Lee Rami, Lee Na-Eun, Choi Sun-Hye, Nam Sung Min, Kim Hyoung-Chun, Rhim Hyewhon, Cho Ik-Hyun, Hwang Sung-Hee, Nah Seung-Yeol
Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul, Republic of Korea.
Neuro Psychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon, Republic of Korea.
Integr Med Res. 2021 Jun;10(2):100475. doi: 10.1016/j.imr.2020.100475. Epub 2020 Jul 15.
Recently, gintonin and gintonin-enriched fraction (GEF) have been isolated from ginseng, a herbal medicine. Gintonin induces [Ca] transition in cultured hippocampal neurons and stimulates acetylcholine release through LPA receptor activation. Oral administration of GEF is linked to hippocampus-dependent cognitive enhancement and other neuroprotective effects; however, effects of its long-term administration on hippocampal gene expression remains unknown. Here, we used next-generation sequence (NGS) analysis to examine changes in hippocampal gene expressions after long-term oral administration of GEF.
C57BL/6 mice were divided into three groups: control group, GEF50 (GEF 50 mg/kg, ), and GEF100 (GEF 100 mg/kg, ). After 22 days, total RNA was extracted from mouse hippocampal tissues. NGS was used for gene expression profiling; quantitative-real-time PCR and western blot were performed to quantify the changes in specific genes and to confirm the protein expression levels in treatment groups.
NGS analysis screened a total of 23,282 genes, analyzing 11-related categories. We focused on the neurogenesis category, which includes four genes for candidate markers: choline acetyltransferase (ChAT) gene, β-adrenergic receptor (Adrb3) gene, and corticotrophin-releasing hormone (Crh) gene, and tryptophan 2,3-dioxygenase (Tdo2) gene. Real-time PCR showed a marked overexpression of ChAT, Adrb3, and Crh genes, while reduced expression of Tdo2. Western blot analysis also confirmed increased ChAT and decreased Tdo2 protein levels.
We found that GEF affects mouse hippocampal gene expressions, associated with memory, cognitive, anti-stress and anti-anxiety functions, and neurodegeneration at differential degree, that might explain the genetic bases of GEF-mediated neuroprotective effects.
最近，从草药人参中分离出了人参皂甙和富含人参皂甙的组分（GEF）。人参皂甙可诱导培养的海马神经元中的[Ca]转换，并通过激活LPA受体刺激乙酰胆碱释放。口服GEF与海马依赖性认知增强及其他神经保护作用有关；然而，长期给药对海马基因表达的影响尚不清楚。在此，我们使用下一代测序（NGS）分析来研究长期口服GEF后海马基因表达的变化。
将C57BL/6小鼠分为三组：对照组、GEF50组（GEF 50mg/kg）和GEF100组（GEF 100mg/kg）。22天后，从小鼠海马组织中提取总RNA。使用NGS进行基因表达谱分析；进行定量实时PCR和蛋白质印迹以量化特定基因的变化并确认治疗组中的蛋白质表达水平。
NGS分析共筛选了23,282个基因，分析了11个相关类别。我们关注神经发生类别，其中包括四个候选标志物基因：胆碱乙酰转移酶（ChAT）基因、β-肾上腺素能受体（Adrb3）基因、促肾上腺皮质激素释放激素（Crh）基因和色氨酸2,3-双加氧酶（Tdo2）基因。实时PCR显示ChAT、Adrb3和Crh基因明显过表达，而Tdo2表达降低。蛋白质印迹分析也证实ChAT增加而Tdo2蛋白质水平降低。
我们发现GEF会影响小鼠海马基因表达，在不同程度上与记忆、认知、抗应激和抗焦虑功能以及神经退行性变相关，这可能解释了GEF介导的神经保护作用的遗传基础。
