br Materials and method br Results br

Materials and method
Results
Discussion Although several studies have showed the crude extracts of K. senegalensis possessed the anti-inflammatory and hypertensive activities (Olayinka et al., 1992). However, the anti-inflammatory effect and underlying molecular mechanisms of its bioactive compounds remain still poorly understood. In the present study, we tried to identify the bioactive compounds from K. senegalensis and clarify the potential anti-inflammatory mechanisms in LPS-stimulated RAW 264.7 and BV-2 cells. Khayandirobilide A (KLA), a new andirobin-type limonoid with modified furan ring, was isolated from the stem barks of K. senegalensis. Moreover, KLA attenuated LPS-induced inflammatory response though suppressing the Mubritinib receptor of pro-inflammatory mediators, like NO, ROS, IL-6, iNOS and COX-2 in RAW 264.7 and BV-2 cells. NF-κB, the nuclear transcription factor, plays a principle role in regulating inflammatory mediators during inflammatory process. The nuclear translocation of NF-κB which dissociated from its inhibitory protein IκB, is the most crucial step for NF-κB signaling activation (Fujioka et al., 2004). It was found that KLA suppressed LPS-activated NF-κB signaling by inhibiting phosphorylation of IκBα and IκBα degradation as well as the subsequent nuclear translocation of NF-κB. Numerous studies have reported that phosphorylation and degradation of IκBα induced by TRAF6/IKKα/β may directly trigger NF-κB activation (Zhao et al., 2014). KLA not only suppressed expression of TRAF6, but also attenuated the phosphorylation of IKKα/β in both cells. Therefore, KLA inhibited nuclear translocation of NF-κB though suppressing the activation of TRAF6-mediated IKKα/β. Notably, the activation of NF-κB for some promoters requires assist from other transcription factors such as AP-1 which is regulated by  the  phosphorylation  of  AKT during inflammatory response (Fujioka et al., 2004, Han et al., 2008) . In consistent with NF-κB, KLA decreased the nuclear levels of LPS-stimulated AP-1. In addition, KLA reduced phosphorylation of AKT induced by LPS. Other studies reported that AKT can simulate the activation of NF-κB through IKKα/β (Sizemore et al., 2002). Taken together, KLA might alleviate the activation of LPS-stimulated NF-κB and AP-1 via preventing the phosphorylation of AKT, which contributed to its inhibitory effects on inflammatory mediators. Accumulated evidence indicates that excessive ROS aggravates the expression of the inflammatory mediators like iNOS and COX-2 eliciting inflammatory process, which may be modulated by HO-1. The degradation of Keap1 induced by autophagy could provoke Nrf2 stabilization and the release of Nrf2 from Keap1, subsequent accumulation in the nucleus, which regulate various target genes like HO-1 and participate in anti-inflammatory response (Kim et al., 2010, Taguchi et al., 2012). As we expect, KLA not only decreased the generation of LPS-stimulated ROS, but also promoted the nuclear translocation of Nrf2 though the autophagic degradation of Keap1, and following induction of HO-1 in both cells. Furthermore, the inhibition of Nrf2 and HO-1 with their specific inhibitors Brusatol and ZnPP, respectively, could partly reverse the suppressive effects of KLA on the release of LPS-induced NO and mRNA expressions of pro-inflammatory genes like iNOS, COX-2 and IL-6, which suggested the Nrf2/HO-1 signaling participated in the anti-inflammatory effect of KLA. It has been reported the activation of MAPKs participates in the induction of Nrf2-mediated HO-1 (Khan et al., 2017). Moreover, the activation of p38 is well recognized in participating in inflammation through mediating Nrf2/HO-1 signaling (Park et al., 2013). Although KLA elevated the phosphorylation of MAPKs, the activation of Nrf2/HO-1 protein level induced by KLA was only decreased by the specific inhibitor SB203580 of p38 in both cells. In addition, the specific inhibitor SB203580 of p38 could inhibit KLA-induced autophagy. Some reports showed that the p38 MAPK is important in regulating autophagy (Sui et al., 2014). Thus, these findings suggested that the activation of p38 MAPK was likely to participate in KLA-induced autophagic degradation of Keap1 and activate Nrf2/HO-1 signaling. More interestingly, the specific inhibitor U0126 of ERK also attenuated the up-regulation of KLA-mediated HO-1 as well as Nrf2 activation in LPS-stimulated RAW 264.7 cells, which differences may be due to the different characteristics of two cell lines and further investigation is required.