Crocetin restored the ETC function of mitochondria in aged
primary astrocytes
We further assessed the functionality of mitochondrion in aged primary
astrocytes (4th passage) after treating them for 15
days with crocetin at 25 μM (Wani et al., 2021). The flow cytometric
analysis of mitochondrial membrane potential (ΔΨm) revealed a
significantly increased number of cells having higher Mito Tracker
(CMXRos) fluorescence in crocetin treated cells than control samples
indicating higher ΔΨm (Figure 5A). We reconfirmed these findings by
using confocal microscopy under similar experimental conditions. Here,
we measured the mean fluorescence intensity (MFI) of individual
mitochondria in each cell. There was a significant increase of Mito
Tracker (CMXRos) MFI in crocetin-treated astrocytes in comparison to
untreated control cells, however, there was no change in number and area
of mitochondria between the samples (Figure 5B and Supplementary Figures
S4A-D). Further analysis for the expression of mitochondrial inner
membrane proteins ND5 and ND6 revealed that cells treated with crocetin
had a significantly higher expression of these proteins than untreated
aged astrocytes (Figure 5C). Furthermore, the aged astrocytes treated
with crocetin also showed same pattern of change in the levels of
NAD+ and ATP as we found in the mice brain, though,
there was no change in NADH and AMP levels between control and treated
astrocytes (Figure 5D).