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Involvement of Nrf2-mediated heme oxygenase-1 expression in anti-inflammatory action of chitosan oligosaccharides through MAPK activation in murine macrophages
European Journal of Pharmacology xx (xxxx) xxxx–xxxx
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Involvement of Nrf2-mediated heme oxygenase-1 expression in antiinflammatory action of chitosan oligosaccharides through MAPK activation in murine macrophages Jun-Ho Hyunga,1, Chang-Bum Ahnb,1, Boo IL Kimc, Kyunghoi Kimd, Jae-Young Jea,
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a
Department of Marine-Bio Convergence Science, Pukyong National University, Busan 48547, Republic of Korea Division of Food and Nutrition, Chonnam National University, Gwangju 61186, Republic of Korea c Specialized Graduate School of Science & Technology Convergence, Pukyong National University, Busan 48547, Republic of Korea d Depatment of Ocean Engineering, Pukyong National University, Busan 48513, Republic of Korea b
A R T I C L E I N F O
A BS T RAC T
Keywords: Chitosan oligosaccharides HO-1 Nrf2 MAPKs Anti-inflammatory
Chitosan and its derivatives have been reported to have anti-inflammatory effects in vitro and in vivo. It is also suggested that chitosan and its derivatives could be up-regulating heme oxygenase-1 (HO-1) in different models. However, the up-regulation of HO-1 by chitosan oligosaccharides (COS) remains unexplored in regard to anti-inflammatory action in lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells). Treatment with COS induced HO-1 expression in LPS-stimulated RAW264.7 cells, whereas the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased. Pretreatment with ZnPP, a HO-1 inhibitor, reduced the COS-mediated anti-inflammatory action. HO-1 induction is mediated by activating the nuclear translocation of NF-E2-related factor 2 (Nrf2) using COS. Moreover, COS increased the phosphorylation of extracellular signal regulated kinase (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), and p38 MAPK. However, specific inhibitors of ERK, JNK, and p38 reduced COSmediated nuclear translocation of Nrf2. Therefore, HO-1 induction also decreased in RAW264.7 cells. Collectively, COS exert an anti-inflammatory effect through Nrf2/MAPK-mediated HO-1 induction.
1. Introduction Inflammation is one of the protective responses that lead to the upregulation of various enzymes and signaling proteins in stimulated cells including macrophages, mononuclear phagocytes, and neutrophils (Abarikwu, 2014; Ahn et al., 2016). Upon stimulation, a series of inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), and interleukin-6 (IL-6) are secreted by inflammatory proteins and stimuli such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and LPS through the activation of transcription factors (Ham et al., 2015; Hayden and Ghosh, 2004; Li and Verma, 2002; Udenigwe et al., 2013). Uncontrolled production of inflammatory mediators leads to inflammatory-mediated diseases such as rheumatoid, arthritis, and atherosclerosis, thus the modulation of inflammatory mediators is an important therapeutic target (Abarikwu, 2014; Heo et al., 2010; Park et al., 2011). Inducible heme oxygenase-1 (HO-1) catalyzes the degradation of
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proinflammatory free heme to carbon monooxide, biliverdin, and ferrous ion (Paine et al., 2010). HO-1 is rapidly induced by a wide variety of stimuli and participates in regulating cell survival, oxidative stress and inflammation (Lee et al., 2010; Paine et al., 2010). A number of studies have suggested that HO-1 has major anti-inflammatory properties that have been demonstrated in HO-1 knockout animal models and human cases with genetic deficiencies (Poss and Tonegawa, 1997; Yachie et al., 1999). Recent studies suggested that natural products could up-regulate HO-1 through activating NF-E2-related factor 2 (Nrf2) (Hu et al., 2009; Jun et al., 2011; Lee et al., 2010). Chitosan, a naturally occurring biopolymer, is composed of glucosamine and N-acetyl-glucosamine units and exhibits unique biological properties such as being biodegradable, biocompatible, and less-toxic. Chitosan oligosaccharides (COS) are derivatives of chitosan through enzymatic or acidic hydrolysis and also exhibit biological activities. Recent advances have provided evidence that chitosan and COS may be useful as therapeutic agents in inflammation (Kim et al., 2002; Liu et al., 2011; Qiao et al., 2011; Wei et al., 2012; Yoon et al., 2007; Yousef
Corresponding author. E-mail address: [email protected] (J.-Y. Je). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejphar.2016.11.002 Received 9 May 2016; Received in revised form 4 November 2016; Accepted 4 November 2016 Available online xxxx 0014-2999/ © 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Hyung, J-H., European Journal of Pharmacology (2016), http://dx.doi.org/10.1016/j.ejphar.2016.11.002
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et al., 2012). Both chitosan and COS exert anti-inflammatory effects by suppressing the production of NO, PGE2, and pro-inflammatory cytokines, and also suppress the expression of iNOS and COX-2 through inactivating nuclear factor-κB (NF-κB) via mitogen-activated protein kinases (MAPKs). However, to the best of our knowledge, there is no information regarding the involvement of HO-1 induction mediated by COS or chitosan in the anti-inflammatory effects of either COS or chitosan in LPS-stimulated murine macrophages. Therefore, the objective of this study is to elucidate the involvement of HO-1 induction by COS in anti-inflammatory action and to determine the underlying signaling pathways.
rated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% bovine serum albumin or skim milk for 1 h. The membranes were incubated with primary antibody overnight at 4 °C. Blots were washed three times with TBS-T (trisbuffered saline containing 0.1% Tween 20) and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h. Blots were developed for visualization using a chemiluminescence (ECL) detection kit (Thermo Scientific, Inc.). Band intensity was determined by densitometry using the public domain software Image J.
2. Materials and methods
The data are presented as the mean ± standard deviation (S.D.) of at least three independent experiments (n=3). Differences between the means of each group were assessed by one-way analysis of variance followed by Duncan's test using PASW Statistics 19.0 software (SPSS, Chicago, IL, USA). A P-value < 0.05 was considered statistically significant.
