Arteannuin B, a sesquiterpene lactone from Artemisia annua, attenuates inflammatory response by inhibiting the ubiquitin-conjugating enzyme UBE2D3-mediated NF-B activation

Background: Anomalous activation of NF-kappa B signaling is associated with many inflammatory disorders, such as ulcerative colitis (UC) and acute lung injury (ALI). NF-kappa B activation requires the ubiquitination of receptorinteracting protein 1 (RIP1) and NF-kappa B essential modulator (NEMO). Therefore, inhibition of ubiquitation of RIP1 and NEMO may serve as a potential approach for inhibiting NF-kappa B activation and alleviating inflammatory disorders. Purpose: Here, we identified arteannuin B (ATB), a sesquiterpene lactone found in the traditional Chinese medicine Artemisia annua that is used to treat malaria and inflammatory diseases, as a potent anti-inflammatory compound, and then characterized the putative mechanisms of its anti-inflammatory action. Methods: Detections of inflammatory mediators and cytokines in LPSor TNF-alpha-stimulated murine macrophages using RT-qPCR, ELISA, and western blotting, respectively. Western blotting, CETSA, DARTS, MST, gene knockdown, LC-MS/MS, and molecular docking were used to determine the potential target and molecular mechanism of ATB. The pharmacological effects of ATB were further evaluated in DSS-induced colitis and LPSinduced ALI in vivo. Results: ATB effectively diminished the generation of NO and PGE2 by down -regulating iNOS and COX2 expression, and decreased the mRNA expression and release of IL-1 beta, IL -6, and TNF-alpha in LPS-exposed RAW264.7 macrophages. The anti-inflammatory effect of ATB was further demonstrated in LPS-treated BMDMs and TNF alpha-activated RAW264.7 cells. We further found that ATB obviously inhibited NF-kappa B activation induced by LPS or TNF-alpha in vitro. Moreover, compared with ATB, dihydroarteannuin B (DATB) which lost the unsaturated double bond, completely failed to repress LPS-induced NO release and NF-kappa B activation in vitro. Furthermore, UBE2D3, a ubiquitin-conjugating enzyme, was identified as the functional target of ATB, but not DATB. UBE2D3 knockdown significantly abolished ATB-mediated inhibition on LPS-induced NO production. Mechanistically, ATB could covalently bind to the catalytic cysteine 85 of UBE2D3, thereby inhibiting the function of UBE2D3 and preventing ubiquitination of RIP1 and NEMO. In vivo, ATB treatment exhibited robust protective effects against DSS-induced UC and LPS-induced ALI. Conclusion: Our findings first demonstrated that ATB exerted anti-inflammatory functions by repression of NF-kappa B pathway via covalently binding to UBE2D3, and raised the possibility that ATB could be effective in the treatment of inflammatory diseases and other diseases associated with abnormal NF-kappa B activation.