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

ABSTRACT: 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.

SUBMITTER: Zhu Q

PROVIDER: S-EPMC4769291 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Zhu Qiuzhen Q Zhang Yuefan Y Liu Yulan Y Cheng Hao H Wang Jing J Zhang Yue Y Rui Yaocheng Y Li Tiejun T

PloS one 20160226 2

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 inflamm ...[more]

PMID: 26918757

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Project description:Oxidative stress plays a key role in cerebral ischemia/reperfusion injury. Artemisinin (ART) has antioxidative stress activity in addition to its powerful antimalarial effects. In this article, we investigated the effect of ART on OGD/R-induced oxidative stress injury and its underlying mechanisms. We used oxygen-glucose deprivation/reoxygenation (OGD/R) to establish an in vitro model of cerebral ischemia/reperfusion (I/R) injury. CCK-8 and lactate dehydrogenase (LDH) release were used to assess cellular damage. Measurement of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and mitochondrial membrane potential (MMP) estimates oxidative stress-induced damage and protection from ART effect. OGD/R treatment aggravated oxidative stress damage, whereas ART reversed the effects of OGD/R. Autophagy is closely related to oxidative stress; in order to confirm whether the antioxidative stress effect of ART is related to PHB2-mediated autophagy, we examined the protein expression of prohibitin 2 (PHB2), TOMM20, p62, and the conversion of microtubule-associated protein light chain 3I (LC3I) to LC3II and found that the protein expression of PHB2, TOMM20, p62, and LC3II/LC3I was significantly correlated with OGD/R treatment. The colocalization of PHB2 and LC3, TOMM20, and LC3 was reduced after OGD/R treatment, and ART reversed this change. After silencing PHB2, the protective effect of ART against OGD/R-induced oxidative stress injury was reduced, the protein expressions of PHB2, TOMM20 and LC3II/LC3I and the colocalization of PHB2 and LC3, TOMM20, and LC3 were decreased. We used chloroquine to block the lysosomal pathway and found that ART increased the conversion of LC3I to LC3II, silencing PHB2 which inhibited the conversion of LC3I to LC3II, and impaired mitophagy. Our findings showed that ART attenuated OGD/R-induced oxidative stress damage through PHB2-mediated mitophagy. To the current knowledge, our study is the first to demonstrate that ART attenuates OGD/R-induced oxidative stress injury through PHB2-mediated autophagy in the human neuroblastoma SH-SY5Y cell line, which provided new insights into the treatment of OGD/R injury.

Project description:BackgroundMaleic acid is a multi-functional chemical widely used in the field of industrial chemistry for producing food additives and food contact materials. As maleic acid may contaminate food by the release from food packages or intentional addition, it raises the concern about the effects of excessive dietary exposure to maleic acid on human health. However, the influence of maleic acid on human health has not been thoroughly studied. In silico toxicogenomics approaches have found the association between maleic acid and nervous system disease in human. The aim of this study is to experimentally explore the effects of maleic acid on human neuronal cells.MethodsA microarray-based transcriptome profiling was performed to offer a better understanding of the effects of maleic acid on human health. Gene expression profiles of human neuroblastoma SH-SY5Y cells exposed to three concentrations of maleic acid (10, 50, and 100 μM) for 24 h were analyzed. Genes which were differentially expressed in dose-dependent manners were identified and further analyzed with an enrichment analysis. The expression profile of selected genes related to the inferred functional changes was validated using quantitative polymerase chain reaction (qPCR). Specific fluorescence probes were applied to observe the inferred functional changes in maleic acid-treated neuronal cells.ResultsA total of 316 differentially expressed genes (141 upregulated and 175 downregulated) were identified in response to the treatment of maleic acid. The enrichment analysis showed that DNA binding and metal ion binding were the significant molecular functions (MFs) of the neuronal cells affected by maleic acid. Maleic acid exposure decreased the expression of genes associated with calcium and thiol levels of the cells in a dose-dependent manner. The levels of intracellular calcium and thiol levels were also affected by maleic acid dose-dependent.DiscussionThe exposure to maleic acid is found to decrease the cellular calcium and thiol levels in human neuronal cells at both transcriptional and functional levels. This study reported the first transcriptomic profiling of human neuronal cells treated with maleic acid. It is also the first experimental validation of chemical effects predicted by in silico toxicogenomics approaches. The proposed approach may be useful in understanding the potential effects of other poorly characterized chemicals on human health.

Dataset Information

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

Publications

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

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