目的:Pleckstrin同源域和富含亮氨酸重复蛋白磷酸酶2(PHLPP2)与各种病理状态有关。然而,PHLPP2是否介导糖尿病性视网膜病变尚未解决.本工作探讨了PHLPP2在调节高糖(HG)引起的视网膜神经节细胞(RGCs)损伤中的生物学功能,研究糖尿病视网膜病变的体外模型。
方法:用HG处理小鼠RGCs以建立细胞模型。通过转染靶向PHLPP2的特异性shRNA来沉默PHLPP2。RT-qPCR,免疫印迹,CCK-8测定,流式细胞术,TUNEL检测,进行ELISA。
结果:在暴露于HG的培养的RGC中观察到PHLPP2水平的显著增加。HG对RGC的严重损害在PHLPP2沉默的RGC中明显减弱,包括提高细胞存活率,细胞凋亡减弱,抑制氧化应激,并禁止促炎反应。PHLPP2的沉默通过调节Akt-GSK-3β轴增强了HG处理的RGC中Nrf2的活化。Akt-GSK-3β轴的中断逆转了PHLPP2沉默引发的Nrf2激活。通过Nrf2抑制,PHLPP2沉默对HG诱导的RGC损伤的保护作用减弱。
结论:通过影响Akt-GSK-3β-Nrf2途径,PHLPP2的丢失对HG损伤的RGC是有益的。这项工作表明PHLPP2在糖尿病性视网膜病变中的可能作用。
OBJECTIVE: Pleckstrin homology domain and leucine-rich repeat protein phosphatase 2 (PHLPP2) is linked to various pathological states. However, whether PHLPP2 mediates diabetic retinopathy is unaddressed. This work explored the biological function of PHLPP2 in modulating high glucose (HG)-elicited damage of retinal ganglion cells (RGCs), an in vitro model for studying diabetic retinopathy.
METHODS: Mouse RGCs were treated with HG to establish a cell model. PHLPP2 was silenced by transfecting specific shRNAs targeting PHLPP2. RT-qPCR, immunoblotting, CCK-8 assay, flow cytometry, TUNEL assay, and ELISA were carried out.
RESULTS: Significant increases in PHLPP2 levels were observed in cultured RGCs exposed to HG. The severe damages evoked by HG to RGCs were remarkably weakened in PHLPP2-silenced RGCs, including improved cell survival, attenuated cell apoptosis, repressed oxidative stress, and prohibited proinflammatory response. The silencing of PHLPP2 strengthened the activation of Nrf2 in HG-treated RGCs via modulation of the Akt-GSK-3β axis. Interruption of the Akt-GSK-3β axis reversed PHLPP2-silencing-elicited Nrf2 activation. The protective effects of PHLPP2 silencing on HG-induced injury of RGCs were diminished by Nrf2 inhibition.
CONCLUSIONS: The loss of PHLPP2 was beneficial for HG-injured RGCs through the effect on the Akt-GSK-3β-Nrf2 pathway. This work suggests a possible role of PHLPP2 in diabetic retinopathy.