Production Optimization, Structural Analysis, and Prebiotic- and Anti-Inflammatory Effects of Gluco-Oligosaccharides Produced by Leuconostoc lactis SBC001

Leuconostoc lactis SBC001, isolated from chive, produces glucansucrase and synthesizes oligosaccharides through its enzymatic activity. This study was conducted to optimize oligosaccharide production using response surface methodology, analyze the structure of purified oligosaccharides, and investigate the prebiotic effect on 24 bacterial and yeast strains and the anti-inflammatory activity using RAW 264.7 macrophage cells. The optimal conditions for oligosaccharide production were a culture temperature of 30 °C and sucrose and maltose concentrations of 9.6% and 7.4%, respectively. Based on 1H-NMR spectroscopic study, the oligosaccharides were identified as gluco-oligosaccharides that consisted of 23.63% α-1,4 glycosidic linkages and 76.37% α-1,6 glycosidic linkages with an average molecular weight of 1137 Da. The oligosaccharides promoted the growth of bacterial and yeast strains, including Lactobacillus plantarum, L. paracasei, L. johnsonii, Leuconostoc mesenteroides, L. rhamnosus, and Saccharomyces cerevisiae. When lipopolysaccharide-stimulated RAW 264.7 cells were treated with the oligosaccharides, the production of nitric oxide was decreased; the expression of inducible nitric oxide synthase, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-10 was suppressed; and the nuclear factor-kappa B signaling pathway was inhibited. In conclusion, the gluco-oligosaccharides obtained from Leu. lactis SBC001 exhibited a prebiotic effect on six bacterial and yeast strains and anti-inflammatory activity in RAW 264.7 macrophage cells.


Introduction
The metabolic activities and microbial composition of intestinal microbiota are associated with a variety of diseases [1]. The intestinal microbiota maintain the normal function of the human digestive system and establish host resistance to pathogens in the intestinal tract [2]. Probiotics are defined as "a live microbial supplement that beneficially affects the host animal by improving its intestinal microbial balance" [3]. Probiotics have beneficial effects on the host such as the suppression of infectious diseases and promotion of digestion and the intestinal flora balance [1,4]. Probiotics include lactic acid bacteria (LAB) such as Lactobacillus sp. and bifidobacteria as well as some yeast species such as Saccharomyces cerevisiae var. boulardii [3].
Some probiotic strains produce oligosaccharides that cannot be digested by human digestive enzymes. These non-digestible oligosaccharides can be used as prebiotics that stimulate the growth of probiotic strains [5]. Oligosaccharides are low molecular weight carbohydrates with a degree of polymerization (DP) from 3 to 10, and prebiotic oligosaccharides consist of inulin, fructo-oligosaccharides (FOSs), galacto-oligosaccharides, isomaltooligosaccharides (IMOs), mannan-oligosaccharides, soybean oligosaccharides, and lac-tulose [6,7]. Oligosaccharides, as prebiotics, improve the growth of lactobacilli and bifidobacteria and act as precursors of the production of short-chain fatty acids (SCFAs), and these SCFAs, such as acetate, lactate, propionate, and butyrate, maintain the homeostasis of the host, including the enhancement of immune function and improvement of the bioavailability of minerals [8][9][10][11][12][13][14].
Leuconostoc sp. is a heterofermentative LAB that produces lactic acid as well as diacetyl, CO 2 , acetoin, acetate, ethanol, and 2, 3-butylenglycol [18]. Leuconostoc sp. is generally found in raw milk and cheese and used as a starter in the dairy industry [19]. Although numerous studies have investigated the oligosaccharides produced by Leu. mesenteroides, only a few studies have been conducted on the oligosaccharides produced by Leu. lactis [16,17,[20][21][22].
Leu. lactis SBC001, which is isolated from chives, produces oligosaccharides in good yield [23]. This strain exhibits glucansucrase activity and produces oligosaccharides using sucrose and maltose as a donor molecule and receptor molecule, respectively [23].
In the present study, we determined the optimum conditions of oligosaccharides production by Leu. lactis SBC001 using response surface methodology (RSM) and also evaluated their structure. We also investigated the prebiotic effect of oligosaccharides obtained from Leu. lactis SBC001 on the growth of microorganisms and the anti-inflammatory effect of oligosaccharides in RAW 264.7 macrophage cells in vitro using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis.

