Authors: Zhang Limin, Zhang Fengjiu, Lin Anling, Peng Xiangdong, Yang Jianling, Yu Lingyan, Wang Haiming, Gao Shikai
To study the pathogenesis and management of diabetic retinopathy (DR) has an important clinical significance.With the development of biomedical photonics in recent years, photobiomodulation therapy has been paid more and more attention.However, the sudy on biological regulation of light to DR is rarely reported.
This study was to explore the photobiomodulating effects on the apoptosis of retinal neuronal cells induced by high glucose environment and tried to offer a basis for the management of DR.
The retinal neurons were isolated from Wistar rats using immunomagnetic beads and primarily cultured in Neurobasal, and the cells were identified by Nissl staining.The cells were divided into normal control group, high-glucose group and high-glucose+ LED group.The glucose at the concentration of 25 mmol/L was added into medium for 48 hours in the high-glucose group, and the cells induced by high-glucose were irradiated in incubator by LED for consecutive 300 seconds per time in a 12-hour interval with the wavelength of 620 nm, maximal power of 1 W, central light radiation exposure of 6.67 mW/cm2 and spot diameter of 2.0 cm.The apoptosis rate of the cells was assayed by flow cytometry; the intracellular Ca2+ content was determined by laser scanning confocal microscope; the relative expression level of phosphorylated serine-threonine kinase (p-AKT) protein in the cells was detected by Western blot.
The cells grew well 2-3 days after cultured with the polygon and oval shape, and nucleolus were visible.More neuronal processes were obtained in 5-7 days after culture.Nissl staining showed the blue violet color in cytoplasma of neurons.The proportion of neurons and glial cells was 91%.The apoptosis rates of the cells were (7.634±3.176)%, (33.642±9.315)% and (23.914±6.375)% in the normal control group, high-glucose group and high-glucose+ LED group, respectively, and the apoptosis rates of high-glucose group and high-glucose+ LED group were significantly higher than that in the normal control group, while the apoptosis rate in the high-glucose+ LED group was lower than that in the high-glucose group (all at P<0.01). The fluorescence of Ca2+ in the cytoplasma was strong in the high-glucose group and weak in the normal control group.The fluorescence pixel values in the high-glucose group and high-glucose+ LED group were significantly higher than that in the normal control group, and that in the high-glucose+ LED group was reduced in comparison with high-glucose group (all at P<0.05). The expressing band of p-AKT protein was strong in the normal control group and weak in the high-glucose group.The relative expressing levels were 10.34±3.18, 2.16±0.46 and 7.15±1.72 in the normal control group, high-glucose group and high-glucose+ LED group, and relative expression level of high-glucose+ LED group was significatly lower than that in the high-glucose group (P<0.05).
High-glucose environment inhibits PI3K/AKT pathway and calcium homeostasis of retinal neurons, which results in cell apoptosis.Low intensity of LED light irradiation activates the anti-apoptotic PI3K/AKT pathway and therefore reduces apoptosis induced by high glucose.