2.6. Statistical analysis
2.1. Materials COS (an average molecular weight of 3.5 kDa and 90% degree of deacetylation) were kindly donated by Kitto Life Co. (Seoul, Korea). Lipopolysaccharides (LPS, from Escherichia coli serotype 026: B6), Griess reagents, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Znpp, antibodies for HO-1, iNOS, COX-2, Nrf2, TNF-α, IL-6, IL-1β, ERK1/2, JNK1/2, and p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL Co. (Grand Island, NY, USA).
3. Result 3.1. COS increased HO-1 expression but decreased iNOS and COX-2 expression in LPS-stimulated murine macrophages The cytotoxic effect of COS was evaluated prior to determining the anti-inflammatory effect and the COS did not show cytotoxicity in murine macrophages (Fig. 1). To investigate the expression of HO-1, iNOS, and COX-2 in LPS-stimulated murine macrophages, cells were pretreated with COS for 1 h followed by addition of LPS and incubated for 24 h. As shown in Fig. 2, HO-1 induction was not detected in the non-treatment cells but was slightly increased by LPS treatment. However, the COS plus LPS cells showed dramatically increased HO1 expression in LPS-stimulated murine macrophages. In basal murine macrophages, iNOS and COX-2 proteins were not detected but LPS treatment caused a significant increase in iNOS and COX-2 induction. However, COS treatment resulted in the decrease of iNOS and COX-2 induction by LPS. The expression pattern of HO-1 by COS was completely different compared to the expression of iNOS and COX-2 in LPS-stimulated murine macrophages.
2.2. Cell culture RAW264.7 cells (murine macrophage) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in DMEM supplemented with 10% FBS and 100 U/ml penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2. 2.3. MTT assay Cells were seeded into a 96-well plate at a density of 5×103 cells/ well and left to adhere for 24 h. The cells were incubated with COS (1– 4 mg/ml) for 24 h. One hundred microliters of MTT solution (1 mg/ml) was added to each well and further incubated for 4 h. The formed formazan crystals in viable cells were dissolved in DMSO and absorbance was measured at 540 nm.
3.2. Effects of COS on HO-1, NO, and pro-inflammatory cytokines in the presence of ZnPP To investigate whether HO-1 induction by COS is involved in the anti-inflammatory effect, the protein levels of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 were examined in the presence of ZnPP (5 µm), a HO-1 inhibitor. As shown in Fig. 3A, HO-1 induction
2.4. Nitric oxide (NO) quantification assay
100 Cell viability (%)
NO level in the culture medium was measured by Griess reagent. Cells were treated with COS for 1 h followed by the addition of LPS (1 μg/ml) for 24 h. The culture supernatant (50 μl) was mixed with 50 μl Griess reagent and then incubated for 20 min. The absorbance was measured using a microplate reader at 540 nm. The NO concentration was determined using a standard curve from sodium nitrite. 2.5. Western blot analysis After COS treatment with or without LPS exposure, total proteins were prepared by using RIPA buffer (Sigma Chemical Co.) containing protease and phosphatase inhibitor cocktails (Roche Diagnostics, Seoul, Korea). Nuclear extracts were prepared using a nuclear extraction kit (NE-PER™ Nuclear and Cytoplasmic Extraction Reagents, Thermo Scientific, Inc. Rockford, IL, USA) according to the manufacturer's protocol. Protein concentration was determined by using a BCA protein assay kit (Thermo Scientific, Inc.). Protein (20 μg) was sepa-
80 60 40 20 0
0
1 2 COS (mg/ml)
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Fig. 1. Effect of chitosan oligosaccharides (COS) on cell viability. Data represents mean ± S.D. of three independent experiments.
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Fig. 2. Effects of chitosan oligosaccharides (COS) on HO-1, iNOS, and COX-2 expression in LPS-stimulated RAW264.7 cells. Cells were treated with COS prior to LPS exporsure for 24 h and protein extracts were subjected to Western blot analysis. The relative density was calculated as the ratio of each protein expression to β-actin expression. * P < 0.05 vs. non-treatment.
by COS was significantly decreased in the presence of ZnPP. TNF-α, IL1β, and IL-6 expression induced by LPS treatment was significantly increased compared to the non-treatment group but this induction was attenuated by COS treatment. However, the inhibitory effects of COS against the production of TNF-α, IL-1β, and IL-6 were decreased by treatment with ZnPP. The concentration of NO, which is produced by iNOS, was also determined and found to be increased by LPS treatment (Fig. 3B). The COS plus LPS cells showed decreased NO production; however this effect was decreased in the presence of ZnPP, indicating that HO-1 induction by COS is an important factor affecting the antiinflammatory effect in LPS-stimulated murine macrophages. 3.3. Effects of COS on Nrf2 and MAPK expression Fig. 3. Effects of chitosan oligosaccharides (COS) on HO-1, TNF-α, IL-1β, and IL-6 expression in the presense of ZnPP (HO-1 inhibitor) in LPS-stimulated RAW264.7 cells (A). Nitric oxide (NO) quantification (B). Cells were treated with ZnPP (5 µm) for 2 h prior to COS and LPS exporsure for 24 h and protein extracts were subjected to Western blot analysis. NO from the medium was measured using Griess reagent. The relative density was calculated as the ratio of each protein expression to β-actin expression. * P < 0.05 vs. LPS treatment.
