蛇床子催眠活性组分对对氯苯丙氨酸致失眠大鼠海马钟基因与氨基酸类神经递质表达的影响
|
摘要
目的观察蛇床子催眠活性组分(SCZ)对失眠大鼠海马神经递质及钟基因表达的影响,探讨其催眠作用机制。方法采用ip对氯苯丙氨酸(PCPA)建立大鼠失眠模型,ig给予低(25 g/kg)、中(50 g/kg)、高(100 g/kg)剂量SCZ,并设阳性对照(地西泮)组,给药3 d,免疫组织化学法检测大鼠海马齿状回γ-氨基丁酸(GABA)和谷氨酸(Glu)受体蛋白的表达;高效液相色谱法和免疫组化法检测大鼠海马GABA和Glu含量的变化;实时荧光定量PCR检测大鼠海马Clock、Bmal1、Cry1、Cry2、Per1、Per2、Per3等生物钟基因的表达水平。结果免疫组化结果显示,与对照组比较,模型组大鼠海马齿状回GABA表达水平显著降低,Glu表达水平显著升高(P<0.05、0.01);与模型组比较,SCZ中、高剂量组大鼠海马齿状回GABA表达水平显著升高,Glu表达水平显著降低(P<0.01)。HPLC结果显示,与对照组比较,模型组大鼠海马GABA表达水平显著降低,Glu表达水平显著升高(P<0.05、0.01);与模型组比较,SCZ中、高剂量组大鼠海马组织Glu表达水平显著降低(P<0.05),SCZ高、低剂量组大鼠GABA表达水平显著升高(P<0.01)。生物钟基因表达水平检测结果显示,与对照组比较,模型组大鼠Clock、Bmal1基因表达水平显著升高(P<0.05),Cry1、Per1、Per2基因表达水平显著降低(P<0.05、0.01)。与模型组比较,SCZ中剂量组大鼠Clock、Bmal1基因表达水平显著降低(P<0.01);SCZ高剂量组大鼠Cry1、Per1、Per2基因表达水平显著增高(P<0.05、0.01),SCZ低剂量组大鼠Per1基因表达水平显著增高(P<0.01)。结论蛇床子催眠活性组分通过降低海马Clock、Bmal1表达,提高Cry1、Per1、Per2基因的表达水平,增加抑制性、减少兴奋性氨基酸类神经递质的表达,从而调节睡眠-觉醒周期,达到治疗失眠的效果。
Objective To study the hypnotic mechanism of Cnidii Fructus hypnotic active constituent(SCZ) on the expression of clock genes and hippocampal neurotransmitters in insomnia rats. Methods The insomnia model was established by intraperitoneal injection of para-chlorophenylalanine(PCPA). SCZ at low(25 g/kg), medium(50 g/kg), and high(100 g/kg) conentrations were ig administrated respectively at the same time with diazepam as positive group, intragastric administration of PCPA was performed in insomnia rats for three days, The expression levels of Clock, Bmal1, Cry1, Cry2, Per1, Per2, and Per3 were detected by real-time PCR. The expression levels of γ-aminobutyric acid(GABA) and glutamic acid(Glu) in hippocampus dentate gyrus were detected by immunohistochemistry. The changes of contents of GABA and Glu were determinated by immunohistochemistry and high performance liquid chromatography. Results The results of immunohistochemistry showed that the expression of GABA in the dentate gyrus of the hippocampus of rats in the model group was significantly reduced compared with the control group, and the expression level of Glu was significantly increased(P < 0.05, 0.01). Compared with the model group, the expression level of GABA in SCZ medium and high dose group was significantly increased, and Glu expression was reduced significantly(P < 0.01). HPLC results showed that the GABA expression level in the hippocampus of the model group was significantly decreased compared with the control group, and the expression level of Glu was significantly increased(P < 0.05, 0.01). Compared with the model group, the expression level of Glu in hippocampus of rats in SCZ medium and high dose group was significantly decreased(P < 0.05), and the expression of GABA in rats with high and low doses of SCZ was significantly increased(P < 0.01). The results of Clock gene expression test showed that the expression of Clock and Bmal1 gene in the model group was significantly increased(P < 0.05) compared with the control group, and the expression levels of Cry1, Per1, and Per2 genes were significantly decreased(P < 0.05, 0.01). Compared with the model group, the expression of Clock and Bmal1 gene in the SCZ medium dose group was significantly decreased(P < 0.01); The expression levels of Cry1, Per1, and Per2 in the SCZ high dose group were significantly increased(P < 0.05, 0.01). The Per1 gene expression in the SCZ low-dose group was significantly increased(P < 0.01). Conclusion Cnidii Fructus hypnotic active components can increase the expression of Cry1, Per1, and Per2 gene by reducing the hypnosis of hippocampal Clock and Bmal1 expression. The increased expression of the inhibitory neurotransmitter GABA and the decreased expression of excitatory neurotransmitter Glu can inhibit the occurrence and development of insomnia, which can regulate the sleep-wake cycle for clinical treatment of insomnia.
