# Involvement of Heme Oxygenase-1 Induction in the Cytoprotective and Immunomodulatory Activities ofViola patrinii in Murine Hippocampal and Microglia Cells

Authors: Bin Li; Dong-Sung Lee; Hyun-Gyu Choi; Kyoung-Su Kim; Gil-Saeng Jeong; Ren Bo An; Youn-Chul Kim
Journal: Evidence-Based Complementary and Alternative Medicine
 (2012)

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## Abstract

A number of diseases that lead to injury of the central nervous system are caused by oxidative stress and inflammation in the brain. In this study, NNMBS275, consisting of the ethanol extract ofViola patrinii, showed potent antioxidative and anti-inflammatory activity in murine hippocampal HT22 cells and BV2 microglia. NNMBS275 increased cellular resistance to oxidative injury caused by glutamate-induced neurotoxicity and reactive oxygen species generation in HT22 cells. In addition, the anti-inflammatory effects of NNMBS275 were demonstrated by the suppression of proinflammatory mediators, including proinflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2) and cytokines (tumor necrosis factor-α and interleukin-1β). Furthermore, we found that the neuroprotective and anti-inflammatory effects of NNMBS275 were linked to the upregulation of nuclear transcription factor-E2-related factor 2-dependent expression of heme oxygenase-1 in HT22 and BV2 cells. These results suggest that NNMBS275 possesses therapeutic potential against neurodegenerative diseases that are induced by oxidative stress and neuroinflammation.


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## Body

## 1. Introduction

Brain tissues are vulnerable to oxidative stress, which may occur physiologically as a component of the aging process, or pathologically as a result of neurodegenerative disease [1, 2]. Oxidative stress, or the accumulation of reactive oxygen species (ROS), not only results in accidental damage to cells, but also actively triggers intracellular signaling pathways that lead to cell death. Neuronal oxidative stress has been postulated to be the underlying basis for neuronal cell death in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [3–5]. Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS). However, at high concentrations, glutamate has been shown to induce neuronal cell death via both receptor-initiated excitotoxicity and non-receptor-mediated oxidative stress [6, 7]. In this study, immortalized mouse hippocampal HT22 cells were used as in vitro models for studying the mechanism of oxidative glutamate toxicity. Glutamate-induced neurotoxicity in HT22 cells has been characterized as an intracellular process attributable to the inhibition of cystine uptake via the cystine/glutamate antiporter system Xc−, eventually leading to programmed cell death due to glutathione depletion and accumulation of ROS [8, 9].Heme oxygenase 1 (HO-1), also referred to as HSP32, belongs to the HSP family and protects mammalian cells from oxidative stress by degrading toxic heme into free iron, carbon monoxide, and biliverdin/bilirubin. Although HO-1 does not directly catalyze an antioxidant reaction, its induction is generally considered an adaptive cytoprotective response against the toxicity of oxidative stress [10–12]. The induction of HO-1 is primarily regulated at the transcriptional level, and its induction by various inducers is related to the nuclear transcription factor-E2-related factor 2 (Nrf2) [13]. Nrf-2 is a basic leucine zipper transcription factor that resides in the cytoplasm bound to its inhibitor protein, Keap 1, and translocates to the nucleus after stimulation. It then forms heterodimers with small oncogene family proteins for the selective recognition of the antioxidant responsive element on target genes, resulting in the regulation of gene expression of phase II detoxifying enzymes, including HO-1 [14, 15]. HO-1 induction occurs in a wide variety of cells, including neuronal and nonneuronal brain cells. Previous studies have reported that the cerebellar granule cells harvested from transgenic mice designed to overexpress HO-1 in neurons appear to be relatively resistant to H2O2-mediated oxidative damage in vitro [16].Furthermore, several researchers have reported that phytochemicals, such as resveratrol, curcumin, and epigallocatechin-3-gallate, exhibit the significant therapeutic benefit in the case of oxidative stress-induced neuronal damage via the induction of HO-1 [17–19].Along with neuronal oxidative stress, neuroinflammation also contributes to the pathogenesis of several neurodegenerative disorders [20, 21]. Microglia, the immunocompetent resident cells of the brain, play the main role in innate immune defense in the CNS. Several studies have shown that excessive activation of microglia increases neurotoxicity and, therefore, contributes to neuronal injury through the release of various proinflammatory cytokines and inflammatory mediators such as nitric oxide (NO), prostaglandins, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β [22–25]. In addition to its antioxidative effects, recent studies have also demonstrated anti-inflammatory effects of HO-1 in a number of inflammation models [26–29]. The anti-inflammatory action of HO-1 is mediated by inhibiting the production of proinflammatory cytokines and chemokines such as TNF-α, IL-1β, and IL-6 in activated macrophages. HO-1 and its product, carbon monoxide, can also suppress the expression of proinflammatory COX-2 and iNOS, thereby reducing the production of COX-2-derived prostaglandin E2 (PGE2) and iNOS-derived NO [30–33]. The anti-inflammatory action of HO-1 in microglia has also been reported. Therefore, regulating the degree of generation of oxidative stress and microglial activation via the upregulation of HO-1 may be considered an important intervention in formulating a strategy for the treatment of neurological diseases.Violae herba has been widely used in traditional Chinese medicine to treat carbuncles, boils, and other cutaneous and subcutaneous pyogenic infections. Sources of violae herba includeViola patrinii, V. hederacea, V. arvensis, and V. odorata. Previous phytochemical studies of Viola species have reported the isolation of cyclotides and several flavonoid glycosides [34–37]. However, little is known about the biological activities of these plants. As a part of ongoing research to identify natural products that can induce HO-1 expression in vitro, we have shown here that the ethanol extract of V. patrinii (NNMBS275) was shown to significantly increase the levels of HO-1 in both BV2 microglia and HT22 cells.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from GIBCO BRL Co. (Grand Island, NY, USA). Tin protoporphyrin IX (SnPP IX; inhibitor of HO activity) was obtained from Porphyrin Products (Logan, UT, USA). Cobalt protoporphyrin IX (CoPP; HO-1 inducer), Trolox, and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). TRIzol reagent and a polymerase chain reaction (PCR) kit were purchased from Invitrogen Corporation (Carlsbad, CA, USA). Primary antibodies, including mouse/goat/rabbit anti-HO-1, anti-COX-2, and anti-iNOS, and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for PGE2, TNF-α, and IL-1β were purchased from R & D Systems, Inc. (Minneapolis, MN, USA).


### 2.2. Plant Material and Preparation of NNMBS275

The aerial parts ofViola patrinii DC (Violaceae) were collected in the Botanical Garden of Wonkwang University, Iksan, Korea, in June 2010. A voucher specimen (no. WP2010-26) was authenticated by Dr. Kyu-Kwan Jang, the Botanical Garden of Wonkwang University, and deposited at the herbarium of the College of Pharmacy, Wonkwang University. Dried and pulverized V. patrinii (50 g) was extracted twice with ethanol (300 mL) at room temperature for 3 days, and the extract was concentrated in vacuo to obtain an ethanol extract (NNMBS275, 720 mg). NNMBS275 was deposited at the Standardized Material Bank for New Botanical Drugs, Wonkwang University. For each experiment, NNMBS275 was dissolved in dimethylsulfoxide (final culture concentration, 0.05%). Serum-free medium was used as a vehicle control. Preliminary studies indicated that the solvent had no effect on cell viability at the concentration used.


