人参皂苷Rg1延缓神经干细胞衰老作用及机理研究
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摘要
干细胞衰老学说是迄今解释机体衰老机制的最新学说。随着对干细胞研究的深入,人们认识到干细胞并非是“长生不老”的细胞,所有衰老现象都反映出成体干细胞衰老的水平。研究证明,随着年龄的增长,人或动物大脑内的神经发生率呈指数下降,其原因可能与神经干细胞(Neural stem cells, NSCs)衰老导致的自我更新和多向分化能力衰退有关。因此,深入研究NSCs衰老和延缓NSCs衰老的现代生物学机理,寻找重新激活NSCs的方法和调控它靶向分化对预防和治疗老年退行性疾病有不可估量的社会价值。
人参作为中医临床“补气”要药已有2000多年的应用历史,现代药理学研究证明,人参皂苷是人参的主要药效成分,具有广泛的药理作用,人参皂苷Rg1是重要的人参单体皂苷。近年研究认为,人参皂苷Rg1对中枢神经系统有明确的调节作用,具促智和益智功效。也有学者的工作证明,人参皂苷Rg1能够提高体内外神经前体细胞的增殖能力,但其作用机制尚不清楚。本课题前期研究发现人参皂苷Rg1能延缓造血干细胞衰老,其机制与调控p16~INK4a-Rb、p19~Arf-p53-p21~Cip1/Waf1信号转导通路以及延缓端粒长度缩短、提高端粒酶活性有关。迄今还未见关于人参皂苷Rg1对体、内外NSCs衰老相关生物学特征的影响及机制报道。本课题将干细胞最新的研究技术与祖国传统医学的抗衰老理论和对干细胞的认识紧密结合起来,摸索复制NSCs体外衰老模型方法及复制D-半乳糖脑衰老动物模型,并在此基础上从体内和体外两条途径系统探讨人参皂苷单体Rg1延缓NSCs衰老的作用及其可能的生物学调控机制。旨在为探寻延缓NSCs衰老以及重新激活衰老NSCs的途径提供理论及实验依据。
1材料和方法
1.1从新生SD大鼠海马组织内分离提取细胞进行原代培养,并通过以下检测鉴定培养细胞是NSCs:
1)NSCs标志物——Nestin免疫细胞化学染色;
2)根据NSCs多向分化特性,诱导培养的NSCs分化并进行神经元特异性核蛋白NeuN、星形胶质细胞标志物GFAP、少突胶质细胞标志物Gal-C免疫细胞化学检测;
3)根据NSCs自我更新特性,进行BrdU掺入实验和BrdU免疫细胞化学染色。
1.2采用不同浓度的D-gal(4、8、10、12、16 mg/mL)分别作用第3代NSCs 24、48、72 h或t-BHP(50、100、150μmol/L)分别作用第
3代NSCs 1 h、2 h、3 h进行如下检测:
1)MTT检测各组NSCs的增殖能力;
2)每毫升培养体系接种3000个NSCs,计数NSCs增殖形成的NSCs克隆球数量;
3)体外诱导分化培养检测NSCs多向分化能力,采用体视学方法对分化形成的神经元样细胞进行计数估计其数密度(单位面积内的细胞数量);
4)衰老特异性β-半乳糖苷酶染色显示衰老神经球,采用体视学方法计数阳性及阴性神经球的数量,以阳性神经球的数量除以两者之和即为SA-β-Gal阳性神经球百分比。
通过以上检测选择诱导NSCs衰老的最佳体外模型。1.3将第3代NSCs分为对照组、衰老组(100μmol/L t-BHP作用2 h)、Rg1组(10μg/ml Rg1作用2 h),Rg1抗衰老组(100μmol/L t-BHP和10μg/ml Rg1共同作用2 h)和Rg1治疗衰老组(按衰老组处理后,再用10μg/ml Rg1作用2 h),通过以下检测探讨人参皂苷Rg1延缓NSCs衰老的体外作用及机制:
1)同方法2 1)- 3)检测指标;
2)体外诱导分化培养检测NSCs多向分化能力,采用体视学方法对分化形成的神经元样细胞、星形胶质细胞样细胞和少突胶质细胞样细胞分别进行计数并分别估计这三种细胞的数密度; 3)检测各组NSCs衰老相关基因p16~INK4a、p21~Cip1/Waf1mRNA的表
达水平。
1.4将30只3月龄雄性SD大鼠随机分组。脑衰老组:大鼠颈背部皮下连续注射D-半乳糖42 d(120 mg.kg~-1/d);Rg1抗脑衰老组:同衰老模型组处理,并从模型复制的第15 d起腹腔注射Rg1(20 mg.kg~-1 /d) ,连续注射28 d;对照组:正常大鼠颈背部皮下连续注射等时和等量生理盐水。各组大鼠在处死前1 d每4 h腹腔注射BrdU 50 mg.kg~-1/次,共注射3次。并进行以下几方面检测:
