从肝论治失眠的文献与实验研究
摘要
目的
失眠作为现代社会的常见疾病,严重影响着人们的生活质量。中西医治疗失眠均着眼于镇静催眠,然而西医存在明显副作用并且长期疗效证据不足,中医方药众多并且疗效证据不足。临床上失眠的反复发作与异常情绪常相互伴随,并且一些针对失眠的方药研究显示从肝论治失眠确有疗效,提示从肝论治失眠具有一定的临床可行性。本研究首先从中医病机和西医发病的角度为从肝论治失眠寻找理论基础,之后一方面采用系统评价手段分析从肝论治失眠的临床有效性,为从肝论治失眠寻找临床证据；一方面采用电生理和分子生物学手段分析从肝论治方治疗失眠的作用靶点,为从肝论治失眠寻找实验室证据。最终使从肝论治失眠理论应用于临床有据可依。
方法
1文献研究
研究一：全面检索从肝论治失眠的临床研究文献,建立从肝论治失眠随机对照临床试验、半随机对照临床试验、自身前后对照试验、病例报告、病例分析文献的数据库。统计近20年每年临床研究各设计类型的数量。
研究二：选取研究一数据库中的从肝论治失眠随机对照临床试验和半随机对照临床试验,制作资料提取表以采集基本信息、设计类型、研究对象、干预措施、结局指标等文献信息,并采用Cochrane偏倚风险评价工具评价文献质量。
研究三：根据伴随精神性疾病、中医证型、干预及对照措施、结局指标将研究二中纳入的文献划分为不同亚组,并采用RevMan5.0软件在异质性允许的条件下对从肝论治失眠疗效进行meta分析。
研究四：选取研究一数据库中的从肝论治失眠自身前后对照试验,制作资料提取表以采集基本信息、设计类型、研究对象、干预措施、结局指标等文献信息。根据伴随精神性疾病、中医证型、结局指标将纳入文献划分为不同亚组,并采用RevMan5.0软件在异质性允许的条件下对从肝论治失眠疗效进行meta分析。
2实验研究
研究一：以空白血清为对照,采用HPLC测定从肝论治方四逆散含药血清的固有成分,并比较4个不同批次含药血清的成分差异。
研究二：根据Springer protocol,采用新生24h内SD大鼠培养皮层神经元,倒置相差显微镜下观察神经元生长状态,并采用免疫荧光技术对培养12d的细胞进行神经元鉴定。
研究三：原代培养SD大鼠皮层神经元培养至第7-12d,分别加入对照组血清、四逆散组血清,使血清终浓度为10%,继续培养3h和24h后,采用全细胞膜片钳技术记录各组GABA诱导的IGABA,描绘浓度-效应曲线、计算IGARA衰减时间常数。
研究四：原代培养SD大鼠皮层神经元培养至第7-12d,分别加入对照组血清、四逆散组血清、归脾汤组血清,使血清终浓度为10%,继续培养24h后分别提取细胞膜蛋白及总蛋白。采用western blot技术检测各组蛋白质中GABA.受体α1、α2、α3、α5亚基的含量。
结果
1文献研究
研究一：最终纳入977篇从肝论治失眠临床研究文献,其中随机对照临床试验占17.81%,半随机对照临床试验占1.23%,自身前后对照试验占5.42%。从肝论治失眠临床研究文献发表数量从1993年的9篇逐渐增多至2013年的103篇,其中,随机/半随机对照临床试验从2003年的4篇增至2013年的28篇,同比增幅达600%。
研究二：最终纳入186篇从肝论治失眠随机/半随机对照临床试验文献,研究对象的年龄、性别、伴随疾病、证型分布以及干预措施、结局指标的选择可以良好的代表临床上的失眠患病人群,外部真实性良好。从随机分配方法、分配方案隐藏、盲法应用、结局损耗四方面判断,93.55%的文献存在高偏倚风险,内部真实性较差
研究三：(1)伴有更年期失眠：肝郁气滞证、肝阴亏虚证从肝论治组量表评分低于西药组或从他脏论治中药组。(2)伴有亚健康失眠：肝郁气滞证、肝郁化火证从肝论治组量表评分低于西药组或安慰剂组。(3)伴有抑郁症失眠：无特定证型从肝论治组量表评分低于西药组。(4)伴有焦虑症失眠：无特定证型从肝论治组量表评分低于从他脏论治中药组。(5)原发性失眠：无特定证型从肝论治中药组或联合西药组量表评分低于西药组、从他脏论治中药组或安慰剂组；肝郁气滞证从肝论治组量表评分低于西药组或从他脏论治中药组；肝郁化火证从肝论治组中药或联合西药组量表评分低于西药或从他脏论治中药组；肝阴亏虚证从肝论治组量表评分低于西药组或从他脏论治中药组。(6)未知伴随精神性疾病失眠：肝郁气滞证、肝郁化火证从肝论治组量表评分低于西药组或从他脏论治中药组。
研究四：最终纳入111篇从肝论治失眠自身前后对照试验文献,研究对象的年龄、性别、伴随疾病、证型分布以及干预措施、结局指标的选择可以良好的代表临床上的失眠患病人群,外部真实性良好。内部真实性无法判断。与治疗前相比,各亚组从肝论治后量表评分均显著降低。
2实验研究
研究一：4份四逆散含药血清的9个特有共有峰的相对保留时间RSD均在1%以内,相关系数均在0.90以上,证明不同批次血清成分比较稳定。但相对峰面积RSD最高达61.041%,证明不同批次血清成分含量存在差异。
