Neoagaro-oligosaccharide monomers inhibit inflammation in LPS-stimulated macrophages through suppression of MAPK and NF-κB pathways

Neoagaro-oligosaccharides derived from agarose have been demonstrated to possess a variety of biological activities, such as anti-bacteria and anti-oxidative activities. In this study, we mainly explored the inhibitory effects and the mechanisms of neoagaro-oligosaccharide monomers against LPS-induced inflammatory responses in mouse macrophage RAW264.7 cells. The results indicated that neoagaro-oligosaccharide monomers especially neoagarotetraose could significantly reduce the production and release of NO in LPS-induced macrophages. Neoagarotetraose significantly suppressed the expression and secretion of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines such as TNF-α and IL-6. The inhibition mechanisms may be associated with the inhibition of the activation of p38MAPK, Ras/MEK/ERK and NF-κB signaling pathways. Thus, neoagarotetraose may attenuate the inflammatory responses through downregulating the MAPK and NF-κB signaling pathways in LPS-stimulated macrophages. In summary, the marine-derived neoagaro-oligosaccharide monomers merit further investigation as novel anti-inflammation agents in the future.

Neoagarotetraose significantly decreased LPS-induced production of NO in RAW264.7 cells. To evaluate the effects of neoagaro-oligosaccharides on the macrophage inflammatory responses induced by LPS, the NO production was evaluated by measuring the content of nitrite accumulated in culture medium based on the Griess reaction as previously described 8 . LPS treatment induced high nitrite production in RAW264.7 cells ( Fig. 2A), while the positive control drug acetylsalicylic acid significantly decreased the nitrite production at the concentrations of 100 and 200 μ g/ml (P < 0.05). Pretreatment with neoagaro-oligosaccharides (NA2, NA4, NA6, NA8, NA10) at indicated concentrations (62.5, 125, 250, 500 μ g/ml) for 2 h markedly decreased the nitrite production in cell culture media compared to that in LPS treated control group (Fig. 2B). Neoagarotetraose (NA4), neoagarohexaose (NA6) and neoagarooctaose (NA8) also significantly decreased the nitrite production at low concentrations (62.5 and 125 μ g/ml), and neoagarotetraose markedly decreased the nitrite production from 6.6 to about 2.5 μ M at 500 μ g/ml, superior to other neoagaro-oligosaccharide monomers (Fig. 2B). Thus, neoagarotetraose was used to further explore the inhibition mechanisms of neoagaro-oligosaccharides against LPS-induced inflammation responses in RAW264.7 cells.
Moreover, we also evaluated the effects of neoagaro-oligosaccharide monomers on RAW264.7 cells without LPS treatment, and found that neoagaro-oligosaccharide monomers did not significantly enhance the production of nitrite in RAW264.7 cells (Fig. 2C), suggesting that neoagaro-oligosaccharide monomers possess anti-inflammatory activities rather than the immune-stimulating activities in RAW264.7 cells.
Furthermore, we also tested the effects of neoagarotetraose on the levels of TNF-α and IL-6 in RAW264.7 cells without LPS treatment by ELISA, and the results showed that neoagarotetraose (62.5-500 μ g/ml) treatment could  5, 125, 250, 500 μ g/ml) for 2 h, the LPS (100 ng) was added to cells and incubated for 16 h. Then the protein levels of pro-inflammatory cytokines TNF-α (A) and IL-6 (B) in cell culture media were measured using ELISA kits in a microplate reader, respectively. Values are the means ± SD (n = 3). Significance: ## P < 0.01 vs. normal control; *P < 0.05, **P < 0.01 vs. LPS treated control. (C,D) The total mRNAs of RAW264.7 cells were collected after treatment with neoagarotetraose (62.5, 125, 250, 500 μ g/ml) for 2 h and 100 ng LPS for 16 h. Then the mRNA levels of TNF-α gene (C) and IL-6 gene (D) were detected by quantitative RT-PCR assay, respectively. The relative amounts of TNF-α and IL-6 mRNAs were determined using the comparative (2 − Δ Δ CT ) method. The mRNA levels for non-drug treated cells (Control) were assigned values of 1.