### 2.3. High-Performance Liquid Chromatography (HPLC)

Chromatographic experiments were performed on a SNKNM series HPLC instrument equipped with a sample injector and a diode-assay UV/Vis detector (DAD) (SYKAM, Germany). For all experiments, an Inertsil ODS C-18 column (4.6 × 150 nm; 5μm, GL Science Inc., Tokyo, Japan) was used as the stationary phase, and the injection volume was 20 μL. The mobile phase was composed of water (A) and acetonitrile (B), with an applied gradient of 20% B increasing to 60% B in 40 min. The column was cleaned with 10% B for 20 min, and then the system was equilibrated for 20 min with the starting conditions. Flow rate was 0.7 mL/min, and the detection wavelength was adjusted to 210 nm.


### 2.4. Cell Culture and Viability Assay

Mouse hippocampal HT22 cells were received from Prof. In-Hee Mook at Seoul National University (Seoul, Korea), and BV2 microglia cells were received from Prof. Hyun Park at Wonkwang University. The cells were maintained at 5 × 105 cells/mL in DMEM medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin G, 100 mg/L streptomycin, and 2 mML-glutamine, and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. For determination of cell viability, 50 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added to 1 mL of cell suspension (1 × 105 cells per 1 mL in 96-well plates) and incubated for 4 h. The formazan formed was dissolved in acidic 2-propanol, and optical density was measured at 590 nm.


### 2.5. Reactive Oxygen Species Measurement

For measurement of ROS, HT22 cells (2.5 × 104 cells/mL in 24-well plates) were treated with 5 mM glutamate in the presence or absence of NNMBS275 or SnPP (HO inhibitor) and incubated for 8 h. After washing with phosphate-buffered saline (PBS), the cells were stained with 10 μM 2′,7′-dichlorofluorescein diacetate in Hanks’ balanced salt solution for 30 min in the dark. The cells were then washed twice with PBS and extracted using 1% Triton X-100 in PBS for 10 min at 37°C. Fluorescence was recorded at an excitation wavelength of 490 nm and emission wavelength of 525 nm (Spectramax Gemini XS; Molecular Devices, Sunnyvale, CA, USA).


### 2.6. Nitrite Assay

The method used for the determination of NO production follows the protocol published by Lee et al. [38]. The nitrite concentration in the medium was measured as an indicator of NO production as per the Griess reaction. 100 μL of each supernatant was mixed with the same volume of Griess reagent, and the absorbance of the mixture at 525 nm was determined with an ELISA plate reader.


### 2.7. PGE2, TNF-α, and IL-1β Assay

BV2 microglia were cultured in 24-well plates, preincubated for 12 h with various concentrations of NNMBS275, and then treated for 18 h with lipopolysaccharide (LPS). Culture medium was collected and the concentration of PGE2, TNF-α, and IL-1β was determined using ELISA kits (R&D Systems) as per the manufacturer’s instructions.


### 2.8. Western Blot Analysis

HT22 and BV2 cells were harvested and pelleted at 200 ×g for 3 min, washed with PBS, and lysed with 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethanesulfonyl fluoride (PMSF), 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). Protein concentration was determined using a Lowry protein assay kit (P5626; Sigma). An equal amount of protein from each sample was resolved using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto a Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk and incubated with anti-HO-1, anti-iNOS, anti-COX-2, anti-Nrf2, anti-IκB-α, anti- phospho-IκB-α, anti-p65, anti-PCNA, or anti-β-actin (all antibodies were used at a 1 : 1000 dilution and were purchased from Santa Cruz Biotechnology, CA, USA) antibodies at 4°C overnight. The immunoreactive bands were visualized by horseradish peroxidase-conjugated secondary antibody (1 : 1000 dilution, Santa Cruz Biotechnology) followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and quantitated using an image program (Image Gauge v3.12 software, Fujifilm, Tokyo, Japan).


### 2.9. Preparation of Nuclear and Cytosolic Fractions

Cells were homogenized (1 : 20, w : v) in PER-mammalian protein extraction buffer (Pierce Biotechnology, Rockford, IL, USA) containing freshly added protease inhibitor cocktail I (EMD Biosciences, San Diego, CA, USA) and 1 mM PMSF. The cytosolic fraction of the cell was prepared by centrifugation at 15,000 ×g for 10 min at 4°C. Nuclear and cytoplasmic extracts of HT22 cells and BV2 cells were prepared, using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology), respectively. After treatment, cells (3 × 106 cells/3 mL in 60 mm dish) were collected and washed with PBS. After centrifugation, cell lysis was performed at 4°C by vigorous shaking for 15 min in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl [pH 7.4], 50 mM glycerophosphate, 20 mM NaF, 20 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, 1 mM Na3VO4, and protease inhibitors). After centrifugation at 14,800 ×g for 15 min, the supernatant was separated and stored at −70°C until further use. Protein content was determined using the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA).


### 2.10. RNA Quantification

Total RNA was extracted from cells harvested for reverse transcription polymerase chain reaction (RT-PCR) using the TRIzol System (Invitrogen). The RNA isolation protocol included a DNase I treatment. We quantified RNA and reverse transcribed cDNAs from 1μg of total RNA per 20 μL RT reaction with Oligo (dT) 12-8 primer and the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). RT-PCR was conducted in a 25 μL solution containing 67.7 mM Tris-HCL (pH 8.8); 16.6 mM (NH4)2SO4; 0.01% Tween-20; 200 nM each of dATP, dCTP, dGTP, and 400 nM dUTP; 4.5 mM MgCl2; 300 nM of each primer; 200 nM probe; 2 U Taq DNA polymerase, and 1/10 (by volume) of the cDNA synthesis reaction. Thermal cycling conditions consisted of 4 min at 95°C followed by 25 cycles of 15 s at 95°C and 1 min at 60°C. PCR was carried out with following primers for HO-1 (375 bp, Forward: 5′-TGA AGG AGG CCA CCA AGG AGG-3′, Reverse: 5′-AGA GGT CAC CCA GGT AGC GGG-3′), and actin (514 bp, Forward: 5′-TGT GAT GGT GGG AAT GGG TCA G-3′, Reverse: 5′-TTT GAT GTC ACG CAC GAT TTC C-3′). The annealing temperatures were 55°C for HO-1 and 58°C for actin.


