Saussurea lappa Clarke extract exhibits potent antioxidant effect and attenuates neuroinflammatory responses in lipopolysaccharide-stimulated microglial cells

Purpose: To investigate the antioxidant and anti-neuroinflammatory potential of Saussurea lappa Clarke (SLC-EA) extract in LPS-stimulated BV-2 microglial cells. Methods: Cell viability was measured by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay while antioxidant activity was evaluated by using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity. Lipopolysaccharide (LPS) was used to stimulate BV-2 microglia. Griess assay was employed to assess nitric oxide (NO) production. iNOS (inducible NO synthase) expression and TNF-α (tumor necrosis factor-alpha) cytokine production were measured by ELISA (enzyme-linked immunosorbent assay) and immuno blot analysis, respectively. Results: Pretreatment of 100 mg/ml of SLC-EA (p < 0.001) was inhibited Nitric Oxide (NO) by 1 ug/ml of LPS-treated murine BV-2 cells. The expression of iNOS and TNF-α were reduced by SLC-EA concentration dependent manner (p < 0.001 at 100 mg/ml). SLC-EA were scavenged 1, 1-diphenyl-2picrylhydrazyl (DPPH) radicals in a dose-dependent manner with an IC50 value of approximately 51.4 μg/ml. Conclusion: The results indicate that SLC-EA extract exhibits strong antioxidant properties and inhibits excessive pro-inflammatory cytokine due probably to the antioxidant phenolic compounds present in SLC-EA extract. Further work in exploring the in-depth mechanisms of SLC-EA extract in regulating inflammatory signaling pathways in treating neuroinflammatory diseases is necessary.


INTRODUCTION
Stress related stimulating cell signaling and untimely generating of the cell death pathways involved in the beginning of some neurological diseases [1]. PD (Parkinson's disease), second most common degenerative dementia is a loss of dopaminergic neurons in the substantia nigra important to subsequent deficiency of dopamine in brain [2]. Accumulating indication intensely support the theory of oxidative stress and mitochondrial defect in the pathogenesis of this disease. The major mitochondrial dysfunction identified in PD is inhibition of respiratory chain complex I [2][3][4].
Microglia activation results in consequent release of different pro-inflammatory mediators and free radicals [5]. It was well documented that reduction of pro-inflammatory mediators such as NO (nitric oxide), iNOS (inducible NO synthase), TNF-α (tumor necrosis factor-alpha) and several other inflammatory cytokines in stimulated microglia could reduce the severity of these disorders [6]. LPS (Lipopolysaccharide), found in the outer membrane of Gram-negative bacteria such as Escherichia coli can activate microglia both in vitro and in vivo and releases various proinflammatory and neurotoxic factors. Therefore, LPS-stimulated microglia is a useful in vitro model for rapid screening of several antineuroinflammatory agents [7].
In current times, various herbal plants and their active physiological role have come to highlight and have been accepted for wide-ranging research. These oriental medicines have been confirmed by traditional usage as compared to current trend supplements. The oriental herbal medicines with reliable ethnopharmacological properties have been demonstrated to have neurotrophic and neuroprotective effects, which can be beneficial in preventing various forms of neuronal cell loss in brain including the nigrostriatal degeneration seen in PD [8,9]. Many reports have confirmed that medicinal herbbased extracts increase the anti-oxidative abilities of the body and can effectively slow the progress of diseases such as PD [10].
Although the health beneficial effects of olive fruit and leaf oils were confirmed in vitro using different cell lines, studies on the S. lappa extracts and their protective effect on neuroinflammatory conditions using microglial cells have not been demonstrated. In the present study the ethyl acetate fraction obtained from S. lappa Clarke (SLC-EA) was investigated for its anti-neuroinflammatory effects in LPS-stimulated BV-2 microglial cells. Further the antioxidant property was also evaluated to substantiate its anti-neuroinflammatory effects.

