阿魏酸钠的镇静催眠作用机制研究
发布时间：2019-05-27 03:38
【摘要】：前言失眠是常见的一种睡眠障碍性疾病,是多种躯体、精神和行为疾病所具有的常见临床表现。流行病学调查资料表明,西方国家约35.2%的人有不同程度的失眠;我国失眠率也高达10%~20%。睡眠障碍可能对人体健康造成严重的危害,长期失眠,不仅导致抑郁,还会造成机体疲劳,导致认知功能障碍,甚至破坏免疫系统功能,对心脏造成损伤,干扰血糖调节、激素分泌等,甚至发展为精神抑郁。失眠的发病原因与遗传因素、生活习惯、环境、疾病和精神心理因素有关。对其发病机制的研究表明,失眠患者5-HT、NE、GABA等中枢神经递质紊乱,边缘-皮质系统环路异常。阿魏酸钠是心脑血管病的常用药物,国外研究和临床观察发现阿魏酸钠具有镇静催眠的作用,然而,阿魏酸钠对中枢神经递质5-HT、NE、GABA以及海马神经细胞有何影响,尚不清楚。目的本研究通过建立失眠大鼠模型,探讨阿魏酸钠对失眠大鼠的睡眠改善作用以及作用机制。材料与方法1.利用对氯苯丙氨酸(PCPA)腹腔注射制备失眠大鼠模型,观察阿魏酸钠对模型大鼠一般状态、体重变化、戊巴比妥钠致大鼠睡眠时间的影响,并对大鼠GABA、5-HT、NE、IL-1β含量及GABAARa1 m RNA表达量进行测定,同时观察大鼠脑组织病理学变化;2.阿魏酸钠改善睡眠作用与5-羟色胺能神经系统相关性的实验研究。观察阿魏酸钠对大鼠前额叶皮质区、下丘脑部位和海马组织5-HT水平的影响;观察阿魏酸钠分别联合5-HT合成的前体物质5-HTP和5-HT合成酶的抑制剂PCPA后对大鼠睡眠潜伏期和睡眠持续时间的影响;Elisa法测定阿魏酸钠对大鼠中枢不同脑区5-HT合成的限速酶色氨酸羟化酶(TPH)和代谢产物5-HIAA的含量的影响;应用RT-PCR技术考察阿魏酸钠对大鼠下丘脑5-HT转运体编码基因Slc6a4和5-HT1A受体编码基因5-HTR1A表达的影响;3.阿魏酸钠对模型大鼠GABA水平影响的机制研究。利用对氯苯丙氨酸(PCPA)腹腔注射制备失眠大鼠模型,分离各组大鼠脑干、下丘脑、额叶皮质和海马组织,Elisa法测定大鼠脑干、下丘脑、额叶皮质和海马组织GABA水平,HPLC法测定Glu水平,Western Blot法测定GAD、GS水平;4.阿魏酸钠对大鼠海马神经细胞的影响及作用机制研究。叠氮钠诱导大鼠海马神经细胞制备线粒体损伤海马神经细胞模型,MTT法考察阿魏酸钠对线粒体损伤海马神经细胞的保护作用。采用腹腔注射对氯苯丙氨酸(PCPA)法制备睡眠剥夺大鼠模型,TUNEL染色法观察阿魏酸钠对大鼠海马神经细胞凋亡的影响,采用透射电子显微镜观察阿魏酸钠对线粒体结构影响,测定阿魏酸钠对线粒体ATP酶活力的影响,RT-PCR法检测各组大鼠海马组织Bcl-2和Bax的m RNA和蛋白表达水平,探讨阿魏酸钠对海马神经细胞的作用机制。结果1.阿魏酸钠对PCPA失眠模型大鼠一般状态,睡眠质量及睡眠相关的神经递质均有较好的调节作用。阿魏酸钠可有效改善模型大鼠的一般状态,缩短睡眠潜伏期、延长睡眠时间、提高GABA、5-HT、IL-1β水平及GABA m RNA相对表达量,同时可有效降低NE水平;2.阿魏酸钠改善睡眠作用与5-羟色胺能神经系统相关性的实验研究。阿魏酸钠可明显提高大鼠前额叶皮质区、下丘脑部位和海马组织5-HT和TPH浓度(P0.05)。阿魏酸钠组与5-HTP联合应用试验中,阿魏酸钠组以及5-HTP组大鼠睡眠潜伏期均有缩短的趋势,在睡眠持续时间上均有延长的趋势,但差异与空白对照组均无统计学意义(P0.05),当阿魏酸钠和5-HTP联合应用时,大鼠的睡眠潜伏期和睡眠持续时间与空白组比较差异均具有统计学意义(P0.05)。阿魏酸钠和PACA联合应用试验中,阿魏酸钠组大鼠睡眠潜伏期明显缩短(P0.05),PCPA组潜伏期明显长于阿魏酸钠组和空白对照组(P0.05),睡眠持续时间实验结果表明,与空白组比较,阿魏酸钠组可以显著延长大鼠睡眠时间(P0.05),PCPA组睡眠持续时间明显缩短(P0.05),联合组与空白组比较差异无统计学意义(P0.05)。阿魏酸钠组大鼠海马区、下丘脑部位以及前额叶皮质区5-HIAA的浓度较空白组均显著升高(P0.05),海马组织与下丘脑5-HIAA/5-HT比值与空白组相比差异具有统计学意义(P0.05)。前额叶皮质区5-HIAA/5-HT比值与空白组相比差异无统计学意义(P0.05)。阿魏酸钠组大鼠下丘脑部位Slc6a4表达明显降低,5-HTR1A表达显著升高;3.与对照组比较,模型组大鼠不同脑区GABA、GAD水平均显著降低,Glu、GS水平均显著升高,阿魏酸钠各剂量组均可提高模型大鼠不同脑区GABA、GAD水平,降低不同脑区Glu水平,降低下丘脑、额叶皮质和海马组织GS水平,对大鼠脑干GS调节效果不明显;4.MTT法考察阿魏酸钠对线粒体损伤海马神经细胞的保护作用研究结果显示,线粒体损伤大鼠海马神经细胞存活率较正常对照组显著下降(P0.05),阿魏酸钠组海马神经细胞存活率高于模型组(P0.05)。TUNEL染色法结果显示阿魏酸钠可降低睡眠剥夺模型大鼠海马神经细胞凋亡率,RT-PCR结果显示阿魏酸钠可以促进Bcl-2 m RNA和蛋白表达,抑制Bax m RNA和蛋白表达,抑制海马神经细胞凋亡。结论1.阿魏酸钠对PCPA失眠模型大鼠一般状态,睡眠质量均有较好的调节作用;该作用可能是通过增加GABA水平,抑制神经元兴奋,提高5-HT,减低NE水平,降低NE水平来抑制NE的维持觉醒功能来实现。2.阿魏酸钠可通过协同5-HT前体物质5-HTP、拮抗脑内5-HT合成酶抑制剂PCPA、提高5-HT能神经系统的兴奋性、抑制5-HT转运体表达、增强5-HT受体表达等机制发挥改善睡眠的作用;3.阿魏酸钠对大鼠GABA水平影响可能是通过影响脑组织各部位中GAD及GS的表达水平,抑制或促进GABA与Glu之间的相互转化,改变Glu/GABA比值平衡实现的;4.阿魏酸钠可以抑制睡眠剥夺引起的海马神经细胞凋亡,从而改善睡眠,其作用机制与促进Bcl-2 m RNA和蛋白表达,抑制Bax m RNA和蛋白表达有关。
[Abstract]:Insomnia is a common type of sleep disorder, which is a common clinical manifestation of various body, mental and behavioral diseases. According to the epidemiological survey, about 35.2% of Western countries have different levels of insomnia, and the rate of insomnia in China is also as high as 10% ~ 20%. The sleep disorder may cause serious harm to human health, long-term insomnia, not only cause depression, but also cause body fatigue, cause cognitive dysfunction, even destroy the function of the immune system, cause damage to the heart, interfere with blood glucose regulation, hormone secretion, etc., And even developed into mental depression. The causes of insomnia are related to the genetic factors, the living habits, the environment, the disease and the mental and psychological factors. The study of its pathogenesis