促红细胞生成素对视网膜Müller细胞光损伤的作用及机制

Effect and mechanism of erythropoietin on light-induced damage in retinal Müller cells

目的探讨促红细胞生成素(EPO)对视网膜Müller细胞紫外线光损伤的作用及机制。方法将培养的小鼠原代Müller细胞分为无紫外线组(对照组)和紫外线组,每组均用不同浓度药物进行处理,采用噻唑蓝(MTT)法检测EPO对小鼠原代Müller细胞抗紫外线的保护作用。采用蛋白质免疫印迹法检测EPO活化Müller细胞中蛋白激酶B(Akt)水平;将Müller细胞分为二甲基亚砜(DMSO)对照组(DMSO组)和Akt抑制剂MK-2206组(MK-2206组)。以上细胞均以3种方式进行处理:①无紫外线组不处理;②紫外线组采用UV辐射(30 mJ/cm^(2))30 min;③紫外线+药物组,采用1μg/mL的EPO预处理30 min,再进行UV(30 mJ/cm^(2))辐射30 min细胞进一步按照指定时间培养,采用MTT检测细胞活性。将24只小鼠分为无注射组(小鼠右眼,n=6)、注射组(小鼠左眼,n=6)、光照组(无注射眼接受光照,n=6)、光照+注射组(注射眼接受光照,n=6)共4小组,对各组小鼠进行闪光视网膜电图检查,以评估视觉功能受损情况。结果紫外线组不同浓度EPO预处理后的细胞MTT检测的吸光度(OD)值与无紫外线组比较,差异有统计学意义(P<0.05);EPO(0.1、1、10μg/mL)预处理后的细胞MTT检测的OD值较未处理细胞大幅提高,差异有统计学意义(P<0.01);EPO诱导的Müller细胞保护作用也随EPO浓度的增高(0.1、1μg/mL)而增强,差异有统计学意义(P<0.05),而1、10μg/mL浓度处理的细胞保护作用比较,差异无统计学意义(P=0.114)。采用指定浓度的EPO(0、0.1、1、10μg/mL)处理Müller细胞1 h,Western blot检测结果表明,p-Akt(Ser-473)和Akt1蛋白表达水平增高,且在0.1、1μg/mL浓度范围存在浓度依赖性。无注射组和注射组振幅比较,差异无统计学意义(P=0.17);无注射组与光照组及光照+注射组振幅比较,差异均有统计学意义(P<0.05)。光照+注射组和光照组振幅比较,差异有统计学意义(P<0.05)。结论EPO通过激活Müller细胞Akt信号通路减轻小鼠视网膜光损伤。

Objective To explore the effect and mechanism of erythropoietin(EPO)on light-induced retinal damage in Müller cells.Methods Primary Müller cells of mice cultured were selected and divided into ultraviolet free group(control group)and ultraviolet group,each group was treated with different concentrations of EPO.The 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to detect cell viability.Western blot was used to detect the rotein kinase B(Akt)levels in EPO-activated Müller cells.Müller cells were divided into dimethyl sulfoxide(DMSO)control group(DMSO group)and inhibitors of Akt MK-2206 group(MK-2206 group).The above cells were treated in three ways:①no ultraviolet group without treatment;②ultraviolet group with ultraviolet radiation(30 mJ/cm2)for 30 min;③ultraviolet+drug group[the cells were pretreated with 1μg/mL EPO for 30 min,and then treated with ultraviolet(30 mJ/cm^(2))radiation for 30 min].The cell viability was detected by MTT.A total of 24 mice were divided into four groups:no injection group(right eye of mice,n=6),injection group(left eye of mice,n=6),illumination group(no injection eye receiving illumination,n=6)and illumination+injection group(injection eye receiving illumination,n=6).The mice in each group were examined by flash electroretinogram to assess impaired visual function.Results Optical density(OD)values of cells pretreated with different concentrations of EPO in the ultraviolet group showed significant differences compared with those in the no ultraviolet group(P<0.05).Compared with untreated cells,OD values of pretreated cells by EPO(0.1,1,10μg/mL)were significantly increased by MTT(P<0.01).The protective effect of Müller cells induced by EPO was also enhanced with the increase of EPO concentration(0.1 and 1μg/mL,respectively,P<0.05),while the protective effects of 1 and 10μg/mL treatments showed no statistically significant differences(P=0.114).After Müller cells were treated with EPO(0,0.1,1,10μg/mL)for 1 h,western blot analysis showed that the protein expression levels of p-Akt(Ser-473)and Akt1 increased,and there was concentration dependence in the range of 0.1 and 1μg/mL.There was no significant difference in amplitude between non-injection group and injection group(P=0.17).There were statistically significant differences in amplitude between the no injection group and injection group(P=0.17).There were statistically significant differences in amplitude between the no injection group and the illumination group,the illumination+injection group(P<0.05).The amplitude of illumination+injection group showed significant difference compared with the illumination group(P<0.05).Conclusion EPO alleviates retinal light damage in mice by activating Müller cell Akt signaling pathway.