MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2

Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury.单核细胞运动抑制因子（Monocyte locomotion inhibitory factor，MLIF）是一种热稳定性五肽，已被证实可在缺血性脑损伤中发挥强效抗炎作用。本研究旨在探究MLIF对氧糖剥夺（oxygen-glucose deprivation，OGD）诱导的人神经母细胞瘤SH-SY5Y细胞损伤的神经保护作用。采用MTT实验评估细胞活力，流式细胞术与Hoechst染色法检测细胞凋亡，乳酸脱氢酶（LDH）实验检测细胞坏死；通过酶联免疫吸附试验（ELISA）检测炎性细胞因子的释放量，利用蛋白质印迹（Western blot）分析测定凋亡相关蛋白的表达水平。为鉴定MLIF的蛋白靶点，本研究开展了pull-down实验（pull-down assay）与质谱分析法（mass spectrometry）。结果显示，MLIF可通过抑制p-JNK、p53、剪切型caspase9及剪切型caspase3的表达，增强细胞存活能力并抑制细胞凋亡与坏死。在小胶质细胞（microglia）中，MLIF可显著降低OGD诱导的炎性细胞因子分泌水平。此外，本研究证实真核翻译延伸因子1A2（eukaryotic translation elongation factor 1A2，eEF1A2）是MLIF的下游靶点。采用短链干扰RNA（short interfering RNA，siRNA）敲低eEF1A2的表达，可几乎完全逆转MLIF在OGD处理的SH-SY5Y细胞中发挥的抗凋亡作用，同时伴随细胞存活率下降以及p-JNK与p53表达水平升高。上述结果表明，MLIF可通过eEF1A2调控p-JNK/p53凋亡信号通路，从而改善OGD诱导的SH-SY5Y神经母细胞瘤细胞损伤。本研究结果提示，eEF1A2或可成为缺血性脑损伤的新型治疗靶点。