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 11
The CM and ALA-CM pretreatment modulates Amyloid β- (Aβ1-42-) induced effects on mitophagy and autophagy. 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 the cotreatment of CM and ALA-CM with Insulin Degrading Enzyme (IDE) to check whether insulin and IGF-I presence of 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 showed that Aβ1-42 significantly increased mRNA levels of markers of mitophagy (PINK-1 (a), PARKIN (b)), and autophagy (ATG5 (c) and LC3β (d)). Whereas pretreatment with CM of the SH-SY5Y cells exposed to Aβ1-42 significantly decreased expression of mitophagy and autophagy markers. IDE increased levels of mitophagy and autophagy markers. The immunocytofluorescence staining showed that the Aβ1-42 treated cells had an increased PARKIN fluorescence intensity, a well-known marker of mitophagy (e, f). Whereas pretreatment with CM and ALA-CM of the SH-SY5Y cells exposed to Aβ1-42 significantly decreased PARKIN fluorescence intensity. A similar effect was observed after the insulin treatment. IDE increased PARKIN fluorescence intensity. Bar graph showed the relative fluorescence intensity of PARKIN. Antibody against PARKIN was used to stain marker of mitophagy in differentiated SH-SY5Y cells (shown as green signals). Hoechst 33342 was used to stain nuclei (shown as blue signals). Scale bar is 20 μm. The results showed that Aβ1-42 exposure has a similar effect to CCCP suggesting that Aβ1-42 induce mitophagy and autophagy. 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 cells and untreated control cells are indicated by asterisks ( for ; for ; for ; # versus the control group; ## versus group).
| (a) |
| (b) |
| (c) |
| (d) |
| (e) |
| (f) |