Neuroprotective Effect of Sargassum thunbergii (Mertens ex Roth) Kuntze in Activated Murine Microglial Cells

Purpose: To evaluate the anti-oxidant and anti-neuroinflammatory effects of the Sargassum thunbergii extract (Mertens ex Roth) Kuntze (STE) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells in vitro. Methods: STE antioxidative activity was evaluated with an Electron Spin Resonance (ESR) spectrometer, which measured 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity. Cell viabilities were estimated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assays. LPS-stimulated BV-2 microglia were used to study the expression and production of inflammatory mediators, such as nitric oxide (NO), inducible NO synthase (iNOS) and tumor necrosis alpha (TNF-α). Results: LPS treatment, following STE pretreatment, decreased NO production by 13 ~ 65% in a dose-dependent manner (p < 0.001 at 20, 40, 80 and 100 μg/mL), and was associated with the down-regulation of inducible nitric oxide synthase (iNOS) expression. STE also attenuated the TNF-α soluble protein by 16 ~ 47% (p < 0.01at 20, 40 and 80 μg/mL) in activated murine microglia. Furthermore, the DPPH-generated free radicals were inhibited by STE concentration-dependently. Conclusion: STE has therapeutic potential in the prevention or treatment of neurodegenerative and oxidative stress-related disorders.


INTRODUCTION
Microglia, as immune cells of the central nervous system (CNS), produce a variety of inflammatory mediators in response to immunological stressors, and thus play a critical role in neuroinflammatory processes [1]. Upon activation by exposure to free radicals and lipopolysaccharides (LPS) [2], microglia secrete various bioactive molecules such as nitric oxide (NO), inducible NO synthase (iNOS), interleukins (IL) and tumor necrosis factor (TNF)-α [3]. Over-production of these inflammatory mediators can cause a number of severe neuro-degenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and Huntington's disease [4,5].
LPS, as the main component of endotoxins, initiates numerous cellular effects that play critical roles in the pathogenesis of inflammatory responses. LPS has been proven to induce microglial activation in response to Gramnegative bacterial infections [6]. These prior experiments indicate that LPS-induced stimulation of microglia may be an potent mechanism to study in vitro. Studies have shown that antioxidant and anti-inflammatory agents may inhibit microglial activation, and protect neurons from cell death, a prominent symptom of various neurodegenerative disorders [6,7].
Brown seaweed is a marine organism with antiinflammatory and anti-oxidative effects [8]. Sargassum thunbergii is found along shallow marine coastlines of Korea, and perennially grows on low tide-level rocks of open and sheltered coasts [9]. Sargassum thunbergii, also known as Hede, is also used as a food additive, an anti-helminthic treatment for lumps, dropsy, or swollen and painful scrotums [10].
Studies on Sargassum thunbergii for its beneficial effects on microglia-mediated neuroinflammatory diseases have not yet been reported. The aim of this study was to investigate whether the ethanol extract of Sargassum thunbergii (STE) exhibits protective effects on LPS-activated neuro-inflammatory processes in murine microglial BV-2 cells.

EXPERIMENTAL Preparation of Sargassum thunbergii extract (STE)
Dried plant materials of S. thunbergii were purchased from a traditional herb market in Seoul, South Korea and authenticated by taxonomist, Professor Jong-Bo Kim at Konkuk University, South Korea. The S. thunbergii was washed in running tap water, dried at 60 °C for 24 h and then ground to a fine powder. The STE was extracted from the fine powder by mixing with 70 % ethanol (v/w) for 2 h in a heating mantle at 70 ~ 80 °C. The filtered extract was concentrated by a rotary evaporator (EYELA NVC-2000, Tokyo, Japan) under reduced pressure and lyophilized to produce the supernatant.

Measurement of DPPH radical scavenging activity
STE antioxidative activity was measured using the stable radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis, MO, USA). Samples composed of a reaction mixture of STE aliquots and a DPPH methanolic solution (described previously) [9], each with 60 µL of STE and 60 µL of DPPH (60 µM) in methanol. After mixing vigorously for 10 s, the mixture was transferred to a 100 µL Teflon capillary tube. The scavenging activity on the DPPH radical was measured in each sample using a JES-FA ESR spectrometer (Jeol Ltd, Tokyo, Japan). Spin adducts were measured by an ESR spectrometer. Experimental conditions were as follows: central field, 3,475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G; microwave power, 5 mW; gain, 6.3 x 10 5 ; and temperature, 298 K.

Nitric oxide assay
NO production assays indicated varying nitrite levels in the STE supernatant using the colorimetric assay with Griess reagent [10]. BV-2 cells were seeded in 6-well plates in 500 µl of the 10 % RPMI-1640 medium, and then stimulated with LPS (1 µg/ml) for 2 h. Fifty microliters of the STE-contained supernatant were combined with the equal volume of Griess reagent (1 % sulfanilamide in 5 % phosphoric acid and 0.1 % naphthyl ethylenediamine dihydrochloride in water). ELISA reader (Bio-Tek Instrument, Winooski, VT, USA) indicated nitrite concentrations (absorbance of 540 nm) in comparison to sodium nitrite as control.

