Discussion
There is no doubt that the global population is aging. Neurodegenerative
diseases have developed into a severe concern to the health of the
world’s elderly population. However, as humans age, NSCs age and
degenerate, limiting the ability of the neurological system to repair
and protect itself, leading to cognitive impairment (Xiang Y et
al,2017). Damage to nerve cells caused by aging is a significant factor
in the development of neurodegenerative disorders. There is currently no
viable strategy for the prevention or treatment of neurodegenerative
disorders. The hippocampus is composed of nerve cells, that perform
memory and spatial positioning tasks. Therefore, postponing nerve cell
aging damage is an efficient strategy for preventing and treating
neurodegenerative diseases. Our previous research report stated that
Ginsenoside Rg1 is the primary component of ginseng and has been shown
to protect the brain and NSCs from the aging effects of D-gal (Xiang Y
et al,2019). However, this process warrants additional investigation.
While a small amount of D-gal can be converted to glucose and
participate in cell metabolism, a significant amount of D-gal will
disrupt cell metabolism disorders and produce a large number of
oxidation products, resulting in cell oxidative damage and senescence
(Wang ZL et al,2016). As a result, in this study, we successfully
extracted, cultured, purified, and identified NSCs using
immunofluorescence. The cells were Nestin (green fluorescence) positive,
and an in vitro model was constructed. CCK-8 assay results showed that
D-gal inhibited cell proliferation, whereas Rg1 promoted cell
proliferation. Trypan blue staining revealed that D-gal decreased cell
survival rate, but Rg1 increased cell survival rate. The results of
SA-β-gal staining showed that Rg1 can antagonized the aging effect of
D-gal on NSCs and effectively reduced the senescence level of NSCs. The
above experimental results demonstrated that D-gal can induced
senescence in NSCs and that Rg1 can protected NSCs against D-gal induced
senescence.To gain a better understanding of what causes this aging, we
measured indicators of oxidative stress which is SOD, CAT, MDA, 4-HNE ,
8-OHdG,these findings suggested that aging was associated with oxidative
stress damage, that D-gal can generated oxidative stress damage in NSCs,
and that Rg1 can reversed the oxidative stress damage caused by D-gal,
hence delaying NSCs aging. Is Rg1’s resistance to oxidative stress
damage related to oxidation-related pathways? As a result of our
investigation into the molecular mechanisms behind oxidation, we
discovered the Keap1-Nrf2/ARE pathway, an antioxidant route. Firstly, we
explored the expression of pathway-related proteins. The Nrf2 and HO-1
proteins were expressed at a lower level in the D-gal group than in the
Control or D-gal + Rg1 groups, although the Keap1 protein content was
increased. The results indicate that Rg1 can down-regulate the
expression of Keap1 protein, promote the dissociation of Nrf2 and Keap1,
while simultaneously up-regulating the expression of Nrf2 and HO-1
proteins, activating this pathway and thereby reducing the aging caused
by D-gal. Then, we examined the levels of expression of genes involved
in this pathway. NQO1, GCLM, GSTM-1, and GCLC are all Nrf2/ARE pathway
target genes. Compared with the D-gal group, the mRNA expression levels
of each gene in the Control group and D-gal+Rg1 group were higher. The
data indicated that Rg1 can activate this pathway, promoting the
association of Nrf2 and ARE sequences and transcription of associated
target genes, thereby alleviating D-gal-induced senescence. The above
findings suggested that Rg1 protect against oxidative stress damage via
the Keap1-Nrf2/ARE pathway, hence delaying NSCs aging.
To validate the aforementioned findings, we conducted relevant in vivo
research. we established an animal model. MWM experiment was used to
assess mice’s brain function, the results indicated that D-gal has a
clear detrimental effect on the cognitive function and spatial memory of
mice and that Rg1 can reverse the cognitive and spatial memory
impairment induced by D-gal. D-gal enhanced hippocampus neuron necrosis,
which Rg1 could ameliorate. The results of Nissl staining showed that
D-galactose could damage the physiological state of neurons, while Rg1
could restore the physiological state of neurons.SA-β-gal staining
indicates that Rg1 can prevent hippocampus neurons from succumbing to
D-gal-induced senescence.The results of transmission electron microscopy
indicated that D-gal causes neuronal damage and that Rg1 can reverse
this effect. P53 protein is a critical aging-related protein, the result
showed that Rg1 down-regulated aging-related protein expression. The
cholinergic test result Rg1 has been shown to inhibit the D-gal
breakdown of the Ach neurotransmitter. The data above demonstrated that
D-gal promoted hippocampal aging in mice and Rg1 delayed D-gal-induced
aging.We continue to measured indicators related to oxidative stress,
which are the same as the results of in vitro experiments ,the results
indicated that the aging of hippocampals caused by D-gal was related to
oxidative stress and that Rg1 was resistant to oxidative stress.
Finally, we determined the indicators of proteins and genes involved in
the Keap1-Nrf2/ARE pathway and obtained same results to those observed
in vitro model. The results indicated that Rg1 can reduce the oxidative
damage in hippocampals via the Keap1-Nrf2/ARE pathway to delay aging.
Ginseng is widely used in traditional Chinese medicine. Among the more
than 30 varieties of ginsenosides, Rg1 is considered to be the main
active ingredient, responsible for the attributes of multi-target
pharmacological effects. Previous research has established that
Ginsenoside Rg1 possesses anti-aging, and anti-oxidant properties, as
well as the ability to promote hematopoiesis, improve immunity, protect
the liver, and other functions. Ginsenoside Rg1 can enhance antioxidant
activity by modulating the Wnt/β-catenin signaling pathway( Qi RJ et
al,2020). Rg1 plays an anti-aging effect by regulating the p19-p53-p21
pathway (Cheng X et al,2019). However, Rg1 needs further research,for
example, study the role of Rg1 in gene knockout, pathway agonists or
inhibitors et al.