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Abstract：
OBJECTIVE: To explore the mechanism of Anemarrhenae Rhizoma in treatment of Alzheimer\'s Disease (AD).
METHODS: The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacology methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriching analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblastoid cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT and CCK-8 methods were used to quantify SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS), and immunofluorescence staining was used to detect the generation of Aβ1-42 in a co-cultured model. Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aβ deposition in the pharynx was detected by Thioflavin S staining.
RESULTS: Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were identified. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP) and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer\'s disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aβ1-42 (both P<0.01), inhibit the expression of β-secretase 1 (BACE1), APP and Aβ1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3β/GSK3β in SKNMC cells (all P<0.05). The in vivo studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aβ deposition.
CONCLUSIONS: Anemarrhenae Rhizoma may reduce the production of Aβ in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/Akt/GSK-3β pathway.
目的: 探索知母治疗阿尔茨海默病（AD）的作用机制。方法: 通过网络药理学筛选知母治疗AD的活性成分、作用靶点，蛋白质-蛋白质相互作用网络构建及核心靶点分析，并进行基因本体（GO）和京都基因和基因组百科全书（KEGG）通路富集分析。提取外周血淋巴细胞并构建淋巴母细胞样细胞系（LCL），建立LCL-SKNMC体外细胞模型。采用MTT法和细胞计数试剂盒8（CCK-8）法分别检测SKNMC细胞和LCL的活性，7 ´-二氯荧光素二乙酸酯（DCFH-DA）探针检测共培养模型中生成的活性氧，免疫荧光染色检测共培养模型中生成的β淀粉样蛋白1-42 （Aβ1-42 ），蛋白质印迹法检测共培养模型中相关蛋白的表达。分别观察氧化应激、正常状态及热应激状态下N2线虫的寿命，DCFH-DA探针检测N2线虫生成的活性氧，瘫痪实验研究CL4176线虫的瘫痪时间，硫黄素S染色检测CL4176线虫咽部沉积的Aβ。 结果 : 知母具有15个潜在活性成分及103个药物-疾病靶点，可能通过白蛋白、Akt1、肿瘤坏死因子、表皮生长因子受体、血管内皮生长因子A、哺乳动物雷帕霉素靶蛋白、淀粉样前体蛋白（APP）等相关靶点发挥抗AD作用。知母药理作用主要涉及阿尔茨海默病、内分泌抵抗、胰岛素抵抗等生物过程，以及神经活性配体-受体相互作用、磷脂酰肌醇3-激酶（PI3K）-Akt信号通路、钙信号通路、糖尿病并发症中的AGE-RAGE信号通路、神经营养因子信号通路等通路。体外实验显示，知母可减少LCL-SKNMC中活性氧生成和Aβ 1-42的产生（均P<0.01），抑制β-分泌酶1、APP、Aβ1-42蛋白的表达（均P <0.05），上调PI3K、Akt、GSK3β蛋白的磷酸化水平（均P <0.05）。体内研究进一步证实，知母可延长应激状态及正常情况下线虫的寿命，减轻活性氧积累及Aβ沉积毒性。结论: 知母可减少AD中Aβ的生成，抑制其诱导的氧化应激作用，这些作用可能是通过调控PI3K/Akt/GSK-3β通路实现的。.
METHODS: The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacology methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriching analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblastoid cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT and CCK-8 methods were used to quantify SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS), and immunofluorescence staining was used to detect the generation of Aβ1-42 in a co-cultured model. Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aβ deposition in the pharynx was detected by Thioflavin S staining.
RESULTS: Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were identified. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP) and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer\'s disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aβ1-42 (both P<0.01), inhibit the expression of β-secretase 1 (BACE1), APP and Aβ1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3β/GSK3β in SKNMC cells (all P<0.05). The in vivo studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aβ deposition.
CONCLUSIONS: Anemarrhenae Rhizoma may reduce the production of Aβ in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/Akt/GSK-3β pathway.
目的: 探索知母治疗阿尔茨海默病（AD）的作用机制。方法: 通过网络药理学筛选知母治疗AD的活性成分、作用靶点，蛋白质-蛋白质相互作用网络构建及核心靶点分析，并进行基因本体（GO）和京都基因和基因组百科全书（KEGG）通路富集分析。提取外周血淋巴细胞并构建淋巴母细胞样细胞系（LCL），建立LCL-SKNMC体外细胞模型。采用MTT法和细胞计数试剂盒8（CCK-8）法分别检测SKNMC细胞和LCL的活性，7 ´-二氯荧光素二乙酸酯（DCFH-DA）探针检测共培养模型中生成的活性氧，免疫荧光染色检测共培养模型中生成的β淀粉样蛋白1-42 （Aβ1-42 ），蛋白质印迹法检测共培养模型中相关蛋白的表达。分别观察氧化应激、正常状态及热应激状态下N2线虫的寿命，DCFH-DA探针检测N2线虫生成的活性氧，瘫痪实验研究CL4176线虫的瘫痪时间，硫黄素S染色检测CL4176线虫咽部沉积的Aβ。 结果 : 知母具有15个潜在活性成分及103个药物-疾病靶点，可能通过白蛋白、Akt1、肿瘤坏死因子、表皮生长因子受体、血管内皮生长因子A、哺乳动物雷帕霉素靶蛋白、淀粉样前体蛋白（APP）等相关靶点发挥抗AD作用。知母药理作用主要涉及阿尔茨海默病、内分泌抵抗、胰岛素抵抗等生物过程，以及神经活性配体-受体相互作用、磷脂酰肌醇3-激酶（PI3K）-Akt信号通路、钙信号通路、糖尿病并发症中的AGE-RAGE信号通路、神经营养因子信号通路等通路。体外实验显示，知母可减少LCL-SKNMC中活性氧生成和Aβ 1-42的产生（均P<0.01），抑制β-分泌酶1、APP、Aβ1-42蛋白的表达（均P <0.05），上调PI3K、Akt、GSK3β蛋白的磷酸化水平（均P <0.05）。体内研究进一步证实，知母可延长应激状态及正常情况下线虫的寿命，减轻活性氧积累及Aβ沉积毒性。结论: 知母可减少AD中Aβ的生成，抑制其诱导的氧化应激作用，这些作用可能是通过调控PI3K/Akt/GSK-3β通路实现的。.
摘要：
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