indicated that 5-HT, NE, GABA and other central neurotransmitters, such as 5-HT, NE, GABA and other central neurotransmitters, were abnormal in the edge-cortex system. Sodium ferulate is a common drug for cardiovascular and cerebrovascular diseases. It is found that sodium ferulate has a sedative and hypnotic effect in foreign research and clinical observation. However, the effect of sodium ferulate on the 5-HT, NE, GABA and hippocampal neurons of the central neurotransmitters is not clear. Objective To study the effect and mechanism of sodium ferulate on the sleep improvement of insomnia rats by establishing a model of insomnia rats. Materials and Methods 1. The effect of sodium ferulate on the sleep time of rats induced by sodium pentobarbital was observed by intraperitoneal injection of p-chlorophenylalanine (PCPA), and the content of GABA,5-HT, NE, IL-1 and the expression of GABAAR1 m were measured. The pathological changes of the brain tissue of the rats were also observed. Experimental study of the relationship between the effect of sodium ferulate on the sleep and the nervous system of 5-hydroxytryptamine. The effects of sodium ferulate on the levels of 5-HT in the prefrontal cortex, the hypothalamus and the hippocampus of the rats were observed, and the effects of sodium ferulate on the sleep latency and sleep duration of the rats were observed with 5-HT-synthesized precursor materials 5-HTP and 5-HT synthetase inhibitor PCPA, respectively. The effects of sodium ferulate on the content of 5-HT (5-HT) and 5-HT1A receptor-encoding gene (5-HT1A) in the hypothalamus of rats were determined by Elisa method. The effect of sodium ferulate on the expression of 5-HT1A receptor-encoding gene, Sc6a4 and 5-HT1A, was investigated by RT-PCR. The mechanism of the effect of sodium ferulate on the level of GABA in the model rats. The rat brain stem, the hypothalamus, the frontal cortex and the hippocampus of the rat brain stem, the hypothalamus, the frontal cortex and the hippocampus were isolated by intraperitoneal injection of p-chlorophenylalanine (PCPA). The levels of GABA in the brain stem, the hypothalamus, the frontal cortex and the hippocampus of the rats were determined by the Elisa method. GS level;4. The effect of sodium ferulate on the rat hippocampal neurons and its mechanism of action. The effects of sodium ferulate on the protective effects of sodium ferulate on the nerve cells of the hippocampus were studied by means of MTT method. The effect of sodium ferulate on the apoptosis of the rat's hippocampal neurons was studied by means of intraperitoneal injection of p-phenylalanine (PCPA), and the effect of sodium ferulate on the structure of the mitochondria was observed by a transmission electron microscope. The effect of sodium ferulate on the activity of the mitochondrial ATP enzyme was determined. The mRNA and protein expression levels of Bcl-2 and Bax in the hippocampus of each group were detected by RT-PCR, and the mechanism of the action of sodium ferulate on the nerve cells in the hippocampus was discussed. Results 1. Sodium ferulate has a good effect on the general status, sleep quality and sleep-related neurotransmitters in the model of PCPA insomnia. Sodium ferulate can effectively improve the normal state of the model rat, shorten the sleep period, prolong the sleep time, improve the relative expression of the GABA,5-HT, IL-1, and the GABA m RNA, and