Flavonoid-Rich Ethanol Extract from the Leaves of Diospyros kaki Attenuates D-Galactose-Induced Oxidative Stress and Neuroinflammation-Mediated Brain Aging in Mice

Abstract

Aging is a major factor that contributes to neurological impairment and neuropathological changes, such as inflammation, oxidative stress, neuronal apoptosis, and synaptic dysfunction. Flavonoids act as protective antioxidant and anti-inflammatory agents against various age-related neurodegenerative diseases. Here, we investigated the protective effect and mechanisms of the flavonoid-rich ethanol extract from the leaves of Diospyros kaki (FELDK) in the cortex and hippocampus of D-galactose- (gal-) aged mice. Our results showed that FELDK treatment restored memory impairment in mice as determined by the Y-maze and Morris water maze tests. FELDK decreased oxidative stress levels via inhibiting reactive oxygen species (ROS) and malondialdehyde (MDA) production and elevating antioxidative enzymes. FELDK also alleviated D-gal-induced neuroinflammation via suppressing the expression of advanced glycation end products (AGEs) and receptor for AGEs (RAGE) and activating microgliosis and astrocytosis, nuclear factor kappa B (NF-κB) nuclear translocation, and downstream inflammatory mediators. Moreover, FELDK inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt and C-jun N-terminal kinase (JNK) apoptotic signaling pathways and ameliorated the impairment of synapse-related proteins. Hence, these results indicate that FELDK exerts neuroprotective effects on D-gal-induced brain aging. Thus, FELDK may be a potential therapeutic strategy for preventing and treating age-related neurodegenerative diseases such as Alzheimer's disease.

Figure 1

Effects of FELDK on memory impairment in D-gal-treated mice. (a) The percentage of spontaneous alteration in the Y-maze test. (b) Comparison of EL during 6 days in the MWM. (c) The number of crossings and (d) the time spent in the target quadrant where the platform had been previously situated during the spatial probe trial on day 6. Data are presented as mean value ± SEM for n = 9, 8, 8, 8. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus NC group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the D-gal group.

Figure 2

Effects of FELDK on AGEs, RAGE, GFAP, and Iba1 expression in the cortex and hippocampus of D-gal-aged mice. (a) AGEs levels were determined by ELISA (n = 9, 8, 8, 8). (b) Representative western blots showing protein expression of RAGE, GFAP, and Iba1 (n = 3). (c) Quantification of western blot analysis of RAGE, GFAP, and Iba1 protein expressed as percent of control. (d) The immunohistochemical images of active microglia and astrocytes in the cortex and hippocampal dentate gyrus (DG) regions of mice (n = 9, 8, 8, 8). Scale bars = 50 μm. All data are presented as mean values ± SEM. ^∗P < 0.05, ^∗∗P < 0.01 versus the NC group; ^#P < 0.05, ^##P < 0.01 versus the D-gal group.

Figure 3

Effects of FELDK on NF-κB nuclear translocation and expression of inflammatory markers in the cortex and hippocampus of D-gal-aged mice. (a) Western blot images showing the expression of cytoplasmic and nuclear NF-κB, TNF-α, IL-1β, COX-2, and iNOS protein levels. (b) Quantification of the western blot analysis of cytoplasmic and nuclear NF-κB, TNF-α, IL-1β, COX-2, and iNOS protein expressed as percent of control. All data are presented as mean values ± SEM for n = 3. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus the NC group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the D-gal group; ^△P < 0.05, ^△△P < 0.01 versus the F1 group.

Figure 4

Effects of FELDK on ROS and MDA production and SOD, GSH-Px, and CAT activity in the cortex and hippocampus of D-gal-aged mice. (a) ROS levels. (b) MDA levels. (c) SOD activity. (d) GSH-Px activity. (e) CAT activity. All data are presented as mean values ± SEM for n = 9, 8, 8, 8. ^∗P < 0.05, ^∗∗∗P < 0.001 versus the NC group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the D-gal group, ^△△P < 0.01 versus the F1 group.

Figure 5

Effects of FELDK on the PI3K-Akt pathway, JNK activation, and Bcl-2 and Bax expression in the cortex and hippocampus of D-gal-aged mice. Western blots and related quantification of PI3K, p-Akt (Ser473), p-JNK, Bcl-2, and Bax expressed as percent of control. All data are presented as mean values ± SEM for n = 3. ^∗P < 0.05, ^∗∗P < 0.01 versus the NC group; ^#P < 0.05, ^##P < 0.01 versus the D-gal group; ^△P < 0.05 versus the F1 group.

Figure 6

Effects of FELDK on synaptic protein expression, including synaptophysin, synaptotagmin, p-CREB, and p-CaMKII, in the cortex and hippocampus of D-gal-aged mice. Western blot and related quantification of synaptophysin, synaptotagmin, p-CREB, and p-CaMKII protein expression expressed as percent of control. All data are presented as mean values ± SEM for n = 3. ^∗P < 0.05, ^∗∗P < 0.01 versus the NC group; ^#P < 0.05, ^##P < 0.01 versus the D-gal group; ^△P < 0.05 versus the F1 group.

Figure 7

Schematic diagram of the protective effects of FELDK on D-gal-induced brain aging.