Anti-Aging Effects Of Ginsenoside Rg1 And It's Mechanisms On Neural Stem Cell

The stem cell aging theory is the latest theory to explain aging mechanism. With the greater depth of research in stem cell, it is recognized that stem cells are not"immortal"cells, all aging phenomena reflects the aging level of adult stem cell. Studies have shown that the neurogenesis declined rapidly in human or animal brain with inceased age. The reason may be related to the degeneration of self-renewal and multi-directional differentiation activity of neural stem cells (NSCs) related with NSCs aging. Therefore, it has an immeasurable social value for the prevention and treatment of chronic diseases to in-depth study of the modern biology mechanism of aging and delay aging in NSCs, to explorate ways to re-activate and regulate the directional differentiation of NSCs.Ginseng has more than 2,000 years history used in Chinese medicine clinical as replenish qi. Modern pharmacological studies found that ginsenoside has wide effects as main pharmaceutical component of Ginseng. Rg1 has some regulatory effects and nootropic effect on the central nervous system. Shen et al showed that ginsenoside Rg1 can improve the reproductive activity of in vivo or in vitro neural precursor cell though it's mechanism was unclear. Our previous studies showed that ginsenoside Rg1 can delay the aging of hematopoietic stem cells, the underlying mechanism may be the regulation of p16~INK4a-Rb and p19Arf-p53-p21~Cip1/Waf1 signal transduction pathways, the telomere length and telomerase activity. However, the regulative effects and mechanisms of ginsenoside Rg1 on the age-related biological characteristics of aging NSCs in vivo or in vitro have not been reported so far. In this study, we take the latest techniques of stem cell closely integrated with the anti-aging theory and the stem cell knowledge of traditional medicine to build NSCs aging model in vivo and in vitro respectively, to explorate the possible mechanisms of Rg1 to delay NSCs senescence, for providing the guidance in theory and experiment to search the methods delaying NSCs senescence.1. Materials and methods1.1 Primary cultured Neural stem cells (NSCs) were isolated from hippocampus of neonatal Sprague-Dawley (SD) rat. To assess the types of the cells present in culture, the expressions of the specific markers-Nestin in the NSCs, NeuN in the neurons, GEAP in the astrocytes and Gal-C in the oligodendrocytes were evaluated by using immunocytochemistry. Proliferation of NSCs was measured by using BrdU (5-bromo-2-deoxyuridine, Bromodeoxyuridine) incorporation and immunocytochemistry. 1.2 To determine the viability of cells and choose the optimal aging model induced in vitro, the MTT assay, neurospheres counting, differentiated neurons counting and senescence-associatedβ-Galactosidase(SA-β-Gal) staining were used after the third generation of NSCs were treated with different concentration of D-gal (4,8,10,12,16 mg/ml) or t-BHP (50,100,150μmol/L) respectively for 24, 48, 72 h or 1 h, 2 h, 3 h.1.3 To explore the anti- aging effects and the underlying mechanisms of Ginsenoside Rg1 on aging model of NSCs in vitro, the third generation of NSCs were divided into 5 groups: control group, aging group (treated with 100μmol/L t-BHP for 2 h), Rg1 group (treated with 10μg/ml Rg1 for 2 h), Rg1 anti-aging group (co-treated with 100μmol/L t-BHP and 10μg/ml Rg1 for 2 h) and Rg1 treat-aging group (treated with 10μg/ml Rg1 for 2 h after treated with 100μmol/L t-BHP for 2 h). Then the MTT assay, neurospheres counting were used to evaluate the reproductive activity of NSCs. Neurons, astrocytes and oligodendrocytes differentiated from NSCs were counted to evaluate the ability of multi-directional differentiation ability of NSCs. The expressions of senescence associated p16~INK4a and p21~Cip1/Waf1mRNA in each group were examined by RT-PCR.1.4 Thirty 12-wk-old male SD rats were randomly divided into control group, aging group and Rg1 anti-aging group. In the aging group and the Rg1 anti-aging group, D-galactose (120 mg/kg/day) was given to rats for 42 days by subcutaneous injection at the nape of the neck. The rats in latter group were additionally administered with Ginsenoside Rg1 (20 mg/kg/day) by intraperitoneal injection for 28 days after 2 weeks. The rats in control group were injected with saline. Twenty-four hours before sacrifice, each rat in three groups was received BrdU (50 mg/kg) by