EXPERIMENTAL Preparation of the Saussurea lappa extract
S. lappa were purchased from the local market, Seoul, South Korea. To obtain the S. lappa extract, olives (100 g) were powdered in a mixer and washed three times with three volumes of ethanol. The residue of S. lappa (fruit pulp) was extracted with 1:10 ratio (w/v) of absolute ethanol (EtOH) for 2 h in heating block at 70 -80 °C. The ethanol extract of S. lappa obtained (the yield was 24.8 %) was resuspended in distilled water:EtOH (9:1, v/v) and separated sequentially with n-hexane, ethyl acetate (EA) and n-butanol to obtain a final production of 15.7, 43 and 21.5 %, respectively. LPS-stimulated murine BV-2 microglial cells was investigated using SLC-EA extract. SLC-EA extract was dissolved in sterile distilled water, filtered on 0.22 µm filters before use.

DPPH radical scavenging activity
The anti-oxidant activity of the SLC-EA extract was determined using the stable radical DPPH (2, 2-diphenyl-1-picrylhydrazyl, Sigma-Aldrich, St. Louis, MO, USA). The radical scavenging capacity was measured by using a reaction mixture set up by aliquots of the SLC-EA extract and a DPPH methanolic solution as described previously [19]. Briefly, a solution of 60 µL of each SLC-EA extract, was added to 60 µL of DPPH (60 µM) in absolute methanol. After mixing strongly for 10 sec, the mixture was then moved into a 100 µL Teflon capillary tube and the scavenging activity of each sample on DPPH radical was measured using a ESR spectrometer (Jeol Ltd, Tokyo, Japan). A spin adduct was measured by ESR spectrometer after 2 min. For viability assay, MTT assay was used as described previously [20].

Immunoblot analysis and antibodies
Cultured cells were washed in cold PBS three times and lysed by lysis buffer(50 mM Tris-HCl, pH 7.4, 1 % (v/v) NP-40, 0.25 % sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 25 mM NaF, 2 mM Na3VO4) and added protease inhibitor cocktail (Complete Mini TM , Roche, Mannheim, Germany) at 4°C. The lysate was cleared by centrifugation at 10,000 x g for 20 min at 4°C to remove the residue of cell pellets [20]. Cell lysates were used for protein content using BCA reagent (Pierce, Rockford, IL, USA). Equal amounts of cellular protein were loaded onto 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cellular proteins were transferred to a nitrocellulose membrain (NC) membrane (S&S, Dassel, Germany) and blocked with 5 % bovine serum albumin in trisbuffered saline (TBS, 50 mM Tris-Cl, pH 7.5. 150 mM NaCl) [20]. To detect protein expression, the blots were probed with the targeted antibodies against iNOS and TNF-α followed by the secondary antibodies coupled to horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Herculus, CA, USA). The detection of βactin protein was used for an internal control protein. The immunoreactive proteins on the NC membrane were detected by chemiluminescence by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ) on X-ray film. The antibodies against iNOS, TNF-α and β-actin were purchased from Cell Signaling Technology INC. (Beverly, MA, USA).

TNF-α assay
Murine BV-2 microglia cells (1 x 10 5 cells/well) were cultured on a flat form of 96 well plates and treated with the SLC-EA extract at indicated concentrations for 1 h and stimulated with 1 µg/mL of LPS. At 4 h of post LPS treatment, the cells were harvested and the supernatants were subjected to evaluate tumor necrosis factor --α (TNF-α) levels using murine TNF-α ELISA kit from BD Biosciences (San Jose, CA, USA).

Statistical analysis
All data are presented as mean ± SEM. Statistical significance (p < 0.05 for all analyses) was assessed by ANOVA using Instat 3.05 (GraphPad, San Diego, CA), followed by Student-Newman-Keuls analysis.