Neoagarotetraose decreased LPS-induced production of iNOS and IL-1β in RAW264.7 cells. The high levels of NO induced by stimulation with LPS are often produced by the inducible isoform of the enzyme nitric oxide synthase (iNOS) 19 . Thus, we further explored whether the inhibitory effect of neoagarotetraose on NO production was due to inhibition of iNOS expression by using real time RT-PCR and ELISA assay. As shown in Fig. 4A, LPS treatment induced upregulation of iNOS mRNA expression while pretreatment with neoagarotetraose (62.5, 125, 250, 500 μ g/ml) reduced iNOS mRNA expression in a dose-dependent manner. Neoagarotetraose significantly decreased the iNOS mRNA level when used at the concentration of > 125 μ g/ml (P < 0.05) (Fig. 4A). In addition, neoagarotetraose also significantly inhibited the LPS-induced iNOS protein expression in a dose-dependent manner at the concentrations of 125-500 μ g/ml (P < 0.05), compared to that in LPS treated control cells (Fig. 4C).
Moreover, the inhibitory effects of neoagarotetraose on the mRNA and protein levels of another cytokine IL-1β were also evaluated by quantitative RT-PCR and ELISA assay. The results showed that the mRNA levels of IL-1β significantly increased upon LPS treatment but this induction was effectively inhibited by neoagarotetraose treatment in a dose-dependent manner (Fig. 4B). Neoagarotetraose could significantly reduce the mRNA expression of IL-1β when used at the concentration > 125 μ g/ml (P < 0.05) (Fig. 4B). Similar was the case of protein expression of IL-1β in LPS-induced RAW264.7 cells. Neoagarotetraose significantly suppressed the LPS-induced IL-1β protein expression in a dose-dependent manner at the concentrations of 125-500 μ g/ml (P < 0.05), compared to that in LPS treated control cells (Fig. 4D). Thus, neoagarotetraose may reduce the production of NO through inhibiting the expression of pro-inflammatory mediators such as iNOS and IL-1β . Neoagarotetraose affected phosphorylation of MAPK in LPS-stimulated RAW 264.7 cells. It was reported that LPS treatment could stimulate cellular MAPK pathways including p38MAPK, Ras/MEK/ERK, and JNK pathways, which was associated with the inflammation responses [20][21][22][23][24] . Thus, we further investigated whether the inhibition effects of neoagarotetraose on inflammation responses were related to the MAPK signaling pathway. Firstly, the influence of neoagarotetraose on phosphorylation of p38MAPK was evaluated by western blot assay. As shown in Fig. 5A,B, LPS stimulation significantly increased the phosphorylation of p38MAPK in macrophage RAW264.7 cells compared to the normal control group (P < 0.01). Pretreatment of neoagarotetraose (125, 250, 500 μ g/ml) significantly suppressed the phosphorylation of p38MAPK from 15.2 to about 9.8, 6.4 and 2.6-fold of the normal control, respectively (P < 0.01), as compared to that in the LPS treated control group (Fig. 5B). However, neoagarotetraose did not significantly reduce the total levels of p38MAPK in LPS-activated macrophages (Fig. 5A,B).
Moreover, the influence of neoagarotetraose on Ras/MEK/ERK pathway was also evaluated by western blot. The results showed that LPS treatment significantly increased the phosphorylation of ERK1/2 compared to the normal control group (P < 0.05) but the total level of ERK1/2 in RAW264.7 cells did not significantly change (Fig. 5C,D). Pretreatment of neoagarotetraose (125, 250, 500 μ g/ml) significantly reduced the phosphorylation of ERK1/2 from 3.9 to about 3.6, 2.1 and 2.0-fold of the normal control, respectively (P < 0.05) (Fig. 5D). However, neoagarotetraose did not significantly decrease the total levels of ERK1/2, which suggested that neoagarotetraose may also inhibit the activation of Ras/MEK/ERK pathway. Taken together, MAPK signal pathways might be effectively blocked by neoagarotetraose in LPS stimulated RAW264.7 cells.
