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.

Fig 1. The effect of MLIF on OGD-induced SH-SY5Y neuroblastoma injury was evaluated using MTT assay, flow cytometry and Hoechst staining assay.

SH-SY5Y cells were exposed to OGD for 6h. After treatment with MLIF (0.1, 1.0, 10 μg/ml), MTT assay was used to measure the cell survival ratio (A). Annexin V/PI labeling, assessed by flow cytometry (B, D), and Hoechst 33258 staining (C, E) were performed to evaluate apoptosis in SH-SY5Y cells. Data are expressed as the mean ± SEM. Results were analyzed with one-way ANOVA; n = 3. **P < 0.01, OGD group vs. control group; ^##P< 0.01, MLIF group vs.OGD group.

Fig 2. The effect of MLIF on the protein levels of apoptotic proteins in SH-SY5Y cells exposed to OGD.

SH-SY5Y cells were exposed to OGD for 6 h with or without incubation with MLIF (0.10 μg/mL). p-JNK (A),p53 (B), p38 (C), ERK (D), caspase3 (E) and caspase9 (F) levels were determined by immunoblotting. Data are expressed as the mean ± SEM. Results were analyzed using one-way ANOVA; n = 3. *P < 0.05, **P < 0.01 or ***P < 0.001, OGD group vs. control group; ^#P < 0.05 or ^##P < 0.01 MLIF group vs.OGD group.

Fig 3. eEF1A2 was identified as the binding protein of MLIF in SH-SY5Y cells.

Pull-down assays were carried out using biotin-conjugated MLIF (bio-MLIF) and cell lysates. Binding proteins were washed and separated by SDS-PAGE (A). The binding protein was analyzed by MALDI-TOF MS after in-gel digestion, and found to be eEF1A2 (B). Western blotting with anti-eEF1A2 antibody confirmed the identity of the binding protein (C). Confocal microscopy revealed co-localization of FITC-labeled MLIF (green) and eEF1A2 (labeled with rabbit anti-eEF1A2; red) in SH-SY5Y cells (D).

Fig 4. MLIF protects SH-SY5Y cells against apoptosis in an eEF1A2-dependent manner.

OGD-exposed SH-SY5Y cells transfected with eEF1A2 siRNA or negative control siRNA (NC) were treated with MLIF (0.1 μg/ml). Cell survival was measured using MTT assay (A). Representative annexin V/PI labeling, assessed by flow cytometry, was used to analyze the ratio of apoptotic SH-SY5Y cells (B, D). Hoechst 33258 staining was used to evaluate the nuclear morphology of SH-SY5Y cells (C, E). Data are expressed as the mean ± SEM. Results were analyzed using one-way ANOVA; n = 3. **P< 0.01 or *P< 0.05, OGD group vs. control group or MLIF group; ^##P < 0.01 or ^#P< 0.05, eEF1A2 siRNA group vs. NC group.

Fig 5. MLIF downregulates p-JNK and p53 levels via eEF1A2.

After transfection with eEF1A2 siRNA or NC, OGD-exposed SH-SY5Y cells were incubated with MLIF (0.1 μg/mL). The levels of p-JNK (A) and p53 (B) were determined by immunoblotting. Data are expressed as the mean ± SEM. Results were analyzed using one-way ANOVA; n = 3. *P< 0.01 siRNA group vs. NC group.