Journal of Food and Nutrition Research
ISSN (Print): 2333-1119 ISSN (Online): 2333-1240 Website: Editor-in-chief: Prabhat Kumar Mandal
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Journal of Food and Nutrition Research. 2016, 4(8), 515-521
DOI: 10.12691/jfnr-4-8-5
Open AccessArticle

Silibinin Suppresses Mediators of Inflammation through the Inhibition of TLR4-TAK1 Pathway in LPS-induced RAW264.7 Cells

Ji-Hyeon Song1, Kui-Jin Kim1 and Boo-Yong Lee1,

1Department of Food Science and Biotechnology, CHA University, Kyonggi 463-400, South Korea

Pub. Date: August 19, 2016

Cite this paper:
Ji-Hyeon Song, Kui-Jin Kim and Boo-Yong Lee. Silibinin Suppresses Mediators of Inflammation through the Inhibition of TLR4-TAK1 Pathway in LPS-induced RAW264.7 Cells. Journal of Food and Nutrition Research. 2016; 4(8):515-521. doi: 10.12691/jfnr-4-8-5


Silibinin is the major bioactive compound of silymarin which is the mixture of flavonolignans extracted from milk thistle. Silibinin has been shown to possess anti-inflammatory activity. However, the underlying mechanisms still remain unclear. The aims of this study were to determine the effect of silibinin on molecular mechanism in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. Here, we observed that silibinin attenuated the production of nitric oxide (NO) and its regulatory protein inducible nitric oxide synthase (iNOS) expression. The pro-inflammatory cytokine interleukin (IL)-1β was inhibited by silibinin in a time dependent manner. Moreover, silibinin decreased the expression of toll-like receptor (TLR)-4, TAK1, and IRF3. TLR- associated MAPK signaling pathway was also dramatically down-regulated in LPS-induced RAW 264.7 cells with presence of silibinin. Silibinin repressed oxidative stress-associated proteins including NOX4, G6PDH, and CuZnSOD, while silibinin increased the expression of GR and catalase in LPS-induced RAW264.7 cells. In the current study, silibinin suppresses the LPS-induced inflammation via modulation of TLR4-TAK1 signaling and subsequently attenuated the production of inflammation mediators in RAW264.7 cells. Therefore, we suggest that silibinin has a potential bioactivity for prevention and intervention of endotoxin-mediated inflammation and TLR4-TAK1-associated chronic diseases.

