Artemisinin inhibits TLR4 signaling by targeting co-receptor MD2 in microglial BV-2 cells and prevents lipopolysaccharide-induced blood-brain barrier leakage in mice.


Zhang T(1)(2), Zhang X(1), Lin C(1), Wu S(1), Wang F(1)(3), Wang H(1), Wang Y(1), Peng Y(4), Hutchinson MR(5)(6), Li H(1), Wang X(1)(7).
Author information:
(1)Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, China.
(2)Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education, Yantai University, Yantai, China.
(3)State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences of Guangxi, Normal University, Guilin, China.
(4)State Key Laboratory for Molecular Biology of Special Economic Animal, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
(5)Discipline of Physiology, Adelaide Medical School, University of Adelaide, South Australia, Australia.
(6)ARC Centre of Excellence for Nanoscale Biophotonics, University of Adelaide, Adelaide, SA, Australia.
(7)Department of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei, China.


Artemisinin and its derivatives have been the frontline drugs for treating malaria. In addition to the antiparasitic effect, accumulating evidence shows that artemisinins can alleviate neuroinflammatory responses in the central nervous system (CNS). However, the precise mechanisms underlying their anti-neuroinflammatory effects are unclear. Herein we attempted to delineate the molecule target of artemisinin in microglia. In vitro protein intrinsic fluorescence titrations and saturation transfer difference (STD)-NMR showed the direct binding of artemisinin to Toll-like receptor TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that artemisinin binding increased MD2 stability, which implies that artemisinin directly binds to MD2 in the cellular context. Artemisinin bound MD2 showed much less collapse during the molecular dynamic simulations, which supports the increased stability of MD2 upon artemisinin binding. Flow cytometry analysis showed artemisinin inhibited LPS-induced TLR4 dimerization and endocytosis in microglial BV-2 cells. Therefore, artemisinin was found to inhibit the TLR4-JNK signaling axis and block LPS-induced pro-inflammatory factors nitric oxide, IL-1β and TNF-α in BV-2 cells. Furthermore, artemisinin restored LPS-induced decrease of junction proteins ZO-1, Occludin and Claudin-5 in primary brain microvessel endothelial cells, and attenuated LPS-induced blood-brain barrier disruption in mice as assessed by Evans blue. In all, this study unambiguously adds MD2 as a direct binding target of artemisinin in its anti-neuroinflammatory function. The results also suggest that artemisinin could be repurposed as a potential therapeutic intervention for inflammatory CNS diseases.