Induction of HO-1 is primarily mediated through nuclear translocation of Nrf2. Therefore, the effect of COS on Nrf2 translocation in murine macrophages was investigated in order to better understand Nrf2-mediated HO-1 induction by COS. As shown in Fig. 4A, COS treatment increased Nrf2 translocation into the nucleus compared to the non-treatment but decreased cytoplasmic Nrf2, indicating that HO1 induction was mediated through nuclear translocation of Nrf2 by COS. MAPK pathways are considered a major mechanism in inflammation and are involved in the regulation of the HO-1 and Nrf2 signaling pathway. Therefore, we examined the effect of COS on the phosphorylation of three MAPKs including p38, ERK1/2, and JNK1/2 in murine macrophages. For time-course experiments, the cells were treated with
COS for 0, 15, 30, 60, and 120 min. The phosphorylation of p38, ERK1/2, and JNK1/2 occurred after treatment with COS and peaked at 15 min (Fig. 4B).
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Fig. 4. Effects of chitosan oligosaccharides (COS) on Nrf2 (A) and MAPKs expression (B) in RAW264.7 cells. Cells were treated with COS for 2 h (Nrf2) and for the indicated time (MAPKs), then the nuclear and cytoplasmic fraction for Nrf2 and the total proteins for MAPKs were subjected to Western blot analysis. * P < 0.05 vs. non-treatment.
κB) and its up-stream signal molecules such as ERK1/2, JNK1/2, and p38. However, the mechanism through which HO-1 induction by chitosan and its derivatives regulates inflammatory mediators and the anti-inflammatory effects are still not elucidated. In this study, therefore, we examined the potential involvement of HO-1 induction by COS in the anti-inflammatory effect in LPS-stimulated murine macrophages. HO-1 is rapidly induced by oxidative stress or other exogenous stimuli such as LPS and is generally expressed at low levels without stimuli (Khodagholi et al., 2010). It is reported that LPS treatment rapidly increased accumulation of intracellular reactive oxygen species such as hydrogen peroxide, superoxide anion, and hydroxyl radicals (Haddad and Land, 2002). As expected, treatment with LPS alone markedly increased HO-1 expression. HO-1 induction by COS was evaluated and COS was found to significantly increase HO-1 expression but inhibited the expression of iNOS and COX-2 proteins in LPSstimulated murine macrophages (Fig. 2). The expression patterns of HO-1 and inflammatory proteins (iNOS and COX-2) were exactly opposite. It is well-characterizied that HO-1 induction has an important role in cytoprotection in different cell types. Recent studies suggested that HO-1 is involved in the modulation of inflammatory responses in murine macrophages and HO-1 induction is achieved by several naturally occurring compounds (Hu et al., 2009; Jin et al., 2012; Kim et al., 2010; Park et al., 2011). Therefore, we investigated whether HO-1 induction by COS is implicated in anti-inflammatory action in LPS-stimulated murine macrophages. Pretreatment with
3.4. COS induce Nrf2-mediated HO-1 expression through MAPK pathway In order to verify which MAPK molecules involved in Nrf2mediated HO-1 induction by COS, we used the specific inhibitors SB203580 (p38 inhibitor), PD98059 (ERK inhibitor), and SP600125 (JNK inhibitor). 10 µm of the inhibitors were treated for 1 h before treatment with COS for 2 h (Nrf2) or 24 h (HO-1). The nuclear fraction and total proteins were subjected to Western blot analysis. As shown in Fig. 5A, the specific inhibitors of three MAPKs reduced COS-mediated Nrf2 translocation into the nucleus in murine macrophages. Similar to Nrf2, HO-1 induction by COS was also reduced by treatment with three MAPK inhibitors (Fig. 5B). These results suggest that phosphorylation of three MAPKs is an important role in the anti-inflammatory effect of COS in murine macrophages.
4. Discussion Previous reports demonstrated that chitosan and its derivatives exhibit anti-inflammatory effects in vitro and in vivo (Ahn et al., 2016; Lee et al., 2009; Qiao et al., 2011; Wei et al., 2012; Yoon et al., 2007; Yousef et al., 2012). They exerted anti-inflammatory effects by suppressing inflammatory mediators as well as through iNOS and COX-2 expression in LPS-stimulated cell culture and animal models. The inhibition was achieved by the inactivation of transcription factor (NF4
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Fig. 5. Effects of chitosan oligosaccharides (COS) on Nrf2 and HO-1 expression in the presence of MAPKs inhibitors. Cells were treaed with SB203580 (p38 inhibitor), PD98059 (ERK inhibitor), or SP600125 (JNK inhibitor) for 1 h and then treated with COS for 2 h (Nrf2) or 24 h (HO-1). Nuclear fraction for Nrf2 and the total proteins for HO-1 were subjected to Western blot analysis. The relative density was calculated as the ratio of each protein expression to lamin B or β-actin expression. * P < 0.05 vs. COS treatment.
prooxidant, and natural products (Lee et al., 2010, 2009; Paine et al., 2010). In this study, we demonstrated that COS-induced Nrf2 accumulation in the nucleus leads to the induction of HO-1 in murine macrophages. MAPKs play a centrol role in the regulating cellular signaling pathways that are involved in cell death, apoptosis, and inflammation (Nakagawa and Maeda, 2012). It is known that the activation of MAPKs regulates Nrf2-mediated HO-1 induction (Furukawa et al., 2010; Kim et al., 2010). Thus, we examined the influence of COS on the phosphorylation of ERK1/2, JNK1/2, and p38, COS induced the phosphorylation of ERK1/2, JNK1/2, and p38. Further, we examined the role of MAPK phosphorylation by using specific MAPK inhibitors in Nrf2 translocation and HO-1 expression. Co-treatment with COS and specific inhibitors of ERK1/2, JNK1/2, and p38 reduced Nrf2 translocation into the nucleus (Fig. 5A), thereby the expression of HO-1 was decreased in murine macrophages (Fig. 5B). There results indicate that suppression one of MAPKs is crucial role in decreasing COS-induced anti-inflammatory action because Nrf2 translocation followed by activation of HO-1 expression was inhibited by suppressing the phosphorylation of MAPKs. An array of studies regarding the anti-inflammatory effects of chitosan and its derivatives have been conducted including the modulation of inflammatory proteins and pro-inflammatory cytokines. In addition, chitosan and its derivatives inactivated the nuclear translocation of NF-κB through the blockade of MAPKs phosphorylation in LPS-stimulated murine macrophages. Although extensive studies were performed previously, we present the first evidence that HO-1 induction by COS plays an important role in anti-inflammation in LPS-stimulated murine macrophages and that COS may reduce inflammatory mediators during exposure to LPS or other inflammatory stimuli. Moreover COS induced Nrf2/MAPK-mediated HO-1 induction in normal condition (without LPS stimulation), indicating that daily consumption of COS may be helpful for preventing inflammation in the human body.
