Brain-derived neurotrophic factor mediates macrophage migration inhibitory factor to protect neurons against oxygen-glucose deprivation

Abstract

Macrophage migration inhibitory factor (MIF) is a chemokine that plays an essential role in immune system function. Previous studies suggested that MIF protects neurons in ischemic conditions. However, few studies are reported on the role of MIF in neurological recovery after ischemic stroke. The purpose of this study is to identify the molecular mechanism of neuroprotection mediated by MIF. Human neuroblastoma cells were incubated in Dulbecco's modified Eagle's medium under oxygen-glucose deprivation (OGD) for 4 hours and then returned to normal aerobic environment for reperfusion (OGD/R). 30 ng/mL MIF recombinant (30 ng/mL) or ISO-1 (MIF antagonist; 50 μM) was administered to human neuroblastoma cells. Then cell cultures were assigned to one of four groups: control, OGD/R, OGD/R with MIF, OGD/R with ISO-1. Cell viability was analyzed using WST-1 assay. Expression levels of brain-derived neurotrophic factor (BDNF), microtubule-associated protein 2 (MAP2), Caspase-3, Bcl2, and Bax were detected by western blot assay and immunocytochemistry in each group to measure apoptotic activity. WST-1 assay results revealed that compared to the OGD/R group, cell survival rate was significantly higher in the OGD/R with MIF group and lower in the OGD/R with ISO-1 group. Western blot assay and immunocytochemistry results revealed that expression levels of BDNF, Bcl2, and MAP2 were significantly higher, and expression levels of Caspase-3 and Bax were significantly lower in the MIF group than in the OGD/R group. Expression levels of BDNF, Bcl2, and MAP2 were significantly lower, and expression levels of Caspase-3 and Bax were significantly higher in the ISO-1 group than in the OGD/R group. MIF administration promoted neuronal cell survival and induced high expression levels of BDNF, MAP2, and Bcl2 (anti-apoptosis) and low expression levels of Caspase-3 and Bax (pro-apoptosis) in an OGD/R model. These results suggest that MIF administration is effective for inducing expression of BDNF and leads to neuroprotection of neuronal cells against hypoxic injury.

Keywords: apoptosis; brain-derived neurotrophic factor; hypoxia; in vitro; ischemic stroke; macrophage migration inhibitory factor; nerve regeneration; neuroprotective effect; reperfusion.

Figure 1

Human neuroblastoma cell line SH-SY5Y (on the confocal microscopy; A) and a schematic diagram of OGD/R model (B). Scale bar in A: 50 μm. ISO-1: MIF antagonist; MIF: macrophage migration inhibitory factor recombinant; OGD/R: oxygen-glucose deprivation and reperfusion.

Figure 2

Cell survival assessed by the water-soluble tetrazolium salts-1 assay relative to the control group. Compared to the OGD/R group, cell survival was significantly higher in MIF-treated cells and lower in ISO-1 treated cells. *P < 0.05, vs. OGD/R group; one-way analysis of variance followed by the Tukey’s honestly significant difference test; n = 5 (number of independent cell culture experiments). ISO-1: MIF antagonist; MIF: macrophage migration inhibitory factor recombinant; OGD/R: oxygen-glucose deprivation and reperfusion.

Figure 3

Western blot results of neuroblastoma cell protein expression under control, OGD/R conditions, and OGD/R conditions treated with exogenous MIF or MIF antagonist ISO-1. (A) The expression levels of immature and mature BDNF and MAP2 in the MIF group were significantly higher than those in the OGD/R group (immature BDNF: P < 0.05; mature BDNF: P < 0.01). (C) The expression levels of Bcl2, Caspase-3, and Bax in the MIF group were significantly lower than those in the OGD/R group (Caspase-3: P < 0.01; Bax: P = 0.02). The expression levels of Bcl2 and MAP2 were significantly higher in the MIF group and lower in the ISO-1 group than those in the OGD/R group (Bcl2: P < 0.01; MAP2: P < 0.05). The expression levels of Caspase-3 and Bax were significantly lower in the MIF group and higher in the ISO-1 group than those in the OGD/R group (Caspase-3: P < 0.01; Bax: P = 0.02). (B and D) The representative bands of each group in western blot are presented. *P < 0.05, **P < 0.01, vs. OGD/R group; one-way analysis of variance followed by the Bonferroni post-hoc analysis (number of independent cell culture experiments (n): immature BDNF, n = 5; mature BDNF, n = 6; Bcl2, n = 6, MAP2, n = 8; Caspase-3, n = 7; Bax, n = 8). BDNF: Brain-derived neurotrophic factor; ISO-1: MIF antagonist; MAP2: microtubule-associated protein 2; MIF: macrophage migration inhibitory factor recombinant; OGD/R: oxygen-glucose deprivation and reperfusion.

Figure 4

ICC findings of neuroblastoma cells under control, OGD/R conditions, and OGD/R conditions treated with exogenous MIF or MIF antagonist ISO-1. (A) Immunoreactivities of BDNF, MAP2, Bcl-2, Caspase-3 and Bax. The expression levels of mature BDNF and MAP2 in the MIF group were significantly higher than those in the OGD/R group (P < 0.01). (C) The expression levels of Caspase-3 and Bax in the MIF group were significantly lower than those in the OGD/R group (P < 0.01). The expression levels of Caspase-3 and Bax in the ISO-1 group were significantly higher than those in the OGD/R group (P < 0.01). The mean expression level of Bcl2 (anti-apoptotic marker) tended to be higher in the MIF group than that in the ISO-1 group (P = 0.03 but not significant in the post-hoc analysis). **P < 0.01, vs. OGD/R group; one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc analysis); (number of independent cell culture experiments (n); mature BDNF, n = 11; Bcl2, n = 5; MAP2, n = 5; Caspase-3, n = 11; Bax, n = 8). (B and D) The representative images of each group in immunocytochemistry are presented. BDNF, red staining in ICC; Bcl2, green staining in ICC; MAP2, red staining in ICC; Caspase-3 and Bax, green staining in ICC; DAPI, blue staining in ICC. BDNF: Brain-derived neurotrophic factor; ICC: immunocytochemistry; ISO-1: MIF antagonist; MAP2: microtubule-associated protein 2; MIF: macrophage migration inhibitory factor recombinant; OGD/R: oxygen-glucose deprivation and reperfusion.