Heme oxygenase-1-mediated anti-inflammatory effect of tussilagonone in RAW 264.7 macrophages

The dried flower buds of Tussilago farfara have been used in traditional medicine, mainly as antitussive in treatment of coughs and other respiratory problems. In the present study, we investigated the anti-inflammatory signaling pathway by up-regulation of heme oxygenase-1 (HO-1) expression in response to tussilagonone (TGN), a sesquiterpene compound isolated from Tussilago farfara. Treatment of RAW 264.7 cells with TGN induced HO-1 protein and mRNA expression. TGN increased protein but not mRNA level of nuclear factor-E2-related factor 2 (Nrf2), suggesting that TGN regulated Nrf2 at the translational level. Nuclear translocation of Nrf2 by TGN is also increased in a time- and dose-dependent manner indicating that TGN induced HO-1 via Nrf2 pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, TGN suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and also reduced the mRNA expression of pro-inflammatory cytokines, including iNOS, COX-2, TNF-α and IL-6, as well as nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Furthermore, the LPS-induced SEAP expression in NF-κB-SEAP-NPT construct-transfected RAW 264.7 macrophages was inhibited by TGN. Moreover, TGN inhibited the phosphorylation and degradation of inhibitory κB (IκB)-α and the nuclear translocation of NF- κB. However, a specific inhibitor of HO-1, SnPP, reversed TGN-mediated suppression of NO production and knockdown of HO-1 by small interfering RNA abrogated inhibitory effects of TGN on iNOS and COX-2 protein expression and NF-κB nuclear translocation in LPS-stimulated RAW 264.7 cells. Taken together, these findings suggest an important role of TGN-induced HO-1 activation in regulating inflammatory responses and TGN is a potent therapeutic candidate targeting the crosstalk between Nrf2/HO-1 and NF-κB signaling pathway for preventing or treating inflammation-associated diseases.