Authors: Hou Wenwen, Shi Huanqi, Zhang Zhen, Zhang Xiaomei
Diabetic retinopathy (DR) is one of the common complications of diabetes.Retinal pigment epithelium (RPE) cells, as important constituent cells of the retina, play important roles in the development and progression of DR.
This study was to investigate the suppressive effects of autophagy inhibitor 3-MA on the proliferation of human retinal pigment epithelium cells (hRPECs) following high glucose culture.
HRPECs were divided into control group, high-glucose group and 3-MA+ high glucose group.The cells were cultured by the DMEM/F12 with 5 mmol/L glucose in the control group, and by the DMEM/F12 with 5 mmol/L glucose in the high glucose group and by the DMEM/F12 with 10 mmol/L 3-MA (for 1 hour firstly) and 30 mmol/L glucose in the 3-MA+ high glucose group.The cells were inoculated into 24-well plate with the content of 1×105/well, and then the cells were consecutively cultured for 24 hours with DMEM/F12 containing 0.5% fetal bovine serum after achieved attached 80% confluence.The morphology and ultrastructure of the cells were examined by optics microscope and transmission electronic microscope.The proliferative rate of the cells was assayed by CCK -8 kit.The expression of autophagy-related gene microtubule associated protein light chain 3B (LC3B) in the cells was detected by Western blot and LC3B-Ⅱ/LC3B-Ⅰ value was quantitatively evaluated among the groups.
The cells grew well with uniform size and regulatory arrangement in the control group.The cells in the high glucose group enlarged and the number of cells evidently increased.In the 3-MA+ high glucose group, the cells decreased with a disorder arrangement.Under the transmission electron microscope, the cells were normal with the round- or oval-like nucleus and normal organelles in the control group, and autolysosome could be seen in the cells in the high glucose group.In the 3-MA+ high glucose group, some autophagic bodies were found.The proliferative rate of the cells was (100.0±2.0)%, (116.9±5.2)% and (103.7±4.7)% in the control group, high glucose group and 3-MA+ high glucose group respectively, showing a significant difference among the groups (F=13.526, P=0.006). The proliferative rate was considerably raised in the high glucose group compared with the control group and 3-MA+ high glucose group (both at P<0.05), but there was no significant difference in the proliferative rate of the cells between the control group and 3-MA+ high glucose group (P>0.05). Compared with the control group, the expressing intensity of LC3B-Ⅰ protein was weakened and that of LC3-Ⅱprotein was enhanced, and the expression intensity of LC3B-Ⅰ and LC3B-Ⅱproteins in the 3-MA+ high glucose group was similar to that in the control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.131±0.065, 2.504±0.097 and 0.274±0.007 in the control group, high glucose group and 3-MA+ high glucose group, respectively, with significant differences among the groups (F=1 694.676, P=0.000), and the LC3-Ⅱ/LC3-Ⅰ ratio was increased in the high glucose group in comparison with the control group and the 3-MA+ high glucose group (all at P<0.05). No significant difference was found in the LC3B-Ⅱ/LC3B-Ⅰ ratio between the control group and 3-MA+ high glucose group (P>0.05).
High glucose culture of hRPECs can activate autophagy process and promote cell proliferation.3-MA, an autophagy inhibitor, suppresses the high glucose-induced growth of HRPECs by inhibiting autophagy process.