Fig. 6. Proposed mechanism for anti-inflammatory action of chitosan oligosaccharides (COS).
ZnPP reduced HO-1 induction by COS and the inhibitory effects of COS on LPS-stimulated NO as well as pro-inflammatory cytokine production were found to be abolished by ZnPP (Fig. 3). These results suggest that the inhibition of HO-1 attenuates the anti-inflammatory effects of COS and the induction of HO-1 is important in COS-mediated antiinflammatory action. The transcription factor Nrf2 is a master regulatory protein and an upstream modulator of HO-1 expression. Sequestered Nrf2 in the cytoplasm is activated by liberating the Nrf2/Keap1 complex. The liberated Nrf2 migrates into the nucleus, binds to the antioxidant response element and induces the expression of target genes such as HO-1 (Kansanen et al., 2013). Nrf2 accumulation in the nucleus is achieved with extra- and intracellular stimuli such as oxidative stress,
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Ethnopharmacol. 133, 524–530. Kansanen, E., Kuosmanen, S.M., Leinonen, H., Levonen, A.L., 2013. The Keap1-Nrf2 pathway: mechanisms of activation and dysregulation in cancer. Redox Biol. 1, 45–49. Khodagholi, F., Eftekharzadeh, B., Maghsoudi, N., Rezaei, P.F., 2010. Chitosan prevents oxidative stress-induced amyloid β formation and cytotoxicity in NT2 neurons: involvement of transcription factors Nrf2 and NF-κB. Mol. Cell. Biochem. 337, 39–51. Kim, K.C., Kang, K.A., Zhang, R., Piao, M.J., Kim, G.Y., Kang, M.Y., Lee, S.J., Lee, N.H., Surh, Y.J., Hyun, J.W., 2010. Up-regulation of Nrf2-mediated heme oxygenase-1 expression by eckol, a phlorotannin compound, through activation of Erk and PI3K/ Akt. Int. J. Biochem. Cell Biol. 42, 297–305. Kim, M.S., Sung, M.J., Seo, S.B., Yoo, S.J., Lim, W.K., Kim, H.M., 2002. Water-soluble chitosan inhibits the production of pro-inflammatory cytokine in human astrocytoma cells activated by amyloid beta peptide and interleukin-1beta. Neurosci. Lett. 321, 105–109. Lee, D.S., Jeong, G.S., Li, B., Park, H., Kim, Y.C., 2010. Anti-inflammatory effects of sulfuretin from Rhus verniciflua Stokes via the induction of heme oxygenase-1 expression in murine macrophages. Int. Immunopharmacol. 10, 850–858. Lee, S.H., Senevirathne, M., Ahn, C.B., Kim, S.K., Je, J.Y., 2009. Factors affecting antiinflammatory effect of chitooligosaccharides in lipopolysaccharides-induced RAW264.7 macrophage cells. Bioorg. Med. Chem. Lett. 19, 6655–6658. Li, Q., Verma, I.M., 2002. NF-kappaB regulation in the immune system. Nat. Rev. Immunol. 2, 725–734. Liu, H.T., Huang, P., Ma, P., Liu, Q.S., Yu, C., Du, Y.G., 2011. Chitosan oligosaccharides suppress LPS-induced IL-8 expression in human umbilical vein endothelial cells through blockade of p38 and Akt protein kinases. Acta Pharmacol. Sin. 32, 478–486. Nakagawa, H., Maeda, S., 2012. Molecular mechanisms of liver injury and hepatocarcinogenesis: focusing on the role of stress-activated MAPK. Pathol. Res. Int. 2012, 172894. Paine, A., Eiz-Vesper, B., Blasczyk, R., Immenschuh, S., 2010. Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential. Biochem. Pharmacol. 80, 1895–1903. Park, S.Y., da, Park, Kim, J., Kim, Y.H., Kim, Y., Shon, S.G., Choi, K.J., Lee, Y.W., S.J, 2011. Upregulation of heme oxygenase-1 via PI3K/Akt and Nrf-2 signaling pathways mediates the anti-inflammatory activity of Schisandrin in Porphyromonas gingivalis LPS-stimulated macrophages. Immunol. Lett. 139, 93–101. Poss, K.D., Tonegawa, S., 1997. Reduced stress defense in heme oxygenase 1-deficient cells. Proc. Natl. Acad. Sci. USA 94, 10925–10930. Qiao, Y., Bai, X.F., Du, Y.G., 2011. Chitosan oligosaccharides protect mice from LPS challenge by attenuation of inflammation and oxidative stress. Int. Immunopharmacol. 11, 121–127. Udenigwe, C.C., Je, J.Y., Cho, Y.S., Yada, R.Y., 2013. Almond protein hydrolysate fraction modulates the expression of proinflammatory cytokines and enzymes in activated macrophages. Food Funct. 4, 777–783. Wei, P., Ma, P., Xu, Q.S., Bai, Q.H., Gu, J.G., Xi, H., Du, Y.G., Yu, C., 2012. Chitosan oligosaccharides suppress production of nitric oxide in lipopolysaccharide-induced N9 murine microglial cells in vitro. Glycoconj. J. 29, 285–295. Yachie, A., Niida, Y., Wada, T., Igarashi, N., Kaneda, H., Toma, T., Ohta, K., Kasahara, Y., Koizumi, S., 1999. Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J. Clin. Investig. 103, 129–135. Yoon, H.J., Moon, M.E., Park, H.S., Im, S.Y., Kim, Y.H., 2007. Chitosan oligosaccharide (COS) inhibits LPS-induced inflammatory effects in RAW 264.7 macrophage cells. Biochem. Biophys. Res. Commun. 358, 954–959. Yousef, M., Pichyangkura, R., Soodvilai, S., Chatsudthipong, V., Muanprasat, C., 2012. Chitosan oligosaccharide as potential therapy of inflammatory bowel disease: therapeutic efficacy and possible mechanisms of action. Pharmacol. Res. 66, 66–79.