研究二：采用classⅢβ-Tubulin抗体进行神经元鉴定显示原代培养皮层神经元纯度较高。显微镜观察提示神经元培养至7-12d活性最好,细胞胞体饱满,光晕明显,分布均匀,神经网络初步形成,适宜进行膜片钳实验。
研究三：经四逆散含药血清孵育后：(1)可增强GABA与GABAA受体的亲和力,随着孵育时间的延长,该作用有所减弱,但GABAA受体对低浓度GABA的敏感性有所增强。(2)可延缓GABAA受体进入失活。但随着孵育时间的延长,该作用有所减弱。(3)皮层神经元细胞膜上GABAA受体的数目增多,并随着孵育时间的延长,该作用有所增强。
研究四：(1)经四逆散含药血清孵育后,皮层神经元总蛋白中α1亚基含量显著升高,α5亚基含量显著下降；皮层神经元膜蛋白中α2、α3亚基含量显著升高,α5亚基含量显著下降。(2)经归脾汤含药血清孵育后,皮层神经元总蛋白中α1亚基含量显著升高；皮层神经元膜蛋白中α1、α3亚基含量显著升高,α5亚基含量显著下降。(3)皮层神经元膜蛋白中α1、α2亚基含量四逆散组和归脾汤组之间存在显著性差异。
结论
1文献研究
与对照组和治疗前相比,从肝论治失眠部分结局指标改善具有统计学意义。但由于文献方法学质量较低；干预措施方案不统一；结局指标中定性指标标准模糊客观性欠佳,定量指标在计算显效、无效的患者例数时标准不一,甚至未进行计算,无法进行进一步的分类变量合并效应量检验,最终均影响亚组meta分析结果临床意义的解释。要证实从肝论治失眠的临床有效性,尚需开展更多高质量的多中心、大样本随机临床对照试验。
2实验研究
从肝论治方通过增强GABAA受体对GABA的亲和性；延缓GABAA受体的失活；提高细胞膜上GABAA受体的表达,增强GABAA受体介导的mIPSP,起到神经元抑制,从而可能改善失眠。从肝论治方通过增加神经元总蛋白中α1亚基含量起到镇静催眠作用；通过增加神经元膜蛋白α2、α3亚基含量起到抗异常情绪作用；通过降低神经元总蛋白和膜蛋白中α5亚基含量避免记忆损害。与所隶属课题中的整体实验和临床试验结果相互印证,从不同角度证实从肝论治方治疗失眠的有效性。
Objective
Insomnia as a common disease of modern society, seriously affecting the quality of life.Western medicine focuses on the treatment of insomnia are sedative and hypnotic,but there are obvious side effects of Western medicine and the lack of long-term efficacy of evidence and lack of evidence of many prescription medicine efficacy. Clinically recurrent insomnia and abnormal emotions often accompany each other, and some herbs for insomnia studies showed indeed from the liver curative treatment of insomnia, insomnia tips from the liver has a certain clinical feasibility.In this study, from the onset of TCM and Western medicine perspective to find the theoretical basis from the liver insomnia, on the one hand the use of the system after the clinical effectiveness evaluation tools from the liver insomnia, insomnia is from the liver to find clinical evidence; On the one hand using electrophysiological and molecular biology analysis of treatment from the liver side effects insomnia targets for the treatment of insomnia from the liver to find laboratory evidence. Eventually be applied from the Liver clinical evidence-based theory of insomnia.