Purification of Oligosaccharides
Oligosaccharides produced by Leu. lactis SBC001 were purified as described in a previous study [17]. The culture supernatant was concentrated 10-fold at 60 • C using a rotary evaporator (SB-1200, EYELA, Miyagi, Japan), and the concentrate was loaded onto Bio-gel P2 resin (Bio-Rad Laboratories, Inc., Hercules, CA, USA) packed in a glass Econo-Column (1.5 × 120 cm, Bio-Rad Laboratories, Inc.). The elutes were collected using a fraction collector (Gilson Inc., Middleton, WI, USA) with a fraction volume of 5 mL per tube with a flow rate of 0.5 mL/min. The fractions were analyzed by thin-layer chromatography (TLC), and the oligosaccharide fractions were pooled and freeze-dried using a freeze dryer (SunilEyela, Seongnam, Korea). The freeze-dried powder was dissolved in distilled water at a concentration of 1% (w/v).

HPAEC-PAD Analysis
The purified oligosaccharides produced by Leu. lactis SBC001 were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (DX 500 Chromatography System, Dionex, Sunnyvale, CA, USA). The sample injection volume was 50 µL, and the oligosaccharides were analyzed using a CarboPac PA-1 column (4 × 250 mm, Dionex) and a CarboPac PA-1 guard column (4 × 50 mm, Dionex). The mobile phase was 150 mM sodium hydroxide for the first 15 min and 600 mM sodium acetate (in 150 mM sodium hydroxide) for the next 5 min, followed by 150 mM sodium hydroxide for the final 10 min at a flow rate of 1.0 mL/min.

Size exclusion HPLC Analysis
The molecular weight of the oligosaccharides was determined using high-performance liquid chromatography (HPLC) (UltiMate TM 3000 RSLCnano system, Thermo Fisher Scientific, Inc., Waltham, MA, USA), with an OHpak SB-802.5 column (8.0 × 300 mm, Shodex, New York, NY, USA). The oligosaccharides were dissolved in distilled water at a concentration of 100 mg/mL, and 100 µL of sample was injected for analysis and detected using an refractive index (RI) (RI-101, Shodex). The column mobile phase was distilled water, the flow rate was 0.4 mL/min, and the column oven temperature was 35 • C. Glucose polymer (DP 1-8, Carbosynth Co.) was used as a standard sugar.

Analysis of Monosaccharide Composition
The oligosaccharides were hydrolyzed by heating at 121 • C for 2 h in 4 M trifluoroacetic acid (TFA). TFA was then removed by N 2 gas flow, and the monosaccharide composition of the oligosaccharides was analyzed by HPAEC-PAD and TLC.

Analysis of Linkage Ratio by 1 H-NMR Spectroscopy
The relative abundance of α-1,4 and α-1,6 linkages in the oligosaccharides was determined by 400 MHz 1 H-NMR spectroscopy (JeolJNM-LA400 with LFG, JEOL, Tokyo, JAPAN) and Delta NMR Processing and Control Software version 5.3 (JEOL USA, Inc., Peabody, MA, USA). The oligosaccharides (10 mg/mL) were dissolved in deuterium oxide (D 2 O) and freeze-dried. The oligosaccharides were then dissolved in D 2 O (10 mg/mL) and analyzed by 1 H-NMR operated at 80 • C. . These strains were cultured in MRS medium in the presence or absence of sugar or prebiotics at 37 • C for 48 h. All lactic acid bacteria were cultured using a capped tube without agitation, and all Bifidobacterium strains were cultured in anaerobic condition using the GasPak TM system (BD). S. cerevisiae KCCM 50549 and Zygosaccharomyces rouxii KCCM 12066 were also obtained from KCCM and cultured using yeast extract malt extract (YM) medium (BD), which consisted of 10 g/L glucose, 3 g/L malt extract, 5 g/L peptone, and 3 g/L yeast extract, at 30 • C for 48 h.

