ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
ABSTRACT
AIM@#To investigate the anti-inflammatory activities of the semen extract of Cuscuta chinensis Lam. (Cuscutae Semen; CS) on the production of inflammatory mediators, nitric oxide (NO), prostaglandin 2 (PGE2), and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV-2 microglia.@*METHOD@#BV-2 cells were treated with CS extract for 30 min, and then stimulated with LPS or without for 24 h. The levels of NO, PGE2 and proinflammatory cytokines were measured by Griess assay and ELISA. The expression of inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 mRNA and protein was determined by RT-PCR and Western blot, respectively. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), and the nuclear expression of nuclear factor (NF)-κB p65 were investigated by Western blot analysis.@*RESULTS@#CS extract significantly decreased the production of NO and PGE2 by suppressing the expression of iNOS and COX-2 in activated microglia. CS extract decreased the production of TNF-α, IL-1β, and IL-6 by down-regulating their transcription levels. In addition, CS extract suppressed the phosphorylation of ERK1/2, JNK, and p38 MAPK, and the nuclear translocation of NF-κB p65 in activated microglia.@*CONCLUSION@#These results indicate that CS extract is capable of suppressing the inflammatory response by microglia activation, suggesting that CS extract has potential in the treatment of brain inflammation.