Qi Y X, Su X J, Wei L L, Zhang J
Department of Ophthalmology, The First Affiliated Hospital of Jiamusi University, Jiamusi, China.
Department of Pediatrics, The First Affiliated Hospital of Jiamusi University, Jiamusi, China.
J Biol Regul Homeost Agents. 2021 Mar-Apr;35(2):547-557. doi: 10.23812/20-646-A.
The aim of this study was to investigate the effect of erythropoietin (EPO) on the apoptosis of retinal ganglion cells (RGCs) induced by high glucose and its mechanism. Rat primary RGCs were extracted to establish high glucose-induced apoptosis models using a 30 mM high-glucose medium. Then flow cytometry, cell counting kit-8 (CCK-8) assay and Western blotting assay were performed to detect the effects of high-, medium- and low-dose EPO on the apoptosis of RGCs induced by high glucose. Next, the molecular mechanism by which EPO suppressed the high glucose-induced apoptosis of RGCs was explored via gene array assay and bioinformatics analysis. The results and mechanism of bioinformatics analysis were verified by Western blotting assay. Finally, the small interfering ribonucleic acid (siRNA) experiment was applied to knock down tyrosine-protein phosphatase non-receptor type 1 (PTPN1) and PTPN11 to verify their roles in the inhibition of EPO on the apoptosis of RGCs triggered by high glucose. Flow cytometry-Annexin V/propidium iodide (PI) staining and CCK-8 assay confirmed that the high-, medium- and low-dose EPO inhibited the apoptosis of RGCs induced by high glucose in a dose-dependent manner (P<0.05). Subsequently, Western blotting assay results manifested that the high-, medium- and low-dose EPO reduced the expression levels of apoptosis-related proteins active-cysteinyl aspartate specific proteinase 3 (Caspase 3) and active- Caspase 9 in a dose-dependent manner (P<0.05). Moreover, according to gene array assay and bioinformatics analysis results, the c-Jun N-terminal kinase (JNK) signaling pathway, PTPN1 and PTPN11 might exert crucial effects in the inhibition of EPO on the apoptosis of RGCs induced by high glucose. Western blotting assay results also demonstrated that, compared with the high-glucose treatment, the high-dose EPO treatment decreased the protein expression level of phosphorylated (p)-JNK1/JNK but increased the protein expression levels of PTPN1 and PTPN11 (P<0.05). Moreover, flow cytometry-Annexin V/PI staining and CCK-8 assay results revealed that in EPO-treated cells, knocking down PTPN1 and PTPN11 significantly reversed the protective effect of EPO against high glucose-induced retinal ganglion cell apoptosis (P<0.05). Lastly, Western blotting assay illustrated that knocking down PTPN1 and PTPN11 significantly abolished the inhibition of high-dose EPO on the JNK signaling pathway. EPO may suppress the JNK signaling pathway by raising the expression levels of PTPN1 and PTPN11, so as to inhibit the apoptosis of RGCs triggered by high glucose.
本研究旨在探讨促红细胞生成素（EPO）对高糖诱导的视网膜神经节细胞（RGCs）凋亡的影响及其机制。提取大鼠原代RGCs，使用30 mM高糖培养基建立高糖诱导的凋亡模型。然后进行流式细胞术、细胞计数试剂盒-8（CCK-8）检测和蛋白质印迹分析，以检测高、中、低剂量EPO对高糖诱导的RGCs凋亡的影响。接下来，通过基因芯片检测和生物信息学分析，探索EPO抑制高糖诱导的RGCs凋亡的分子机制。通过蛋白质印迹分析验证生物信息学分析的结果和机制。最后，应用小干扰核糖核酸（siRNA）实验敲低非受体型1酪氨酸蛋白磷酸酶（PTPN1）和PTPN11，以验证它们在EPO抑制高糖触发的RGCs凋亡中的作用。流式细胞术-膜联蛋白V/碘化丙啶（PI）染色和CCK-8检测证实，高、中、低剂量EPO均以剂量依赖性方式抑制高糖诱导的RGCs凋亡（P<0.05）。随后，蛋白质印迹分析结果表明，高、中、低剂量EPO均以剂量依赖性方式降低凋亡相关蛋白活性半胱氨酸天冬氨酸特异性蛋白酶3（Caspase 3）和活性Caspase 9的表达水平（P<0.05）。此外，根据基因芯片检测和生物信息学分析结果，c-Jun氨基末端激酶（JNK）信号通路、PTPN1和PTPN11可能在EPO抑制高糖诱导的RGCs凋亡中发挥关键作用。蛋白质印迹分析结果还表明，与高糖处理组相比，高剂量EPO处理组降低了磷酸化（p）-JNK1/JNK的蛋白表达水平，但增加了PTPN1和PTPN11的蛋白表达水平（P<0.05）。此外，流式细胞术-膜联蛋白V/PI染色和CCK-8检测结果显示，在EPO处理的细胞中，敲低PTPN1和PTPN11显著逆转了EPO对高糖诱导的视网膜神经节细胞凋亡的保护作用（P<0.05）。最后，蛋白质印迹分析表明，敲低PTPN1和PTPN11显著消除了高剂量EPO对JNK信号通路的抑制作用。EPO可能通过提高PTPN1和PTPN11的表达水平来抑制JNK信号通路，从而抑制高糖触发的RGCs凋亡。