Media for Bacterial and Yeast Strains for the Determination of Prebiotic Effect
To determine the prebiotic effect of oligosaccharides, bacterial and yeast strains were incubated with medium in the presence or absence of sugar or oligosaccharides as a sole carbon source. Modified MRS (m-MRS) and modified YM (m-YM), which had no carbon sources, were prepared as described in a previous study [17]. For LAB, m-MRS was used as a negative control, and m-MRS containing 1% (w/v) dextrose was used as a positive control. For yeast strains, modified YM (m-YM) was used as a negative control, and YM containing 1% (w/v) dextrose was used as a positive control. Oligosaccharides were added to the liquid growth medium at a concentration of 1% (w/v). FOSs (from chicory, Cat. # F8052, Sigma-Aldrich) were also added to the growth medium at a concentration of 1% (w/v) as a reference.

Determination of Viable Cell Number of Bacterial and Yeast Strains
A colony of LAB and Bifidobacterium sp. and a colony of yeast were seeded in MRS broth and YM broth, respectively, and incubated at 37 • C and at 30 • C at 250 rpm, respectively, for 24 h. Culture broth (1% (v/v)) was inoculated into the above-described media (described in Section 2.5.2). The culture broth was sampled at 0, 6, 12, 24, and 48 h. Bifidobacterium sp. and LAB were spread onto an MRS agar plate, and yeast cells were spread onto a YM agar plate. After the plates were incubated at 37 • C for 24 h, the viable cell number was calculated by counting the number of colonies on each plate and expressed as log CFU/mL.

cDNA Synthesis and Real-time PCR
Real-time PCR was used to measure the expression levels of inducible nitric oxide synthase (iNOS) gene, cytokine genes, including interleukin (IL)-1β and IL-6, and tumor necrosis factor (TNF)-α gene, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as the housekeeping gene. After treating RAW 264.7 cells, which were cultured in a 6-well plate with oligosaccharides for 1 h, RAW 264.7 cells were treated with LPS (1 µg/mL) for 24 h and RNA was isolated using the easy-BLUE TM Total RNA Extraction kit (iNtRON Biotechnology, Inc., Seongnam, Korea) according to the manufacturer's instructions. Subsequently, cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Real-time PCR was conducted according to the manufacturer's instructions (LightCycler96, Roche, Basel, Switzerland) using the FastStart Essential DNA Green Master Kit (Roche, Basel, Switzerland). The primer sequences used for the analysis of mRNA expression are shown in Table 1. The expression levels of cytokine mRNA relative to those of GAPDH mRNA were analyzed using the 2 −ddCT method. RAW 264.7 cells were plated in a 6-well plate (1 × 10 6 cells/well), pretreated with various concentrations (10, 100, and 1000 µg/mL) of oligosaccharides for 1 h, and then treated with 1 µg/mL LPS as a control for 24 h. After treatments, the cells were washed with phosphate-buffered saline (PBS) and lysed with radioimmunoprecipitation assay (RIPA) cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). The supernatant was collected after centrifugation at 14,000× g for 10 min at 4 • C. The protein content was measured using the BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Proteins (50 µg) for each sample were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5% skim milk at room temperature for 2 h and then incubated overnight with primary antibodies (1:1000; Cell Signalling Technology, Danvers, MA, USA) at 4 • C. After washing three times with Tris-buffered saline containing 0.5% Tween 20 (TBST), the membranes were treated with the corresponding secondary antibody (1:3000; anti-rabbit IgG, Cell Signalling Technology) at 37 • C for 2 h. The membranes were again washed three times with TBST, after which the chemiluminescent signals emitted after incubation with secondary antibodies were detected with EzWestLumi, plus chemiluminescent substrate (ATTO Corporation, Tokyo, Japan), using Odyssey LCI Image software (LI-COR Biosciences, Lincoln, NE, USA).

Statistical Analysis
Data were expressed as mean ± standard deviation (SD) from triplicate experiments. Statistical analyses were conducted using SPSS 23 (SPSS Inc., Chicago, IL, USA). Statistical significance between groups was determined by a one-way analysis of variance (ANOVA), followed by Duncan's multiple range test (p < 0.05).

