LED光源照射及PI3K抑制剂对人视网膜色素上皮细胞自噬和凋亡的影响

Effects of LED light irradiation and PI3K inhibitor on autophagy and apoptosis of human retinal pigment epithelial cells

目的探讨LED光源照射对人视网膜色素上皮(RPE)细胞自噬和凋亡的影响,以及磷脂酰肌醇3激酶(PI3K)抑制剂LY294002在光照下对RPE细胞自噬和凋亡的影响。方法体外培养人ARPE-19细胞,随机分为6 h对照组、6 h模型组、6 h LY294002组,12 h对照组、12 h模型组、12 h LY294002组,24 h对照组、24 h模型组、24 h LY294002组,分别给于设定时间的LED冷光照射或加入LY294002后的光照。采用噻唑蓝法检测各组细胞存活率;流式细胞术检测各组细胞凋亡率;透射电子显微镜观察各组细胞超微结构;构建稳定表达红色荧光蛋白-绿色荧光蛋白-微管相关蛋白1轻链3(LC3)的RPE细胞株,激光共聚焦合倒置荧光显微镜分析细胞自噬流变化;Western blot法检测苄氯素1(Beclin 1)、LC3和p62自噬相关蛋白表达水平。结果与对照组比较,6、12、24 h模型组细胞存活率均下降(均P<0.05),12、24 h LY294002组细胞存活率均下降(均P<0.01);与模型组比较,12、24 h LY294002组细胞存活率均升高(均P<0.05)。与对照组比较,6、12、24 h模型组和LY294002组细胞凋亡率均升高(均P<0.05);与模型组比较,6、12 h LY294002组细胞凋亡率均下降(均P<0.05)。模型组RPE细胞在光照24 h后,胞内可见较多的自噬泡,LY294002干预后也可见聚集性分布的自噬囊泡。观察自噬流发现,对照组少见红色亮点,模型组红色亮点荧光随光照时间延长数量逐渐增多,Merge图中黄色亮点数量增多明显,LY294002干预后,红色亮点与黄色亮点呈增多趋势。与对照组比较,6、12、24 h模型组和LY294002组细胞中Beclin 1、LC3Ⅱ/LC3Ⅰ蛋白表达水平均升高,p62蛋白表达水平均下降,差异均有统计学意义(均P<0.05);与模型组比较,6、12、24 h LY294002组细胞中Beclin 1、LC3Ⅱ/LC3Ⅰ蛋白表达均升高,12、24 h LY294002组细胞中p62蛋白表达水平均下降,差异均有统计学意义(均P<0.05)。结论LED光源照射可刺激ARPE-19细胞发生自噬,增加细胞凋亡率;PI3K抑制剂LY294002能上调细胞的自噬活性,减缓凋亡。

Objective To investigate the effects of LED light irradiation on autophagy and apoptosis of human retinal pigment epithelial(RPE)cells,and the effects of phosphatidylinositol 3-kinase(PI3K)inhibitor LY294002 on autophagy and apoptosis of RPE cells under light exposure.Methods Human ARPE-19 cells were cultured in vitro and randomly divided into 6 h control,6 h model and 6 h LY294002 groups;12 h control,12 h model and 12 h LY294002 groups;and 24 h control,24 h model and 24 h LY294002 groups,which were given cold light irradiation by LEDs for a set time or light irradiation with the addition of LY294002,respectively.Methylthiazolyl diphenyl-tetrazolium bromide(MTT)method and flow cytometry(FCM)were used to detect cell proliferation and apoptosis,respectively.The ultrastructure of cells was observed by transmission electron microscope.RPE cell lines with stable expression of monomeric red fluorescent protein(mRFP)-enhanced green fluorescent protein(eGFP)-microtubule-associated protein1 light chain 3(LC3)were constructed,and the changes of autophagy flow were analyzed by confocal laser combined with inverted fluorescence microscopy.The expression levels of Beclin 1,LC3 and p62 autophagy related proteins were detected by Western blot.Results Compared with the control group,the cell survival rate in the 6,12 and 24 h model groups decreased significantly(all P<0.05);same results were achieved in the 12 and 24 h LY294002 groups(both P<0.01).Compared with the model groups,the cell survival rate of 12 and 24 h LY294002 groups was elevated(both P<0.05).Compared with the control group,the apoptosis rate in the 6,12 and 24 h model groups was significantly higher(all P<0.01);same results were achieved in 6,12 and 24 h LY294002 groups(all P<0.05).Compared with the model group,the apoptosis rate decreased in 6 and 12 h LY294002 groups(both P<0.05).More autophagic vesicles were seen intracellularly in RPE cells of the model group after 24 h of light exposure,and aggregated distribution of autophagic vesicles was also seen after LY294002 intervention.Observation of autophagic flow revealed few red bright spots in the control groups,increased number of red bright spots fluorescence in the model group with the light exposure time,significantly increased number of yellow bright spots in the merge graph,and the increased red and yellow bright spots after the intervention of LY294002.Compared with the control group,the protein expression levels of Beclin 1 and LC3II/LC3I in the cells of the model groups and LY294002 groups increased at 6,12 and 24 h,while the protein expression level of p62 all decreased(all P<0.05);compared with the model groups,the protein expression levels of Beclin 1 and LC3II/LC3I in the cells of the 6,12 and 24 h LY294002 groups all increased,while the protein expression level of p62 decreased in 12 and 24 h LY294002 groups(all P<0.05).Conclusion LED light irradiation can stimulate autophagy of ARPE-19 cells and increase apoptosis rate.PI3K inhibitor LY294002 can up-regulate autophagy activity of cells and reduce apoptosis.