In summary, the present results demonstrate that COS exert antiinflammatory effects in LPS-stimulated murine macrophages through the up-regulation of HO-1. This occurs through the phosphorylation of MAPKs, which are responsible for the nuclear translocation of Nrf2 and subsequent the up-regulating of HO-1 expression (Fig. 6). Conflict of interests The authors declare that there is no conflict of interests. Acknowledgment This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation of Korea (NRF2014H1C1A1066586). References Abarikwu, S.O., 2014. Kolaviron, a natural flavonoid from the seeds of Garcinia kola, reduces LPS-induced inflammation in macrophages by combined inhibition of IL-6 secretion, and inflammatory transcription factors, ERK1/2, NF-kappaB, p38, Akt, pc-JUN and JNK. Biochim. Biophys. Acta 1840, 2373–2381. Ahn, C.B., Jung, W.K., Park, S.J., Kim, Y.T., Kim, W.S., Je, J.Y., 2016. Gallic Acid-gChitosan Modulates Inflammatory Responses in LPS-Stimulated RAW264.7 Cells Via NF-kappaB, AP-1, and MAPK Pathways. Inflammation 39, 366–374. Furukawa, Y., Urano, T., Minamimura, M., Nakajima, M., Okuyama, S., Furukawa, S., 2010. 4-Methylcatechol-induced heme oxygenase-1 exerts a protective effect against oxidative stress in cultured neural stem/progenitor cells via PI3 kinase/Akt pathway. Biomed. Res. 31, 45–52. Haddad, J.J., Land, S.C., 2002. Redox/ROS regulation of lipopolysaccharide-induced mitogen-activated protein kinase (MAPK) activation and MAPK-mediated TNF-α biosynthesis. Br. J. Pharm. 135, 520–536. Ham, Y.-M., Ko, Y.-J., Song, S.-M., Kim, J., Kim, K.-N., Yun, J.-H., Cho, J.-H., Ahn, G., Yoon, W.-J., 2015. Anti-inflammatory effect of litsenolide B2 isolated from Litsea japonica fruit via suppressing NF-kB and MAPK pathways in LPS-induced RAW264.7 cells. J. Funct. Foods 17, 434–448. Hayden, M.S., Ghosh, S., 2004. Signaling to NF-kappaB. Genes Dev. (18), 2195–2224. Heo, S.K., Yi, H.S., Yun, H.J., Ko, C.H., Choi, J.W., Park, S.D., 2010. Ethylacetate extract from Draconis Resina inhibits LPS-induced inflammatory responses in vascular smooth muscle cells and macrophages via suppression of ROS production. Food Chem. Toxicol. 48, 1129–1136. Hu, C.M., Liu, Y.H., Cheah, K.P., Li, J.S., Lam, C.S., Yu, W.Y., Choy, C.S., 2009. Heme oxygenase-1 mediates the inhibitory actions of brazilin in RAW264.7 macrophages stimulated with lipopolysaccharide. J. Ethnopharmacol. 121, 79–85. Jin, G.H., Park, S.Y., Kim, E., Ryu, E.Y., Kim, Y.H., Park, G., Lee, S.J., 2012. Antiinflammatory activity of Bambusae Caulis in Taeniam through heme oxygenase-1 expression via Nrf-2 and p38 MAPK signaling in macrophages. Environ. Toxicol. Pharmacol. 34, 315–323. Jun, M.S., Ha, Y.M., Kim, H.S., Jang, H.J., Kim, Y.M., Lee, Y.S., Kim, H.J., Seo, H.G., Lee, J.H., Lee, S.H., Chang, K.C., 2011. Anti-inflammatory action of methanol extract of Carthamus tinctorius involves in heme oxygenase-1 induction. J.
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Involvement of Nrf2-mediated heme oxygenase-1 expression in antiinflammatory action of chitosan oligosaccharides through MAPK activation in murine macrophages Jun-Ho Hyunga,1, Chang-Bum Ahnb,1, Boo IL Kimc, Kyunghoi Kimd, Jae-Young Jea,
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a
Department of Marine-Bio Convergence Science, Pukyong National University, Busan 48547, Republic of Korea Division of Food and Nutrition, Chonnam National University, Gwangju 61186, Republic of Korea c Specialized Graduate School of Science & Technology Convergence, Pukyong National University, Busan 48547, Republic of Korea d Depatment of Ocean Engineering, Pukyong National University, Busan 48513, Republic of Korea b
A R T I C L E I N F O
A BS T RAC T
Keywords: Chitosan oligosaccharides HO-1 Nrf2 MAPKs Anti-inflammatory
Chitosan and its derivatives have been reported to have anti-inflammatory effects in vitro and in vivo. It is also suggested that chitosan and its derivatives could be up-regulating heme oxygenase-1 (HO-1) in different models. However, the up-regulation of HO-1 by chitosan oligosaccharides (COS) remains unexplored in regard to anti-inflammatory action in lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells). Treatment with COS induced HO-1 expression in LPS-stimulated RAW264.7 cells, whereas the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased. Pretreatment with ZnPP, a HO-1 inhibitor, reduced the COS-mediated anti-inflammatory action. HO-1 induction is mediated by activating the nuclear translocation of NF-E2-related factor 2 (Nrf2) using COS. Moreover, COS increased the phosphorylation of extracellular signal regulated kinase (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), and p38 MAPK. However, specific inhibitors of ERK, JNK, and p38 reduced COSmediated nuclear translocation of Nrf2. Therefore, HO-1 induction also decreased in RAW264.7 cells. Collectively, COS exert an anti-inflammatory effect through Nrf2/MAPK-mediated HO-1 induction.