Optimization of Oligosaccharide Production
Leu. lactis SBC001 was isolated from chives in our laboratory and selected as an oligosaccharide-producing strain by detecting the formation of slimy colonies on sucrosecontaining agar medium and by measuring the glucansucrase activity [23].
To optimize the conditions for oligosaccharide production by Leu. lactis SBC001, RSM was used with a central composite design. From the preliminary experiments, the initial pH, sucrose concentration, and culture temperature were chosen as factors for optimization. As shown in Table 2, when the initial pH (X 1 ) was 7.0, sucrose concentration (X 2 ) was 0.4 M and culture temperature (X 3 ) was 29 • C, the maximum SBC-oligosaccharide production was obtained, with 209.44 as a relative peak area. The mathematical regression equation was given as follows: where Y denotes oligosaccharide production, X 1 is the initial pH, X 2 is the sucrose concentration, and X 3 indicates the culture temperature. The appropriateness of the experimental model used in this study was confirmed assuming that a significance of 0.000 was suitable for the model, and in the lack-of-fit test, the significance was 0.000 (Table 3). Figure 1 shows the three-dimensional plot for the effect of each variable on the production of oligosaccharides from Leu. lactis SBC001. Using these optimum conditions, oligosaccharides were produced by Leu. lactis SBC001 and purified by Bio-gel P2 size exclusion column chromatography. The resulting purified oligosaccharides had a DP of 4 to 9, as shown in Figure 1D. A previous study demonstrated that the maximum production of gluco-oligosaccharides from Leu. lactis CCK940 could be obtained using a culture temperature of 30 • C and sucrose and maltose concentrations of 0.28 and 0.22 M, respectively, as determined by the RSM study [13].

Analysis of the Monosaccharide Composition of Oligosaccharides
The molecular weight of the purified oligosaccharides was measured by size exclusion chromatography. Among the oligosaccharides, the molecular weight of the oligosaccharide with the highest concentration was determined as 1137 Da, which corresponded to DP 7 of glucose unit (Figure 2). In the previous study on gluco-oligosaccharides produced by Leu. lactis CCK940, the molecular weight of the oligosaccharide with the highest concentration was 942 Da, which was smaller than that of the oligosaccharides produced by Leu. lactis SBC001 [17]. It has also been reported that the molecular weight range of chitosan oligosaccharides was 1000-1600 Da [24], and that of pectin oligosaccharides was 1000-3000 Da [25]. As shown in Figure 3, the hydrolysates consisted of glucose, which indicated that the oligosaccharides produced by Leu. lactis SBC001 were gluco-oligosaccharides that consisted of only glucose. The oligosaccharides produced by Leu. lactis CCK940 were also gluco-oligosaccharides [17].

1 H-NMR Analysis
1 H-NMR spectroscopy is a useful method for the determination of linkage type, DP, and structural analysis of polysaccharides and oligosaccharides [33,34]. When the linkage ratio of the oligosaccharides produced by Leu. lactis SBC001 was analyzed by 1 H-NMR spectroscopy, the chemical shifts of oligosaccharides, maltose, and waxy corn starch (WCS) were at 5.2-6.0 ppm, which are characteristic of the α-linkages ( Figure S1). The results demonstrated that the oligosaccharides produced by Leu. lactis SBC001 consisted of 23.63% α-1,4 linkages and 76.37% α-1,6 linkages (Table 4).

