Effects of Flower Buds Extract of Tussilago farfara on Focal Cerebral Ischemia in Rats and Inflammatory Response in BV2 Microglia

Abstract

Objective: To investigate the effects of the flower buds extract of Tussilago farfara Linne (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- alpha ), and hypoxia-inducible factor-1 alpha (HIF-1 alpha) 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-kappa B 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-alpha, and HIF-1 alpha expression in ischemic brains. FF extract (0.2 and 0.5 mg/mL, P<0.01) and tussilagone 20 and 50 mu mol/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-alpha, IL-1 beta, and IL-6 mRNA, and also suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK and the nuclear expression of NF- kappa B in a dose-dependent manner. Conclusion: 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.