1. Introduction Inflammation is one of the protective responses that lead to the upregulation of various enzymes and signaling proteins in stimulated cells including macrophages, mononuclear phagocytes, and neutrophils (Abarikwu, 2014; Ahn et al., 2016). Upon stimulation, a series of inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), and interleukin-6 (IL-6) are secreted by inflammatory proteins and stimuli such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and LPS through the activation of transcription factors (Ham et al., 2015; Hayden and Ghosh, 2004; Li and Verma, 2002; Udenigwe et al., 2013). Uncontrolled production of inflammatory mediators leads to inflammatory-mediated diseases such as rheumatoid, arthritis, and atherosclerosis, thus the modulation of inflammatory mediators is an important therapeutic target (Abarikwu, 2014; Heo et al., 2010; Park et al., 2011). Inducible heme oxygenase-1 (HO-1) catalyzes the degradation of
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proinflammatory free heme to carbon monooxide, biliverdin, and ferrous ion (Paine et al., 2010). HO-1 is rapidly induced by a wide variety of stimuli and participates in regulating cell survival, oxidative stress and inflammation (Lee et al., 2010; Paine et al., 2010). A number of studies have suggested that HO-1 has major anti-inflammatory properties that have been demonstrated in HO-1 knockout animal models and human cases with genetic deficiencies (Poss and Tonegawa, 1997; Yachie et al., 1999). Recent studies suggested that natural products could up-regulate HO-1 through activating NF-E2-related factor 2 (Nrf2) (Hu et al., 2009; Jun et al., 2011; Lee et al., 2010). Chitosan, a naturally occurring biopolymer, is composed of glucosamine and N-acetyl-glucosamine units and exhibits unique biological properties such as being biodegradable, biocompatible, and less-toxic. Chitosan oligosaccharides (COS) are derivatives of chitosan through enzymatic or acidic hydrolysis and also exhibit biological activities. Recent advances have provided evidence that chitosan and COS may be useful as therapeutic agents in inflammation (Kim et al., 2002; Liu et al., 2011; Qiao et al., 2011; Wei et al., 2012; Yoon et al., 2007; Yousef
Corresponding author. E-mail address: [email protected] (J.-Y. Je). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejphar.2016.11.002 Received 9 May 2016; Received in revised form 4 November 2016; Accepted 4 November 2016 Available online xxxx 0014-2999/ © 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Hyung, J-H., European Journal of Pharmacology (2016), http://dx.doi.org/10.1016/j.ejphar.2016.11.002
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et al., 2012). Both chitosan and COS exert anti-inflammatory effects by suppressing the production of NO, PGE2, and pro-inflammatory cytokines, and also suppress the expression of iNOS and COX-2 through inactivating nuclear factor-κB (NF-κB) via mitogen-activated protein kinases (MAPKs). However, to the best of our knowledge, there is no information regarding the involvement of HO-1 induction mediated by COS or chitosan in the anti-inflammatory effects of either COS or chitosan in LPS-stimulated murine macrophages. Therefore, the objective of this study is to elucidate the involvement of HO-1 induction by COS in anti-inflammatory action and to determine the underlying signaling pathways.
rated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% bovine serum albumin or skim milk for 1 h. The membranes were incubated with primary antibody overnight at 4 °C. Blots were washed three times with TBS-T (trisbuffered saline containing 0.1% Tween 20) and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h. Blots were developed for visualization using a chemiluminescence (ECL) detection kit (Thermo Scientific, Inc.). Band intensity was determined by densitometry using the public domain software Image J.
2. Materials and methods
The data are presented as the mean ± standard deviation (S.D.) of at least three independent experiments (n=3). Differences between the means of each group were assessed by one-way analysis of variance followed by Duncan's test using PASW Statistics 19.0 software (SPSS, Chicago, IL, USA). A P-value < 0.05 was considered statistically significant.
2.6. Statistical analysis
2.1. Materials COS (an average molecular weight of 3.5 kDa and 90% degree of deacetylation) were kindly donated by Kitto Life Co. (Seoul, Korea). Lipopolysaccharides (LPS, from Escherichia coli serotype 026: B6), Griess reagents, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Znpp, antibodies for HO-1, iNOS, COX-2, Nrf2, TNF-α, IL-6, IL-1β, ERK1/2, JNK1/2, and p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL Co. (Grand Island, NY, USA).
3. Result 3.1. COS increased HO-1 expression but decreased iNOS and COX-2 expression in LPS-stimulated murine macrophages The cytotoxic effect of COS was evaluated prior to determining the anti-inflammatory effect and the COS did not show cytotoxicity in murine macrophages (Fig. 1). To investigate the expression of HO-1, iNOS, and COX-2 in LPS-stimulated murine macrophages, cells were pretreated with COS for 1 h followed by addition of LPS and incubated for 24 h. As shown in Fig. 2, HO-1 induction was not detected in the non-treatment cells but was slightly increased by LPS treatment. However, the COS plus LPS cells showed dramatically increased HO1 expression in LPS-stimulated murine macrophages. In basal murine macrophages, iNOS and COX-2 proteins were not detected but LPS treatment caused a significant increase in iNOS and COX-2 induction. However, COS treatment resulted in the decrease of iNOS and COX-2 induction by LPS. The expression pattern of HO-1 by COS was completely different compared to the expression of iNOS and COX-2 in LPS-stimulated murine macrophages.
