Research Article
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 6
The CM and ALA-CM pretreatment reversed the Amyloid β1-42- (Aβ1-42-) induced cytotoxicity in differentiated SH-SY5Y cells. The LDH assay (a, c) and double-stained with Hoechst 33342 and propidium iodide (PI) (b, d, e) results indicated that Aβ1-42 significantly increased cell death of differentiated SH-SY5Y cells. Whereas, the CM pretreatment decreased cell death in the SH-SY5Y cells treated with Aβ1-42. Besides, ALA preactivated CM significantly intensified the protective effect of CM. In contrast, Insulin Degrading Enzyme (IDE) significantly attenuated the protective effect of CM and ALA-CM. The insulin pretreatment reversed the Aβ1-42-induced cytotoxicity 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 Aβ1-42 for the next 24 h. The SH-SY5Y cells were also exposed to cotreatment of CM and ALA-CM with 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). Next, cells were subjected to vital double staining with PI and Hoechst 33342 (see Materials and Methods section). The PI/Hoechst ratio was calculated by dividing the PI by Hoechst Relative fluorescence units (RFUs). One-way ANOVA test for cytotoxicity followed by Tukey’s multiple comparisons was used to analyse data. Statistical differences between the treated cells and untreated control cells are indicated by asterisks ( for ; for ; for ; # versus the control group). Results are of three independent experiments. The vital double staining cells were also analyzed in inverted fluorescence microscopy (see Materials and Methods section). Scale bar is 50 μm.
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