### 2.11. DNA-Binding Activity of NF-κB

BV2 cells were pretreated for 12 h with the indicated concentrations of NNMBS275 and stimulated for 1 h with LPS. The DNA-binding activity of NF-κB in nuclear extracts was measured (TransAM kit; Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, the following procedure was used: add 30 μL complete-binding buffer (DTT, herring sperm DNA, and binding buffer AM3) to each well. Add 20 μL of samples, the indicated concentrations of NNMBS275, and stimulated for 1 h with LPS on BV2 cells, diluted in complete lysis buffer per well (20 μg of nuclear extract diluted in complete lysis buffer). Incubate for 1 h at room temperature with mild agitation (100 rpm on a rocking platform). After washing each well with wash buffer, 100 μL of diluted NF-κB antibody (1 : 1000 dilution in 1 × Antibody Binding Buffer) was added to each well and then incubated for 1 h with mild agitation (100 rpm on a rocking platform). After washing each well with wash buffer, 100 μL of diluted HRP-conjugated antibody (1 : 1000 dilution in 1 × Antibody Binding Buffer) was added to each well and then incubated for 1 h with mild agitation (100 rpm on a rocking platform). Developing solution was added to each well and left to react for 5 min followed by a wash to remove the supernatant. The 450-nm absorbance of each sample was then read on a spectrophotometer within 5 min.


### 2.12. Statistical Analysis

Data were expressed in terms of the mean ± S.D. of at least 3 independent experiments. To compare 3 or more groups, one-way analysis of variance was used followed by a Newman-Keuls post hoc test. Statistical analysis was performed using GraphPad Prism software version 3.03 (GraphPad Software Inc, San Diego, CA, USA).


## 2.1. Chemicals and Reagents

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from GIBCO BRL Co. (Grand Island, NY, USA). Tin protoporphyrin IX (SnPP IX; inhibitor of HO activity) was obtained from Porphyrin Products (Logan, UT, USA). Cobalt protoporphyrin IX (CoPP; HO-1 inducer), Trolox, and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). TRIzol reagent and a polymerase chain reaction (PCR) kit were purchased from Invitrogen Corporation (Carlsbad, CA, USA). Primary antibodies, including mouse/goat/rabbit anti-HO-1, anti-COX-2, and anti-iNOS, and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for PGE2, TNF-α, and IL-1β were purchased from R & D Systems, Inc. (Minneapolis, MN, USA).


## 2.2. Plant Material and Preparation of NNMBS275

The aerial parts ofViola patrinii DC (Violaceae) were collected in the Botanical Garden of Wonkwang University, Iksan, Korea, in June 2010. A voucher specimen (no. WP2010-26) was authenticated by Dr. Kyu-Kwan Jang, the Botanical Garden of Wonkwang University, and deposited at the herbarium of the College of Pharmacy, Wonkwang University. Dried and pulverized V. patrinii (50 g) was extracted twice with ethanol (300 mL) at room temperature for 3 days, and the extract was concentrated in vacuo to obtain an ethanol extract (NNMBS275, 720 mg). NNMBS275 was deposited at the Standardized Material Bank for New Botanical Drugs, Wonkwang University. For each experiment, NNMBS275 was dissolved in dimethylsulfoxide (final culture concentration, 0.05%). Serum-free medium was used as a vehicle control. Preliminary studies indicated that the solvent had no effect on cell viability at the concentration used.


## 2.3. High-Performance Liquid Chromatography (HPLC)

Chromatographic experiments were performed on a SNKNM series HPLC instrument equipped with a sample injector and a diode-assay UV/Vis detector (DAD) (SYKAM, Germany). For all experiments, an Inertsil ODS C-18 column (4.6 × 150 nm; 5μm, GL Science Inc., Tokyo, Japan) was used as the stationary phase, and the injection volume was 20 μL. The mobile phase was composed of water (A) and acetonitrile (B), with an applied gradient of 20% B increasing to 60% B in 40 min. The column was cleaned with 10% B for 20 min, and then the system was equilibrated for 20 min with the starting conditions. Flow rate was 0.7 mL/min, and the detection wavelength was adjusted to 210 nm.


## 2.4. Cell Culture and Viability Assay

Mouse hippocampal HT22 cells were received from Prof. In-Hee Mook at Seoul National University (Seoul, Korea), and BV2 microglia cells were received from Prof. Hyun Park at Wonkwang University. The cells were maintained at 5 × 105 cells/mL in DMEM medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin G, 100 mg/L streptomycin, and 2 mML-glutamine, and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. For determination of cell viability, 50 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added to 1 mL of cell suspension (1 × 105 cells per 1 mL in 96-well plates) and incubated for 4 h. The formazan formed was dissolved in acidic 2-propanol, and optical density was measured at 590 nm.


## 2.5. Reactive Oxygen Species Measurement

For measurement of ROS, HT22 cells (2.5 × 104 cells/mL in 24-well plates) were treated with 5 mM glutamate in the presence or absence of NNMBS275 or SnPP (HO inhibitor) and incubated for 8 h. After washing with phosphate-buffered saline (PBS), the cells were stained with 10 μM 2′,7′-dichlorofluorescein diacetate in Hanks’ balanced salt solution for 30 min in the dark. The cells were then washed twice with PBS and extracted using 1% Triton X-100 in PBS for 10 min at 37°C. Fluorescence was recorded at an excitation wavelength of 490 nm and emission wavelength of 525 nm (Spectramax Gemini XS; Molecular Devices, Sunnyvale, CA, USA).


## 2.6. Nitrite Assay

The method used for the determination of NO production follows the protocol published by Lee et al. [38]. The nitrite concentration in the medium was measured as an indicator of NO production as per the Griess reaction. 100 μL of each supernatant was mixed with the same volume of Griess reagent, and the absorbance of the mixture at 525 nm was determined with an ELISA plate reader.


## 2.7. PGE2, TNF-α, and IL-1β Assay

BV2 microglia were cultured in 24-well plates, preincubated for 12 h with various concentrations of NNMBS275, and then treated for 18 h with lipopolysaccharide (LPS). Culture medium was collected and the concentration of PGE2, TNF-α, and IL-1β was determined using ELISA kits (R&D Systems) as per the manufacturer’s instructions.


## 2.8. Western Blot Analysis

HT22 and BV2 cells were harvested and pelleted at 200 ×g for 3 min, washed with PBS, and lysed with 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethanesulfonyl fluoride (PMSF), 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). Protein concentration was determined using a Lowry protein assay kit (P5626; Sigma). An equal amount of protein from each sample was resolved using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto a Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk and incubated with anti-HO-1, anti-iNOS, anti-COX-2, anti-Nrf2, anti-IκB-α, anti- phospho-IκB-α, anti-p65, anti-PCNA, or anti-β-actin (all antibodies were used at a 1 : 1000 dilution and were purchased from Santa Cruz Biotechnology, CA, USA) antibodies at 4°C overnight. The immunoreactive bands were visualized by horseradish peroxidase-conjugated secondary antibody (1 : 1000 dilution, Santa Cruz Biotechnology) followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and quantitated using an image program (Image Gauge v3.12 software, Fujifilm, Tokyo, Japan).


