Aims
Dihydroartemisinin (DHA), a derivative of artemisinin, suppresses angiogenesis by regulating endothelial cell phenotypes. In this study, we investigated the effect of DHA on endothelial cell autophagy and the underlying mechanisms.
Conclusion
DHA induces autophagy in HUVECs by inhibition of the Akt/mTOR pathway.
Methods
Human umbilical vein endothelial cells (HUVECs) were treated with DHA. Formation of autophagosomes in HUVECs was observed by fluorescence microscope after pcDNA3.1-green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) plasmids transfection. Dichlorofluorescein diacetate (DCFH-DA) staining was used to detect intracellular reactive oxygen species (ROS). Western blot was performed to detect the protein levels of LC3, p62, beclin 1, autophagy-related protein (Atg) 5, p-Akt (protein kinase B), p-mTOR (mammalian target of rapamycin), p-4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1), and p-p70S6K (p70 ribosomal S6 kinase).
Results
DHA increased LC3-II and the number of fluorescent GFP-LC3 puncta in HUVECs. Silencing ATG5 by siRNA interference attenuated DHA-induced LC3-II elevation. DHA enhanced ROS production, but pretreatment with antioxidant N-acety-l-cysteine (NAC) failed to reduce DHA-induced autophagy in HUVECs. Pretreatment with PD98059, SP600125 and SB203580, the inhibitors of ERK, JNK, and p38 MAPK, did not reverse autophagy in DHA-treated HUVECs. DHA significantly reduced phosphorylation of Akt, mTOR, p70S6K, 4E-BP1 in HUVECs. Rapamycin, an mTOR antagonist, compromised DHA-induced autophagy.