摘要
目的:观察蛇床子催眠活性组分对体外培养原代海马神经元细胞神经递质及钟基因表达的影响,探讨其催眠作用机制。方法:体外培养SD乳鼠原代海马神经元细胞,CCK-8法检测细胞活性,绘制细胞生长曲线,在细胞生长状态最佳时,经神经元特异性烯醇化酶(NSE)免疫组化鉴定纯度并给药。将海马神经元细胞随机分为为空白对照组、地西泮(25μg/mL)阳性对照组及蛇床子催眠活性组分低(25μg/mL)、中(50μg/mL)、高(100μg/mL)浓度组共5组,给药24 h后流式细胞术分析海马神经元细胞早期凋亡,酶联免疫吸附法(ELISA)检测细胞上清液γ-氨基丁酸(GABA)、5-羟色胺(5-HT)、去甲肾上腺素(NA)等神经递质的含量,实时荧光定量PCR检测细胞内GABA、5-HT相关受体基因及钟基因Clock、Bmal1、Cry1、Per1、Per2 mRNA的表达。结果:体外原代培养第7天海马神经元细胞生长状态良好,鉴定纯度大于90%。与空白对照组比较,蛇床子催眠活性组分低、中浓度组海马神经元细胞凋亡率显著降低,中、高浓度组神经递质GABA、5-HT分泌及Gabra1、Gabra5、Gabbr1、5-HT1A(B)、5-HT1A(C)、5-HT1B、5-HT1D、Cry1、Per1、Per2 mRNA表达显著升高,蛇床子催眠活性组分高浓度组5-HT1A(A) mRNA表达显著升高,Bmal1 mRNA表达显著降低(P<0.05或P<0.01)。结论:蛇床子催眠活性组分能降低体外培养的海马神经元细胞凋亡率,增强抑制性神经递质分泌与表达,并能调节钟基因。
Objective:To observe the effects of hypnotic active components from Cnidii Fructus(SCZ)on the neurotransmitters and clock genes expression in primary hippocampal neurons and to explore the hypnotic mechanism.Methods:The primary hippocampal neurons of neonatal SD rats were cultured in vitro and the cell activity was detected by CCK-8 method,the cell growth curve was drawn.At the optimum cell growth state,the purity was identified by neuron-specific enolase(NSE)immunohistochemistry and the drug was administered.Hippocampal neurons were randomly divided into blank control group,diazepam positive control group(25 μg/mL),and low-(25 μg/mL),moderate-(50 μg/mL)and high-(100 μg/mL)concentrations of SCZ groups.Early apoptosis of hippocampus neurons was detected using flow cytometry technique after 24 h administration,and the contents of γ-aminobutyric acid(GABA),5-hydroxytryptamine(5-HT)and noradrenaline(NA)in cell supernatant were detected using enzyme-linked immunosorbent.The mRNA expressions of receptor genes related to GABA and 5-HT and clock genes including Clock,Bmal1,Cry1,Per1 and Per2 were detected by real-time quantitative PCR.Results:On the 7 th day of primary culture in vitro,hippocampal neurons were in a good growth state and the purity was greater than 90%.Apoptosis rates of hippocampal neurons in the low-and moderate-concentrations of SCZ groups were significantly lower than that in the blank control group(P<0.01).The secretion of neurotransmitters GABA and 5-HT and the mRNA expressions of Gabra1,Gabra5,Gabbr1,5-HT1 A(B),5-HT1 A(C),5-HT1 B,5-HT1 D,Cry1,Per1 and Per2 in moderate-and high-concentrations of SCZ groups were significantly increased,the expression of Bmal1 mRNA in high concentration group was significantly decreased while the 5-HT1 A(A) mRNA expression significantly increased(P<0.05 or P<0.01).Conclusion:The hypnotic active components of Cnidii Fructus can reduce the apoptosis rate of cultured hippocampal neurons in vitro,enhance the secretion and expression of inhibitory neurotransmitters and regulate clock genes.
作者
郝雨蒙
王若瑜
仲启明
仝立国
宋美卿
杨钤
冯玛莉
HAO Yu-meng;WANG Ruo-yu;ZHONG Qi-ming;TONG Li-guo;SONG Mei-qing;YANG Qian;FENG Ma-li(Shanxi Academy of Traditional Chinese Medicine,Taiyuan 030012,China)
出处
《中药材》
CAS
北大核心
2019年第5期1133-1138,共6页
Journal of Chinese Medicinal Materials
基金
国家自然科学基金(81173574)
山西省实验动物专项资金项目[2013(K05),2014(K02)].
关键词
蛇床子
海马神经元细胞
体外培养
细胞凋亡
神经递质
钟基因
Cnidii Fructus
Hippocampal neurons
Culture in vitro
Cell apoptosis
Neurotransmitter
Clock gene
作者简介
郝雨蒙(1992-),女,在读硕士研究生,专业方向:中药药理与内分泌疾病研究,Tel:0351-4174328,E-mail:630380521@qq.com;通讯作者:冯玛莉,Tel:0351-4174328,E-mail:fengmali1@163.com。