Effect of SLC-EA extract on DPPH radical scavenging activity
The capacity of SLC-EA to scavenge DPPH was measured by electron spin resonance (ESR) spectroscopy. As shown in Figure 1 A, SLC-EA showed significant DPPH radical scavenging activity in a dose-dependent manner presenting a maximum effect at 100 µg/mL of concentration (p < 0.001). The ESR spectroscopy data showed in Figure 1 B. Figure 1: Effect of SLC-EA extract on DPPH radical scavenging activity. The capacity to scavenge the free radical DPPH by different concentrations of SLC-EA extract (A) and ESR spectra (B). BV-2 cells were treated with or without SLC-EA extract at the various concentrations (10, 50 and 100 µg/mL). The scavenging activity of each sample on DPPH radical was measured using a JES-FA electron spin resonance (ESR) spectrometer. A spin adduct was measured by ESR spectrometer accurately 2 min later. Data are showed as mean ± SEM (n = 3) for three independent experiments; *p < 0.05 and ***p < 0.001, compared with control group by Student t-test. SLC-EA = Saussurea lappa-ethyl acetate

Effect of SLC-EA on BV-2 cell viability
The cytotoxicity of SLC-EA extract was evaluated based on their effects on cell growth using MTT assay (> 95 % cell viability). As shown in Figure  2, treatment with SLC-EA at concentrations ranging from 10 μg/mL to 100 μg/mL did not affect cell viability nor did they exhibit any cytotoxicity in BV2 microglia (Figure 2).

SLC-EA extract reduces NO production in LPS-stimulated murine BV-2 cells
In the next step, we examined the SLC-EA for its suppressive effects on NO release in LPSstimulated BV-2 microglia cells. As shown in Figure 3, LPS alone treated cells significantly increased the NO levels (p < 0.001). Pretreatment with SLC-EA extract significantly inhibited the LPS-stimulated increased NO release in BV-2 cells in a dosage dependent manner compared to LPS only treated cells. The maximum effect was observed at 100 µg/mL concentration (p < 0.001). The other concentrations of SLC-EA (10, 25 and 50 µg/mL) also significantly and dose-dependently inhibited the release of NO in LPS-stimulated BV-2 cells.

SLC-EA extract reduces iNOS expression in LPS-stimulated murine BV-2 cells
Immuno-blot analysis showed that the protein expression of Inducible nitric oxide synthase (iNOS) in the LPS-stimulated murine BV-2 cells was also suppressed in a concentrationdependent manner (Figure 4). Although LPSstimulated BV-2 cells are associated with the increased iNOS expression, pretreatment with SLC-EA extract suppressed the increased iNOS protein expressional levels.

Effect of SLC-EA extract on TNF-α production in LPS-stimulated BV-2 cells
The suppressive effect of SLC-EA on the expression of tumor necrosis factor-α (TNF-α) in protein levels was also confirmed by Immunoblot analysis using a targeted antibody against murine TNF-α. Murine BV-2 cell culture supernatant was harvested 4 h after administrating LPS for analysis of TNF-α production by enzyme-linked immunosorbent assay (ELISA). As shown Figure 5, the level of TNF-α increased significantly after LPS treatment when compared to those in untreated cells. However, the SLC-EA extract at indicated concentrations (10, 20, 40 and 80 µg/mL) significantly inhibited TNF-α production in a dose-dependent manner.
The SLC-EA extracted sample was filtered through a 0.45 μm poly syringe-tip filter. the sample was analyzed using a reversed-phase HPLC system (Agilent 1200, MA, USA), a quaternary pump, and a vacuum degasser. For HPLC analyses of quercetin contents in the SLC-EA, a ZORBAX C18 column (XDB-C18, 4.6×150 mm, 5μm) was used. Two mobile phases were used: solvent A, 5% acetic acid; B, 100% acetonitrile.  The analytical High-Performance Liquid Chromatography (HPLC) chromatograms of SLC-EA containing quercetin were presented in Figure 6. The calibration curves for the quercetin standards have been established. Content of quercetin in the SLC-EA was 6.103 μg/mg.

