Protection against Oxygen-Glucose Deprivation/Reperfusion Injury in Cortical Neurons by Combining Omega-3 Polyunsaturated Acid with Lyciumbarbarum Polysaccharide

Abstract

Ischemic stroke, characterized by the disturbance of the blood supply to the brain, is a severe worldwide health threat with high mortality and morbidity. However, there is no effective pharmacotherapy for ischemic injury. Currently, combined treatment is highly recommended for this devastating injury. In the present study, we investigated neuroprotective effects of the combination of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) and Lyciumbarbarum polysaccharide (LBP) on cortical neurons using an in vitro ischemic model. Our study demonstrated that treatment with docosahexaenoic acid (DHA), a major component of the ω-3 PUFAs family, significantly inhibited the increase of intracellular Ca(2+) in cultured wild type (WT) cortical neurons subjected to oxygen-glucose deprivation/reperfusion (OGD/R) injury and promoted their survival compared with the vehicle-treated control. The protective effects were further confirmed in cultured neurons with high endogenous ω-3 PUFAs that were isolated from fat-1 mice, in that a higher survival rate was found in fat-1 neurons compared with wild-type neurons after OGD/R injury. Our study also found that treatment with LBP (50 mg/L) activated Trk-B signaling in cortical neurons and significantly attenuated OGD/R-induced cell apoptosis compared with the control. Notably, both combining LBP treatment with ω-3 PUFAs administration to WT neurons and adding LBP to fat-1 neurons showed enhanced effects on protecting cortical neurons against OGD/R injury via concurrently regulating the intracellular calcium overload and neurotrophic pathway. The results of the study suggest that ω-3 PUFAs and LBP are promising candidates for combined pharmacotherapy for ischemic stroke.

Keywords: Ca2+; DHA; LBP; OGD/R; Trk-B; cortical neurons; neuroprotection.

Figure 1

Identification of primary cultured neurons. Cultures were prepared from the cortex of E16.5 fat-1 and WT embryos and examined at 7 DIV. (A) An image showing the cortical tissue in the embryonic brain; (B) gel electrophoresis of PCR products using primers for fat-1 gene. Wild-type controls (lanes 3, 6 and 7) and positive fat-1 specimens (lanes 1, 2, 4 and 5); (C,D) examples of phase contrast images of cultured primary neurons; and (E,F) images showing immunostainning on WT and fat-1 neurons respectively (Green, β-III tubulin; Red, GFAP; Blue, DAPI). Scale bar: 50 µm.

Figure 2

Primary cortical neurons were protected against OGD/R injury after LBP and ω-3 PUFAs treatment. Phase contrast images showing the morphological changes of the primary cultured neurons prior or post OGD/R injury. Scale bar: 50 µm.

Figure 3

LBP and exogenous DHA (10 μM) significantly prevent OGD/R-induced neuronal apoptosis respectively via intracellular Ca²⁺ handling or neurotrophic pathway activation: (A) TUNEL staining; (B) fluorescent micrographs showing intracellular Ca²⁺ levels as stained by the Fluo4-AM dye; (C) statistic of cell viability; (D) statistic of TUNEL positive cells; (E) results of relative fluorescence intensity analysis of intracellular Ca²⁺; and (F) expression levels of Trk-B and Bcl-2 measured by Western blot. Data are presented as mean ± SEM, ** p < 0.01, *** p < 0.001 indicate significant difference compared with the WT OGD group; p < 0.05 indicates significant difference compared with the WT DHA + LBP group (t-test). Scale bar: 50 µm.

Figure 4

LBP and endogenous ω-3 PUFAs significantly prevent OGD/R-induced neuronal apoptosis respectively via intracellular Ca²⁺ handling or neurotrophic pathway activation: (A) TUNEL staining; (B) fluorescent micrographs showing intracellular Ca²⁺ levels as stained by the Fluo4-AM dye; (C) statistic of cell viability; (D) statistic of TUNEL positive cells; (E) results of relative fluorescence intensity analysis of intracellular Ca²⁺; and (F) expression levels of Trk-B and Bcl-2 measured by Western blot. Data are presented as mean ± SEM, ** p < 0.01, *** p < 0.001 indicate significant difference compared with the WT OGD group; p < 0.05, p < 0.01 indicates significant difference compared with the WT DHA + LBP group (t-test). Scale bar: 50 µm.