Prebiotic Effect of Oligosaccharides on the Growth of Bacterial and Yeast Strains
To determine the prebiotic effect of oligosaccharides produced by Leu. lactis SBC001, 24 representative bacterial and yeast strains were selected and their growth was determined using the modified culture media supplemented with different carbon sources. Among the 24 strains, six strains, including L. plantarum, L. paracasei, L. johnsonii, Leu. mesenteroides, L. rhamnosus, and S. cerevisiae, showed higher viable cell numbers in glucose-free MRS supplemented with oligosaccharides at 48 h of incubation when compared with FOS that was used as a reference ( Figure 5). For example, the viable cell numbers of L. plantarum were 8.90 ± 0.18 and 8.40 ± 0.28 log CFU/mL in oligosaccharide medium and FOS medium, respectively, at 48 h ( Figure 5A). The viable cell numbers of strains were significantly increased by the supplementation of oligosaccharides compared with glucose-free MRS or glucose-free YM during 48 h of incubation. In the case of S. cerevisiae, the viable cell numbers were 8.09 ± 0.07 and 8.47 ± 0.33 log CFU/mL in oligosaccharide medium and FOS medium, respectively, at 48 h, which showed the prebiotic effect of oligosaccharides on yeast cells ( Figure 5F). The results demonstrated that the oligosaccharides produced by Leu. lactis SBC001 exhibited a higher prebiotic effect on some strains than FOSs that are widely used as prebiotics. In the previous study, the gluco-oligosaccharides produced by Leu. lactis CCK940 exhibited prebiotic effects on Lactobacillus casei, L. pentosus, L. plantarum, W. cibaria, B. animalis, and S. cerevisiae, which was different from this study result [17]. Several other studies have shown that each prebiotic oligosaccharide has its own prebiotic effect on specific probiotic strains [36][37][38]. The results of these studies indicate that the prebiotic effects of lactic acid strains vary depending on the type and structure of the oligosaccharides produced.

Effect of Oligosaccharides on RAW 264.7 Cell Viability and NO Production
Because some oligosaccharides exhibit anti-inflammatory activity on macrophages, we explored the anti-inflammatory effect of the oligosaccharides produced by Leu. lactis SBC001 on RAW 264.7 macrophage cells.
The viability of RAW 264.7 cells was analyzed in the presence of different concentrations of purified oligosaccharides. When RAW 264.7 cells were treated with the oligosac-charides, the oligosaccharides did not exert cytotoxic effects on RAW 264.7 cells up to a concentration of 1000 µg /mL ( Figure 6). Therefore, oligosaccharide concentrations of 10, 100, and 1000 µg/mL were used for further experiments. NO is a free radical molecule synthesized from L-arginine by NO synthase; the production of NO promotes the inflammatory response, and NO inhibitors can prevent the inflammatory response [39]. NO is not stable, and in the presence of water and oxygen, it converted into nitrite and nitrate, which can be detected using Griess reagent. When the cells were treated with LPS alone, NO production was stimulated, and its relative amount was set to 100%. The production of NO was decreased by 20% upon treatment with oligosaccharides at a concentration of 1000 µg/mL, whereas treatment with 1 mg/mL FOS did not decrease the NO production ( Figure 6). This result indicated that the oligosaccharides produced by Leu. lactis SBC001 exhibited a higher anti-inflammatory activity than FOSs. FOSs are oligosaccharides which consist of fructose residues with a glucose residue at the end of the chain linked by beta-(2,1) glycosidic bonds, the structure of which is quite different from oligosaccharides produced by Leu. lactis SBC001. Even though their structural properties are different, FOSs also have anti-inflammatory activity similar to oligosaccharides produced by Leu. lactis SBC001 [40,41]
The activation of iNOS, inducible NO synthase, has been demonstrated to contribute to septic shock [43]. iNOS inhibitors regulate as therapeutic agents to influence gastrointestinal diseases and arthritis [44]. IL-6 acts as a multifunctional cytokine that increases phagocytosis and complements production and is defined as a B cell differentiation factor [45]. TNF-α, an important mediator cytokine, is involved in host defense against pathogens [46]. TNF-α induces apoptosis and is involved in the development of humoral immune response. IL-1β is released by macrophages and plays a role in the pathophysiology of rheumatoid arthritis [47].
In the present study, the expression of iNOS and other proinflammatory cytokines was evaluated in RAW 264.7 cells treated with the oligosaccharides produced by Leu. lactis SBC001. As depicted in Figure 7, the mRNA expression levels of TNF-α, IL-1β, and IL-6 decreased in a dose-dependent manner after treatment with the oligosaccharides. Moreover, the iNOS mRNA expression levels were reduced after oligosaccharide treatment. These results indicated that the anti-inflammatory activity of the oligosaccharides was due to the repression of iNOS and other proinflammatory cytokines; however, the underlying mechanism needs to be elucidated in a further study. Relative mRNA expression of inducible NO synthase (iNOS) and inflammatory cytokines upon treatment with oligosaccharides in RAW 264.7 cells. RAW 264.7 cells were pretreated with oligosaccharides for 1 h and then stimulated with 1 µg/mL LPS. One-way ANOVA was used to compare group mean values, followed by Duncan's t-test. Different letters represent significant differences among groups at p < 0.05 (n ≥ 3).

