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).