Objective To study the hypnotic mechanism of Cnidii Fructus hypnotic active constituent(SCZ) on the expression of clock genes and hippocampal neurotransmitters in insomnia rats. Methods The insomnia model was established by intraperitoneal injection of para-chlorophenylalanine(PCPA). SCZ at low(25 g/kg), medium(50 g/kg), and high(100 g/kg) conentrations were ig administrated respectively at the same time with diazepam as positive group, intragastric administration of PCPA was performed in insomnia rats for three days, The expression levels of Clock, Bmal1, Cry1, Cry2, Per1, Per2, and Per3 were detected by real-time PCR. The expression levels of γ-aminobutyric acid(GABA) and glutamic acid(Glu) in hippocampus dentate gyrus were detected by immunohistochemistry. The changes of contents of GABA and Glu were determinated by immunohistochemistry and high performance liquid chromatography. Results The results of immunohistochemistry showed that the expression of GABA in the dentate gyrus of the hippocampus of rats in the model group was significantly reduced compared with the control group, and the expression level of Glu was significantly increased(P < 0.05, 0.01). Compared with the model group, the expression level of GABA in SCZ medium and high dose group was significantly increased, and Glu expression was reduced significantly(P < 0.01). HPLC results showed that the GABA expression level in the hippocampus of the model group was significantly decreased compared with the control group, and the expression level of Glu was significantly increased(P < 0.05, 0.01). Compared with the model group, the expression level of Glu in hippocampus of rats in SCZ medium and high dose group was significantly decreased(P < 0.05), and the expression of GABA in rats with high and low doses of SCZ was significantly increased(P < 0.01). The results of Clock gene expression test showed that the expression of Clock and Bmal1 gene in the model group was significantly increased(P < 0.05) compared with the control group, and the expression levels of Cry1, Per1, and Per2 genes were significantly decreased(P < 0.05, 0.01). Compared with the model group, the expression of Clock and Bmal1 gene in the SCZ medium dose group was significantly decreased(P < 0.01); The expression levels of Cry1, Per1, and Per2 in the SCZ high dose group were significantly increased(P < 0.05, 0.01). The Per1 gene expression in the SCZ low-dose group was significantly increased(P < 0.01). Conclusion Cnidii Fructus hypnotic active components can increase the expression of Cry1, Per1, and Per2 gene by reducing the hypnosis of hippocampal Clock and Bmal1 expression. The increased expression of the inhibitory neurotransmitter GABA and the decreased expression of excitatory neurotransmitter Glu can inhibit the occurrence and development of insomnia, which can regulate the sleep-wake cycle for clinical treatment of insomnia.
引文
[1]侯小斌,宋美卿,贾力莉,等.蛇床子催眠活性组分对睡眠剥夺大鼠学习记忆的影响[J].中华中医药杂志,2015,30(3):837-840.
[2]仝立国,谢君,冯玛莉.蛇床子中佛手柑内酯和异虎耳草素含量的高效液相色谱-二级管阵列检测法同时测定[J].时珍国医国药,2014,25(3):536-538.
[3]Nolte C,Staiger D.RNA around the clock-regulation at the RNA level in biological timing[J].Front Plant Sci,2015,doi:10.3389/fpls.2015.00311.
[4]何芳辉,尹蓉莉,刘杨,等.大孔树脂纯化白芷总香豆素工艺研究[J].时珍国医国药,2008,19(3):710-712.
[5]刘乃慧,张莉,邵颖,等.在体心脏灌流术在大鼠脑组织切片观察中的应用[J].实验动物科学与管理,2005,22(2):52-53.