Furthermore, the influence of neoagarotetraose on JNK pathway was also evaluated by western blot. The results showed that LPS treatment significantly increased the phosphorylation of JNK compared to the normal control group (P < 0.01) but the total level of JNK in RAW cells did not significantly change (Fig. 5E,F). Pretreatment of neoagarotetraose (125, 250, 500 μ g/ml) significantly reduced the phosphorylation of JNK from 5.3 to about 4.4, 3.2 and 2.4-fold of the normal control, respectively (P < 0.01) (Fig. 5F). However, neoagarotetraose did not significantly influence the total levels of JNK, which suggested that neoagarotetraose may also inhibit the activation of JNK pathway. Taken together, MAPK signal pathways might be effectively blocked by neoagarotetraose in LPS stimulated RAW264.7 cells.

Effects of neoagarotetraose on NF-κB pathway in LPS-stimulated RAW264.7 cells. NF-κ B
pathway was reported to be related to the immune responses and inflammation responses in macrophages 25,26 . The activation of NF-κ B is often required for the upregulation of pro-inflammatory mediators, such as, iNOS, TNF-α , and IL-6, in LPS-induced RAW264.7 macrophages, so the effect of neoagarotetraose on LPS-induced NF-κ B activation was also evaluated by western blot. As shown in Fig. 6A,B, LPS stimulation significantly increased the phosphorylation of NF-κ B p65 subunit in macrophage RAW264.7 cells compared to the normal control group (P < 0.01). Pretreatment of neoagarotetraose (125, 250, 500 μ g/ml) significantly suppressed the phosphorylation of p65 from 2.5 to about 2.1, 1.8 and 1.4-fold of normal control group, respectively (P < 0.05) (Fig. 6B). However, neoagarotetraose could not significantly reduce the total levels of p65 in LPS-activated macrophages (Fig. 6A,B). Moreover, the influence of neoagarotetraose on phosphorylated IKK which was the upstream signal molecule of NF-κ B was also evaluated (Fig. 6C,D). The results showed that LPS treatment significantly increased the phosphorylation of IKK compared to the normal control group (P < 0.01) (Fig. 6C). Pretreatment of neoagarotetraose (125, 250, 500 μ g/ml) significantly reduced the phosphorylation of IKK from 1.9 to about 1.7, 1.6 and 1.5-fold of the normal control, respectively (P < 0.05) (Fig. 6D). Therefore, these results indicated a crucial role of NF-κ B signaling in the anti-inflammation actions of neoagarotetraose.
In summary, neoagarotetraose may inhibit LPS induced inflammation responses through downregulating the MAPK and NF-κ B pathways.

Discussion
Recently, agaro-oligosaccharides have been reported to possess a variety of physiological activities, such as antioxidative activities and anti-inflammation effects 12,14 , suggesting that these oligosaccharides have great potential in development of functional foods. In the present study, the inhibitory effects and mechanisms of neoagaro-oligosaccharides against LPS-induced inflammatory response were investigated. The results showed that neoagarotetraose significantly inhibited LPS-induced inflammatory responses in RAW264.7 cells. The inhibition action may be due to the reduction of LPS-induced iNOS and IL-1β expression through downregulating both MAPK and NF-κ B signaling pathways.