can effectively reduce the NE level; Experimental study of the relationship between the effect of sodium ferulate on the sleep and the nervous system of 5-hydroxytryptamine. Sodium ferulate significantly increased the concentration of 5-HT and TPH in the prefrontal cortex, the hypothalamus and the hippocampus of the rats (P0.05). During the combined application of sodium ferulate and 5-HTP, the sleep latency of the sodium ferulate group and the 5-HTP group had a tendency to be shortened, but there was no significant difference in the duration of the sleep (P0.05). When the sodium ferulate and the 5-HTP were used in combination, The sleep latency and sleep duration of the rats were significantly different from those in the blank group (P0.05). In the combined application of sodium ferulate and PACA, the sleep latency of the sodium ferulate group was significantly shorter than that of the sodium ferulate group (P0.05). The latency of the PCPA group was significantly longer than that of the sodium ferulate group and the blank control group (P0.05). The experimental results of the sleep duration showed that compared with the blank group, The group of sodium ferulate could significantly prolong the sleep time of the rats (P0.05). The sleep duration of the PCPA group was significantly shortened (P0.05), and the difference between the combined group and the blank group was not statistically significant (P0.05). The concentration of 5-HIAA in the hippocampus, the hypothalamus and the prefrontal cortex of the sodium ferulate group was significantly higher than that in the blank group (P0.05). The ratio of the 5-HIAA/5-HT in the hippocampus and the hypothalamus was statistically significant (P0.05). The 5-HIAA/5-HT ratio in the prefrontal cortex was not statistically significant (P0.05). The expression of Sc6a4 in the hypothalamus of the sodium ferulate group was significantly decreased, and the expression of 5-HTR1A was significantly increased. Compared with the control group, the levels of GABA and GAD in different brain regions of the model group were significantly decreased, and the levels of Glu and GS increased significantly, and the levels of GABA and GAD in different brain regions of the model rats can be improved by the dosage groups of sodium ferulate, and the levels of Glu and GAD in different brain regions can be reduced, and the hypothalamus can be reduced. The results of the study of the protective effect of sodium ferulate on the nerve cells in the hippocampus of the rats with mitochondrial injury showed that the survival rate of the neurons in the hippocampus of the rats with the mitochondrial injury was significantly lower than that in the normal control group (P0.05). The results of the TUNEL staining showed that the sodium ferulate could decrease the apoptosis rate of the hippocampal neurons in the sleep deprivation model. The results of RT-PCR showed that the sodium ferulate could promote the expression of Bcl-2 mRNA and protein, and inhibit the expression of Bax mRNA and protein. And the apoptosis of the hippocampal neurons is inhibited. Conclusion 1. The effect of sodium ferulate on the general status and the quality of sleep in the model of PCPA insomnia may be achieved by increasing the level of GABA, inhibiting the excitation of neurons, increasing the 5-HT, reducing the NE level, and decreasing the NE level to inhibit the maintenance of NE. Sodium