Salicornia bigelovii Torr Attenuates Neuro-Inflammatory Responses in Lipopolysaccharide-Induced BV-2 Microglia by Regulation of NF-kappa B Signaling

Purpose: To investigate the anti-oxidant and anti-neuroinflammatory effects of Salicornia bigelovii extract (SBE) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Methods: Anti-oxidant activity was measured using 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay. Cell viability was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium bromide (MTT) assay. BV- microglial cells were stimulated with LPS to study the protein expression and production of inflammatory mediators, determined by Western blot analysis. Results: SBE significantly inhibited the DPPH-generated free radicals showing maximum inhibition at 40 µg/mL (p < 0.001). SBE alone did not exhibit any signs of cytotoxicity to BV-2 cells up to 200 µg/mL concentration. The LPS-induced increase in the production of nitric oxide was concentration-dependently suppressed by SBE (p < 0.05 for 10 µg/mL, p < 0.01 at 20 µg/mL and p < 0.001 at 40 µg/mL, respectively). SBE also inhibited the LPS-induced increase in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions. Further, the production of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6 by LPS-stimulation in BV-2 cells was inhibited by SBE pretreatment. Mechanistic study revealed that SBE acts by regulation of nuclear factor kappa-B signaling pathway in LPS-stimulated BV-2 microglial cells. Conclusion: This study revealed for the first time that SBE possesses anti-oxidant and anti-neuroinflammatory effects and can be developed as a potential therapeutic target in ameliorating microglia-mediated neuroinflammation.


INTRODUCTION
Salicornia bigelovii Torr. (S. bigelovii) commonly known as "Dwarf saltwort" from the family Amaranthaceae, is a leafless annual salt-marsh herb with green jointed and succulent stems [1]. S. bigelovii has an exceptional salt tolerance, adaptation to marginal lands and hot climates, therefore has great potential as a domesticated biomass, oilseed, and forage crop plant [2]. S. bigelovii has been successfully cultivated as an oilseed and vegetable crop in the desert coastlines of Mexico, India, the Middle East, Africa and in Southeast China [3]. The seed is rich in oil (30%) and protein (35%) with a high content of polyunsaturated linoleic (75%) and linolenic (omega-3) fatty acids. In addition to its value in human diet, the oil can be used for the production of biodiesel [4]. Earlier studies suggested that Salicornia species has been used as a folk medicine to treat a variety of diseases such as atherosclerosis, hypertension, tumors and claimed as one of the most promising halophytes as immunomodulators [3,5]. However, till date there have been no reports on the antioxidant and anti-neuroinflammatory properties of S. bigelovii.
Microglia, the resident immune cells of the central nervous system, become activated thereby inducing significant and highly detrimental neurotoxic effects by excessively producing a large array of cytotoxic and proinflammatory factors [6]. Microglia-mediated neuroinflammation appears to play an essential role in the pathogenesis of various neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis [7]. Previous reports strongly demonstrated that regulation of microglial activation could reduce neuroinflammation and further neuronal cell damage [8].
Lipopolysaccharide (LPS) is a common toxin used to investigate the impact of inflammation on neuronal death. LPS can directly activate microglia triggering the production of inflammatory mediators, such as nitric oxide (NO), cyclooxygenase (COX)-2, proinflammatory cytokines and leukotrienes [9]. Therefore, LPS-induced inflammatory mediators in vitro can be considered as one of the important tools to evaluate new and existing agents for their anti-neuroinflammatory actions. Recent studies have shown a convincing link between reactive oxygen species (ROS) and neuroinflammation. Inhibition of ROS by several anti-oxidants may suppress microglial activation and thus protect neuronal cell death [10,11]. In this study we evaluated the antioxidant potential and anti-neuroinflammatory properties of S. bigelovii extract in LPS-stimulated BV-2 microglial cells.

EXPERIMENTAL Preparation of S. bigelovii extract
The dried whole plant of S. bigelovii (5 kg) collected during May 2012, were purchased from a local market in South Korea and authenticated by Prof Jong-Bo Kim, a taxonomist at Konkuk University, South Korea. A voucher specimen (SB-KU2012) has been kept in our laboratory herbarium, Konkuk University, South Korea, for future reference. To obtain the S. bigelovii extract, the dried plant material was ground in a blender and defatted three times with three volumes of ethanol. The residue was extracted with absolute ethanol at 1:10 ratio (w/v) for 2 h in a heating mantle at 70 -80 °C, and the supernatant was filtered and concentrated in a vacuum evaporator system at 50°C. For further fractionation, the extract (1 kg) was partitioned into hexane, chloroform and ethyl acetate fractions to yield 220 mg, 50 mg and 456 mg, respectively. The active ethyl acetate fraction of S. bigelovii extract (SBE, 45.6%) was lyophilized and stored in a refrigerator (-20 °C) until use. SBE extract was re-dissolved in distilled water and filtered on 0.22 μm filters to evaluate its antioxidant and anti-neuroinflammatory activities.