[6]Udoh U S,Valcin J A,Gamble K L,et al.The molecular circadian clock and alcohol-induced liver injury[J].Biomolecules,2015,5(4):2504-2537.
[7]Chicasta?eda D,Ortega A.Clock genes in glia cells:A rhythmic history[J].Asn Neuro,2016,8(5):1-13.
[8]聂玉芝.沙苑子对运动训练大鼠脑组织中氨基酸类神经递质及相关基因表达影响的研究[D].西安:陕西师范大学,2005.
[9]刘振华,王世军.针刺四神聪、百会对失眠大鼠脑组织钟基因及氨基酸类神经递质表达的影响[J].中国老年学杂志,2015(35):6067-6069.
[10]富祯祯.乌龙丹对慢性脑缺血大鼠钟基因及氨基酸类神经递质表达的影响[D].广州:南方医科大学,2012.
[11]张舜波,王平,田代志,等.酸枣仁总皂苷对失眠老年大鼠脑氨基酸类神经递质及受体表达的影响[J].中国实验方剂学杂志,2014,20(4):124-127.
[12]阮继源.电针对失眠大鼠脑内谷氨酸、γ-氨基丁酸含量及γ-氨基丁酸A型受体表达的影响[J].中华中医药杂志,2013(12):3657-3660.
[13]Morioka N,Sugimoto T,Sato K,et al.The induction of Per1 expression by the combined treatment with glutamate,5-hydroxytriptamine and dopamine initiates a ripple effect on Bmal1 and Cry1 m RNA expression via the ERK signaling pathway in cultured rat spinal astrocytes[J].Neurochem Inter,2015,doi:10.1016/j.neuint.2015.06.013.
[14]Dudek M,Gossan N,Nan Y,et al.The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity[J].J Clin Invest,2016,126(1):365-376.
[2]仝立国,谢君,冯玛莉.蛇床子中佛手柑内酯和异虎耳草素含量的高效液相色谱-二级管阵列检测法同时测定[J].时珍国医国药,2014,25(3):536-538.
[3]Nolte C,Staiger D.RNA around the clock-regulation at the RNA level in biological timing[J].Front Plant Sci,2015,doi:10.3389/fpls.2015.00311.
[4]何芳辉,尹蓉莉,刘杨,等.大孔树脂纯化白芷总香豆素工艺研究[J].时珍国医国药,2008,19(3):710-712.
[5]刘乃慧,张莉,邵颖,等.在体心脏灌流术在大鼠脑组织切片观察中的应用[J].实验动物科学与管理,2005,22(2):52-53.
[6]Udoh U S,Valcin J A,Gamble K L,et al.The molecular circadian clock and alcohol-induced liver injury[J].Biomolecules,2015,5(4):2504-2537.
[7]Chicasta?eda D,Ortega A.Clock genes in glia cells:A rhythmic history[J].Asn Neuro,2016,8(5):1-13.
[8]聂玉芝.沙苑子对运动训练大鼠脑组织中氨基酸类神经递质及相关基因表达影响的研究[D].西安:陕西师范大学,2005.
[9]刘振华,王世军.针刺四神聪、百会对失眠大鼠脑组织钟基因及氨基酸类神经递质表达的影响[J].中国老年学杂志,2015(35):6067-6069.
[10]富祯祯.乌龙丹对慢性脑缺血大鼠钟基因及氨基酸类神经递质表达的影响[D].广州:南方医科大学,2012.
[11]张舜波,王平,田代志,等.酸枣仁总皂苷对失眠老年大鼠脑氨基酸类神经递质及受体表达的影响[J].中国实验方剂学杂志,2014,20(4):124-127.
[12]阮继源.电针对失眠大鼠脑内谷氨酸、γ-氨基丁酸含量及γ-氨基丁酸A型受体表达的影响[J].中华中医药杂志,2013(12):3657-3660.
[13]Morioka N,Sugimoto T,Sato K,et al.The induction of Per1 expression by the combined treatment with glutamate,5-hydroxytriptamine and dopamine initiates a ripple effect on Bmal1 and Cry1 m RNA expression via the ERK signaling pathway in cultured rat spinal astrocytes[J].Neurochem Inter,2015,doi:10.1016/j.neuint.2015.06.013.
[14]Dudek M,Gossan N,Nan Y,et al.The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity[J].J Clin Invest,2016,126(1):365-376.