Inflammation is a host response to infectious microbes or injured tissues 1,27 and involves recruitment and activation of neutrophils and macrophages 2 . During this process, some toxins such as LPS can stimulate the macrophages to induce a high production of NO by the inducible enzyme iNOS 28,29 . Herein, we found that neoagarotetraose significantly inhibited the mRNA expression of iNOS and IL-1β in LPS induced RAW264.7 cells, and, thus, inhibited the production and release of NO (Fig. 4). In addition, neoagarotetraose also concentration-dependently inhibited the expression of TNF-α and IL-6 at transcription level, and reduced the production and secretion of TNF-α and IL-6 in LPS stimulated cells (Fig. 3). Therefore, neoagarotetraose possessed inhibition actions on the production of key mediators in inflammation such as iNOS, TNF-α and other cytokines in LPS-stimulated RAW 264.7 macrophages.
It was reported that LPS treatment can stimulate cellular MAPK pathways including p38MAPK, Ras/MEK/ ERK, and JNK pathways, which are associated with the inflammation responses [20][21][22][23][24] . In this study, neoagarotetraose oligosaccharide monomers inhibited the activation of p38MAPK, ERK1/2 and JNK in a dose-dependent manner in LPS-stimulated RAW264.7 cells, suggesting that neoagarotetraose may inhibit inflammation responses mainly through downregulating Ras/MEK/ERK, p38MAPK and JNK signaling pathways. It was reported that JNK signaling regulates the expression of iNOS, whereas Ras/MEK/ERK and p38MAPK signaling upregulate the production of iNOS and proinflammatory cytokines such as TNF-α and IL-6 in LPS-stimulated macrophages 20,21 . Thus, neoagarotetraose may inhibit MAPK signaling pathways to reduce the production of proinflammatory cytokines.
NF-κ B pathway was reported to be related to the immune responses and inflammation responses in macrophages 25,26 . The activation of NF-κ B which mediated by the NF-κ B translocation dependent pathway 30 or the phosphorylation of MAPK signaling 31 is required for the upregulation of pro-inflammatory mediators in LPS-induced RAW264.7 cells. In the present study, we found that neoagarotetraose significantly decreased the After pretreated with neoagarotetraose (125, 250, 500 μ g/ml) for 2 h, RAW264.7 cells were exposed to 100 ng LPS for 1 h. Then the expression levels of p38MAPK and phosphorylated p38MAPK were detected by western blot, respectively. Blots were also probed for β -actin as loading controls. The result shown is a representative of three separate experiments with similar results. (B) Quantification of immunoblot for the ratio of p38MAPK or phosphorylated p38MAPK to β -actin. The ratio for non-treated control cells was assigned a value of 1.0 and the data presented as mean ± SD (n = 3). Significance: ## P < 0.01 vs. normal control; **P < 0.01 vs. LPS treated control. (C) After treatment, the expression levels of ERK1/2 and phosphorylated ERK1/2 were detected by western blot, respectively. (D) Quantification of immunoblot for the ratio of total ERK1/2 or phosphorylated ERK1/2 to β -actin. The ratio for non-treated normal control cells was assigned a value of 1.0 and the data presented as mean ± SD (n = 3). Significance: # P < 0.05 vs. normal control; *P < 0.05 vs. LPS treated control.
SCiEnTiFiC RePoRtS | 7:44252 | DOI: 10.1038/srep44252 LPS-induced phosphorylation of p65NF-κ B rather than the expression level of total NF-κ B. Moreover, neoagarotetraose also significantly inhibited the phosphorylation of IKK in LPS treated RAW cells (Fig. 6), suggesting that neoagarotetraose may also inhibit NF-κ B pathway. Thus, neoagarotetraose may be able to inhibit both MAPK and NF-κ B signaling pathways in LPS-induced RAW264.7 cells. Moreover, some anti-inflammatory peptides were reported to be able to suppress LPS-induced activation of macrophages through the interaction with LPS 32 . It was reported that low molecular-weight oligosaccharides could be internalized into cells to affect intracellular signal pathways 33,34 . Therefore, neoagarotetraose may inhibit LPS-induced inflammatory responses through direct interaction with LPS or affecting the MAPK and NF-κ B pathways in macrophages.
