The Effects of Alpha-Linolenic Acid on the Secretory Activity of Astrocytes and β Amyloid-Associated Neurodegeneration in Differentiated SH-SY5Y Cells: Alpha-Linolenic Acid Protects the SH-SY5Y cells against β Amyloid Toxicity

Figure 7

The CM and ALA-CM pretreatment reversed Amyloid β- (Aβ_1-42-) induced synaptic toxicity in differentiated SH-SY5Y cells. On the day 6th, the SH-SY5Y cells (differentiated) were pretreated for 1 h with CM or ALA-CM before the addition of 5 μM Aβ_1-42 for the next 24 h. The SH-SY5Y cells were also exposed to the cotreatment of CM and ALA-CM with Insulin Degrading Enzyme (IDE) to check whether insulin and IGF-I presence in CM and ALA-CM was responsible for the neuroprotective effect. Positive controls were the SH-SY5Y cells treated with insulin and carbonyl-cyano-m-chlorophenylhydrazone-CCCP (10 μM). RT-qPCR results indicated that Aβ_1-42 significantly decreased mRNA levels of Synaptophysin (a) and PSD95 (b), well-known synaptic markers. The CM and ALA-CM pretreatment reversed the effect of Aβ_1-42 when compared with group. The IDE treatment of CM and ALA-CM reduced this effect. The immunocytofluorescence staining showed that the Aβ_1-42 treated cells had a decreased Synaptophysin (c, e) and TUJ 1 (β3-Tubulin) (d, f) fluorescence intensity and increased neurites fragmentation. Moreover, results showed that the CM and ALA-CM pretreatment reversed the Aβ_1-42–induced synaptic toxicity in differentiated SH-SY5Y cells. A similar effect of TUJ 1 fluorescence intensity was observed after the treatment of differentiated SH-SY5Y cells with insulin (d, f). The IDE treatment of CM and ALA-CM reduced this effect. The cells were subjected to immunocytofluorescence staining with antibodies against Synaptophysin and TUJ 1. TUJ 1 was used as a marker to stain differentiated SH-SY5Y cells (show as green signals). Synaptophysin was used to stain synaptic in differentiated SH-SY5Y cells (show as red signals). Hoechst 33342 was used to stain nuclei (show as blue signals) (see Materials and Methods section). Bar graphs (e, f) showed the relative fluorescence intensity of Synaptophysin and TUJ 1. Scale bar is 20 μm. One-way ANOVA followed by Tukey’s multiple comparisons test at the 0.05 level was used to determine differences between the treated cells and untreated control cells. Results are presented as (). RT-qPCR fold increase and the fluorescence intensity were calculated according to the formula described in the Materials and Methods section. Statistical differences between the treated group and untreated control cells are indicated by asterisks ( for ; for ; for ; ^# versus the control group; ^## versus group).

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