ferulate can play an important role in improving sleep by combining 5-HT precursor substance 5-HTP, antagonizing the 5-HT synthetase inhibitor PCPA in the brain, increasing the excitability of the 5-HT energy nervous system, inhibiting the expression of 5-HT transporter, and enhancing the expression of 5-HT receptor. The effect of sodium ferulate on the level of GABA in rats may be by influencing the expression level of GAD and GS in various parts of the brain tissue, inhibiting or promoting the mutual transformation between GABA and Glu, and changing the balance of Glu/ GABA ratio; Sodium ferulate can inhibit the apoptosis of the hippocampal neurons caused by sleep deprivation, so as to improve the sleep, and the mechanism of action is related to the promotion of the expression of Bcl-2 mRNA and protein and the inhibition of the expression of Bax mRNA and protein.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R740
本文编号：2485881
[Abstract]:Insomnia is a common type of sleep disorder, which is a common clinical manifestation of various body, mental and behavioral diseases. According to the epidemiological survey, about 35.2% of Western countries have different levels of insomnia, and the rate of insomnia in China is also as high as 10% ~ 20%. The sleep disorder may cause serious harm to human health, long-term insomnia, not only cause depression, but also cause body fatigue, cause cognitive dysfunction, even destroy the function of the immune system, cause damage to the heart, interfere with blood glucose regulation, hormone secretion, etc., And even developed into mental depression. The causes of insomnia are related to the genetic factors, the living habits, the environment, the disease and the mental and psychological factors. The study of its pathogenesis indicated that 5-HT, NE, GABA and other central neurotransmitters, such as 5-HT, NE, GABA and other central neurotransmitters, were abnormal in the edge-cortex system. Sodium ferulate is a common drug for cardiovascular and cerebrovascular diseases. It is found that sodium ferulate has a sedative and hypnotic effect in foreign research and clinical observation. However, the effect of sodium ferulate on the 5-HT, NE, GABA and hippocampal neurons of the central neurotransmitters is not clear. Objective To study the effect and mechanism of sodium ferulate on the sleep improvement of insomnia rats by establishing a model of insomnia rats. Materials and Methods 1. The effect of sodium ferulate on the sleep time of rats induced by sodium pentobarbital was observed by intraperitoneal injection of p-chlorophenylalanine (PCPA), and the content of GABA,5-HT, NE, IL-1 and the expression of GABAAR1 m were measured. The pathological changes of the brain tissue of the rats were also observed. Experimental study of the relationship between the effect of sodium ferulate on the sleep and the nervous system of 5-hydroxytryptamine. The effects of sodium ferulate on the levels of 5-HT in the prefrontal cortex, the hypothalamus and the hippocampus of the rats were observed, and the effects of sodium ferulate on the sleep latency and sleep duration of the rats were observed with 5-HT-synthesized precursor materials 5-HTP and 5-HT synthetase inhibitor PCPA, respectively. The effects of sodium ferulate on the content of 5-HT (5-HT) and 5-HT1A receptor-encoding gene (5-HT1A) in the hypothalamus of rats were determined by Elisa