Abstract
Objective
To investigate the effects of the flower buds extract of Tussilago farfara Linné (Farfarae Flos; FF) on focal cerebral ischemia through regulation of inflammatory responses in activated microglia.
Methods
Brain ischemia was induced in Sprague-Dawley rats by a transient middle cerebral artery occlusion (tMCAO) for 90 min and reperfusion for 24 h. Twenty rats were randomly divided into 4 groups (n=5 per group): normal, tMCAO-induced ischemic control, tMCAO plus FF extract 300 mg/kg-treated, and tMCAO plus MK-801 1 mg/kg-treated as reference drug. FF extract (300 mg/kg, p.o.) or MK-801 (1 mg/kg, i.p.) was administered after reperfusion. Brain infarction was measured by 2,3,5,-triphenyltetrazolium chloride staining. Neuronal damage was observed by haematoxylin eosin, Nissl staining and immunohistochemistry using anti-neuronal nuclei (NeuN), anti-glial fibrillary acidic protein (GFAP), and anti-CD11b/c (OX42) antibodies in ischemic brain. The expressions of inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF-α), and hypoxia-inducible factor-1a (HIF-1α) were determined by Western blot. BV2 microglial cells were treated with FF extract or its main bioactive compound, tussilagone with or without lipopolysaccharide (LPS). Nitric oxide (NO) production was measured in culture medium by Griess assay. The expressions of iNOS, COX-2 and pro-inflammatory cytokines mRNA were analyzed by reverse transcription-polymerase chain reaction. The expression of iNOS, and COX-2 proteins, the phosphorylation of ERK1/2, JNK, and p38 MAPK and the nuclear expression of NF-κB p65 in BV2 cells were determined by Western blot.
Results
FF extract significantly decreased brain infarctions in ischemic rats (P<0.01). The neuronal death and the microglia/astrocytes activation in ischemic brains were inhibited by FF extract. FF extract also suppressed iNOS, TNF-α, and HIF-1α expression in ischemic brains. FF extract (0.2 and 0.5 mg/mL, P<0.01) and tussilagone 20 and 50 μmol/L, P<0.01) significantly decreased LPS-induced NO production in BV2 microglia through downregulation of iNOS mRNA and protein expression. FF extract and tussilagone significantly inhibited LPS-induced expression of TNF-α, IL-1β, and IL-6 mRNA, and also suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK and the nuclear expression of NF-κB in a dose-dependent manner.
Conclusions
FF extract has a neuroprotective effect in ischemic stroke by the decrease of brain infarction, and the inhibition of neuronal death and microglial activation-mediated inflammatory responses.
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References
Ip FC, Zhao YM, Chan KW, Cheng EY, Tong EP, Chandrashekar O, et al. Neuroprotective effect of a novel Chinese herbal decoction on cultured neurons and cerebral ischemic rats. BMC Complement Altern Med 2016;16:437.
Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. J Leukoc Biol 2010;87:779–789.
Amantea D, Tassorelli C, Petrelli F, Certo M, Bezzi P, Micieli G. Understanding the multifaceted role of inflammatory mediators in ischemic stroke. Curr Med Chem 2014;21:2098–2117.
Jordán, J, Segura T, Brea D, Galindo MF, Castillo J. Inflammation as therapeutic objective in stroke. Curr Pharm 2008;14:3549–3564.
Zhi HJ, Qin XM, Sun HF, Zhang LZ, Guo XQ, Li ZY. Metabolic fingerprinting of Tussilago farfara L. using 1H-NMR spectroscopy and multivariate data analysis. Phytochem Anal 2012;23:492–501.
Cho JS, Kim HM, Ryu JH, Jeong YS, Lee YS, Jin CB. Neuroprotective and antioxidant effects of the ethyl acetate fraction prepared from Tussilago farfara L. Biol Pharm Bull 2005;28:440–455.
Kokoska L, Polesny Z, Rada V, Nepovim A, Vanek T. Screening of some Siberian medicinal plants for antimicrobial activity. J Ethnopharmacol 2002;82:51–53.
Lim HJ, Lee HS, Ryu JH. Suppression of inducible nitric oxide synthase and cyclooxygenase-2 expression by tussilagone from Farfarae flos in BV-2 microglial cells. Arch Pharm Res 2008;31:645–652.
Hwangbo C, Lee HS, Park J, Choe J, Lee JH. The anti-inflammatory effect of tussilagone, from Tussilago farfara, is mediated by the induction of heme oxygenase-1 in murine macrophages. Int Immunopharmacol 2009;9:1578–1584.
Li W, Huang X, Yang XW. New sesquiterpenoids from the dried flower buds of Tussilago farfara and their inhibition on NO production in LPS-induced RAW264.7 cells. Fitoterapia 2012;83:318–322.
Lim HJ, Dong GZ, Lee HJ, Ryu JH. In vitro neuroprotective activity of sesquiterpenoids from the flower buds of Tussilago farfara. J Enzyme Inhib Med Chem 2015;30:852–856.
Jang H, Lee JW, Lee C, Jin Q, Choi JY, Lee D, et al. Sesquiterpenoids from Tussilago farfara inhibit LPS-induced nitric oxide production in macrophage RAW 264.7 cells. Arch Pharm Res 2016;39:127–132.