DPPH radical scavenging activity
The anti-oxidant activity of the SBE was determined using the stable radical 2, 2diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis, MO, USA). The radical scavenging capacity was evaluated by employing a reaction mixture constituted by aliquots of the SBE extract and a DPPH methanolic solution as described previously [12]. Briefly, a sample solution of 60 µl of each OFP-EA extract, was added to 60 µl of DPPH (60 µM) in methanol. After mixing vigorously for 10 s, the mixture was then transferred into a 100 µl Teflon capillary tube and the scavenging activity of each sample on DPPH radical was measured using a JES-FA ESR spectrometer (Jeol Ltd., Tokyo, Japan). A spin adduct was measured on an ESR spectrometer exactly after 2 min. 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
The amount of stable nitrite, the end product of NO generation, by activated microglia was determined by a colorimetric assay as previously described [14]. Briefly,BV-2 cells (2 x 10 5 cells/ml) were seeded in 6-well plates in 500 μl complete culture medium and treated with the SBE extract at indicated concentrations (10, 20 and 40 μg/ml) for 1 h prior stimulation with LPS (1 μg/ml) for 2 h. 50 μl of culture supernatant was mixed with an equal volume of Griess reagent and incubated at room temperature for 10 min. The absorbance at 540 nm was read using a PowerWavex Microplate Scanning spectrophotometer (Bio-Tek Instrument, Winooski, VT, USA). Nitrite concentration was determined by extrapolation from a sodium nitrite standard curve.

IL-6 assay
BV-2 microglia cells (1 x 10 5 cells/well) were cultured on 96-well plates and treated with SBE at indicated concentrations with or without LPS (1μg/ml). At 4 h of post LPS treatment, the cells were collected and the supernatants were subjected to assay of IL-6 contents using a murine IL-6 ELISA kit from BD Biosciences (San Jose, CA, USA) according to the manufacturer's instruction.

TNF-α assay
BV-2 microglia cells (1 x 10 5 cells/well) were cultured on 96-well plates and treated with the SBE at indicated concentrations for 1 h and stimulated with LPS (1 μg/ml). At 4 h post-LPS treatment, the cells were collected and the supernatants were evaluated for TNF-α level using a murine TNF-α ELISA kit from BD Biosciences (San Jose, CA, USA) according to the manufacturer's instructions.

Statistical analysis
All data are represented as the mean ± SEM 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.

Effect of SBE extract on DPPH radical scavenging activity
As shown in Fig 1A, SBE exhibited significant DPPH radical scavenging activity in a concentration-dependent manner showing a maximum effect at 40 µg/ml of concentration (p < 0.001). The ESR spectroscopy data is represented in Fig 1B.

Effect of SBE on BV-2 microglial cell viability
As shown in Fig. 2, SBE treatment for 24 hr at various concentrations ranging from 0.1 µg/ml to 200 µg/ml did not exhibit any significant cytotoxicity on BV-2 microglial cells. Effect of SBE on DPPH radical scavenging activity. The capacity to scavenge DPPH free radical by different concentrations of SBE (A) and ESR spectra (B) was measured. BV-2 cells were treated with or without SBE at the various concentrations (10, 20, 40 and 80 µg/ml). The scavenging activity of each sample on DPPH radical was measured using a JES-FA ESR spectrometer. A spin adduct was measured on an ESR spectrometer exactly 2 min later. Data are presented as the mean ± SEM (n = 3); **p < 0.01 and ***p < 0.001, compared with control group by one-way analysis of variance, followed by Dunnett's multiple range tests. SBE = Salicornia bigelovii extract.

Effect of SBE on LPS-induced NO production in BV-2 microglial cells
As shown in Fig 3, cells treated with LPS alone significantly increased the NO levels (p < 0.001). Pretreatment with SBE (10, 20, 40 and 80 μg/ml) significantly suppressed the LPS-stimulated increased NO release in BV-2 cells in a dosedependent manner compared to LPS only treated cells. The maximum effect was observed at a concentration of 100 μg/ml (p < 0.001).