Methods
1Literature study
Study Ⅰ:A comprehensive clinical research literature retrieved from the liver treatment of insomnia, the establishment of a randomized controlled clinical trial of the treatment of insomnia from the liver, quasi-randomized controlled clinical trials, self controlled trials, case reports, case analysis data base literature.Statistical quantities of each design type of clinical research for nearly20years annually.
Study Ⅱ:Select a database study of randomized controlled trials and quasi-randomized controlled clinical trials on the treatment of insomnia from the liver to produce data to collect basic information mentioned in the table, design type, object of study, interventions, outcomes and other literature information, and the use of Cochrane risk of bias assessment tool for evaluating the quality of the literature.
Study Ⅲ:According to accompany mental disorders, syndromes, intervention and control measures, outcome indicators will be included in the research literature is divided into two different subgroups, using RevMan5.0software in heterogeneous conditions allowed from the liver on the efficacy of treatment of insomnia meta-analysis.
Study IV:Select a database study controlled trials from the liver after treatment of insomnia itself, making the data referred to collect basic information table, design type, object of study, interventions, outcomes and other literature information.According accompanied by mental disorders, syndromes,outcome measures will be included in the literature is divided into different subgroups,us ing RevMan5.0software in heterogeneous conditions allowed for the treatment of insomnia effects meta-analysis from the liver.
2Experimental Study
Study Ⅰ:Blank serum as control, were determined by HPLC from the liver side Sini serum containing natural ingredients, and compare four different batches of serum containing composition differences.
Study Ⅱ:According to Springer protocol, using newborn SD rats within24h cultured cortical neurons, neuronal growth inverted phase contrast microscope to observe state and immunofluorescence techniques12d cells were cultured neurons identification.
Study Ⅲ:SD primary cul tured rat cor t ical neurons cul tured million to7-12d, respectively, to join the control group, serum, Sini serum, serum final concentration of10%, continue to develop after3h and24h, using whole-cell patch clamp technique in each group GABA-induced IGABA, depicting the concentration-response curve, calculated IGABA decay time constant.
Study IV:SD primary cul tured rat cortical neurons cultured million to7-12d, respectively, to join the control group, serum, serum Sini, spleen soup serum, serum at a final concentration of10%, were cultured for24h extraction of membrane proteins and total protein. Detected by western blot in each group GABAA receptor al, α2, α3, α5subunit content.
Resul ts
1Literature study
Study I:The final study included977clinical literature from the liver treatment of insomnia, which accounted for randomized controlled clinical trials17.81%,quasi-randomized controlled clinical trials accounted1.23%, self controlled trials accounted for5.42%. From the liver of insomnia clinical research literature published from nine the number gradually increased to103in2013, which randomized/ quasi-randomized controlled clinical trials of28to2013from2003's4, an increase of1993600%.