1)Morris水迷宫评估各组大鼠的空间学习记忆能力;
2)比色法测定脑皮质内的SOD活性及MDA含量;
3)实时荧光定量RT-PCR检测海马组织内p16~INK4a、p21~Cip1/Waf1 mRNA的表达水平;
4)Western blotting检测衰老相关基因P16~INK4a、P21~Cip1/Waf1蛋白的表达水平;
5)各组动物制作海马及SVZ区的脑组织石蜡切片,分别用NeuN、BrdU免疫组织化学显色显示神经元细胞核和脑组织内的增殖细胞,镜下观察各组动物海马及SVZ区形态学特征,采用体视学新技术——光学体视框估计SVZ区BrdU阳性细胞的数密度(单位体积SVZ区内的细胞数量)。
2结果
2.1体外培养的NSCs呈Nestin免疫细胞化学染色阳性反应;诱导分化形成的细胞呈NeuN(显示神经元)、GFAP(显示星形胶质细胞)和Gal-C(显示少突胶质细胞)免疫细胞化学染色阳性反应;BrdU掺入实验呈BrdU免疫细胞化学染色阳性反应。
2.2不同浓度的D-gal或t-BHP作用于体外培养的NSCs,均可引起NSCs的增殖能力、NSCs克隆球形成率和分化形成的神经元数密度显著下降,β-半乳糖苷酶染色阳性神经球比率显著升高。
2.3与衰老组(t-BHP 100μmol/L作用NSCs 2 h)比较,抗衰老组(10μg/ml Rg1作用NSCs 2 h)及治疗衰老组(100μmol/L t-BHP作用2 h ,再用10μg/ml Rg1作用NSCs 2 h)的NSCs MTT吸光度值分别升高了35%和78%;NSCs克隆球形成数分别显著增加了29%和35%;衰老特异性SA-β-Gal染色阳性神经球百分比显著降低了40%和58%;衰老相关基因p16~INK4a、p2~1Cip1/Waf1mRNA表达水平显著下降。与衰老组比较,Rg1抗衰老组的神经元样细胞、星形胶质细胞样细胞和少突胶质细胞样细胞的数密度显著增加,分别是衰老组的2.2倍、1.9倍和1.7倍; Rg1治疗衰老组的三种细胞的数密度显著增加,分别是衰老组的2.7倍、6.3倍和2.7倍。
2.4与D-半乳糖脑衰老组相比,Rg1抗脑衰老组大鼠学习记忆能力显著提高;脑组织内SOD活性显著升高,MDA含量显著降低;p16~INK4a、p21~ Cip1/Waf1 mRNA及蛋白的表达水平显著降低;NeuN免疫组织化学切片显示齿状回区神经元大小较一致,排列较紧密,细胞核着色较均匀、轮廓较清楚;SVZ区BrdU圆形和椭圆形阳性细胞的数量显著增加到脑衰老组的10.4倍和10.8倍。
3结论3.1本研究体外培养的细胞经鉴定为NSCs。3.2 D-半乳糖或t-BHP均能在体外诱导NSCs衰老,以D-gal 10 mg/ml作用48 h和t-BHP 100μmol/L作用2 h为衰老模型的较好选择。3.3人参皂苷Rg1可以增强已经衰老NSCs的增殖和分化能力,减少NSCs溶酶体的数量,提示其具有抗衰老和治疗衰老的作用,其机制可能与下调衰老相关基因p16~INK4a、p21~Cip1/Waf1 mRNA的表达有关。3.4 Rg1具有延缓D-半乳糖所致大鼠脑衰老作用,表现为能提高脑衰老大鼠的空间学习记忆能力、促进SVZ区的NSCs增殖。其机制可能与增强脑组织的抗氧化能力,下调脑组织内p16~INK4a、p21~Cip1/Waf1 mRNA及蛋白的表达水平有关。
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 methods
1.1 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. Results
2.1 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 Conclusion
3.1 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.