TNF-α assay
Murine microglial BV-2 cells (1 x 10 5 cells/well) were cultured on 96-well plates treated with the STE at indicated concentrations for 1 h and stimulated with LPS (1 µg/mL). 24 h post-LPS treatment, TNF-α production was determined with cell supernatant using assay kits (BD Biosciences, San Jose, CA, USA) per manufacturer's instructions at room temperature. The optical absorbance was measured at 450 nm with the ELISA reader.

Statistical analysis
All data is presented as the mean ± S.E.M of at least three independent experiments. Statistical analyses were performed using SAS statistical software (SAS Institute, Cray, NC, USA) using one-way analysis of variance, followed by Dunnett's multiple range tests. P < 0.05 was considered statistically significant.

STE's effect on DPPH radical-scavenging activity
As shown in Fig 1A, STE exhibited significant DPPH radical scavenging activity in a dosedependent manner, showing a maximum effect at a concentration of 1 mg/mL. The ESR spectroscopy data at 0.01, 0.1 and 1 mg/ml was represented in Fig 1B.

STE's Effect on LPS-induced NO production
STE treatment did not result in cytotoxic overproduction of NO in BV-2 microglial cells treated for 24 h at concentrations up to 200 µg/mL. In all cases of STE treatment, cell viability was above 96 % (Fig 2). In contrast, LPS treatment resulted in excessive production of NO. Pretreatment with STE prior to LPS treatment, however, significantly decreased (by 13 ~ 65 %) the production of NO in comparison to LPS-only treatment in a dose-dependent manner (Fig 3). The maximum effect was observed at 100 µg/mL (p < 0.001).

Effect of STE on LPS-induced expressional levels of iNOS
STE treatment of murine microglial BV-2 cells exhibited a broad spectrum of inhibitory effects on the expression of iNOS, in contrast to LPS treatment, which enhanced iNOS expression (Fig  4).

Effect of STE on TNF-α production in LPSstimulated BV-2 cells
STE significantly inhibited the production of the pro-inflammatory cytokine, TNF-α, in a concentration-dependent manner in LPSstimulated BV-2 cells (p < 0.05 at 20 µg/mL and p < 0.01 at 40 and 80 µg/mL, respectively). In the LPS-only treatment (1 µg/mL), TNF-α levels were significantly higher than those of the untreated cell control sample (p < 0.001) (Fig 5).

DISCUSSION
The present study has shown a few antineuroinflammatory effects of STE in murine microglial BV-2 cells, in tandem with LPS (neuroinflammatory) treatment. Although LPS activates TLR4 receptors of microglia to secrete various cytokines, STE treatment STE treatment affects NO, iNOS and TNF-α production and expression in dose-dependent manner. These results show that STE extract has antineuroinflammatory and antioxidative effects. STE exhibited significant antioxidant activity, as evidenced by the DPPH free radical scavenging method. The DPPH radical assay is a widely used method for evaluating the free radical scavenging activities of several antioxidants in a relatively short period of time [12]. Free radicals and reactive oxygen species (ROS) are important causative factors in the development of age-related neuro-inflammatory and neurodegenerative diseases, [11] so neutralization of free radicals by antioxidants and radical scavengers reduces neuro-inflammation. In our present study, STE significantly affected free radical scavengers, indicating its potential as an antioxidative agent. Many common antioxidants have been effective in reducing neuroinflammation [11], but STE has not studied in activated murine microglial BV-2 cells. Our results present STE's strong potential in reducing neuroinflammatory activity in various neurodegenerative disorders.
In BV-2 microglia cells, NO is generated by the inducible isoform of NO synthase (iNOS), and has been identified as a neurotoxic substance contributing to central nervous system inflammation [13]. High levels of NO are produced from L-arginine by iNOS activation in the brain, which prolongs microglial cell activation, and this mechanism is associated with the progression of various neuro-degenerative diseases [14]. Our results clearly show that STE's effect on two elements of this mechanism, as STE attenuates LPS-induced iNOS expression and decreases NO production. STE acts principally on NO generation by downregulating iNOS gene expression at the posttranscriptional level, and can thus prevent the progression of neuro-inflammation.
STE treatment's significant effects on TNF-α expression also indicate STE's therapeutic potential to treat chronic neuroinflammatory diseases. TNF-α is a pro-inflammatory cytokine that initiates the inflammatory response, and its over-production is a possible etiological factor of most neurological disorders [16]. Microglial cell activation by LPS produces various cytokines, including TNF-α, which in turn attracts neutrophils and causes the accumulation of neutrophil-secreted proteases and ROS at sites of inflammation. Data from our study shows that STE attenuates production of TNF-α, an initiator of the inflammatory response, thereby inhibiting NO production and iNOS expression levels.

CONCLUSION
These results demonstrate that STE has antiinflammatory properties in LPS-induced BV-2 microglial activation through the down-regulation of inflammation-related gene expression, including iNOS and the proinflammatory cytokine, TNF-α. STE's ability to down-regulate key proteins involved in the neuroinflammatory response, even combined with LPS, shows its strong potential as a therapeutic agent. STE can be considered as an effective therapeutic and preventative herbal extract for the treatment of several neurodegenerative and oxidative stressrelated diseases.