silibinin TLR4 TAK1 MAPK Inflammation LPS RAW264.7

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[1]  Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition. International immunology. 2009; 21(4): 317-37.
[2]  Campbell DE, Boyle RJ, Thornton CA, Prescott SL. Mechanisms of allergic disease - environmental and genetic determinants for the development of allergy. Clin Exp Allergy. 2015;45(5):844-58.
[3]  Lumeng CN, Saltiel AR. Inflammatory links between obesity and metabolic disease. J Clin Invest. 2011;121(6):2111-7.
[4]  Hameed I, Masoodi SR, Mir SA, Nabi M, Ghazanfar K, Ganai BA. Type 2 diabetes mellitus: From a metabolic disorder to an inflammatory condition. World J Diabetes. 2015;6(4):598-612.
[5]  Xie C, Liu C, Wu B, Lin Y, Ma T, Xiong H, et al. Effects of IRF1 and IFN-beta interaction on the M1 polarization of macrophages and its antitumor function. Int J Mol Med. 2016;38(1):148-60.
[6]  Sostres C, Gargallo CJ, Arroyo MT, Lanas A. Adverse effects of non-steroidal anti-inflammatory drugs (NSAIDs, aspirin and coxibs) on upper gastrointestinal tract. Best Pract Res Clin Gastroenterol. 2010;24(2):121-32.
[7]  Williams CD, Antoine DJ, Shaw PJ, Benson C, Farhood A, Williams DP, et al. Role of the Nalp3 inflammasome in acetaminophen-induced sterile inflammation and liver injury. Toxicol Appl Pharmacol. 2011;252(3):289-97.
[8]  Martinez MJ, Molano VJ, Henao RS. [Gastroduodenal mucosal injuries by aspirine. Management of the risks]. Rev Gastroenterol Peru. 2016;36(2):129-34.
[9]  Curiel RV, Katz JD. Mitigating the cardiovascular and renal effects of NSAIDs. Pain Med. 2013;14 Suppl 1:S23-8.
[10]  Hwang J-H, Kim K-J, Ryu S-J, Lee B-Y. Caffeine prevents LPS-induced inflammatory responses in RAW264. 7 cells and zebrafish. Chemico-biological interactions. 2016;248:1-7.
[11]  Ryu S-J, Choi H-S, Yoon K-Y, Lee O-H, Kim K-J, Lee B-Y. Oleuropein suppresses LPS-induced inflammatory responses in RAW 264.7 cell and zebrafish. Journal of agricultural and food chemistry. 2015;63(7):2098-105.
[12]  Tursun X, Zhao Y, Alat Z, Xin X, Tursun A, Abdulla R, et al. Anti-Inflammatory Effect of Rosa rugosa Flower Extract in Lipopolysaccharide-Stimulated RAW264.7 Macrophages. Biomol Ther (Seoul). 2016;24(2):184-90.
[13]  Wang Q, Xia M, Liu C, Guo H, Ye Q, Hu Y, et al. Cyanidin-3-O-β-glucoside inhibits iNOS and COX-2 expression by inducing liver X receptor alpha activation in THP-1 macrophages. Life sciences. 2008;83(5):176-84.
[14]  Kim EJ, Lee MY, Jeon YJ. Silymarin Inhibits Morphological Changes in LPS-Stimulated Macrophages by Blocking NF-kappaB Pathway. Korean J Physiol Pharmacol. 2015;19(3):211-8.
[15]  El-Elimat T, Raja HA, Graf TN, Faeth SH, Cech NB, Oberlies NH. Flavonolignans from Aspergillus iizukae, a fungal endophyte of milk thistle (Silybum marianum). J Nat Prod. 2014;77(2):193-9.
[16]  Ligeret H, Brault A, Vallerand D, Haddad Y, Haddad PS. Antioxidant and mitochondrial protective effects of silibinin in cold preservation–warm reperfusion liver injury. Journal of ethnopharmacology. 2008;115(3):507-14.
[17]  Singh RP, Agarwal R. Prostate cancer prevention by silibinin. Current cancer drug targets. 2004;4(1):1-11.
[18]  Polyak SJ, Morishima C, Lohmann V, Pal S, Lee DY, Liu Y, et al. Identification of hepatoprotective flavonolignans from silymarin. Proceedings of the National Academy of Sciences. 2010;107(13):5995-9.
[19]  Giorgi VSI, Peracoli MTS, Peracoli JC, Witkin SS, Bannwart-Castro CF. Silibinin modulates the NF-κb pathway and pro-inflammatory cytokine production by mononuclear cells from preeclamptic women. Journal of Reproductive Immunology. 2012;95(1-2):67-72.
[20]  Zhou GY, Yi YX, Jin LX, Lin W, Fang PP, Lin XZ, et al. The protective effect of juglanin on fructose-induced hepatitis by inhibiting inflammation and apoptosis through TLR4 and JAK2/STAT3 signaling pathways in fructose-fed rats. Biomed Pharmacother. 2016;81:318-28.
[21]  Kim KJ, Lee OH, Lee BY. Fucoidan, a sulfated polysaccharide, inhibits adipogenesis through the mitogen-activated protein kinase pathway in 3T3-L1 preadipocytes. Life sciences. 2010; 86 (21-22): 791-7.
[22]  Maelfait J, Vercammen E, Janssens S, Schotte P, Haegman M, Magez S, et al. Stimulation of Toll-like receptor 3 and 4 induces interleukin-1beta maturation by caspase-8. J Exp Med. 2008;205(9):1967-73.
[23]  Le Goff C, Rogers C, Le Goff W, Pinto G, Bonnet D, Chrabieh M, et al. Heterozygous Mutations in MAP3K7, Encoding TGF-beta-Activated Kinase 1, Cause Cardiospondylocarpofacial Syndrome. Am J Hum Genet. 2016.
[24]  Deng S, Yu K, Zhang B, Yao Y, Wang Z, Zhang J, et al. Toll-Like Receptor 4 Promotes NO Synthesis by Upregulating GCHI Expression under Oxidative Stress Conditions in Sheep Monocytes/Macrophages. Oxid Med Cell Longev. 2015; 2015: 359315.
[25]  Bartels K, Grenz A, Eltzschig HK. Hypoxia and inflammation are two sides of the same coin. Proc Natl Acad Sci U S A. 2013; 110(46): 18351-2.