## 2.9. Preparation of Nuclear and Cytosolic Fractions

Cells were homogenized (1 : 20, w : v) in PER-mammalian protein extraction buffer (Pierce Biotechnology, Rockford, IL, USA) containing freshly added protease inhibitor cocktail I (EMD Biosciences, San Diego, CA, USA) and 1 mM PMSF. The cytosolic fraction of the cell was prepared by centrifugation at 15,000 ×g for 10 min at 4°C. Nuclear and cytoplasmic extracts of HT22 cells and BV2 cells were prepared, using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology), respectively. After treatment, cells (3 × 106 cells/3 mL in 60 mm dish) were collected and washed with PBS. After centrifugation, cell lysis was performed at 4°C by vigorous shaking for 15 min in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl [pH 7.4], 50 mM glycerophosphate, 20 mM NaF, 20 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, 1 mM Na3VO4, and protease inhibitors). After centrifugation at 14,800 ×g for 15 min, the supernatant was separated and stored at −70°C until further use. Protein content was determined using the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA).


## 2.10. RNA Quantification

Total RNA was extracted from cells harvested for reverse transcription polymerase chain reaction (RT-PCR) using the TRIzol System (Invitrogen). The RNA isolation protocol included a DNase I treatment. We quantified RNA and reverse transcribed cDNAs from 1μg of total RNA per 20 μL RT reaction with Oligo (dT) 12-8 primer and the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). RT-PCR was conducted in a 25 μL solution containing 67.7 mM Tris-HCL (pH 8.8); 16.6 mM (NH4)2SO4; 0.01% Tween-20; 200 nM each of dATP, dCTP, dGTP, and 400 nM dUTP; 4.5 mM MgCl2; 300 nM of each primer; 200 nM probe; 2 U Taq DNA polymerase, and 1/10 (by volume) of the cDNA synthesis reaction. Thermal cycling conditions consisted of 4 min at 95°C followed by 25 cycles of 15 s at 95°C and 1 min at 60°C. PCR was carried out with following primers for HO-1 (375 bp, Forward: 5′-TGA AGG AGG CCA CCA AGG AGG-3′, Reverse: 5′-AGA GGT CAC CCA GGT AGC GGG-3′), and actin (514 bp, Forward: 5′-TGT GAT GGT GGG AAT GGG TCA G-3′, Reverse: 5′-TTT GAT GTC ACG CAC GAT TTC C-3′). The annealing temperatures were 55°C for HO-1 and 58°C for actin.


## 2.11. DNA-Binding Activity of NF-κB

BV2 cells were pretreated for 12 h with the indicated concentrations of NNMBS275 and stimulated for 1 h with LPS. The DNA-binding activity of NF-κB in nuclear extracts was measured (TransAM kit; Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, the following procedure was used: add 30 μL complete-binding buffer (DTT, herring sperm DNA, and binding buffer AM3) to each well. Add 20 μL of samples, the indicated concentrations of NNMBS275, and stimulated for 1 h with LPS on BV2 cells, diluted in complete lysis buffer per well (20 μg of nuclear extract diluted in complete lysis buffer). Incubate for 1 h at room temperature with mild agitation (100 rpm on a rocking platform). After washing each well with wash buffer, 100 μL of diluted NF-κB antibody (1 : 1000 dilution in 1 × Antibody Binding Buffer) was added to each well and then incubated for 1 h with mild agitation (100 rpm on a rocking platform). After washing each well with wash buffer, 100 μL of diluted HRP-conjugated antibody (1 : 1000 dilution in 1 × Antibody Binding Buffer) was added to each well and then incubated for 1 h with mild agitation (100 rpm on a rocking platform). Developing solution was added to each well and left to react for 5 min followed by a wash to remove the supernatant. The 450-nm absorbance of each sample was then read on a spectrophotometer within 5 min.


## 2.12. Statistical Analysis

Data were expressed in terms of the mean ± S.D. of at least 3 independent experiments. To compare 3 or more groups, one-way analysis of variance was used followed by a Newman-Keuls post hoc test. Statistical analysis was performed using GraphPad Prism software version 3.03 (GraphPad Software Inc, San Diego, CA, USA).


## 3. Results

### 3.1. HPLC Analysis of NNMBS275

Data from the HPLC analysis of NNMBS275 was recorded in the form of chromatograms by monitoring detector responses at 210 nm. As shown in Figure1, according to the HPLC fingerprint, there were 3 peaks in the extract. Although V. patriniihas been long used as a traditional herbal medicine in China and Korea, there are few phytochemical studies of this plant. We intend to pursue an investigation of the chemical components of V. patrinii in future detailed studies, in order to identify the biological activities of this plant.Figure 1
HPLC chromatogram of EtOH extract fromV. patrinii.


### 3.2. Effects of NNMBS275 on Cell Viability

Initially, the cytotoxic effects of NNMBS275 on BV2 microglia and HT22 cells were measured. The MTT assay performed at 200μg/mL NNMBS275 revealed no cytotoxic effects in either BV2 microglia or HT22 cells (Figure 2). Thus, for further experiments, the cells were treated with NNMBS275 in the concentration range of 25–200 μg/mL.Effects of NNMBS275 on cell viability. (a) BV2 microglia and (b) HT22 cells were incubated for 12 h with various concentrations of NNMBS275 (25–300μg/mL). Cell viability was determined as described in Section 2. Bar represents the mean ± S.D. of 3 independent experiments.
(a)(b)


### 3.3. Effects of NNMBS275 on Proinflammatory Enzyme Expression and Cytokines Production in LPS-Stimulated BV2 Microglia

To investigate the effects of NNMBS275 on iNOS and COX-2 expression in LPS-stimulated BV2 microglia, cells were treated with LPS (0.5μg/mL) in the presence or absence of NNMBS275 at noncytotoxic concentrations (25–200 μg/mL). Pretreatment of BV2 microglia with NNMBS275 for 12 h resulted in suppressed iNOS expression (Figure 3(a)) and iNOS-derived NO production (Figure 3(c)). Under the same conditions, NNMBS275 also decreased COX-2 expression (Figure 3(b)), and COX-2-derived PGE2 production (Figure 3(d)). Since our results showed that NNMBS275 suppressed LPS-induced proinflammatory mediators such as NO, iNOS, PGE2, and COX-2, we further determined the effect of NNMBS275 on LPS-induced TNF-α and IL-1β production. The levels of these cytokines were examined by an enzyme immunoassay in which BV2 microglia were preincubated with NNMBS275 for 12 h followed by LPS stimulation. As shown in Figures 3(e) and 3(f), pretreatment with NNMBS275 resulted in a significant and concentration-dependent decrease in TNF-α and IL-1β production.Effects of NNMBS275 on LPS-induced inducible iNOS and COX-2 protein expression and NO, PGE2, and proinflammatory cytokine levels in BV2 microglia. BV2 microglia were pretreated for 12 h with indicated concentrations of NNMBS275 and stimulated for 18 h with LPS (0.5 μg/mL). Western blot analyses for iNOS and COX-2 expression (a and b) were performed as described in Section 2. Representative blots of 3 independent experiments are shown. The concentrations of NO, PGE2, tumor necrosis TNF-α, and IL-1β (c, d, e, and f) were determined as described under Section 2. Data represent mean values of 3 experiments ± S.D. *P<0.05 compared to the group treated with LPS.
(a)(b)(c)(d)(e)(f)


