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 9

The CM and ALA-CM pretreatment reversed Amyloid β (Aβ1-42) induced a reduction in mitochondrial mass in differentiated SH-SY5Y cells. The fluorescence intensity indicating mitochondrial mass was calculated by immunocytofluorescence staining of the translocase of outer mitochondrial membrane 20 (TOMM20) in differentiated SH-SY5Y cells. Representative fluorescence images (a) and the relative fluorescence intensity of TOMM20 (b) showed that Aβ1-42 induced a reduction of TOMM20 fluorescence intensity. However, the decrease in TOMM20 fluorescence intensity was improved by the CM pretreatment of differentiated SH-SY5Y cells before Aβ1-42 exposure. Besides, ALA-preactivated CM intensified this effect. In contrast, cotreatment of CM and ALA-CM with Insulin Degrading Enzyme (IDE) markedly decreased the immunoreactivity of TOMM20-positive mitochondrial. 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. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a mitochondrial membrane potential disruptor. Insulin was used as a positive control. Next, cells were subjected to immunocytofluorescence staining with antibodies against TOMM20 (see Materials and Methods section). TOMM20 was used to stain mitochondria in differentiated SH-SY5Y cells (shown as red signals). Hoechst 33342 was used to stain nuclei (shown as blue signals). Bar graph showed the relative fluorescence intensity of TOMM20. The fluorescence intensity of TOMM20 was 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 DM + Aβ1-42 group). Scale bar is 20 μm.

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