Effect of Oligosaccharides on the MAPK Signaling Pathway of RAW 264.7 Cells
Nuclear factor-kappa B (NF-κB) is an important target transcription factor in the inflammatory response [48]. The NF-κB signaling pathway plays an important role in the LPS-stimulated expression of inflammatory-associated genes and cytokines and is associated with toll-like receptor 4 (TLR4) that activates the downstream signaling pathways [49]. The inhibitor of κB (IκB) that is maintained in the cytoplasm regulates the activation of NF-κB. When LPS stimulates the degradation of IκB, phospho-IκB (p-IκB) results in the nuclear translocation of NF-κB. NF-κB in the nucleus stimulates the transcription of proinflammatory genes such as iNOS, TNF-α, IL-1β, and IL-6 [50].
In the present study, RAW 264.7 cells were treated with the oligosaccharides produced by Leu. lactis SBC001, and the expression of NF-κB and IκB was evaluated ( Figure 8A). Treatment with oligosaccharides suppressed the expression of NF-κB and IκB in a dosedependent manner. The mitogen-activated protein kinase (MAPK) signaling pathways include extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), c-Jun N-terminal kinases (JNKs), and p38 MAPK [46]. The MAPK signaling pathways are associated with cell growth, differentiation, and death through regulation of the expression of numerous genes, and the LPS-stimulated expression of inflammatory mediators, including iNOS, COX-2, and inflammatory cytokines, is involved in the MAPK signaling pathways [51]. In addition, inhibition of MAPK phosphorylation can reduce inflammatory diseases. As shown in Figure 8B, when LPS-stimulated RAW 264.7 cells were pre-treated with the oligosaccharides, the expression of phosphorylated p38, ERK, and JNK was decreased. Based on these results, it is predicted that treatment of RAW 264.7 cells with the oligosaccharides reduces the inflammatory response by suppressing the inflammatory-associated genes and cytokines in the NF-κB signaling pathway and MAPK signaling pathways.
Numerous studies have reported about the anti-inflammatory activities of oligosaccharides. Yoon et al. demonstrated that chitosan-oligosaccharides (COS) suppressed the LPS-induced secretion of TNF-α and IL-6 as well as NO in the medium, and this antiinflammatory effect of COS was achieved by the modulation of the TNF-α pathway [52]. Pangestuti et al. reported about the effects of COS on the modulation of inflammatory mediators in LPS-stimulated BV microglial cells. In their study, it was shown that COS suppressed the production of NO and prostaglandin E2 by inhibiting the expression of iNOS and COX-2, and this effect was due to the suppression of the phosphorylation of JNK and p38 MAPK [53]. Chung also reported that Eubacterium eligens, a species belonging to Firmicutes, utilizes apple pectin as a prebiotic source and promotes the production of the anti-inflammatory cytokine IL-10 in host cells [54]. Although some studies have explored the immunomodulatory activity of gluco-oligosaccharides, there has been no study on the anti-inflammatory activity of gluco-oligosaccharides [16].

Conclusions
There are several oligosaccharides that exhibit prebiotic effects on bacterial and yeast strains such as Lactobacillus sp., Bifidobacterium sp., and S. cerevisiae. There are also several oligosaccharides that exhibit anti-inflammatory or immunomodulatory activity. However, to our knowledge, there has been no report on the anti-inflammatory activity of glucooligosaccharides produced by Leu. lactis, and this is the first report on this aspect. FOSs are one of the most used prebiotic oligosaccharides worldwide, and it is meaningful that the prebiotic and anti-inflammatory activities of gluco-oligosaccharides produced by Leu. lactis SBC001 are higher than those of FOSs. This implies that gluco-oligosaccharides produced by Leu. lactis SBC001 could be used as a substitute for FOSs. It is predicted that their anti-inflammatory activity was due to the reduction in the inflammatory response by suppressing the inflammatory-associated genes and cytokines in the NF-κB signaling pathway and the MAPK signaling pathways.