We previously demonstrated that 2-hydroxypropyltrimethyl ammonium chloride chitosan (HACC) promoted the production of nitric oxide (NO) and proinflammatory cytokines by activating the mitogen-activated protein kinases (MAPK) and Janus kinase (JAK)/STAT pathways in RAW 264.7 cells, indicating good immunomodulatory activity of HACC. In this study, to further investigate the immunomodulatory mechanisms of HACC, we determined the roles of phosphatidylinositol 3-kinase (PI3K)/Akt, activating protein (AP-1) and nuclear factor kappa B (NF-κB) in HACC-induced activation of RAW 264.7 cells by the western blotting. The results suggest that HACC promoted the phosphorylation of p85 and Akt. Furthermore, c-Jun and p65 were also increased after the treatment of RAW 264.7 cells with HACC, indicating the translocation of NF-κB and AP-1 from cytoplasm to nucleus. In addition, as scanning electron microscopy (SEM) analysis shows, the cell morphology changed after HACC treatment. These findings indicate that HACC activated MAPK, JAK/STAT, and PI3K/Akt signaling pathways dependent on AP-1 and NF-κB activation in RAW 264.7 cells, ultimately leading to the increase of NO and cytokines.
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Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Cantley L C. 2002. The phosphoinositide 3-kinase pathway. Science, 296 (5573): 1655–1657.
Chae H S, Kang O H, Lee Y S, Choi J G, Oh Y C, Jang H J, Kim M S, Kim J H, Jeong S I, Kwon D Y. 2009. Inhibition of LPS-induced iNOS, COX-2 and inflammatory mediator expression by paeonol through the MAPKs inactivation in RAW 264.7 cells. Am. J. Chinese Med., 37 (1): 181–194.
Cheever M L, Sato T K, de Beer T, Kutateladze T G, Emr S D, Overduin M. 2001. Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes. Nature Cell Biology3 (7): 613–618.
Chen J J, Huang W C, Chen C C. 2005. Transcriptional regulation of cyclooxygenase-2 in response to proteasome inhibitors involves reactive oxygen species-mediated signaling pathway and recruitment of CCAAT/enhancer-binding protein d and CREB-binding protein. Mol Biol Cell, 16 (12): 5579–5591.
Fang R H, Zhang L F. 2016. Nanoparticle-based modulation of the immune system. Annu Rev Chem Biomol Eng., 7 (1): 305–326.
Gugasyan R, Grumont R, Grossmann M, Nakamura Y, Pohl T, Nesic D, Gerondakis S. 2000. Rel/NF-κB transcription factors: key mediators of B-cell activation. Immunol Rev., 176 (1): 134–140.
Guha M, Mackman N. 2001. LPS induction of gene expression in human monocytes. Cellular Signalling, 13 (2): 85–94.
Gukovsky I, Gukovskaya A S, Blinman T A, Zaninovic V, Pandol S J. 1998. Early NF-κB activation is associated with hormone-induced pancreatitis. Am J Physiol., 275 (6): G1402–G1414.
Hartley J W, Evans L H, Green K Y, Naghashfar Z, Macias A R, Zerfas P M, Ward J M. 2008. Expression of infectious murine leukemia viruses by RAW264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line. Retrovirology, 5: 1.
Hattori Y, Hattori S, Kasai K. 2003. Lipopolysaccharide activates Akt in vascular smooth muscle cells resulting in induction of inducible nitric oxide synthase through nuclear factor-kappa B activation. European Journal of Pharmacology, 481 (2-3): 153–158.
Johnson G L, Lapadat R. 2002. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 298 (5600): 1911–1912.
Kao S J, Lei H C, Kuo C T, Chang M S, Chen B C, Chang Y C, Chiu W T, Lin C H. 2005. Lipoteichoic acid induces nuclear factor-kappaB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages. Immunology, 115 (3): 366–374.
Karin M, Liu Z G, Zandi E. 1997. AP-1 function and regulation. Curr. Opin. Cell Biol., 9 (2): 240–246.
Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield M D. 2001. Cellular function of phosphoinositide 3-kinases: implications for development, immunity, homeostasis, and cancer. Annual Review of Cell and Developmental Biology, 17 (1): 615–675.
Koyasu S. 2003. The role of PI3K in immune cells. Nat Immunol., 4 (4): 313–319.
Kurita K. 2006. Chitin and chitosan: functional biopolymers from marine crustaceans. Mar Biotechno., 8 (3): 203–226.
Lee C G, Da Silva C A, Lee J Y, Hartl D, Elias J A. 2008. Chitin regulation of immune responses: an old molecule with new roles. Curr. Opin. Immunol., 20 (6): 684–689.
Li K K, Shen S S, Deng X Y, Shiu H T, Siu W S, Leung P C, Ko C H, Cheng B H. 2018. Dihydrofisetin exerts its anti-inflammatory effects associated with suppressing ERK/ p38 MAPK and Heme Oxygenase-1 activation in lipopolysaccharide-stimulated RAW 264.7 macrophages and carrageenan-induced mice paw edema. International Immunopharmacology, 54: 366–374.
Li L, Wang L Y, Wu Z Q, Yao L J, Wu Y H, Huang L, Liu K, Zhou X, Gou D M. 2014. Anthocyanin-rich fractions from red raspberries attenuate inflammation in both RAW264.7 macrophages and a mouse model of colitis. Sci Rep., 4: 6234.
Li Y, Qin Y K, Liu S, Li P C, Xing R E. 2016. Preparation, characterization, and antifungal activity of hymexazol-linked chitosan derivatives. Chinese Journal of Oceanology and Limnology, 35 (5): 1079–1085.