2.2. Cell culture RAW264.7 cells (murine macrophage) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in DMEM supplemented with 10% FBS and 100 U/ml penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2. 2.3. MTT assay Cells were seeded into a 96-well plate at a density of 5×103 cells/ well and left to adhere for 24 h. The cells were incubated with COS (1– 4 mg/ml) for 24 h. One hundred microliters of MTT solution (1 mg/ml) was added to each well and further incubated for 4 h. The formed formazan crystals in viable cells were dissolved in DMSO and absorbance was measured at 540 nm.
3.2. Effects of COS on HO-1, NO, and pro-inflammatory cytokines in the presence of ZnPP To investigate whether HO-1 induction by COS is involved in the anti-inflammatory effect, the protein levels of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 were examined in the presence of ZnPP (5 µm), a HO-1 inhibitor. As shown in Fig. 3A, HO-1 induction
2.4. Nitric oxide (NO) quantification assay
100 Cell viability (%)
NO level in the culture medium was measured by Griess reagent. Cells were treated with COS for 1 h followed by the addition of LPS (1 μg/ml) for 24 h. The culture supernatant (50 μl) was mixed with 50 μl Griess reagent and then incubated for 20 min. The absorbance was measured using a microplate reader at 540 nm. The NO concentration was determined using a standard curve from sodium nitrite. 2.5. Western blot analysis After COS treatment with or without LPS exposure, total proteins were prepared by using RIPA buffer (Sigma Chemical Co.) containing protease and phosphatase inhibitor cocktails (Roche Diagnostics, Seoul, Korea). Nuclear extracts were prepared using a nuclear extraction kit (NE-PER™ Nuclear and Cytoplasmic Extraction Reagents, Thermo Scientific, Inc. Rockford, IL, USA) according to the manufacturer's protocol. Protein concentration was determined by using a BCA protein assay kit (Thermo Scientific, Inc.). Protein (20 μg) was sepa-
80 60 40 20 0
0
1 2 COS (mg/ml)
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Fig. 1. Effect of chitosan oligosaccharides (COS) on cell viability. Data represents mean ± S.D. of three independent experiments.
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Fig. 2. Effects of chitosan oligosaccharides (COS) on HO-1, iNOS, and COX-2 expression in LPS-stimulated RAW264.7 cells. Cells were treated with COS prior to LPS exporsure for 24 h and protein extracts were subjected to Western blot analysis. The relative density was calculated as the ratio of each protein expression to β-actin expression. * P < 0.05 vs. non-treatment.
by COS was significantly decreased in the presence of ZnPP. TNF-α, IL1β, and IL-6 expression induced by LPS treatment was significantly increased compared to the non-treatment group but this induction was attenuated by COS treatment. However, the inhibitory effects of COS against the production of TNF-α, IL-1β, and IL-6 were decreased by treatment with ZnPP. The concentration of NO, which is produced by iNOS, was also determined and found to be increased by LPS treatment (Fig. 3B). The COS plus LPS cells showed decreased NO production; however this effect was decreased in the presence of ZnPP, indicating that HO-1 induction by COS is an important factor affecting the antiinflammatory effect in LPS-stimulated murine macrophages. 3.3. Effects of COS on Nrf2 and MAPK expression Fig. 3. Effects of chitosan oligosaccharides (COS) on HO-1, TNF-α, IL-1β, and IL-6 expression in the presense of ZnPP (HO-1 inhibitor) in LPS-stimulated RAW264.7 cells (A). Nitric oxide (NO) quantification (B). Cells were treated with ZnPP (5 µm) for 2 h prior to COS and LPS exporsure for 24 h and protein extracts were subjected to Western blot analysis. NO from the medium was measured using Griess reagent. The relative density was calculated as the ratio of each protein expression to β-actin expression. * P < 0.05 vs. LPS treatment.
Induction of HO-1 is primarily mediated through nuclear translocation of Nrf2. Therefore, the effect of COS on Nrf2 translocation in murine macrophages was investigated in order to better understand Nrf2-mediated HO-1 induction by COS. As shown in Fig. 4A, COS treatment increased Nrf2 translocation into the nucleus compared to the non-treatment but decreased cytoplasmic Nrf2, indicating that HO1 induction was mediated through nuclear translocation of Nrf2 by COS. MAPK pathways are considered a major mechanism in inflammation and are involved in the regulation of the HO-1 and Nrf2 signaling pathway. Therefore, we examined the effect of COS on the phosphorylation of three MAPKs including p38, ERK1/2, and JNK1/2 in murine macrophages. For time-course experiments, the cells were treated with
COS for 0, 15, 30, 60, and 120 min. The phosphorylation of p38, ERK1/2, and JNK1/2 occurred after treatment with COS and peaked at 15 min (Fig. 4B).
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Fig. 4. Effects of chitosan oligosaccharides (COS) on Nrf2 (A) and MAPKs expression (B) in RAW264.7 cells. Cells were treated with COS for 2 h (Nrf2) and for the indicated time (MAPKs), then the nuclear and cytoplasmic fraction for Nrf2 and the total proteins for MAPKs were subjected to Western blot analysis. * P < 0.05 vs. non-treatment.
