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Studies confirmed that hydroxycamptothecin cause the apoptosis of human Tenon capsule fibroblasts (HTFs) by protein kinase R-like endoplasmic reticulum stress kinase (PERK) single pathway.Autophagy and apoptosis are programmed cell death following stress reaction, so they remain a close association.However, the effect of hydroxycamptothecin on the autophagy of HTFs and its mechanism are still unclear.
This study was to explore the promoting effect of PERK signal pathway on hydroxycamptothecin inducing the autophagy of HTFs.
This study procedure was approval by Ethic Committee of Nanjing Medical University.Human Tenon capsule tissue was obtained from fresh adult donors.HTFs were cultured and passaged by explant-culture method and identified by immunofluorescence for vimentin and keratin.pLVX-PERK lentiviral packed by 293T cells was transfected into HTFs to obtain stable PERK-knockout cell line by puromycin selection.Then the HTFs were treated with 0.10 g/L of hydroxycamptothecin for 5 minutes and consecutively cultivated for 24 hours, and the untreated cells were used as the control group.Western blot assay was used to detect the expressions of autophagy specific proteins in the cells, including autophagy related gene 5(ATG-5), Beclin-1, light chain 3(LC-3). Cyto-ID staining was used to identify the autophagosome in the cells.The experimental results were analyzed and compared between different treating groups.
The gray scales for the expressions of Beclin 1, ATG-5, LC-3-Ⅰand LC-3-Ⅱ proteins in HTFs were 0.365±0.045, 0.765±0.055, 0.120±0.030 and 0.215±0.035 in the control group, and those in the hydroxycamptothecin treated group were 0.980±0.070, 1.495±0.095, 0.585±0.025 and 0.785±0.055, showing a significant decline in the hydroxycamptothecin treated group(P=0.018, 0.022, 0.007, 0.013). The green fluorescence of the autophagosome was stronger in the hydroxycamptothecin treated group compared with the control group.Western blot revealed that the gray scale of PERK expression in the cells was 0.130±0.030 in the PERK-knockout group, with a significant reduce in comparison with 0.765±0.055 of the control group (P=0.010). However, no obvious distinctions were seen in the band intensities of the expressions of Beclin-1, ATG-5 and LC-3 proteins between the two groups.Western blot indicated that the grey scale of the PERK expression in the cells was 1.790±0.060 in the 0.10 g/L hydroxycamptothecin group, which was significantly higher than 0.880±0.070 of the control group (P=0.010). Expression levels (gray scales) of Beclin-1, ATG-5, LC-3-Ⅰand LC-3-Ⅱ in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group were 0.475±0.045, 0.390±0.040, 0.055±0.015 and 0.075±0.025, which were significantly lowed in comparison with 0.955±0.065, 0.765±0.055, 0.155±0.015 and 0.280±0.030 of the control+ 0.10 g/L hydroxycamptothecin group (P=0.026, 0.031, 0.042, 0.034). In addition, the fluorescence intensity of autophagosomes was weaker in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group compared with the control+ 0.10 g/L hydroxycamptothecin group.
Hydroxycamptothecin induces the autophagy of HTFs by PERK signal pathway.