In conclusion, neoagaro-oligosaccharide monomers especially neoagarotetraose substantially suppressed the pro-inflammatory mediators such as iNOS and IL-1β as well as various cytokines (IL-6 and TNF-α ) in LPS stimulated RAW264.7 cells by blocking both MAPK and NF-κ B signaling pathways. Therefore, the neoagarotetraose merits further investigation as a novel therapeutic agent against inflammation related diseases in the future.

Methods
Preparation of neoagaro-oligosaccharide monomers. Neoagarobiose, neoagarotetraose, neoagarohexaose, neoagarooctaose, and neoagarodecaose (≥ 98% purity) (Fig. 1A) were prepared in our laboratory as described previously [15][16][17][18] . β -agarases used for preparation of neoagaro-oligosaccharides were all shown in Table 1. In brief, 100 ml 0.25% low-melting point agarose and corresponding 1600 U recombinant agarase were mixed and incubated at the optimum temperature for 48 h. Then the enzyme hydrolysis solution was heated in boiling water for 10 min and concentrated using vacuum-rotary evaporation at 55 °C. The concentrate was transferred into centrifuge tubes and mixed with 3 times volume of absolute ethanol. The supernatant was collected by centrifugation After pretreated with neoagarotetraose (125, 250, 500 μ g/ml) for 2 h, the LPS (100 ng) was added to cells and incubated for 1 h. Then the expression levels of phosphorylated NF-κ B and total NF-κ B were detected by western blot analysis, respectively. Blots were also probed for β -actin as loading controls. The result shown is a representative of three separate experiments with similar results. (B) Quantification of immunoblot for the ratio of total NF-κ B and phosphorylated NF-κ B to β -actin. The ratio for non-treated normal control cells was assigned a value of 1.0 and the data presented as mean ± SD (n = 3). Significance: ## P < 0.01 vs. normal control; *P < 0.05, **P < 0.01 vs. LPS treated control. (C) After pretreated with neoagarotetraose (125, 250, 500 μ g/ml) for 2 h, the LPS (100 ng) was added to cells and incubated for 1 h. Then the levels of phosphorylated IKK were detected by western blot. Blots were also probed for GAPDH as loading controls. (D) Quantification of immunoblot for the ratio of phosphorylated IKK to GAPDH. The ratio for non-treated normal control cells was assigned a value of 1.0 and the data presented as mean ± SD (n = 3). Significance: ## P < 0.01 vs. normal control; *P < 0.05, **P < 0.01 vs. LPS treated control.
Cytotoxicity assay. The cytotoxicity of neoagaro-oligosaccharides on RAW264.7 cells was measured by MTT assay 35 . The cells were cultured in 96-well plates at density of 1 × 10 4 cells/well. After 24 h, the cells were added with 62.5, 125, 250, 500 and 1000 μ g/ml of neoagaro-oligosaccharide monomers and then incubated at 37 °C for 24 h. Then the MTT solution was added to each well and further incubated for 4 h at 37 °C. The medium was discarded and DMSO was added to dissolve the formazan dye. The optical density was determined at 540 nm. The cell viability was expressed as a percentage of non-treated control.
Measurement of nitrite in culture media. The nitrite accumulated in culture medium was measured as an indication of nitric oxide (NO) production based on the Griess reaction as previously described 8 . Briefly, on a 96-well plate, the cell supernatant (100 μ l) was mixed with the Griess reagent (100 μ l), which was prepared as follows: 1:1 (v/v) of 0.1% N-1-naphthyl-ethylenediamine in distilled water and 1% sulfanilamide in 5% phosphoric acid. After 10 min incubation, the absorbance was measured at 550 nm, and the amount of nitrite was calculated from the NaNO 2 standard curve.

Statistical analysis.
All data are representative of at least three independent experiments. All data are represented as the mean ± S.D. Statistical significance was calculated by SPSS 10.0 software using the two-tailed unpaired t-test analysis and the variance analysis (ANOVA). P < 0.05 was considered statistically significant.