Brazilin Limits Inflammatory Responses through Induction of Prosurvival Autophagy in Rheumatoid Fibroblast-Like Synoviocytes.

Brazilin is an active compound of Caesalpinia sappan L. (Leguminosae), which possesses pro-apoptotic and anti-inflammation potentials depending on the specific cell type. However, it is largely unknown whether autophagy is implicated in the mechanism underlying its chemotherapeutic and anti-inflammatory effects in rheumatoid arthritis (RA). Here, we show that treatment of RA fibroblast-like synoviocytes (FLS) with brazilin results in enhanced level of autophagic flux, evidenced by accumulation of autophagosome and increased level of lipidated LC3 (LC3-II), which is mainly mediated by enhanced production of reactive oxygen species (ROS). Interestingly, long-term exposure of brazilin was able to restore cell survival against the cytotoxity, exclusively in RA FLS, but not in normal fibroblast. Importantly, such a restoration from brazilin-induced cytotoxity in RA FLS was completely abrogated after co-treatment with autophagy inhibitors including NH4Cl or chloroquine. Furthermore, we found that the pretreatment of RA FLS with brazilin reduced LPS- or TNF-induced NF-κB activation and the secretion of inflammatory cytokines in parallel with the enhanced autophagic flux. Such anti-NF-κB potentials of brazilin were drastically masked in RA FLS when autophagy was suppressed. These results suggest that brazilin is capable of activating autophagy exclusively in RA FLS, and such inducible autophagy promotes cell survival and limits inflammatory response.

Brazilin selectively disrupts proximal IL-1 receptor signaling complex formation by targeting an IKK-upstream signaling components.

The ligation of interleukin-1 receptor (IL-1R) or tumor necrosis factor receptor 1 (TNFR1) induces the recruitment of adaptor proteins and their concomitant ubiquitination to the proximal receptor signaling complex, respectively. Such are upstream signaling events of IKK that play essential roles in NF-κB activation. Thus, the discovery of a substance that would modulate the recruitment of key proximal signaling elements at the upstream level of IKK has been impending in this field of study. Here, we propose that brazilin, an active compound of Caesalpinia sappan L. (Leguminosae), is a potent NF-κB inhibitor that selectively disrupts the formation of the upstream IL-1R signaling complex. Analysis of upstream signaling events revealed that brazilin markedly abolished the IL-1ß-induced polyubiquitination of IRAK1 and its interaction with IKK-γ counterpart. Notably, pretreatment of brazilin drastically interfered the recruitment of the receptor-proximal signaling components including IRAK1/4 and TRAF6 onto MyD88 in IL-1R-triggerd NF-κB activation. Interestingly, brazilin did not affect the TNF-induced RIP1 ubiquitination and the recruitment of RIP1 and TRAF2 to TNFR1, suggesting that brazilin is effective in selectively suppressing the proximal signaling complex formation of IL-1R, but not that of TNFR1. Moreover, our findings suggest that such a disruption of IL-1R-proximal complex formation by brazilin is not mediated by affecting the heterodimerization of IL-1R and IL-1RAcP. Taken together, the results suggest that the anti-IKK activity of brazilin is induced by targeting IKK upstream signaling components and subsequently disrupting proximal IL-1 receptor signaling complex formation.