Study II:Final included186from the liver insomnia random/quasi-randomized controlled clinical trial literature, the study of age, gender,concomitant diseases,syndromes can be a good representative of the distribution of the population prevalence of insomnia. From the random allocation method, allocation concealment, blinding application, determine the outcome of the loss in four areas,93.55%of the literature there is a high risk of bias.
Study III:(1) Associated with menopause insomnia:liver qi stagnation, liver deficiency syndrome from the liver group scores lower than western medicine or medicine from his dirty set of governance.(2) Accompanied by insomniacs:liverqistagnation, stagnation of the fire permit from the liver group scores lower than western medicine or placebo.(3)Accompanied by depression,insomnia:No specific syndromes from the liver group scores lower than western medicine.(4) Accompanied by anxiety insomnia:No specific syndromes from the liver group scores lower than from his dirty Treating medicine group.(5) Primary insomnia: No specific syndromes from the liver combined with western medicine group or group scores lower than western medicine, traditional Chinese medicine theory of governance from his dirty or placebo; liver qi stagnation from the liver governance group scores lower than western medicine or medicine from his dirty Treating group; stagnation of the fire certificate less than western medicine group from the liver or combined western medicine group scores or Treating from his dirty medicine group; liver deficiency syndrome from the liver group scores lower than we stern medicine group or groups of governance from his dirty.(6) Unknown insomnia associated with mental illness:liver qi stagnation, stagnation of the fire permit from the liver group scores lower than western medicine or medicine from his dirty set of governance.
Study IV:Final included111control trials, the study of the treatment of insomnia itself from the liver after age, gender, concomitant diseases, syndromes can be a good representative of the distribution of the population prevalence of insomnia. Compared with before treatment, scores were significantly lower in the subgroups from the Liver.
2Experimental Study
Study I:Relative retention time four Sini serum containing nine unique peaks of RSD were less than1%, with correlation coefficients above0.90, different batches of serum components proved relatively stable.But the relative peak area RSD up to61.041%, to prove the existence of differences between different batches of serum ingredients.
Study Ⅱ:The class Ⅲ p-Tubulin antibody identified neurons in primary culture displayed a higher purity of cortical neurons. Microscope tips neuronal cultures to7-12d best activity, cell body plump, Halo obviously, evenly distributed, the initial formation of the neural network, suitable for patch clamp experiments.
Study Ⅲ:After Sini after incubation in serum-containing drugs (1) Can enhance the affinity of GABA and GABAA receptors, with the extension of incubation time, the role has been weakened, but the GABAA receptor sensitivity to low concentrations of GABA are the enhancement.(2) May delay the GABAA receptor into inactivation. But with the extension of incubation time, the effect weakened.(3) The number of neurons in the cortex membrane GABA., receptors increase, and with prolonged incubation time, the role has been enhanced.
Study IV:(1) After incubation in serum-containing drug Sini, cortical neurons in a1subunit total protein content was significantly increased, decreased significantly α5subunit content:cortical neuronal membrane proteins α2, α3subunits were significantly elevated, α5subunits were significantly decreased.(2) The spleen soup after incubation in serum-containing drugs, cortical neurons was significantly elevated total protein content of a1subunit, cortical neuronal membrane proteins α1, α3subunits were significantly increased, decreased significantly α5subunit content.(3) There are significant differences between the α1, α2subunit content Sini group and spleen soup group of membrane proteins in cortical neurons.
Conclus ion
1Literature study
Compared with the control group and the treatment of the former with the treatment of insomnia from the liver statistically significant improvement in some outcome measures, but because of lower methodological quality literature; intervention programs are not uniform; outcome indicators objectivity fuzzy qualitative indicators of poor standard, quantitative in dicators lack individual patient clinically important change assessment.Ultimately affect the results of the sub group meta-analysis of clinical significance explanation.From the liver to confirm the clinical effectiveness of the treatment of insomnia, and still need to carry out more high-quality multi-center, large randomized controlled clinical trials.