[26]  Wang C, Wang Z, Zhang X, Zhang X, Dong L, Xing Y, et al. Protection by silibinin against experimental ischemic stroke: Up-regulated pAkt, pmTOR, HIF-1α and Bcl-2, down-regulated Bax, NF-κB expression. Neuroscience letters. 2012;529(1):45-50.
[27]  Kim S, Kim SH, Hur SM, Lee SK, Kim WW, Kim JS, et al. Silibinin prevents TPA-induced MMP-9 expression by down-regulation of COX-2 in human breast cancer cells. J Ethnopharmacol. 2009; 126(2):252-7.
[28]  Li L, Zeng J, Gao Y, He D. Targeting silibinin in the antiproliferative pathway. Expert Opin Investig Drugs. 2010; 19(2): 243-55.
[29]  Sanchavanakit N, Saengtong W, Manokawinchoke J, Pavasant P. TNF-alpha stimulates MMP-3 production via PGE2 signalling through the NF-kB and p38 MAPK pathway in a murine cementoblast cell line. Arch Oral Biol. 2015;60(7):1066-74.
[30]  Zhao F, Chen L, Bi C, Zhang M, Jiao W, Yao X. In vitro anti-inflammatory effect of picrasmalignan A by the inhibition of iNOS and COX2 expression in LPSactivated macrophage RAW 264.7 cells. Mol Med Rep. 2013;8(5):1575-9.
[31]  Park Y, Yoo SA, Kim WU, Cho CS, Woo JM, Yoon CH. Anti-inflammatory effects of essential oils extracted from Chamaecyparis obtusa on murine models of inflammation and RAW 264.7 cells. Mol Med Rep. 2016;13(4):3335-41.
[32]  Mishra BB, Rathinam VA, Martens GW, Martinot AJ, Kornfeld H, Fitzgerald KA, et al. Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent processing of IL-1 [beta]. Nature immunology. 2013;14(1):52-60.
[33]  Chen K-H, Weng M-S, Lin J-K. Tangeretin suppresses IL-1β-induced cyclooxygenase (COX)-2 expression through inhibition of p38 MAPK, JNK, and AKT activation in human lung carcinoma cells. Biochemical pharmacology. 2007;73(2):215-27.
[34]  Zhao F, Wang L, Liu K. In vitro anti-inflammatory effects of arctigenin, a lignan from Arctium lappa L., through inhibition on iNOS pathway. Journal of ethnopharmacology. 2009; 122(3): 457-62.
[35]  Álvarez-Soria MA, Largo R, Santillana J, Sánchez-Pernaute O, Calvo E, Hernandez M, et al. Long term NSAID treatment inhibits COX-2 synthesis in the knee synovial membrane of patients with osteoarthritis: differential proinflammatory cytokine profile between celecoxib and aceclofenac. Annals of the rheumatic diseases. 2006;65(8):998-1005.
[36]  Rao P, Knaus EE. Evolution of nonsteroidal anti-inflammatory drugs (NSAIDs): cyclooxygenase (COX) inhibition and beyond. Journal of Pharmacy & Pharmaceutical Sciences. 2008; 11(2): 81-110s.
[37]  Zwolinska-Wcislo M, Krzysiek-Maczka G, Ptak-Belowska A, Karczewska E, Pajdo R, Sliwowski Z, et al. Antibiotic treatment with ampicillin accelerates the healing of colonic damage impaired by aspirin and coxib in the experimental colitis. Importance of intestinal bacteria, colonic microcirculation and proinflammatory cytokines. J Physiol Pharmacol. 2011; 62(3): 357-68.
[38]  Kong F, Laryea G, Liu Z, Bhattacharyya S. Transforming growth factor-beta-activated kinase 1 resistance limits glucocorticoid responsiveness to Toll-like receptor 4-mediated inflammation. Immunology. 2015;145(1):136-49.
[39]  Kim YJ, Kim HC, Ko H, Amor EC, Lee JW, Yang HO. Stercurensin inhibits nuclear factor-kappaB-dependent inflammatory signals through attenuation of TAK1-TAB1 complex formation. Journal of cellular biochemistry. 2011;112(2):548-58.
[40]  Xu X, Qi X, Shao Y, Li Y, Fu X, Feng S, et al. Blockade of TGF-beta-activated kinase 1 prevents advanced glycation end products-induced inflammatory response in macrophages. Cytokine. 2016;78:62-8.
[41]  Chou ST, Peng HY, Hsu JC, Lin CC, Shih Y. Achillea millefolium L. essential oil inhibits LPS-induced oxidative stress and nitric oxide production in RAW 264.7 Macrophages. Int J Mol Sci. 2013;14(7):12978-93.
[42]  Ibitoye R, Kemp K, Rice C, Hares K, Scolding N, Wilkins A. Oxidative stress-related biomarkers in multiple sclerosis: a review. Biomark Med. 2016;10(8):889-902.
[43]  Muthumani M, Prabu SM. Silibinin potentially attenuates arsenic-induced oxidative stress mediated cardiotoxicity and dyslipidemia in rats. Cardiovascular toxicology. 2014;14(1):83-97.
[44]  Hashem RM, Hassanin KM, Rashed LA, Mahmoud MO, Hassan MG. Effect of silibinin and vitamin E on the ASK1-p38 MAPK pathway in D-galactosamine/lipopolysaccharide induced hepatotoxicity. Experimental biology and medicine. 2016; 241(11): 1250-7.
[45]  Guan J, Wu X, Arons E, Christou H. The p38 mitogen-activated protein kinase pathway is involved in the regulation of heme oxygenase-1 by acidic extracellular pH in aortic smooth muscle cells. J Cell Biochem. 2008;105(5):1298-306.
[46]  Okajima F. Regulation of inflammation by extracellular acidification and proton-sensing GPCRs. Cell Signal. 2013; 25(11): 2263-71.
[47]  Gautam M, Benson C, Sluka KA. Increased response of muscle sensory neurons to decreases in pH after muscle inflammation. Neuroscience. 2010;170(3):893-900.