Zhu Qiuzhen, Zhang Yuefan, Liu Yulan, Cheng Hao, Wang Jing, Zhang Yue, Rui Yaocheng, Li Tiejun
Department of Pharmacology, College of Pharmacy, Second Military Medical University, Shanghai, China.
College of Pharmacy, Anhui University of Chinese Medicine, Hefei, Anhui, China.
PLoS One. 2016 Feb 26;11(2):e0149965. doi: 10.1371/journal.pone.0149965. eCollection 2016.
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.
单核细胞移动抑制因子（MLIF）是一种热稳定的五肽，已被证明在缺血性脑损伤中发挥强大的抗炎作用。在本研究中，我们调查了MLIF对人神经母细胞瘤SH-SY5Y细胞氧糖剥夺（OGD）诱导损伤的神经保护作用。采用MTT法评估细胞活力，流式细胞术和Hoechst染色评估细胞凋亡。采用LDH法检测坏死情况。通过ELISA检测炎性细胞因子的释放。通过蛋白质印迹分析测量凋亡相关蛋白的水平。为了鉴定MLIF的蛋白质靶点，进行了下拉试验和质谱分析。我们观察到，MLIF通过抑制p-JNK、p53、c-caspase9和c-caspase3的表达来提高细胞存活率并抑制细胞凋亡和坏死。在小胶质细胞中，在存在MLIF的情况下，OGD诱导的炎性细胞因子分泌明显减少。此外，我们发现真核翻译延伸因子1A2（eEF1A2）是MLIF的下游靶点。使用小干扰RNA（siRNA）敲低eEF1A2几乎完全消除了MLIF对遭受OGD的SH-SY5Y细胞的抗凋亡作用，同时细胞存活率降低，p-JNK和p53的表达增加。这些结果表明，MLIF通过eEF1A2抑制p-JNK/p53凋亡信号通路，改善OGD诱导的SH-SY5Y神经母细胞瘤损伤。我们的研究结果表明，eEF1A2可能是缺血性脑损伤的一个新的治疗靶点。
