V. Inflammation in neurological disorders: a help or a hindrance? Neuroscientist, 16, 408–420 (2010).

2) Harry GJ, Kraft AD. Neuroinflammation and microglia: consider-ations and approaches for neurotoxicity assessment. Expert Opin. Drug Metab. Toxicol., 4, 1265–1277 (2008).

3) Zheng Z, Yenari MA. Post-ischemic inflammation: molecular mech-anisms and therapeutic implications. Neurol. Res., 26, 884–892 (2004).

4) Danton GH, Dietrich WD. Inflammatory mechanisms after ischemia and stroke. J. Neuropathol. Exp. Neurol., 62, 127–136 (2003).

5) McGeer EG, McGeer PL. Neuroinflammation in Alzheimer’s disease and mild cognitive impairment: a field in its infancy. J.

Alz heimer’s Dis., 19, 355–361 (2010). 6) Dauer W, Przedborski S. Parkinson’s disease: mechanisms and

models. Neuron, 39, 889–909 (2003). 7) Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: multiple con-

stituents and multiple actions. Biochem. Pharmacol., 58, 1685–1693 (1999).

8) Tian J, Fu F, Geng M, Jiang Y, Yang J, Jiang W, Wang C, Liu K. Neuroprotective effect of 20(S)-ginsenoside Rg3 on cerebral isch-emia in rats. Neurosci. Lett., 374, 92–97 (2005).

9) Tian J, Zhang S, Li G, Liu Z, Xu B. 20(S)-ginsenoside Rg3, a neu-roprotective agent, inhibits mitochondrial permeability transition pores in rat brain. Phytother. Res., 23, 486–491 (2009).

10) Kim S, Nah SY, Rhim H. Neuroprotective effects of ginseng sapo-nins against L-type Ca2+ channel-mediated cell death in rat cortical neurons. Biochem. Biophys. Res. Commun., 365, 399–405 (2008).

11) Kim JH, Cho SY, Lee JH, Jeong SM, Yoon IS, Lee BH, Lee JH, Pyo MK, Lee SM, Chung JM, Kim S, Rhim H, Oh JW, Nah SY. Neuroprotective effects of ginsenoside Rg3 against homocysteine-induced excitotoxicity in rat hippocampus. Brain Res., 1136, 190–199 (2007).

12) Kang KS, Kim HY, Yamabe N, Park JH, Yokozawa T. Preventive effect of 20(S)-ginsenoside Rg3 against lipopolysaccharide-induced hepatic and renal injury in rats. Free Radic. Res., 41, 1181–1188 (2007).

13) Bae EA, Kim EJ, Park JS, Kim HS, Ryu JH, Kim DH. Ginseno-sides Rg3 and Rh2 inhibit the activation of AP-1 and protein kinase A pathway in lipopolysaccharide/interferon-gamma-stimulated BV-2 microglial cells. Planta Med., 72, 627–633 (2006).

14) Joo SS, Yoo YM, Ahn BW, Nam SY, Kim YB, Hwang KW, Lee I. Prevention of inflammation-mediated neurotoxicity by Rg3 and its role in microglial activation. Biol. Pharm. Bull., 31, 1392–1396 (2008).

15) Jeong HK, Jou I, Joe EH. Systemic LPS administration induces brain inflammation but not dopaminergic neuronal death in the sub-stantia nigra. Exp. Mol. Med., 42, 823–832 (2010).

16) Ito D, Imai Y, Ohsawa K, Nakajima K, Fukuuchi Y, Kohsaka S. Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res. Mol. Brain Res., 57, 1–9 (1998).

17) Zhu J, Jiang Y, Wu L, Lu T, Xu G, Liu X. Suppression of local in-flammation contributes to the neuroprotective effect of ginsenoside Rb1 in rats with cerebral ischemia. Neuroscience, 202, 342–351 (2012).

