Artesunate attenuates pro-inflammatory cytokine release of macrophage by inhibiting TLR4-mediated autophagic activation via TRAF6-BECN1-Vps34 pathway

抄録

Background/Aims: Lipopolysaccharide (LPS) plays a critical role in excessive inflammatory cytokine production in the patients with bacterial sepsis. Previously, artesunate (AS) was reported to protect mice from LPS challenge by reducing pro-inflammatory cytokine release. Herein, the possible mechanism of the anti-inflammatory effect of AS is investigated. Methods: ELISA was applied to detect TNF-α and IL-6 release from macrophages. Specific siRNA was used to knock-down mRNA expression of target gene. Transmission electron microscope and confocal microscope were used to observe autophagy alterations. Western blot was performed to detect protein expressions of TRAF6, BECN1, total or phosphorylated Vps34, ATG5, LC3BI/II and SQSTM1. The interactions between TRAF6-BECN1 and BECN1-Vps34 were detected by fluorescent co-localization and co-immunoprecipitation. Results: AS attenuated LPS-induced release of TNF-α and IL-6 from RAW264.7 cells, mouse bone marrow-derived monocytes (BMDMs) and peritoneal macrophages (PMs). However, the inhibition of AS on LPS-induced cytokine release was decreased by inhibiting autophagy using 3-MA, bafilomycin A1 or siRNA of atg5 gene. AS showed an inhibitory effect on LPS induced autophagic activation not degradation. Whereas, these effects of AS were lost in macrophages lacking TLR4 and decreased in macrophages down-regulating TRAF6, indicating AS inhibits LPS-induced cytokine release and autophagic activation via a TLR4-TRAF6 signaling. Western blot showed that AS played its role by inhibiting TRAF6 thereby decreasing the expressions of BECN1 and Vps34. Moreover, AS inhibited LPS-induced K63 ubiquitination of BECN1 by acting on TRAF6, thereby interrupting TRAF6-BECN1 interaction. In addition, AS could also inhibit BECN1-Vps34 interaction. Conclusion: AS attenuates LPS-induced cytokine release from macrophages by inhibiting autophagic activation via down-regulating TRAF6-BECN1-Vps34 pathway, decreasing K63 ubiquitination of BECN1 and interrupting BECN1-Vps34 interaction. These findings reveal multiple mechanisms of AS's anti-inflammatory effect and potentially targets for sepsis treatment.