To investigate the inhibitory effects of resveratrol (Res) on the apoptosis of retinal neurons of diabetic rats and ARPE-19 cells induced by high glucose.
Streptozotocin was intraperitoneally injected to establish a diabetes model in 25 adult male SD rats, and 20 successful models were randomized into diabetic model group and Res-administered group according to random number table method.Another 10 matched normal rats were served as normal control group.Res was intragastrically given to the rats in the Res-administered group with the dose of 40 mg/(kg·d), and an equivalent volume of normal saline solution was used in the same way in the diabetic model group and normal control group.The body weight and blood glucose level were measured on the 0th, 4th, 8th, 12th week of administration.The rats were sacrificed on the 12th week by over-anesthesia and the eyeballs were enucleated.The ultrastructure of the retinal ganglion cells (RGCs) were examined under the transmission electron microscope.The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay was to assess the apoptosis of retinal neurons.ARPE-19 cells were divided into normal control group, high glucose group and Res-treated group and cultured for 48 hours in medium containing 5.5 mmol/L glucose, 30 mmol/L glucose, 30 mmol/L glucose and 10 mol/L Res, respectively.Apoptosis rate was detected by a flow cytometry.The distribution and expression of bax and bcl-2 in the cells was detected by immunofluorescence and Western blot assay, respectively.This study protocol was approved by an Experimental Animal Ethic Committee of Hubei University of Science and Technology, and the use and care of the animals complied with the Statement of ARVO.
Compared with the diabetic model group, the body weight was increased at 4-12 weeks and the blood glucose level was lowered at 8-12 weeks of Res administration in the Res-administered group (both at P<0.01). The chromatin condensation, mitochondrial swelling and vacuolation in cytoplasm were obviously slight and the apoptosis rate was reduced in the Res-administered group in comparison with the diabetic model group.The apoptosis indexes of the retinal ganglion cell layer cells and inner nuclear layer cells in the Res-administered group were (18.36±3.37)% and (23.67±8.98)%, respectively, which were significantly lower than (83.91±9.8)% and (64.26±10.66)% in the diabetic model group (both at P<0.01). The apoptosis rate of the ARPE-19 cells in the normal control group, high glucose group and Res-treated group was (3.11±0.26)%, (11.41±1.06)% and (5.38±0.58)%, respectively, and the apoptosis rate in the Res-treated group was significantly lower than that in the high glucose group (all at P<0.01). Immunofluorescence assay showed that the fluorescence of bax in the cell nucleus of Res-treated group was obviously enhanced in comparison with the normal control group and weaker in comparison with the high glucose group.The fluorescence of bcl-2 protein in the Res-interfered group was weaker in comparison with the normal control group and enhanced in comparison with the high glucose group.The relative expressions level of bax protein in the Res-treated group was 0.21±0.08, which was significantly higher than 0.15±0.06 in the normal control group and lower than 0.31±0.09 in the high glucose group (both at P<0.05). The relative expressions of bcl-2 protein was 0.66±0.25 in the Res-treated group, which was significantly lower than 0.80±0.14 in the normal control group and higher than 0.23±0.09 in the high glucose group (both at P<0.05). The bcl-2/bax ratio in the Res-treated group was significantly higher than that in the high glucose group (P<0.01).
Res can inhibit the apoptosis of RGCs of diabetic rats and high glucose-induced RPE cells in vitro.