18) Konat GW, Kielian T, Marriott I. The role of Toll-like receptors in CNS response to microbial challenge. J. Neurochem., 99, 1–12 (2006).

19) Qin L, Wu X, Block ML, Liu Y, Breese GR, Hong JS, Knapp DJ, Crews FT. Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia, 55, 453–462 (2007).

20) Sheng JG, Bora SH, Xu G, Borchelt DR, Price DL, Koliatsos VE. Lipopolysaccharide-induced-neuroinflammation increases intracel-lular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice. Neurobiol. Dis., 14, 133–145 (2003).

21) Hirsch EC, Hunot S. Neuroinflammation in Parkinson’s disease: a target for neuroprotection? Lancet Neurol., 8, 382–397 (2009).

22) Bae EA, Han MJ, Shin YW, Kim DH. Inhibitory effects of Korean red ginseng and its genuine constituents ginsenosides Rg3, Rf, and Rh2 in mouse passive cutaneous anaphylaxis reaction and contact dermatitis models. Biol. Pharm. Bull., 29, 1862–1867 (2006).

23) Hayden MS, Ghosh SNF. NF-κB in immunobiology. Cell Res., 21, 223–244 (2011).

24) Streit WJ, Walter SA, Pennell NA. Reactive microgliosis. Prog. Neurobiol., 57, 563–581 (1999).

25) Consonni A, Morara S, Codazzi F, Grohovaz F, Zacchetti D. Inhibi-tion of lipopolysaccharide-induced microglia activation by calcito-nin gene related peptide and adrenomedullin. Mol. Cell. Neurosci.,

1552 Vol. 35, No. 9

48, 151–160 (2011).26) Zhang D, Hu X, Qian L, Chen SH, Zhou H, Wilson B, Miller DS,

Hong JS. Microglial MAC1 receptor and PI3K are essential in me-diating β-amyloid peptide-induced microglial activation and subse-quent neurotoxicity. J. Neuroinflammation, 8, 3–17 (2011).

27) Kanazawa H, Ohsawa K, Sasaki Y, Kohsaka S, Imai Y. Macro-phage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma-dependent pathway. J. Biol. Chem., 277, 20026–20032 (2002).

28) Ito D, Tanaka K, Suzuki S, Dembo T, Fukuuchi Y. Enhanced ex-pression of Iba1, ionized calcium-binding adapter molecule 1, after transient focal cerebral ischemia in rat brain. Stroke, 32, 1208–1215 (2001).

29) Sheng W, Zong Y, Mohammad A, Ajit D, Cui J, Han D, Hamil-ton JL, Simonyi A, Sun AY, Gu Z, Hong JS, Weisman GA, Sun GY. Pro-inflammatory cytokines and lipopolysaccharide induce changes in cell morphology, and upregulation of ERK1/2, iNOS and

sPLA2-IIA expression in astrocytes and microglia. J. Neuroinflam-mation, 8, 121–136 (2011).

30) Li P, Lu J, Kaur C, Sivakumar V, Tan KL, Ling EA. Expression of cyclooxygenase-1/-2, microsomal prostaglandin-E synthase-1 and E-prostanoid receptor 2 and regulation of inflammatory mediators by PGE(2) in the amoeboid microglia in hypoxic postnatal rats and murine BV-2 cells. Neuroscience, 164, 948–962 (2009).

31) Possel H, Noack H, Putzke J, Wolf G, Sies H. Selective upregula-tion of inducible nitric oxide synthase (iNOS) by lipopolysaccharide (LPS) and cytokines in microglia: in vitro and in vivo studies. Glia, 32, 51–59 (2000).

32) Aïd S, Bosetti F. Targeting cyclooxygenases-1 and -2 in neuroin-flammation: Therapeutic implications. Biochimie, 93, 46–51 (2011).

33) Shie FS, Montine KS, Breyer RM, Montine TJ. Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity. Glia, 52, 70–77 (2005).