2Experimental Study
From the liver side by enhancing GABAA receptor affinity for GABA; slow GABAA recept or in activation; enhance GABAA receptor expression on the cell membrane, enhance GABAA receptor-mediated mlPSP, played neuronal inhibition, there by may improve insomnia. From the liver side play sedative and hypnotic effects by increasing the total protein in neurons αl subunit content; through increased neuronal membrane proteina2, a3subunit content to play the role of anti-abnormal mood; neurons by reducing the total protein and membrane proteins the α5subunit content to avoid memory impairment. But away from the cell body functional status and mode of administration and differences insomatic cells, with the overall results still need to refer to each other confirmed.
失眠作为现代社会的常见疾病,严重影响着人们的生活质量。中西医治疗失眠均着眼于镇静催眠,然而西医存在明显副作用并且长期疗效证据不足,中医方药众多并且疗效证据不足。临床上失眠的反复发作与异常情绪常相互伴随,并且一些针对失眠的方药研究显示从肝论治失眠确有疗效,提示从肝论治失眠具有一定的临床可行性。本研究首先从中医病机和西医发病的角度为从肝论治失眠寻找理论基础,之后一方面采用系统评价手段分析从肝论治失眠的临床有效性,为从肝论治失眠寻找临床证据；一方面采用电生理和分子生物学手段分析从肝论治方治疗失眠的作用靶点,为从肝论治失眠寻找实验室证据。最终使从肝论治失眠理论应用于临床有据可依。
方法
1文献研究
研究一：全面检索从肝论治失眠的临床研究文献,建立从肝论治失眠随机对照临床试验、半随机对照临床试验、自身前后对照试验、病例报告、病例分析文献的数据库。统计近20年每年临床研究各设计类型的数量。
研究二：选取研究一数据库中的从肝论治失眠随机对照临床试验和半随机对照临床试验,制作资料提取表以采集基本信息、设计类型、研究对象、干预措施、结局指标等文献信息,并采用Cochrane偏倚风险评价工具评价文献质量。
研究三：根据伴随精神性疾病、中医证型、干预及对照措施、结局指标将研究二中纳入的文献划分为不同亚组,并采用RevMan5.0软件在异质性允许的条件下对从肝论治失眠疗效进行meta分析。
研究四：选取研究一数据库中的从肝论治失眠自身前后对照试验,制作资料提取表以采集基本信息、设计类型、研究对象、干预措施、结局指标等文献信息。根据伴随精神性疾病、中医证型、结局指标将纳入文献划分为不同亚组,并采用RevMan5.0软件在异质性允许的条件下对从肝论治失眠疗效进行meta分析。
2实验研究
研究一：以空白血清为对照,采用HPLC测定从肝论治方四逆散含药血清的固有成分,并比较4个不同批次含药血清的成分差异。
研究二：根据Springer protocol,采用新生24h内SD大鼠培养皮层神经元,倒置相差显微镜下观察神经元生长状态,并采用免疫荧光技术对培养12d的细胞进行神经元鉴定。
研究三：原代培养SD大鼠皮层神经元培养至第7-12d,分别加入对照组血清、四逆散组血清,使血清终浓度为10%,继续培养3h和24h后,采用全细胞膜片钳技术记录各组GABA诱导的IGABA,描绘浓度-效应曲线、计算IGARA衰减时间常数。
研究四：原代培养SD大鼠皮层神经元培养至第7-12d,分别加入对照组血清、四逆散组血清、归脾汤组血清,使血清终浓度为10%,继续培养24h后分别提取细胞膜蛋白及总蛋白。采用western blot技术检测各组蛋白质中GABA.受体α1、α2、α3、α5亚基的含量。
结果
1文献研究
研究一：最终纳入977篇从肝论治失眠临床研究文献,其中随机对照临床试验占17.81%,半随机对照临床试验占1.23%,自身前后对照试验占5.42%。从肝论治失眠临床研究文献发表数量从1993年的9篇逐渐增多至2013年的103篇,其中,随机/半随机对照临床试验从2003年的4篇增至2013年的28篇,同比增幅达600%。
研究二：最终纳入186篇从肝论治失眠随机/半随机对照临床试验文献,研究对象的年龄、性别、伴随疾病、证型分布以及干预措施、结局指标的选择可以良好的代表临床上的失眠患病人群,外部真实性良好。从随机分配方法、分配方案隐藏、盲法应用、结局损耗四方面判断,93.55%的文献存在高偏倚风险,内部真实性较差
研究三：(1)伴有更年期失眠：肝郁气滞证、肝阴亏虚证从肝论治组量表评分低于西药组或从他脏论治中药组。(2)伴有亚健康失眠：肝郁气滞证、肝郁化火证从肝论治组量表评分低于西药组或安慰剂组。(3)伴有抑郁症失眠：无特定证型从肝论治组量表评分低于西药组。(4)伴有焦虑症失眠：无特定证型从肝论治组量表评分低于从他脏论治中药组。(5)原发性失眠：无特定证型从肝论治中药组或联合西药组量表评分低于西药组、从他脏论治中药组或安慰剂组；肝郁气滞证从肝论治组量表评分低于西药组或从他脏论治中药组；肝郁化火证从肝论治组中药或联合西药组量表评分低于西药或从他脏论治中药组；肝阴亏虚证从肝论治组量表评分低于西药组或从他脏论治中药组。(6)未知伴随精神性疾病失眠：肝郁气滞证、肝郁化火证从肝论治组量表评分低于西药组或从他脏论治中药组。