Seo UM, Zhao BT, Kim WI, Seo EK, Lee JH, Min BS, et al. Quality evaluation and pattern recognition analyses of bioactive marker compounds from Farfarae Flos using HPLC/PDA. Chem Pharm Bull (Tokyo) 2015;63:546–553.
Fluri F, Schuhmann MK, Kleinschnitz C. Animal models of ischemic stroke and their application in clinical research. Drug Des Devel Ther 2015;9:3445–3454.
Cunningham C, Wilcockson DC, Campion S, Lunnon K, Perry VH. Central and systemic endotoxin challenges exacerbate the local inflammatory response and increase neuronal death during chronic neurodegeneration. J Neurosci 2005;25:9275–9284.
Rock RB, Peterson PK. Microglia as a pharmacological target in infectious and inflammatory diseases of the brain. J Neuroimmune Pharmacol 2006;1:117–126.
Block ML, Zecca L, Hong JS. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 2007;8:57–69.
Garden GA, and Möller T. Microglia biology in health and disease. J Neuroimmune Pharmacol 2006;1:127–137.
Bora KS, Shri R, Monga J. Cerebroprotective effect of Ocimum gratissimum against focal ischemia and reperfusion-induced cerebral injury. Pharm Biol 2011;49:175–181.
Kim MS, Bang JH, Lee J, Han JS, Kang HW, Jeon WK. Fructus mume ethanol extract prevents inflammation and normalizes the septohippocampal cholinergic system in a rat model of chronic cerebral hypoperfusion. J Med Food 2016;19:196–204.
Ayala GX, Tapia R. Late N-methyl-D-aspartate receptor blockade rescues hippocampal neurons from excitotoxic stress and death after 4-aminopyridine-induced epilepsy. Eur J Neurosci 2005;22:3067–3076.
Kocaeli H, Korfali E, Ozturk H, Kahveci N, Yilmazlar S. MK-801 improves neurological and histological outcomes after spinal cord ischemia induced by transient aortic cross-clipping in rats. Surg Neurol 2005;64:S22–S26.
Han RZ, Hu JJ, Weng YC, Li DF, Huang Y. NMDA receptor antagonist MK-801 reduces neuronal damage and preserves learning and memory in a rat model of traumatic brain injury. Neurosci Bull 2009;25:367–375.
Ikonomidou C, Turski L. Why did NMDA receptor antagonists fail clinical trials for stroke and traumatic brain injury? Lancet. Neurol 2002;1:383–386.
Gao HM, Hong JS, Zhang W, Liu B. Synergistic dopaminergic neurotoxicity of the pesticide rotenone and inflammogen lipopolysaccharide: relevance to the etiology of Parkinson’s disease. J Neurosci 2003;23:1228–1236.
Boje KM, Arora PK. Microglia-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res 1992;53:236–244.
Tzeng SF, Hsiao HY, Mak OT. Prostaglandins and cyclooxygenases in glial cells during brain inflammation. Curr Drug Targets Inflamm Allergy 2005;4:335–340.
Scali C, Giovannini MG, Prosperi C, Bellucci A, Pepeu G, Casamenti F. The selective cyclooxygenase-2 inhibitor rofecoxib suppresses brain inflammation and protects cholinergic neurons from excitotoxic degeneration in vivo. Neuroscience 2003;117:909–919.
Hoozemans JJ, Veerhuis R, Rosemuller AJ, Eikelenboom P. Non-steroidal anti-inflammatory drugs and cyclooxygenase in Alzheimer’s disease. Curr Drug Targets 2003;4:461–468.
Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature 2007;449:819–826.
Kaminska B, Gozdz A, Zawadzka M, Ellert-Miklaszewska A, Lipko M. MAPK signal transduction underlying brain inflammation and gliosis as therapeutic target. Anat Rec (Hoboken) 2009;292:1902–1913.
Svensson C, Fernaeus SZ, Part K, Reis K, Land T. LPS-induced iNOS expression in Bv-2 cells is suppressed by an oxidative mechanism acting on the JNK pathway—a potential role for neuroprotection. Brain Res 2010;1322:1–7.
Park Y, Ryu HS, Lee HK, Kim JS, Yun J, Kang JS, et al. Tussilagone inhibits dendritic cell functions via induction of heme oxygenase-1. Int Immunopharmacol 2014;22:400–408.
Li H, Lee HJ, Ahn YH, Kwon HJ, Jang CY, Kim WY, et al. Tussilagone suppresses colon cancer cell proliferation by promoting the degradation of β-catenin. Biochem Biophys Res Commun 2014;443:132–137.
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Supported by the grant from Ministry of Food and Drug Safety in 2014 (No. 12172MFDS989), Republic of Korea
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Hwang, J.H., Kumar, V.R., Kang, S.Y. et al. Effects of Flower Buds Extract of Tussilago farfara on Focal Cerebral Ischemia in Rats and Inflammatory Response in BV2 Microglia. Chin. J. Integr. Med. 24, 844–852 (2018). https://doi.org/10.1007/s11655-018-2936-4
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DOI: https://doi.org/10.1007/s11655-018-2936-4