### 3.4. Effect of NNMBS275 on Glutamate-Induced Cytotoxicity and Inhibition of ROS Generation in HT22 Cells

To evaluate thein vitro neuroprotective effect of NNMBS275, we tested its protective effect on glutamate-induced cytotoxicity in HT22 cells. Treatment with glutamate for 12 h increased HT22 cell death by 55% compared to untreated cells, whereas NNMBS275, at noncytotoxic concentrations (25, 50, 100, and 200 μg/mL), caused a dose-dependent increase in the viability of these cells (Figure 4(a)). Glutamate also doubled ROS production, and NNMBS275 effectively suppressed this induction (Figure 4(b)). Trolox, well known for its antioxidative efficiency, was used as a positive control, and it showed a significant cytoprotective effect and ROS-scavenging activity at a concentration of 50 μM.Effects of NNMBS275 on glutamate-induced oxidative neurotoxicity, and inhibition of ROS generation in HT22 cells. (a) Cells were pretreated for 12 h with indicated concentrations of NNMBS275 and then incubated for 12 h with glutamate (5 mM). (b) Exposure of HT22 cells to 5 mM glutamate for 12 h increased ROS production. NNMBS275 effectively inhibited glutamate-mediated ROS production. Data represent mean values of 3 experiments ± S.D. *P<0.05 compared to the group treated with glutamate. Trolox (50 μM) was used as a positive control.
(a)(b)


### 3.5. Effects of NNMBS275 on HO-1 Protein and mRNA Expression

We examined whether noncytotoxic concentrations (25–200μg/mL) of NNMBS275 affected HO-1 protein and mRNA expression, by treating BV2 microglia and HT22 cells with this agent for 12 h. NNMBS275 caused a dose-dependent increase in HO-1 expression in BV2 microglia (Figure 5(a)) and HT22 cells (Figure 5(c)). The induction of HO-1 by NNMBS275 reached a peak at 200 μg/mL. The HO-1 inducer CoPP, used as a positive control, increased HO-1 expression at a dose of 20 μM. At an NNMBS275 concentration of 200 μg/mL, HO-1 expression showed an increase at 6 h, peaked at around 18 h, and decreased after 24 h in BV2 microglia and HT22 cells (Figures 5(b) and 5(d)). Consistently, NNMBS275-induced mRNA expression levels of HO-1 also increased in a dose-dependent manner in both BV2 microglia and HT22 cells, as assessed by RT-PCR analysis (Figure 6).Effects of NNMBS275 on HO-1 expression in BV2 microglia (a and b) and HT22 (c and d). BV2 microglia and HT22 cells were incubated for 12 h with indicated concentrations of NNMBS275 (a and c) and periods with 200μg/mL of NNMBS275 (b and d). The HO-1 inducer CoPP, increased HO-1 expression at 20 μM (a and c). Western blot analysis for HO-1 expression was performed as described in Section 2, and representative blots of 3 independent experiments are shown. Data represent the mean values of 3 experiments ± S.D. *P<0.05 compared to the control group.
(a)
BV2 microglia(b)
BV2 microglia(c)
HT22 cells(d)
HT22 cellsEffects of NNMBS275 on mRNA expression of HO-1 in BV2 microglia and HT22. (a) BV2 microglia and (b) HT22 cells were incubated for 6 h with indicated concentrations of NNMBS275. RNA quantification was performed as described in Section2, and representative blots of three independent experiments are shown.
(a)
BV2 microglia(b)
HT22 cells


### 3.6. Effects of HO-1 Expression on Glutamate-Induced Oxidative Neurotoxicity and Inhibition of Proinflammatory Mediators by NNMBS275

Recent reports have described the expression of HO-1 as an adaptive and protective response against inflammation and oxidative insult in a wide variety of cells, including glial and neuronal cells, such as BV2 and HT22. We examined whether NNMBS275-induced HO-1 expression mediated its cytoprotective effects in HT22 cells. HT22 cells were cotreated with 200μg/mL of NNMBS275 for 12 h in the absence or presence of SnPP, an inhibitor of HO-1 activity. SnPP significantly inhibited NNMBS275-mediated cytoprotection (Figure 7(a)). NNMBS275-induced HO-1 expression was also required for suppressing glutamate-induced ROS generation (Figure 7(b)). On the basis of our findings that preincubation of BV2 microglia with NNMBS275 markedly inhibited LPS-induced proinflammatory enzymes and proinflammatory cytokines (Figure 3), and that NNMBS275 was able to induce HO-1 expression (Figure 5) in BV2 microglia, we examined whether NNMBS275-mediated HO-1 induction might be responsible for the inhibition of iNOS-derived NO, COX-2-derived PGE2, TNF-α, and IL-1β production (Figures 7(c), 7(d), 7(e), and 7(f)). BV2 microglia were pretreated with NNMBS275 for 12 h in the presence of SnPP, followed by LPS stimulation. As shown in Figure 7, SnPP treatment partially reversed the inhibitory effects of NNMBS275 on NO, PGE2, TNF-α, and IL-1β production.Figure 7
HO-1 mediates the suppressive effect of NNMBS275 on LPS-stimulated proinflammatory mediator production, and glutamate-induced oxidative neurotoxicity. (a and b) HT22 cells were pretreated for 6 h with NNMBS275 (200μg/mL) in the presence or absence of SnPP (50 μM), and then incubated for 12 h with glutamate (5 mM). (c, d, e, and f) BV2 cells were pretreated for 3 h with NNMBS275 (200 μg/mL) in the presence or absence of SnPP (50 μM) and stimulated for 24 h with LPS (0.5 μg/mL). The concentrations of NO, PGE2, TNF-α, and IL-1β were determined as described in Section 2. SnPP was pretreated with BV2 microglia and HT22 cells for 3 h in this experiment. Data represent mean values of 3 experiments ± S.D. *P<0.05.


### 3.7. Effects of NNMBS275 on Nrf2 Nuclear Translocation

Several studies have reported that nuclear translocation of activated Nrf2 is an important upstream contributor to the mechanism of HO-1 expression. Therefore, we investigated whether treatment of BV2 microglia and HT22 cells with NNMBS275 induced the translocation of Nrf2 to the nucleus. Using western blot analysis, we tested the presence of Nrf2 proteins in nuclear compartments of BV2 microglia and HT22 cells. Cells were incubated with 200μg/mL NNMBS275 for 0.5, 1, and 1.5 h. As shown in Figure 8, the nuclear fractions of NNMBS275-treated BV2 microglia and HT22 cells showed a gradual increase in Nrf2 levels, whereas Nrf2 levels decreased concomitantly in the cytoplasmic fractions.Effects of NNMBS275 on nuclear translocation of Nrf2 in BV2 cells and HT22. (a) BV2 cells and (b) HT22 cells were treated with 200μg/mL of NNMBS275 for 0.5, 1, and 1.5 h. The nuclei were fractionated from the cytosol using PER-mammalian protein extraction buffer as described in Section 2. Nrf2 protein was detected by western blot analysis, and representative blots of 3 independent experiments are shown.
(a)
BV2 microglia(b)
HT22 cells