DISCUSSION
In the present study, these results showed that the ethyl acetate fraction of Saussurea lappa extract significantly inhibited the increased production of NO, suppressed the expression of iNOS protein level and attenuated the increased TNF-α production and protein expression in LPS-simulated BV-2 microglial cells. Further the SLC-EA extract significantly scavenged the DPPH free radicals.
Previous studies have been demonstrated to identify the various diseases, and to understand the physiological role involved in the cell death of dopaminergic neuronal cell in order to ascertain the mechanisms of conventional and novel agents performing through anti-oxidative and anti-apoptotic mechanisms [21]. Stimulation of neuronal cell death involving oxidative stress and mitochondrial dysfunction are the key known neurodegenerative processes in PD [22]. Therefore, inhibition of death of dopaminergic neuronal cell by adaptable intracellular reactive oxygen species (ROS) and modifying the apoptotic pathway may have therapeutic value, which may lead to mitigation of the progression of neurodegeneration.
Thus, free radicals are important mediators that provoke or sustain inflammatory responses and their neutralization by antioxidants and radical scavengers can reduce neuroinflammation. It was well known that DPPH radical assay is one of the generally used methods for calculating the free radical scavenging activities of antioxidants [23].
The beneficial effects of polyphenolic compounds are directly related to the radical scavenging capability with the formation of less-reactive flavonoid phenoxyl radicals. In our present study, the ethyl acetate fraction obtained from the Olive fruit pulp extract also exhibited significant free radical scavenging effect indicating that the fruit pulp extract also contained potential antioxidant agents.
In light of existing reports that antioxidants can reduce neuroinflammation, we used SLC-EA extract to study for its anti-neuroinflammatory activity in LPS-stimulated BV-2 cells. Significant reports now exist that inflammatory mediators such as NO and iNOS are responsible for the symptoms of various neuroinflammatory diseases. Furthermore, the neuroinflammatory response was represented by stimulated microglia, producing elevated levels of proinflammatory cytokines. Thus, suppression of cytokine production or function serves as a key role in the control of inflammatory responses in neurodegeneration. The present results that the SLC-EA extract significantly inhibited the increased NO production and iNOS protein expression in LPS-stimulated murine BV-2 cells.
TNF-α, an inflammatory cytokine, mainly produced by activated macrophages is involved in systemic inflammation that stimulate the acute phase reaction. TNF-α, not only amplifies the inflammatory cascade, but also causes the inflammatory injury. TNF-α expression was found to be upregulated in the brains of patients with neurodegenerative diseases such as AD and PD suggesting a causative role in neurodegenerative disorders [24,25]. Oxidative stress could induce neuronal damage in Parkinsonian brain, and modulate intracellular signaling, ultimately leading to neuronal cell death by apoptosis or necrosis [26]. Generation of ROS causes severe damage of cellular functions affecting the apoptotic pathway and is suggested to play an important role in the development of neurodegenerative disorders [27]. From these results, we could affirm that SLC-EA acts by suppressing TNF-α and thereby inhibiting the NO and iNOS expressional levels in LPS-stimulated BV-2 cells, which are involved in the neuroinflammatory events.
Previous studies have shown that SLC contains a large amount of two main sesquiterpene lactones, costunolide (CTL) and dehydrocostus lactone (DTL) [12]12]. This study showed quantitative HPLC chromatograms of SLC-EA containing quercetin were presented in Figure 6. From this results, SLC ethyl acetate extract contains more polyphenols.

CONCLUSION
This study demonstrates a significant role of SLC-EA in mitigating neuroinflammatory responses in LPS-stimulated BV-2 microglial cells. The SLC-EA extract also showed potent antioxidant effects and could be developed as a potential therapeutic herbal medicine for improving neurodegenerative diseases.