method. The effect of sodium ferulate on the expression of 5-HT1A receptor-encoding gene, Sc6a4 and 5-HT1A, was investigated by RT-PCR. The mechanism of the effect of sodium ferulate on the level of GABA in the model rats. The rat brain stem, the hypothalamus, the frontal cortex and the hippocampus of the rat brain stem, the hypothalamus, the frontal cortex and the hippocampus were isolated by intraperitoneal injection of p-chlorophenylalanine (PCPA). The levels of GABA in the brain stem, the hypothalamus, the frontal cortex and the hippocampus of the rats were determined by the Elisa method. GS level;4. The effect of sodium ferulate on the rat hippocampal neurons and its mechanism of action. The effects of sodium ferulate on the protective effects of sodium ferulate on the nerve cells of the hippocampus were studied by means of MTT method. The effect of sodium ferulate on the apoptosis of the rat's hippocampal neurons was studied by means of intraperitoneal injection of p-phenylalanine (PCPA), and the effect of sodium ferulate on the structure of the mitochondria was observed by a transmission electron microscope. The effect of sodium ferulate on the activity of the mitochondrial ATP enzyme was determined. The mRNA and protein expression levels of Bcl-2 and Bax in the hippocampus of each group were detected by RT-PCR, and the mechanism of the action of sodium ferulate on the nerve cells in the hippocampus was discussed. Results 1. Sodium ferulate has a good effect on the general status, sleep quality and sleep-related neurotransmitters in the model of PCPA insomnia. Sodium ferulate can effectively improve the normal state of the model rat, shorten the sleep period, prolong the sleep time, improve the relative expression of the GABA,5-HT, IL-1, and the GABA m RNA, and can effectively reduce the NE level; Experimental study of the relationship between the effect of sodium ferulate on the sleep and the nervous system of 5-hydroxytryptamine. Sodium ferulate significantly increased the concentration of 5-HT and TPH in the prefrontal cortex, the hypothalamus and the hippocampus of the rats (P0.05). During the combined application of sodium ferulate and 5-HTP, the sleep latency of the sodium ferulate group and the 5-HTP group had a tendency to be shortened, but there was no significant difference in the duration of the sleep (P0.05). When the sodium ferulate and the 5-HTP were used in combination, The sleep latency and sleep duration of the rats were significantly different from those in the blank group (P0.05). In the combined application of sodium ferulate and PACA, the sleep latency of the sodium ferulate group was significantly shorter than that of the sodium ferulate group (P0.05). The latency of the PCPA group was significantly longer than that of the sodium ferulate group and the blank control group (P0.05). The experimental results of the sleep duration showed that compared with the blank group, The group of sodium ferulate could significantly prolong the sleep time of the rats (P0.05). The sleep duration of the PCPA group was significantly shortened (P0.05), and the difference between the combined group and the blank group was not statistically significant (P0.05). The concentration of 5-HIAA in the hippocampus, the hypothalamus and the prefrontal cortex of the sodium ferulate group was significantly higher than that in the blank group (P0.05). The ratio of the 