人参作为中医临床“补气”要药已有2000多年的应用历史,现代药理学研究证明,人参皂苷是人参的主要药效成分,具有广泛的药理作用,人参皂苷Rg1是重要的人参单体皂苷。近年研究认为,人参皂苷Rg1对中枢神经系统有明确的调节作用,具促智和益智功效。也有学者的工作证明,人参皂苷Rg1能够提高体内外神经前体细胞的增殖能力,但其作用机制尚不清楚。本课题前期研究发现人参皂苷Rg1能延缓造血干细胞衰老,其机制与调控p16~INK4a-Rb、p19~Arf-p53-p21~Cip1/Waf1信号转导通路以及延缓端粒长度缩短、提高端粒酶活性有关。迄今还未见关于人参皂苷Rg1对体、内外NSCs衰老相关生物学特征的影响及机制报道。本课题将干细胞最新的研究技术与祖国传统医学的抗衰老理论和对干细胞的认识紧密结合起来,摸索复制NSCs体外衰老模型方法及复制D-半乳糖脑衰老动物模型,并在此基础上从体内和体外两条途径系统探讨人参皂苷单体Rg1延缓NSCs衰老的作用及其可能的生物学调控机制。旨在为探寻延缓NSCs衰老以及重新激活衰老NSCs的途径提供理论及实验依据。
1材料和方法
1.1从新生SD大鼠海马组织内分离提取细胞进行原代培养,并通过以下检测鉴定培养细胞是NSCs:
1)NSCs标志物——Nestin免疫细胞化学染色;
2)根据NSCs多向分化特性,诱导培养的NSCs分化并进行神经元特异性核蛋白NeuN、星形胶质细胞标志物GFAP、少突胶质细胞标志物Gal-C免疫细胞化学检测;
3)根据NSCs自我更新特性,进行BrdU掺入实验和BrdU免疫细胞化学染色。
1.2采用不同浓度的D-gal(4、8、10、12、16 mg/mL)分别作用第3代NSCs 24、48、72 h或t-BHP(50、100、150μmol/L)分别作用第
3代NSCs 1 h、2 h、3 h进行如下检测:
1)MTT检测各组NSCs的增殖能力;
2)每毫升培养体系接种3000个NSCs,计数NSCs增殖形成的NSCs克隆球数量;
3)体外诱导分化培养检测NSCs多向分化能力,采用体视学方法对分化形成的神经元样细胞进行计数估计其数密度(单位面积内的细胞数量);
4)衰老特异性β-半乳糖苷酶染色显示衰老神经球,采用体视学方法计数阳性及阴性神经球的数量,以阳性神经球的数量除以两者之和即为SA-β-Gal阳性神经球百分比。
通过以上检测选择诱导NSCs衰老的最佳体外模型。1.3将第3代NSCs分为对照组、衰老组(100μmol/L t-BHP作用2 h)、Rg1组(10μg/ml Rg1作用2 h),Rg1抗衰老组(100μmol/L t-BHP和10μg/ml Rg1共同作用2 h)和Rg1治疗衰老组(按衰老组处理后,再用10μg/ml Rg1作用2 h),通过以下检测探讨人参皂苷Rg1延缓NSCs衰老的体外作用及机制:
1)同方法2 1)- 3)检测指标;
2)体外诱导分化培养检测NSCs多向分化能力,采用体视学方法对分化形成的神经元样细胞、星形胶质细胞样细胞和少突胶质细胞样细胞分别进行计数并分别估计这三种细胞的数密度; 3)检测各组NSCs衰老相关基因p16~INK4a、p21~Cip1/Waf1mRNA的表
达水平。
1.4将30只3月龄雄性SD大鼠随机分组。脑衰老组:大鼠颈背部皮下连续注射D-半乳糖42 d(120 mg.kg~-1/d);Rg1抗脑衰老组:同衰老模型组处理,并从模型复制的第15 d起腹腔注射Rg1(20 mg.kg~-1 /d) ,连续注射28 d;对照组:正常大鼠颈背部皮下连续注射等时和等量生理盐水。各组大鼠在处死前1 d每4 h腹腔注射BrdU 50 mg.kg~-1/次,共注射3次。并进行以下几方面检测:
1)Morris水迷宫评估各组大鼠的空间学习记忆能力;
2)比色法测定脑皮质内的SOD活性及MDA含量;
3)实时荧光定量RT-PCR检测海马组织内p16~INK4a、p21~Cip1/Waf1 mRNA的表达水平;
