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.
This paper contains the experiment data on the emodin, mice and cell survival rate, mice intestinal tissue H&E and TUNEL staining, the expression of p53 protein in mice small intestine, cell apoptosis, the expression of protein and RNA in vitro included. Data was worked out through MTT assay, Flow cytometry, Western blot, Real-time PCR and Staining.
ObjectiveThis study aimed to evaluate the potential mechanism by which Monocyte locomotion inhibitory factor (MLIF) improves the outcome of ischemic stroke (IS) inflammatory injury.MethodsPotential MLIF-related targets were predicted using Swiss TargetPrediction and PharmMapper, while IS-related targets were found from GeneCards, PharmGKB, and Therapeutic Target Database (TTD). After obtaining the intersection from these two datasets, the Search Tool for Retrieval of Interacting Genes/Protein (STRING11.0) database was used to analyze the protein-protein interaction (PPI) network of the intersection and candidate genes for MLIF treatment of IS. The candidate genes were imported into the Metascape database for Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The top 20 core genes and the “MLIF-target-pathway” network were mapped using the Cytoscape3.9.1. Using AutoDock Vina1.1.2, the molecular docking validation of the hub targets and MLIF was carried out. In the experimental part, transient middle cerebral artery occlusion (tMCAO) and oxygen and glucose deprivation (OGD) models were used to evaluate the protective efficacy of MLIF and the expression of inflammatory cytokines and the putative targets.ResultsMLIF was expected to have an effect on 370 targets. When these targets were intersected with 1,289 targets for ischemic stroke, 119 candidate therapeutic targets were found. The key enriched pathways were PI3K-Akt signaling pathway and MAPK signaling pathway, etc. The GO analysis yielded 1,677 GO entries (P < 0.01), such as hormone stimulation, inflammatory response, etc. The top 20 core genes included AKT1, EGFR, IGF1, MAPK1, MAPK10, MAPK14, etc. The result of molecular docking demonstrated that MLIF had the strong binding capability to JNK (MAPK10). The in vitro and in vivo studies also confirmed that MLIF protected against IS by lowering JNK (MAPK10) and AP-1 levels and decreasing pro-inflammatory cytokines (IL-1, IL-6).ConclusionMLIF may exert a cerebral protective effect by inhibiting the inflammatory response through suppressing the JNK/AP-1 signaling pathway.
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