### 3.8. Effects of NNMBS275 on the Phosphorylation and Degradation of IκB-α and NF-κB Activation

NF-κB has been implicated in the transcriptional regulation of LPS-induced iNOS expression. Therefore, we examined the effects of NNMBS275 on the protein expression level of IκB-α phosphorylation and degradation of IκB-α, an inhibitor associated with NF-κB in the cytoplasm. As shown in Figure 9(a), IκB-α was degraded after treatment with LPS in BV2 cells for 1 h, and this degradation was markedly inhibited by Pretreatment of the cells with NNMBS275 (25–200 μg/mL) for 12 h. These results suggest that NNMBS275 inhibited the nuclear translocation of NF-κB (p65) by the stimulation of LPS through prevention of IκB-α degradation. Moreover, shown in our data, the levels of nuclear p65 protein in BV2 cells were increased after treating cells with LPS for 1 h, while the level of p65 declined in response to LPS Pretreatment with NNMBS275 (25–200 μg/mL) for 12 h. We also observed the appearance of NF-κB-DNA-binding activity in nuclear extracts of BV2 cells stimulated with LPS for 1 h. As shown in Figure 9(b), treatment with LPS alone increased the levels of NF-κB-DNA-binding activity. However, NNMBS275 suppressed this increase in a dose-dependent manner.Effects of NNMBS275 on NF-κB activation and NF-κB DNA-binding activity in BV2 cells. Cells were pretreated for 12 h with indicated concentrations of NNMBS275, and stimulated for 1 h with LPS (0.5 μg/mL). (a) Western blot analysis for IκB-α and p-IκB-α in the cytoplasm and NF-κB expression in the nucleus was performed as described in Section 2, and representative blots of 3 independent experiments are shown. (b) A commercially available NF-κB ELISA (active motif) was then used to test nuclear extracts and determine degree of NF-κB binding. Data represent the mean values of 3 experiments ± S.D. *P<0.05 compared to the group treated with LPS.
(a)(b)


## 3.1. HPLC Analysis of NNMBS275

Data from the HPLC analysis of NNMBS275 was recorded in the form of chromatograms by monitoring detector responses at 210 nm. As shown in Figure1, according to the HPLC fingerprint, there were 3 peaks in the extract. Although V. patriniihas been long used as a traditional herbal medicine in China and Korea, there are few phytochemical studies of this plant. We intend to pursue an investigation of the chemical components of V. patrinii in future detailed studies, in order to identify the biological activities of this plant.Figure 1
HPLC chromatogram of EtOH extract fromV. patrinii.


## 3.2. Effects of NNMBS275 on Cell Viability

Initially, the cytotoxic effects of NNMBS275 on BV2 microglia and HT22 cells were measured. The MTT assay performed at 200μg/mL NNMBS275 revealed no cytotoxic effects in either BV2 microglia or HT22 cells (Figure 2). Thus, for further experiments, the cells were treated with NNMBS275 in the concentration range of 25–200 μg/mL.Effects of NNMBS275 on cell viability. (a) BV2 microglia and (b) HT22 cells were incubated for 12 h with various concentrations of NNMBS275 (25–300μg/mL). Cell viability was determined as described in Section 2. Bar represents the mean ± S.D. of 3 independent experiments.
(a)(b)


## 3.3. Effects of NNMBS275 on Proinflammatory Enzyme Expression and Cytokines Production in LPS-Stimulated BV2 Microglia

To investigate the effects of NNMBS275 on iNOS and COX-2 expression in LPS-stimulated BV2 microglia, cells were treated with LPS (0.5μg/mL) in the presence or absence of NNMBS275 at noncytotoxic concentrations (25–200 μg/mL). Pretreatment of BV2 microglia with NNMBS275 for 12 h resulted in suppressed iNOS expression (Figure 3(a)) and iNOS-derived NO production (Figure 3(c)). Under the same conditions, NNMBS275 also decreased COX-2 expression (Figure 3(b)), and COX-2-derived PGE2 production (Figure 3(d)). Since our results showed that NNMBS275 suppressed LPS-induced proinflammatory mediators such as NO, iNOS, PGE2, and COX-2, we further determined the effect of NNMBS275 on LPS-induced TNF-α and IL-1β production. The levels of these cytokines were examined by an enzyme immunoassay in which BV2 microglia were preincubated with NNMBS275 for 12 h followed by LPS stimulation. As shown in Figures 3(e) and 3(f), pretreatment with NNMBS275 resulted in a significant and concentration-dependent decrease in TNF-α and IL-1β production.Effects of NNMBS275 on LPS-induced inducible iNOS and COX-2 protein expression and NO, PGE2, and proinflammatory cytokine levels in BV2 microglia. BV2 microglia were pretreated for 12 h with indicated concentrations of NNMBS275 and stimulated for 18 h with LPS (0.5 μg/mL). Western blot analyses for iNOS and COX-2 expression (a and b) were performed as described in Section 2. Representative blots of 3 independent experiments are shown. The concentrations of NO, PGE2, tumor necrosis TNF-α, and IL-1β (c, d, e, and f) were determined as described under Section 2. Data represent mean values of 3 experiments ± S.D. *P<0.05 compared to the group treated with LPS.
(a)(b)(c)(d)(e)(f)


## 3.4. Effect of NNMBS275 on Glutamate-Induced Cytotoxicity and Inhibition of ROS Generation in HT22 Cells

To evaluate thein vitro neuroprotective effect of NNMBS275, we tested its protective effect on glutamate-induced cytotoxicity in HT22 cells. Treatment with glutamate for 12 h increased HT22 cell death by 55% compared to untreated cells, whereas NNMBS275, at noncytotoxic concentrations (25, 50, 100, and 200 μg/mL), caused a dose-dependent increase in the viability of these cells (Figure 4(a)). Glutamate also doubled ROS production, and NNMBS275 effectively suppressed this induction (Figure 4(b)). Trolox, well known for its antioxidative efficiency, was used as a positive control, and it showed a significant cytoprotective effect and ROS-scavenging activity at a concentration of 50 μM.Effects of NNMBS275 on glutamate-induced oxidative neurotoxicity, and inhibition of ROS generation in HT22 cells. (a) Cells were pretreated for 12 h with indicated concentrations of NNMBS275 and then incubated for 12 h with glutamate (5 mM). (b) Exposure of HT22 cells to 5 mM glutamate for 12 h increased ROS production. NNMBS275 effectively inhibited glutamate-mediated ROS production. Data represent mean values of 3 experiments ± S.D. *P<0.05 compared to the group treated with glutamate. Trolox (50 μM) was used as a positive control.
(a)(b)