Liang N, Sang Y X, Liu W H, Yu W L, Wang X H. 2018. Anti-Inflammatory effects of gingerol on lipopolysaccharide-stimulated RAW 264.7 cells by inhibiting NF-κB signaling pathway. Inflammation, 41 (3): 835–845.
Liaqat F, Eltem R. 2018. Chitooligosaccharides and their biological activities: a comprehensive review. Carbohydr Polym., 184: 243–259.
Ma P, Liu H T, Wei P, Xu Q S, Bai X F, Du Y G, Yu C. 2011. Chitosan oligosaccharides inhibit LPS-induced over-expression of IL-6 and TNF-α in RAW264.7 macrophage cells through blockade of mitogen-activated protein kinase (MAPK) and PI3K/Akt signaling pathways. Carbohydr. Polym., 84 (4): 1391–1398.
Musti A M, Treier M, Bohmann D. 1997. Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP Kinases. Science, 275 (5298): 400–402.
Nishimura K, Nishimura S, Nishi N, Saiki I, Tokura S, Azuma I. 1984. Immunological activity of chitin and its derivatives. Vaccine, 2 (1): 93–99.
Nyati K K, Masuda K, Zaman M M U, Dubey P K, Millrine D, Chalise J P, Higa M, Li S L, Standley D M, Saito K, Hanieh H, Kishimoto T. 2017. TLR4-induced NF-κB and MAPK signaling regulate the IL-6 mRNA stabilizing protein Arid5a. Nucleic Acids Res., 45 (5): 2687–2703.
Pillai C K S, Paul W, Sharma C P. 2009. Chitin and chitosan polymers: chemistry, solubility and fiber formation. Progress in Polymer Science, 34 (7): 641–678.
Poltorak A, He X L, Smirnova I, Liu M Y, van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B. 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science, 282 (5396): 2085–2088.
Raschke W C, Baird S, Ralph P, Nakoinz I. 1978. Functional macrophage cell lines transformed by abelson leukemia virus. Cell, 15 (1): 261–267.
Schindler C, Levy D E, Decker T. 2007. JAK-STAT signaling: from interferons to cytokines. J Biol Chem., 282 (28): 20059–20063.
Shen T, Yang W S, Yi Y S, Sung G H, Rhee M H, Poo H, Kim M Y, Kim K W, Kim J H, Cho J Y. 2013. AP-1/IRF-3 targeted anti-Inflammatory activity of andrographolide isolated from Andrographis paniculata Evid Based Complement Alternat. Med., 2013 (4): 210 736.
Sun H X, Zhang J, Chen F Y, Chen X F, Zhou Z H, Wang H. 2015. Activation of RAW264.7 macrophages by the polysaccharide from the roots of Actinidia eriantha and its molecular mechanisms. Carbohydr. Polym., 121: 388–402.
Tang B, Li X, Ren Y, Wang J, Xu D, Hang Y, Zhou T, Li F, Wang L. 2017. MicroRNA-29a regulates lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophages through the Akt1/ NF-kappaB pathway. Exp. Cell Res., 360 (2): 74–80.
Wen Q, Mei L Y, Ye S, Liu X, Xu Q, Miao J F, Du S H, Chen D F, Li C, Li H. 2018. Chrysophanol demonstrates anti-inflammatory properties in LPS-primed RAW 264.7 macrophages through activating PPAR-ψ. International Immunopharmacology, 56: 90–97.
Wymann M P, Pirola L. 1998. Structure and function of phosphoinositide 3-kinases. BBA- Mol Cell Biol Lipids1436 (1-2): 127–150.
Yang Y, Xing R E, Liu S, Qin Y K, Li K C, Yu H H, Li P C. 2019. Hydroxypropyltrimethyl ammonium chloride chitosan activates RAW 264.7 macrophages through the MAPK and JAK-STAT signaling pathways. Carbohydr Polym., 205: 401–409.
Youn G S, Lee K W, Choi S Y, Park J. 2016. Overexpression of HDAC6 induces pro-inflammatory responses by regulating ROS-MAPK-NF-κB/AP-1 signaling pathways in macrophages. Free Radical Biology and Medicine97: 14–23.
Yu Y, Shen M Y, Wang Z J, Wang Y X, Xie M Y, Xie J H. 2017. Sulfated polysaccharide from Cyclocarya paliurus enhances the immunomodulatory activity of macrophages. Carbohydr. Polym., 174: 669–676.
Zhang Q, Wang L R, Chen B H, Zhuo Q, Bao C Y, Lin L. 2017. Propofol inhibits NF-κB activation to ameliorate airway inflammation in ovalbumin (OVA)-induced allergic asthma mice. International Immunopharmacology, 51: 158–164.
Zhang Y, Igwe O J. 2018. Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage. Biochem. Pharmacol., 147: 104–118.
We gratefully acknowledge Dr. Weicheng HU for proving cell culture room in Huaiyin Normal University (Jiangsu, China).
Supported by the National Key R&D Program of China (No. 2018YFC0311305) and the Key Research and Development Program of Shandong Province (Nos. 2019GHY112015, 2019YYSP028)
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Yang, Y., Xing, R., Liu, S. et al. PI3K/Akt pathway is involved in the activation of RAW 264.7 cells induced by hydroxypropyltrimethyl ammonium chloride chitosan. J. Ocean. Limnol. 38, 834–840 (2020). https://doi.org/10.1007/s00343-019-9013-0
- hydroxypropyltrimethyl ammonium chloride chitosan
- RAW 264.7 cells
- PI3K/Akt pathway
- nuclear factor-κB
- activating protein 1