κB) and its up-stream signal molecules such as ERK1/2, JNK1/2, and p38. However, the mechanism through which HO-1 induction by chitosan and its derivatives regulates inflammatory mediators and the anti-inflammatory effects are still not elucidated. In this study, therefore, we examined the potential involvement of HO-1 induction by COS in the anti-inflammatory effect in LPS-stimulated murine macrophages. HO-1 is rapidly induced by oxidative stress or other exogenous stimuli such as LPS and is generally expressed at low levels without stimuli (Khodagholi et al., 2010). It is reported that LPS treatment rapidly increased accumulation of intracellular reactive oxygen species such as hydrogen peroxide, superoxide anion, and hydroxyl radicals (Haddad and Land, 2002). As expected, treatment with LPS alone markedly increased HO-1 expression. HO-1 induction by COS was evaluated and COS was found to significantly increase HO-1 expression but inhibited the expression of iNOS and COX-2 proteins in LPSstimulated murine macrophages (Fig. 2). The expression patterns of HO-1 and inflammatory proteins (iNOS and COX-2) were exactly opposite. It is well-characterizied that HO-1 induction has an important role in cytoprotection in different cell types. Recent studies suggested that HO-1 is involved in the modulation of inflammatory responses in murine macrophages and HO-1 induction is achieved by several naturally occurring compounds (Hu et al., 2009; Jin et al., 2012; Kim et al., 2010; Park et al., 2011). Therefore, we investigated whether HO-1 induction by COS is implicated in anti-inflammatory action in LPS-stimulated murine macrophages. Pretreatment with
3.4. COS induce Nrf2-mediated HO-1 expression through MAPK pathway In order to verify which MAPK molecules involved in Nrf2mediated HO-1 induction by COS, we used the specific inhibitors SB203580 (p38 inhibitor), PD98059 (ERK inhibitor), and SP600125 (JNK inhibitor). 10 µm of the inhibitors were treated for 1 h before treatment with COS for 2 h (Nrf2) or 24 h (HO-1). The nuclear fraction and total proteins were subjected to Western blot analysis. As shown in Fig. 5A, the specific inhibitors of three MAPKs reduced COS-mediated Nrf2 translocation into the nucleus in murine macrophages. Similar to Nrf2, HO-1 induction by COS was also reduced by treatment with three MAPK inhibitors (Fig. 5B). These results suggest that phosphorylation of three MAPKs is an important role in the anti-inflammatory effect of COS in murine macrophages.
4. Discussion Previous reports demonstrated that chitosan and its derivatives exhibit anti-inflammatory effects in vitro and in vivo (Ahn et al., 2016; Lee et al., 2009; Qiao et al., 2011; Wei et al., 2012; Yoon et al., 2007; Yousef et al., 2012). They exerted anti-inflammatory effects by suppressing inflammatory mediators as well as through iNOS and COX-2 expression in LPS-stimulated cell culture and animal models. The inhibition was achieved by the inactivation of transcription factor (NF4
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Fig. 5. Effects of chitosan oligosaccharides (COS) on Nrf2 and HO-1 expression in the presence of MAPKs inhibitors. Cells were treaed with SB203580 (p38 inhibitor), PD98059 (ERK inhibitor), or SP600125 (JNK inhibitor) for 1 h and then treated with COS for 2 h (Nrf2) or 24 h (HO-1). Nuclear fraction for Nrf2 and the total proteins for HO-1 were subjected to Western blot analysis. The relative density was calculated as the ratio of each protein expression to lamin B or β-actin expression. * P < 0.05 vs. COS treatment.
prooxidant, and natural products (Lee et al., 2010, 2009; Paine et al., 2010). In this study, we demonstrated that COS-induced Nrf2 accumulation in the nucleus leads to the induction of HO-1 in murine macrophages. MAPKs play a centrol role in the regulating cellular signaling pathways that are involved in cell death, apoptosis, and inflammation (Nakagawa and Maeda, 2012). It is known that the activation of MAPKs regulates Nrf2-mediated HO-1 induction (Furukawa et al., 2010; Kim et al., 2010). Thus, we examined the influence of COS on the phosphorylation of ERK1/2, JNK1/2, and p38, COS induced the phosphorylation of ERK1/2, JNK1/2, and p38. Further, we examined the role of MAPK phosphorylation by using specific MAPK inhibitors in Nrf2 translocation and HO-1 expression. Co-treatment with COS and specific inhibitors of ERK1/2, JNK1/2, and p38 reduced Nrf2 translocation into the nucleus (Fig. 5A), thereby the expression of HO-1 was decreased in murine macrophages (Fig. 5B). There results indicate that suppression one of MAPKs is crucial role in decreasing COS-induced anti-inflammatory action because Nrf2 translocation followed by activation of HO-1 expression was inhibited by suppressing the phosphorylation of MAPKs. An array of studies regarding the anti-inflammatory effects of chitosan and its derivatives have been conducted including the modulation of inflammatory proteins and pro-inflammatory cytokines. In addition, chitosan and its derivatives inactivated the nuclear translocation of NF-κB through the blockade of MAPKs phosphorylation in LPS-stimulated murine macrophages. Although extensive studies were performed previously, we present the first evidence that HO-1 induction by COS plays an important role in anti-inflammation in LPS-stimulated murine macrophages and that COS may reduce inflammatory mediators during exposure to LPS or other inflammatory stimuli. Moreover COS induced Nrf2/MAPK-mediated HO-1 induction in normal condition (without LPS stimulation), indicating that daily consumption of COS may be helpful for preventing inflammation in the human body.
Fig. 6. Proposed mechanism for anti-inflammatory action of chitosan oligosaccharides (COS).
ZnPP reduced HO-1 induction by COS and the inhibitory effects of COS on LPS-stimulated NO as well as pro-inflammatory cytokine production were found to be abolished by ZnPP (Fig. 3). These results suggest that the inhibition of HO-1 attenuates the anti-inflammatory effects of COS and the induction of HO-1 is important in COS-mediated antiinflammatory action. The transcription factor Nrf2 is a master regulatory protein and an upstream modulator of HO-1 expression. Sequestered Nrf2 in the cytoplasm is activated by liberating the Nrf2/Keap1 complex. The liberated Nrf2 migrates into the nucleus, binds to the antioxidant response element and induces the expression of target genes such as HO-1 (Kansanen et al., 2013). Nrf2 accumulation in the nucleus is achieved with extra- and intracellular stimuli such as oxidative stress,
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