## 3.5. Effects of NNMBS275 on HO-1 Protein and mRNA Expression

We examined whether noncytotoxic concentrations (25–200μg/mL) of NNMBS275 affected HO-1 protein and mRNA expression, by treating BV2 microglia and HT22 cells with this agent for 12 h. NNMBS275 caused a dose-dependent increase in HO-1 expression in BV2 microglia (Figure 5(a)) and HT22 cells (Figure 5(c)). The induction of HO-1 by NNMBS275 reached a peak at 200 μg/mL. The HO-1 inducer CoPP, used as a positive control, increased HO-1 expression at a dose of 20 μM. At an NNMBS275 concentration of 200 μg/mL, HO-1 expression showed an increase at 6 h, peaked at around 18 h, and decreased after 24 h in BV2 microglia and HT22 cells (Figures 5(b) and 5(d)). Consistently, NNMBS275-induced mRNA expression levels of HO-1 also increased in a dose-dependent manner in both BV2 microglia and HT22 cells, as assessed by RT-PCR analysis (Figure 6).Effects of NNMBS275 on HO-1 expression in BV2 microglia (a and b) and HT22 (c and d). BV2 microglia and HT22 cells were incubated for 12 h with indicated concentrations of NNMBS275 (a and c) and periods with 200μg/mL of NNMBS275 (b and d). The HO-1 inducer CoPP, increased HO-1 expression at 20 μM (a and c). Western blot analysis for HO-1 expression was performed as described in Section 2, and representative blots of 3 independent experiments are shown. Data represent the mean values of 3 experiments ± S.D. *P<0.05 compared to the control group.
(a)
BV2 microglia(b)
BV2 microglia(c)
HT22 cells(d)
HT22 cellsEffects of NNMBS275 on mRNA expression of HO-1 in BV2 microglia and HT22. (a) BV2 microglia and (b) HT22 cells were incubated for 6 h with indicated concentrations of NNMBS275. RNA quantification was performed as described in Section2, and representative blots of three independent experiments are shown.
(a)
BV2 microglia(b)
HT22 cells


## 3.6. Effects of HO-1 Expression on Glutamate-Induced Oxidative Neurotoxicity and Inhibition of Proinflammatory Mediators by NNMBS275

Recent reports have described the expression of HO-1 as an adaptive and protective response against inflammation and oxidative insult in a wide variety of cells, including glial and neuronal cells, such as BV2 and HT22. We examined whether NNMBS275-induced HO-1 expression mediated its cytoprotective effects in HT22 cells. HT22 cells were cotreated with 200μg/mL of NNMBS275 for 12 h in the absence or presence of SnPP, an inhibitor of HO-1 activity. SnPP significantly inhibited NNMBS275-mediated cytoprotection (Figure 7(a)). NNMBS275-induced HO-1 expression was also required for suppressing glutamate-induced ROS generation (Figure 7(b)). On the basis of our findings that preincubation of BV2 microglia with NNMBS275 markedly inhibited LPS-induced proinflammatory enzymes and proinflammatory cytokines (Figure 3), and that NNMBS275 was able to induce HO-1 expression (Figure 5) in BV2 microglia, we examined whether NNMBS275-mediated HO-1 induction might be responsible for the inhibition of iNOS-derived NO, COX-2-derived PGE2, TNF-α, and IL-1β production (Figures 7(c), 7(d), 7(e), and 7(f)). BV2 microglia were pretreated with NNMBS275 for 12 h in the presence of SnPP, followed by LPS stimulation. As shown in Figure 7, SnPP treatment partially reversed the inhibitory effects of NNMBS275 on NO, PGE2, TNF-α, and IL-1β production.Figure 7
HO-1 mediates the suppressive effect of NNMBS275 on LPS-stimulated proinflammatory mediator production, and glutamate-induced oxidative neurotoxicity. (a and b) HT22 cells were pretreated for 6 h with NNMBS275 (200μg/mL) in the presence or absence of SnPP (50 μM), and then incubated for 12 h with glutamate (5 mM). (c, d, e, and f) BV2 cells were pretreated for 3 h with NNMBS275 (200 μg/mL) in the presence or absence of SnPP (50 μM) and stimulated for 24 h with LPS (0.5 μg/mL). The concentrations of NO, PGE2, TNF-α, and IL-1β were determined as described in Section 2. SnPP was pretreated with BV2 microglia and HT22 cells for 3 h in this experiment. Data represent mean values of 3 experiments ± S.D. *P<0.05.


## 3.7. Effects of NNMBS275 on Nrf2 Nuclear Translocation

Several studies have reported that nuclear translocation of activated Nrf2 is an important upstream contributor to the mechanism of HO-1 expression. Therefore, we investigated whether treatment of BV2 microglia and HT22 cells with NNMBS275 induced the translocation of Nrf2 to the nucleus. Using western blot analysis, we tested the presence of Nrf2 proteins in nuclear compartments of BV2 microglia and HT22 cells. Cells were incubated with 200μg/mL NNMBS275 for 0.5, 1, and 1.5 h. As shown in Figure 8, the nuclear fractions of NNMBS275-treated BV2 microglia and HT22 cells showed a gradual increase in Nrf2 levels, whereas Nrf2 levels decreased concomitantly in the cytoplasmic fractions.Effects of NNMBS275 on nuclear translocation of Nrf2 in BV2 cells and HT22. (a) BV2 cells and (b) HT22 cells were treated with 200μg/mL of NNMBS275 for 0.5, 1, and 1.5 h. The nuclei were fractionated from the cytosol using PER-mammalian protein extraction buffer as described in Section 2. Nrf2 protein was detected by western blot analysis, and representative blots of 3 independent experiments are shown.
(a)
BV2 microglia(b)
HT22 cells


## 3.8. Effects of NNMBS275 on the Phosphorylation and Degradation of IκB-α and NF-κB Activation

NF-κB has been implicated in the transcriptional regulation of LPS-induced iNOS expression. Therefore, we examined the effects of NNMBS275 on the protein expression level of IκB-α phosphorylation and degradation of IκB-α, an inhibitor associated with NF-κB in the cytoplasm. As shown in Figure 9(a), IκB-α was degraded after treatment with LPS in BV2 cells for 1 h, and this degradation was markedly inhibited by Pretreatment of the cells with NNMBS275 (25–200 μg/mL) for 12 h. These results suggest that NNMBS275 inhibited the nuclear translocation of NF-κB (p65) by the stimulation of LPS through prevention of IκB-α degradation. Moreover, shown in our data, the levels of nuclear p65 protein in BV2 cells were increased after treating cells with LPS for 1 h, while the level of p65 declined in response to LPS Pretreatment with NNMBS275 (25–200 μg/mL) for 12 h. We also observed the appearance of NF-κB-DNA-binding activity in nuclear extracts of BV2 cells stimulated with LPS for 1 h. As shown in Figure 9(b), treatment with LPS alone increased the levels of NF-κB-DNA-binding activity. However, NNMBS275 suppressed this increase in a dose-dependent manner.Effects of NNMBS275 on NF-κB activation and NF-κB DNA-binding activity in BV2 cells. Cells were pretreated for 12 h with indicated concentrations of NNMBS275, and stimulated for 1 h with LPS (0.5 μg/mL). (a) Western blot analysis for IκB-α and p-IκB-α in the cytoplasm and NF-κB expression in the nucleus was performed as described in Section 2, and representative blots of 3 independent experiments are shown. (b) A commercially available NF-κB ELISA (active motif) was then used to test nuclear extracts and determine degree of NF-κB binding. Data represent the mean values of 3 experiments ± S.D. *P<0.05 compared to the group treated with LPS.
(a)(b)


