Abstract:

Objective To observe autophagy of human tenon′s capsule fibroblasts（HTFs）induced by arte⁃ sunate（Art）and explore the possible mechanism. Methods The cultured in vitro HTFs were divided into control group and Art（50，100，150，200 μg/mL）groups. After treatment for 24 and 48 h，cell activity was determined by methyl thiazol tetrazolium（MTT）assay. 48 h later，quantitative reverse transcriptase PCR（qRT⁃PCR）and Western Blot analysis were employed to assay the relative expression of microtubule⁃associated protein light chain 3（LC3）⁃ Ⅱ and Becline ⁃1 in HTFs. The autophagy activity of HTFs was evaluated by a Cyto ⁃ ID autophagy detection kit， and then the autophagic flux was evaluated by counting the Cyto ⁃ ID positive cells. Results The results of MTT showed that Art could significantly inhibit the growth of fibroblasts in vitro in a concentration and time dependent manner（P < 0.05），and the inhibition rate increased from 4.7% to 68.6% with increase of concentration and prolongation of time. qRT⁃PCR results showed that LC3⁃Ⅱand beclin⁃l mRNA relative expression increased after Art treatment，from（1.367 ± 0.214）to（4.765 ± 0.371），and（1.235 ± 0.164）to（3.487 ± 0.356），respectively. The difference between the groups had statistical significance（FL = 20.920，P < 0.01；FB = 26.370，P < 0.01）. Western Blot results showed that LC3⁃Ⅱand beclin⁃l protein relative expression increased after Art treatment，from（0.101 ± 0.025）to（0.808 ± 0.102），and（0.098 ± 0.036）to（0.784 ± 0.064），respectively. The difference between the groups had statistical significance（FL = 32.852，P < 0.01；FB = 29.874，P < 0.01）. Cyto⁃ID staining showed that the percentage of autophagic cell increased dramatically from（13.302 ± 2.301）% to（53.274 ± 7.851）% upon the exposure of HTFs to 150 μg/mL Art（t = 8.804，P < 0.01）. Conclusion Artesunate can effectively promote the production of LC3⁃Ⅱand Beclin⁃l，increase the Ievel of autophagy in HTFs，and inhibit the growth of fibroblasts.

Key words: artesunate,  , human Tenon′s capsule fibroblasts,  , proliferation,  , autophagy,  , glaucoma