4)Western blotting检测衰老相关基因P16~INK4a、P21~Cip1/Waf1蛋白的表达水平;
5)各组动物制作海马及SVZ区的脑组织石蜡切片,分别用NeuN、BrdU免疫组织化学显色显示神经元细胞核和脑组织内的增殖细胞,镜下观察各组动物海马及SVZ区形态学特征,采用体视学新技术——光学体视框估计SVZ区BrdU阳性细胞的数密度(单位体积SVZ区内的细胞数量)。
2结果
2.1体外培养的NSCs呈Nestin免疫细胞化学染色阳性反应;诱导分化形成的细胞呈NeuN(显示神经元)、GFAP(显示星形胶质细胞)和Gal-C(显示少突胶质细胞)免疫细胞化学染色阳性反应;BrdU掺入实验呈BrdU免疫细胞化学染色阳性反应。
2.2不同浓度的D-gal或t-BHP作用于体外培养的NSCs,均可引起NSCs的增殖能力、NSCs克隆球形成率和分化形成的神经元数密度显著下降,β-半乳糖苷酶染色阳性神经球比率显著升高。
2.3与衰老组(t-BHP 100μmol/L作用NSCs 2 h)比较,抗衰老组(10μg/ml Rg1作用NSCs 2 h)及治疗衰老组(100μmol/L t-BHP作用2 h ,再用10μg/ml Rg1作用NSCs 2 h)的NSCs MTT吸光度值分别升高了35%和78%;NSCs克隆球形成数分别显著增加了29%和35%;衰老特异性SA-β-Gal染色阳性神经球百分比显著降低了40%和58%;衰老相关基因p16~INK4a、p2~1Cip1/Waf1mRNA表达水平显著下降。与衰老组比较,Rg1抗衰老组的神经元样细胞、星形胶质细胞样细胞和少突胶质细胞样细胞的数密度显著增加,分别是衰老组的2.2倍、1.9倍和1.7倍; Rg1治疗衰老组的三种细胞的数密度显著增加,分别是衰老组的2.7倍、6.3倍和2.7倍。
2.4与D-半乳糖脑衰老组相比,Rg1抗脑衰老组大鼠学习记忆能力显著提高;脑组织内SOD活性显著升高,MDA含量显著降低;p16~INK4a、p21~ Cip1/Waf1 mRNA及蛋白的表达水平显著降低;NeuN免疫组织化学切片显示齿状回区神经元大小较一致,排列较紧密,细胞核着色较均匀、轮廓较清楚;SVZ区BrdU圆形和椭圆形阳性细胞的数量显著增加到脑衰老组的10.4倍和10.8倍。
3结论3.1本研究体外培养的细胞经鉴定为NSCs。3.2 D-半乳糖或t-BHP均能在体外诱导NSCs衰老,以D-gal 10 mg/ml作用48 h和t-BHP 100μmol/L作用2 h为衰老模型的较好选择。3.3人参皂苷Rg1可以增强已经衰老NSCs的增殖和分化能力,减少NSCs溶酶体的数量,提示其具有抗衰老和治疗衰老的作用,其机制可能与下调衰老相关基因p16~INK4a、p21~Cip1/Waf1 mRNA的表达有关。3.4 Rg1具有延缓D-半乳糖所致大鼠脑衰老作用,表现为能提高脑衰老大鼠的空间学习记忆能力、促进SVZ区的NSCs增殖。其机制可能与增强脑组织的抗氧化能力,下调脑组织内p16~INK4a、p21~Cip1/Waf1 mRNA及蛋白的表达水平有关。
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 methods
1.1 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. Results
2.1 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 Conclusion
3.1 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.
引文
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