## 4. Discussion

Oxidative stress and inflammation are common in a variety of neurodegenerative diseases, including stroke, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Therefore, naturally occurring compounds having both antioxidative and anti-inflammatory effects may offer a promising strategy for therapeutic application. Previous studies have demonstrated that several natural products such as macelignan and xanthorrhizol have antioxidant and anti-inflammatory activities in hippocampal and microglia cells [39, 40]. HO-1, an enzyme essential for heme degradation, has been shown to play an important role in regulating biological responses, including oxidative stress and inflammation. In particular, HO-1 is inducible in many neuronal cells, including HT22 and BV2 cells. As a part of our ongoing research to identify phytochemicals isolated from natural sources that can induce HO-1 in vitro, the ethanol extract of V. patrinii (NNMBS275) was shown to significantly induce HO-1 expression in BV2 microglia and HT22 cells.Microglia, a resident population of macrophage-like brain cells are believed to play a major role in host defense and tissue repair in the CNS. Microglia are activated by a variety of agents, including LPS, interferon (IFN)-γ, and TNF-α [41–43]. Upon activation, microglia also promote neuronal injury through the release of various proinflammatory cytokines and inflammatory mediators, such as TNF-α and IL-1β. Several studies have also shown that the expression of iNOS and COX-2, the key enzymes for NO and PGE2 production, is upregulated in activated glial cells. Pretreatment with 25–200 μg/mL of NNMBS275 suppressed iNOS and COX-2 expression in LPS-stimulated BV2 microglia, thereby inhibiting iNOS-derived NO as well as COX-2-derived PGE2 production. In addition, NNMBS275 also inhibited LPS-induced TNF-α and IL-1β production. This finding suggests that NNMBS275, at least in LPS-stimulated microglia, exerts its anti-inflammatory effects by inhibiting the expression of proinflammatory enzymes and the secretion of proinflammatory cytokines.Glutamate toxicity, specifically glutamate-induced oxidative damage, is a major contributor to pathological cell death within the nervous system and has been observed in neuronal cells, including primary neuronal cells and tissue slices. In this study, we examined the protective effects of NNMBS275 against glutamate-induced cytotoxicity in HT22 cells. At noncytotoxic concentrations (25–200μg/mL), NNMBS275 inhibited glutamate-induced cell death in a dose-dependent manner 12 h after treatment and also effectively suppressed glutamate-induced ROS generation. We have provided evidence for the induction of HO-1 expression by NNMBS275 in BV2 microglia and HT22 cells and shown that NNMBS275-induced HO-1 protein and mRNA expression occurred in a concentration- and time-dependent manner. Furthermore, preincubation of HT22 cells with NNMBS275 resulted in enhanced resistance to glutamate-induced oxidative damage; this effect was partly attributable to HO-1 expression as SnPP, which inhibits HO enzyme activity, significantly reduced the cytoprotection afforded by NNMBS275. The induction of HO-1 expression was also required to suppress glutamate-induced ROS generation. We also examined whether the inhibition of PGE2, NO, TNF-α, and IL-1β production induced by preincubation with NNMBS275 is mediated through HO-1 expression in BV2 cells. Indeed, SnPP also partially reversed the inhibitory effects of NNMBS275 on NO, PGE2, TNF-α, and IL-1β production suggesting that HO-1 expression is involved in glutamate- or LPS-induced cell responses.HO-1 induction by low-molecular weight compounds might play an important role in the neuroprotection of CNS neurons [44, 45]. Recent reports have suggested that phytochemicals can activate Nrf2 by directly binding to Keap1 through covalent linkages, resulting in the induction of some cytoprotective proteins, including HO-1. In the present study, we showed that NNMBS275 significantly increased Nrf2 levels and efficiently promoted its translocation into the nucleus in both BV2 microglia and HT22 cells. This suggests that Nrf2 may play an essential role in NNMBS275-induced HO-1 expression. Since NNMBS275, as shown in this study, can actively induce the expression of HO-1 in mouse hippocampal HT22 cells and BV2 microglia, it is possible that the antioxidative and anti-inflammatory effects of NNMBS275 are partly mediated by the products of the HO-1 enzyme reaction, namely, CO, bilirubin and/or biliverdin.It is also well known that NF-κB is a significant role factor regulating the expression of inflammation-associated enzymes and cytokine genes, such as iNOS, COX-2, TNF-α, and IL-1β, which contain NF-κB-binding motifs within their respective promoters [46]. Many anti-inflammatory agents exhibit their potency by suppressing NF-κB signaling [47, 48]. In this study, we examined the effects of NNMBS275 on the phosphorylation of IκBα and the nuclear translocation of a NF-κB subunit, p65, in BV2 cells. As shown in Figure 9, the phosphorylation and degradation of IκBα and the nuclear translocation of p65 induced by LPS were significantly reduced after pretreatment with NNMBS275. Furthermore, we also observed the appearance of NF-κB-DNA-binding activity of NF-κB in nuclear extracts of BV2 cells stimulated with LPS. These results suggest that NNMBS275, at least in LPS-stimulated BV2 cells, exerted its anti-inflammatory effects by suppressing the expression of the proinflammatory enzymes as well as the production of the proinflammatory cytokines.In conclusion, NNMBS275, the ethanol extract ofV. patrinii, showed potent anti-inflammatory effects on microglial activation by LPS, possibly through the Nrf2 pathway-dependent expression of HO-1 and suppression of NF-κB signaling. Furthermore, we investigated the cytoprotective effects of NNMBS275 on glutamate-induced neurotoxicity in mouse hippocampal HT22 cells via the HO-1 expression (Figure 10). The results of our study demonstrate the importance of HO-1 in mediating the antioxidative and anti-inflammatory effects of NNMBS275 in mouse microglial and hippocampal cells and reveal the possible therapeutic value of NNMBS275 in preventing the progression of neurodegenerative disease. Further detailed chemical studies of NNMBS275 would help to clarify its therapeutic potential.Figure 10
The pathway scheme of the anti-inflammatory and cytoprotective effects of NNMBS275 in BV2 microglia and HT22 cells. NNMBS275 increased cellular resistance to oxidative injury caused by glutamate-induced oxidative cytotoxicity in HT22 cells, via Nrf2-dependent HO-1 expression. In BV2 cells, NNMBS275 suppressed the LPS-induced expression of proinflammatory enzymes and inflammatory mediators through its interference of NF-κB activation and Nrf2-dependent HO-1 expression. This study indicates that NNMBS275 effectively modulates the regulation of antioxidative and anti-inflammatory action, via Nrf2 pathway dependent HO-1 expression in BV2 microglia and HT22 cells.



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*Source: 128019-2012-03-24.xml*