intraperitoneal injection at intervals of 4 hours for 3 times. Learning and memory abilities were measured by a Morris water maze test 42 days after modeling. The activities of SOD and the amounts of MDA in brain were quantified by chromatometry. The changes in the expressions of p16INK4a and p21Cip1/Waf1 mRNA or protein in brain were determined by real time quantitative RT-PCR or Western blotting respectively.1.5 Paraffin sections of hippocampus and subventricular zone (SVZ) of rats from each group were used for immunohistochemistry to detect the proliferating cells which are BrdU -positive cells within the brain tissue. The morphological characteristics of the cells in each group were observed under the microscope. With the new stereological technology, optical frame counting method, we evaluated the density of BrdU -positive cells within SVZ (the number of cells per unit volume of SVZ). 2. Results2.1 Hippocampus-derived cells in culture proliferated and formed floating neurospheres. Immunocytochemistry analysis of cultured cells revealed that the NSCs specific protein nestin was found. Immunocytochemistry analysis also showed that induction of differentiation in the primary cultured cells, NSCs can differentiate into the appropriate expression of specific markers of terminal cells, namely, the NeuN expression in neurons, the GFAP expression in astrocytes, and the Gal-C expression in oligodendrocytes; the same time, the proliferating cells were stained with the immunocytochemical reaction for BrdU.2.2 The absorbance of MTT assay, the proliferation rate of neurospheres and the density of neurons differentiated from NSCs in cultured cells were significantly reduced with NSCs exposed to various concentrations of D-gal or t-BHP, while the ratio of the neurospheres found to beβ-galactosidase positive is significantly increased.2.3 Compared to aging group (treated with 100μmol/L t-BHP for 2 h), the absorbances of MTT assay in cultured NSCs from Rg1 group (treated with 10μg/ml Rg1 for 2 h) and Rg1 treat - aging group (treated with 10μg/ml Rg1 for 2 h after treated with 100μmol/L t-BHP for 2 h) were increased by 35% and 78% respectively, the numbers of forming colony of NSCs were significantly increased by 29% and 35% respectively, the percentage of senescence-associated SA-β-Gal positive neurospheres was significantly decreased by 40% and 58% respectively and the expressions of p16INK4a and p21Cip1/Waf1 mRNA were significantly reduced. In the meantime, the number of neurons, astrocytes and oligodendrocytes of Rg1 group was 2.2 times, 1.9 times and 1.7 times larger respectively than that of aging group and the same of Rg1 treat - aging group was 2.7 times, 6.3 times and 2.7 times respectively as many as that of aging group.2.4 Compared with the aging group, the spatial learning and memory capacities were significantly enhanced; the SOD activities were significantly increased while the MDA level was significantly reduced and the expressions of the P16~INK4a or P21~Cip1/Waf1 mRNA and protein in brain were reduced in the Rg1 anti-aging group. Immunohistochemical staining for NeuN in the dentate gyrus showed the neurons are more consistent and the arrangement of these cells are closely. The number of round and oval BrdU positive cells in SVZ of Rg1 anti-aging group significantly increased 10.4 and 10.8 folds, respectively, compared with that of aging group.3 Conclusion3.1 In this study, cultured cells were identified as NSCs.3.2 The NSCs can be induced aging by D-galactose or t-BHP in vitro. The optimal option for aging is established by culturing the NSCs with 10 mg/ml D-galactose or 100μmol/L t-BHP for 2 h. 3.3 Ginsenoside Rg1 can delay the aging process of NSCs, restore the ability of proliferation and differentiation in aging NSCs and reduce the number of lysosome in NSCs which indicate that Rg1 is capable of anti-aging and treating aging. The underlying mechanisms maybe relevant to the down-regulation of senescence-associated gene, such as the down-regulation of expressions of the p16~INK4a and p21~Cip1/Waf1 mRNA.3.4 Rg1 could delay the aging effects in rat brain induced by D-galactose. It can promote the spatial learning and memory capacities , the proliferation of NSCs in SVZ of brain aging rats. The underlying mechanisms maybe relevant to enhancing the antioxidant capacity of brain tissue and down-regulation of expressions of the senescence-associated p16~INK4a and p21~Cip1/Waf1 mRNA.