5-HIAA/5-HT in the hippocampus and the hypothalamus was statistically significant (P0.05). The 5-HIAA/5-HT ratio in the prefrontal cortex was not statistically significant (P0.05). The expression of Sc6a4 in the hypothalamus of the sodium ferulate group was significantly decreased, and the expression of 5-HTR1A was significantly increased. Compared with the control group, the levels of GABA and GAD in different brain regions of the model group were significantly decreased, and the levels of Glu and GS increased significantly, and the levels of GABA and GAD in different brain regions of the model rats can be improved by the dosage groups of sodium ferulate, and the levels of Glu and GAD in different brain regions can be reduced, and the hypothalamus can be reduced. The results of the study of the protective effect of sodium ferulate on the nerve cells in the hippocampus of the rats with mitochondrial injury showed that the survival rate of the neurons in the hippocampus of the rats with the mitochondrial injury was significantly lower than that in the normal control group (P0.05). The results of the TUNEL staining showed that the sodium ferulate could decrease the apoptosis rate of the hippocampal neurons in the sleep deprivation model. The results of RT-PCR showed that the sodium ferulate could promote the expression of Bcl-2 mRNA and protein, and inhibit the expression of Bax mRNA and protein. And the apoptosis of the hippocampal neurons is inhibited. Conclusion 1. The effect of sodium ferulate on the general status and the quality of sleep in the model of PCPA insomnia may be achieved by increasing the level of GABA, inhibiting the excitation of neurons, increasing the 5-HT, reducing the NE level, and decreasing the NE level to inhibit the maintenance of NE. Sodium ferulate can play an important role in improving sleep by combining 5-HT precursor substance 5-HTP, antagonizing the 5-HT synthetase inhibitor PCPA in the brain, increasing the excitability of the 5-HT energy nervous system, inhibiting the expression of 5-HT transporter, and enhancing the expression of 5-HT receptor. The effect of sodium ferulate on the level of GABA in rats may be by influencing the expression level of GAD and GS in various parts of the brain tissue, inhibiting or promoting the mutual transformation between GABA and Glu, and changing the balance of Glu/ GABA ratio; Sodium ferulate can inhibit the apoptosis of the hippocampal neurons caused by sleep deprivation, so as to improve the sleep, and the mechanism of action is related to the promotion of the expression of Bcl-2 mRNA and protein and the inhibition of the expression of Bax mRNA and protein.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R740
【参考文献】
相关期刊论文 前10条
1 李莉;叶晓川;宋小英;杨楠;孙志猛;刘焱文;;UPLC-DAD-ELSD切换波长法同时测定复方酸枣仁颗粒中8种成分的含量[J];药物分析杂志;2016年10期
2 赵宏杰;樊官伟;罗毅杨;高笑雨;齐记;柳占彪;郭利平;;参竹精片协同戊巴比妥钠对小鼠睡眠及5-HT的影响[J];天津中医药;2016年05期
3 胡海燕;席东泽;雷磊;陈翔;王文花;;清心开窍方含药脑脊液对谷氨酸致PC12细胞损伤的保护机制[J];中国中药杂志;2013年12期
4 田星月;陈璇;;2型糖尿病患者睡眠质量及其影响因素调查分析[J];护理实践与研究;2013年09期
5 黄俏庭;刘亚平;廖继武;石顺治;马萍;潘集阳;;原发性失眠患者的血清P物质水平及其影响因素[J];中国神经精神疾病杂志;2013年02期
6 刘雅雯;熊建萍;张凌;项晓军;乐微;饶敏超;;634例化疗患者失眠的流行病学特征及相关危险因素分析[J];药品评价;2012年12期
7 朱蕾;张茹;李廷利;;刺五加对睡眠剥夺大鼠学习记忆及海马单胺类神经递质的影响[J];中国实验方剂学杂志;2012年04期
8 王希林;李省会;;失眠与抑郁障碍522例主诉失眠患者临床特点及HAMD分析[J];中国医学创新;2011年19期
9 夏胜利;韩俊;史晓红;谢志强;申晓靖;高晨;周伟;张杰文;张锦;董小平;许汴利;;河南省致死性家族失眠症家系遗传生物学分析[J];中国公共卫生;2011年06期
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