研究四：最终纳入111篇从肝论治失眠自身前后对照试验文献,研究对象的年龄、性别、伴随疾病、证型分布以及干预措施、结局指标的选择可以良好的代表临床上的失眠患病人群,外部真实性良好。内部真实性无法判断。与治疗前相比,各亚组从肝论治后量表评分均显著降低。
2实验研究
研究一：4份四逆散含药血清的9个特有共有峰的相对保留时间RSD均在1%以内,相关系数均在0.90以上,证明不同批次血清成分比较稳定。但相对峰面积RSD最高达61.041%,证明不同批次血清成分含量存在差异。
研究二：采用classⅢβ-Tubulin抗体进行神经元鉴定显示原代培养皮层神经元纯度较高。显微镜观察提示神经元培养至7-12d活性最好,细胞胞体饱满,光晕明显,分布均匀,神经网络初步形成,适宜进行膜片钳实验。
研究三：经四逆散含药血清孵育后：(1)可增强GABA与GABAA受体的亲和力,随着孵育时间的延长,该作用有所减弱,但GABAA受体对低浓度GABA的敏感性有所增强。(2)可延缓GABAA受体进入失活。但随着孵育时间的延长,该作用有所减弱。(3)皮层神经元细胞膜上GABAA受体的数目增多,并随着孵育时间的延长,该作用有所增强。
研究四：(1)经四逆散含药血清孵育后,皮层神经元总蛋白中α1亚基含量显著升高,α5亚基含量显著下降；皮层神经元膜蛋白中α2、α3亚基含量显著升高,α5亚基含量显著下降。(2)经归脾汤含药血清孵育后,皮层神经元总蛋白中α1亚基含量显著升高；皮层神经元膜蛋白中α1、α3亚基含量显著升高,α5亚基含量显著下降。(3)皮层神经元膜蛋白中α1、α2亚基含量四逆散组和归脾汤组之间存在显著性差异。
结论
1文献研究
与对照组和治疗前相比,从肝论治失眠部分结局指标改善具有统计学意义。但由于文献方法学质量较低；干预措施方案不统一；结局指标中定性指标标准模糊客观性欠佳,定量指标在计算显效、无效的患者例数时标准不一,甚至未进行计算,无法进行进一步的分类变量合并效应量检验,最终均影响亚组meta分析结果临床意义的解释。要证实从肝论治失眠的临床有效性,尚需开展更多高质量的多中心、大样本随机临床对照试验。
2实验研究
从肝论治方通过增强GABAA受体对GABA的亲和性；延缓GABAA受体的失活；提高细胞膜上GABAA受体的表达,增强GABAA受体介导的mIPSP,起到神经元抑制,从而可能改善失眠。从肝论治方通过增加神经元总蛋白中α1亚基含量起到镇静催眠作用；通过增加神经元膜蛋白α2、α3亚基含量起到抗异常情绪作用；通过降低神经元总蛋白和膜蛋白中α5亚基含量避免记忆损害。与所隶属课题中的整体实验和临床试验结果相互印证,从不同角度证实从肝论治方治疗失眠的有效性。
Objective
Insomnia as a common disease of modern society, seriously affecting the quality of life.Western medicine focuses on the treatment of insomnia are sedative and hypnotic,but there are obvious side effects of Western medicine and the lack of long-term efficacy of evidence and lack of evidence of many prescription medicine efficacy. Clinically recurrent insomnia and abnormal emotions often accompany each other, and some herbs for insomnia studies showed indeed from the liver curative treatment of insomnia, insomnia tips from the liver has a certain clinical feasibility.In this study, from the onset of TCM and Western medicine perspective to find the theoretical basis from the liver insomnia, on the one hand the use of the system after the clinical effectiveness evaluation tools from the liver insomnia, insomnia is from the liver to find clinical evidence; On the one hand using electrophysiological and molecular biology analysis of treatment from the liver side effects insomnia targets for the treatment of insomnia from the liver to find laboratory evidence. Eventually be applied from the Liver clinical evidence-based theory of insomnia.
Methods
1Literature study
Study Ⅰ:A comprehensive clinical research literature retrieved from the liver treatment of insomnia, the establishment of a randomized controlled clinical trial of the treatment of insomnia from the liver, quasi-randomized controlled clinical trials, self controlled trials, case reports, case analysis data base literature.Statistical quantities of each design type of clinical research for nearly20years annually.
