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.