Effect of SBE on LPS-induced expression of iNOS and COX levels in BV-2 microglial cells
SBE exhibited a broad spectrum of inhibitory effect on the expression of pro-inflammatory mediators and reduced the LPS-stimulated increase of protein expression such as iNOS and inducible COX-2 in a concentration-dependent manner. However, constitutive COX-1 protein expressional levels were uninterrupted (Fig 4).

Fig 6:
Effect of SBE on pro-inflammatory cytokine IL-6 expression in LPS-stimulated BV-2 cells. BV-2 cells were treated with SBE at indicated concentrations (10, 20 and 40 µg/ml) with or without LPS (1 µg/ml) for 4 hr. The IL-6 in the culture supernatant was evaluated using a murine IL-6 ELISA kit from BD Sciences according to the manufacturer's instruction. Data are presented as the mean ± SEM (n = 3). # p<0.001, when compared with control group. *p < 0.05, **p < 0.01, ***p < 0.001, compared with LPS alone treated group by one-way analysis of variance, followed by Dunnett's multiple range tests. SBE = Salicornia bigelovii extract.

Effect of SBE on NF-κB in LPS-stimulated BV-2 microglial cells
SBE inhibited the LPS-induced phosphorylation and degradation of IκB-α, and nuclear translocation of p65 NF-κB in a concentration dependent manner (Fig 7).

DISCUSSION
In the present study, proinflammatory stimulus by LPS to BV-2 cells resulted in excessive production of NO. Earlier studies revealed that prolonged activation of microglial cells leads to increased release of NO by iNOS in the brain. NO, an important regulatory mediator involved in cell survival and death exerts a number of proinflammatory effects during several physiological and pathological processes leading to increased inflammatory reaction. It was well known that COX-1 is constitutively expressed in many cell types and COX-2 is normally not present in most cells, but its expression is induced in response to inflammatory cytokines linked to pathological events [15]. COX-2 is upregulated in response to various inflammatory stimuli including LPS in BV-2 microglia. Therefore, agents that inhibit the release of NO and attenuate iNOS and COX-2 expression could be beneficial for preventing and delaying the progression of neuroinflammatory disease. [16]. Data from our study clearly showed that SBE attenuated LPS-induced iNOS and COX-2 expression and downstream NO production. However, SBE has no influence on the constitutive COX-1 expression.
Pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 play central roles in microgliamediated inflammation [17]. In particular, increased levels of brain TNF-α and IL-6 has been associated with severe cognitive impairments, neuronal damage and neuroinflammation [17]. Therefore, the effects of SBE on proinflammatory cytokine TNF-α and IL-6 production in LPS-stimulated BV-2 microglial cells were evaluated. LPS-stimulation increased the levels of TNF-α and IL-6 in BV-2 cells.
However, pretreatment with SBE suppressed the increased TNF-α and IL-6 production indicating that SBE may convincingly be an effective antineuroinflammatory agent.
NF-κB, a mammalian transcription factor, activated by LPS, is known to control the expression of cell survival genes as well as proinflammatory enzymes and cytokines [18]. Our result showed that SBE inhibited the LPSinduced phosphorylation/degradation of IκB-α and translocation of NF-κB/p65 sub unit in a concentration-dependent manner. Considering the above data, we can conclude that NF-κB is a major target of SBE. However, the exact molecular target of SBE on NF-κB activation remains to be elucidated.
The mechanism of neuro-inflammation is partly attributed, to release of toxic free radicals and ROS from activated microglia which may participate in the neurodegenerative process. DPPH radical assay is one of the widely used methods for screening the free radical scavenging activities of several agents in a relatively short period of time. In the present study, SBE significantly scavenged the DPPH free radicals. Reports from earlier studies indicated that S. bigelovii posses active constituents such as triterpenoids, flavones, glycosides, saponins, vitamins and minerals [3,19]. Salicornia species were also reported to posses' strong anti-oxidant properties [20,21]. In light of such reports, the strong anti-oxidant activity exhibited by SBE supports the notion that SBE might play a promising role in exhibiting anti-neuroinflammatory properties in LPSstimulated BV-2 cells.

CONCLUSION
This study revealed for the first time that SBE inhibits neuro-inflammatory responses via NF-κB signaling in LPS-stimulated BV-2 microglial cells. Further, the antioxidant potential of SBE might partly be involved for the observed effects.