FIGURE 3.

Melatonin promotes autophagy via the PI3K/ATK/mTOR signalling pathway in HCAECs. (A) KEGG analysis showing the enriched pathways of the differentially expressed genes in HCAECs stimulated with TNF‐α (20 ng/ml, 12 h), with or without melatonin pre‐treatment (0.5 mM, 6 h). (B) Genes related to autophagy were upregulated, while those related to apoptosis and vascular endothelial cell damage were downregulated, after melatonin treatment. (C) Relative expression of ATG3 after melatonin (0.5 mM, 6 h) pre‐treatment and TNF‐α (20 ng/ml, 12 h) stimulation, as assessed using RT‐qPCR. (D) The protein levels of LC3‐II, LC3‐I, and p62 were quantified using western blot. (E) HCAECs were infected with an adenoviral vector expressing mRFP‐GFP‐LC3, and the kinetics of autophagic flux was detected using a fluorescence microscope. (F) Western blot showing the protein levels of AKT, p‐AKT, mTOR and p‐mTOR in HCAECs stimulated with TNF‐α (20 ng/ml), with or without melatonin pre‐treatment (0.5 mM, 6 h). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant. AKT, protein kinase B; ATG3, autophagy‐related gene‐3; GFP, green fluorescent protein; KEGG, Kyoto Encyclopedia of Genes and Genomes; LC3, microtubule‐associated protein 1 light chain 3; mRFP, monomeric red fluorescent protein; mTOR, mammalian target of rapamycin