Study Ⅱ:Select a database study of randomized controlled trials and quasi-randomized controlled clinical trials on the treatment of insomnia from the liver to produce data to collect basic information mentioned in the table, design type, object of study, interventions, outcomes and other literature information, and the use of Cochrane risk of bias assessment tool for evaluating the quality of the literature.
Study Ⅲ:According to accompany mental disorders, syndromes, intervention and control measures, outcome indicators will be included in the research literature is divided into two different subgroups, using RevMan5.0software in heterogeneous conditions allowed from the liver on the efficacy of treatment of insomnia meta-analysis.
Study IV:Select a database study controlled trials from the liver after treatment of insomnia itself, making the data referred to collect basic information table, design type, object of study, interventions, outcomes and other literature information.According accompanied by mental disorders, syndromes,outcome measures will be included in the literature is divided into different subgroups,us ing RevMan5.0software in heterogeneous conditions allowed for the treatment of insomnia effects meta-analysis from the liver.
2Experimental Study
Study Ⅰ:Blank serum as control, were determined by HPLC from the liver side Sini serum containing natural ingredients, and compare four different batches of serum containing composition differences.
Study Ⅱ:According to Springer protocol, using newborn SD rats within24h cultured cortical neurons, neuronal growth inverted phase contrast microscope to observe state and immunofluorescence techniques12d cells were cultured neurons identification.
Study Ⅲ:SD primary cul tured rat cor t ical neurons cul tured million to7-12d, respectively, to join the control group, serum, Sini serum, serum final concentration of10%, continue to develop after3h and24h, using whole-cell patch clamp technique in each group GABA-induced IGABA, depicting the concentration-response curve, calculated IGABA decay time constant.
Study IV:SD primary cul tured rat cortical neurons cultured million to7-12d, respectively, to join the control group, serum, serum Sini, spleen soup serum, serum at a final concentration of10%, were cultured for24h extraction of membrane proteins and total protein. Detected by western blot in each group GABAA receptor al, α2, α3, α5subunit content.
