发光二极管照射对高糖培养的视网膜神经元凋亡的光生物调节作用及其机制

背景 研究糖尿病视网膜病变(DR)的发病机制及防治具有重要的临床意义.近年来随着生物医学光子学研究的发展,国际上利用光生物调节进行疾病防治的研究越来越受到重视,但关于光生物调节对DR的防治作用研究鲜见报道. 目的 探讨光生物调节对高糖环境下视网膜神经元凋亡的抑制作用及其机制,为光生物调节在DR治疗方面的应用提供依据.方法 采用免疫磁珠分离法分离Wistar大鼠视网膜神经元并进行传代培养,采用Nissl染色法对培养的细胞进行鉴定.将培养的细胞分为正常对照组、高糖模型组和高糖+发光二极管(LED)组,正常对照组细胞采用Neurobasal培养基进行培养,高糖模型组细胞在Neurobasal培养基中添加25 mmol/L葡萄糖,高糖+LED组细胞造模后48 h在培养箱中用LED红光光源进行照射,光源波长为620 nm,最大功率为1W,中心光辐射照度为6.67 mW/cm2.光源置于细胞上方2 cm处,光斑直径为2.0cm,使光斑完全覆盖1个培养孔,每次连续照射300 s,12h后重复照射1次,共照射3次.培养后48 h采用流式细胞仪测定各组细胞凋亡情况;采用激光扫描共焦显微镜观察各组细胞内Ca2+浓度变化;Western blot法检测各组细胞中磷酸化丝氨酸-苏氨酸激酶(p-AKT)蛋白的相对表达量.结果 培养后2～3d,倒置显微镜下可见细胞呈多边形和椭圆形,可见细胞核及核仁.培养后5～7d神经元突起增多,经Nissl染色后细胞质呈蓝紫色,神经元与神经胶质细胞的比例达91％.正常对照组、高糖模型组和高糖+LED组细胞凋亡率分别为(7.634_±3.176)％、(33.642±9.315)％和(23.914±6.375)％,其中高糖模型组和高糖+LED组细胞凋亡率均明显高于正常对照组,高糖+ LED组细胞凋亡率明显低于高糖模型组,差异均有统计学意义(均P＜0.0l).激光扫描共焦显微镜下可见高糖模型组和高糖+LED组细胞中Ca2+荧光像素值均明显高于正常对照组,高糖+LED组细胞中Ca2+荧光像素值明显低于高糖模型组,差异均有统计学意义(均P＜0.05).Westen blot法检测显示正常对照组、高糖模型组和高糖+LED组细胞中p-AKT蛋白相对表达量分别为10.34±3.18、2.16±0.46和7.15±1.72,高糖+LED组细胞中p-AKT蛋白相对表达量明显低于高糖模型组,差异有统计学意义(P＜0.05).结论 高糖环境抑制抗凋亡的PI3K/AKT通路活性并影响视网膜神经元钙稳态,导致细胞凋亡.低强度LED光照射可激活PI3K/AKT通路,减少高糖引起的细胞凋亡.

Photobiomodulation of light emitting diode irradiation on apoptosis of retinal neuronal cells induced by high-glucose

Background 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.Objective 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.Methods 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.Results 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 highglucose group and weak in the normal control group.The fluorescence pixel values in the high-glucose group and highglucose+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).Conclusions 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.