Resul ts
1Literature study
Study I:The final study included977clinical literature from the liver treatment of insomnia, which accounted for randomized controlled clinical trials17.81%,quasi-randomized controlled clinical trials accounted1.23%, self controlled trials accounted for5.42%. From the liver of insomnia clinical research literature published from nine the number gradually increased to103in2013, which randomized/ quasi-randomized controlled clinical trials of28to2013from2003's4, an increase of1993600%.
Study II:Final included186from the liver insomnia random/quasi-randomized controlled clinical trial literature, the study of age, gender,concomitant diseases,syndromes can be a good representative of the distribution of the population prevalence of insomnia. From the random allocation method, allocation concealment, blinding application, determine the outcome of the loss in four areas,93.55%of the literature there is a high risk of bias.
Study III:(1) Associated with menopause insomnia:liver qi stagnation, liver deficiency syndrome from the liver group scores lower than western medicine or medicine from his dirty set of governance.(2) Accompanied by insomniacs:liverqistagnation, stagnation of the fire permit from the liver group scores lower than western medicine or placebo.(3)Accompanied by depression,insomnia:No specific syndromes from the liver group scores lower than western medicine.(4) Accompanied by anxiety insomnia:No specific syndromes from the liver group scores lower than from his dirty Treating medicine group.(5) Primary insomnia: No specific syndromes from the liver combined with western medicine group or group scores lower than western medicine, traditional Chinese medicine theory of governance from his dirty or placebo; liver qi stagnation from the liver governance group scores lower than western medicine or medicine from his dirty Treating group; stagnation of the fire certificate less than western medicine group from the liver or combined western medicine group scores or Treating from his dirty medicine group; liver deficiency syndrome from the liver group scores lower than we stern medicine group or groups of governance from his dirty.(6) Unknown insomnia associated with mental illness:liver qi stagnation, stagnation of the fire permit from the liver group scores lower than western medicine or medicine from his dirty set of governance.
Study IV:Final included111control trials, the study of the treatment of insomnia itself from the liver after age, gender, concomitant diseases, syndromes can be a good representative of the distribution of the population prevalence of insomnia. Compared with before treatment, scores were significantly lower in the subgroups from the Liver.
2Experimental Study
Study I:Relative retention time four Sini serum containing nine unique peaks of RSD were less than1%, with correlation coefficients above0.90, different batches of serum components proved relatively stable.But the relative peak area RSD up to61.041%, to prove the existence of differences between different batches of serum ingredients.
Study Ⅱ:The class Ⅲ p-Tubulin antibody identified neurons in primary culture displayed a higher purity of cortical neurons. Microscope tips neuronal cultures to7-12d best activity, cell body plump, Halo obviously, evenly distributed, the initial formation of the neural network, suitable for patch clamp experiments.
Study Ⅲ:After Sini after incubation in serum-containing drugs (1) Can enhance the affinity of GABA and GABAA receptors, with the extension of incubation time, the role has been weakened, but the GABAA receptor sensitivity to low concentrations of GABA are the enhancement.(2) May delay the GABAA receptor into inactivation. But with the extension of incubation time, the effect weakened.(3) The number of neurons in the cortex membrane GABA., receptors increase, and with prolonged incubation time, the role has been enhanced.
Study IV:(1) After incubation in serum-containing drug Sini, cortical neurons in a1subunit total protein content was significantly increased, decreased significantly α5subunit content:cortical neuronal membrane proteins α2, α3subunits were significantly elevated, α5subunits were significantly decreased.(2) The spleen soup after incubation in serum-containing drugs, cortical neurons was significantly elevated total protein content of a1subunit, cortical neuronal membrane proteins α1, α3subunits were significantly increased, decreased significantly α5subunit content.(3) There are significant differences between the α1, α2subunit content Sini group and spleen soup group of membrane proteins in cortical neurons.
Conclus ion
1Literature study
Compared with the control group and the treatment of the former with the treatment of insomnia from the liver statistically significant improvement in some outcome measures, but because of lower methodological quality literature; intervention programs are not uniform; outcome indicators objectivity fuzzy qualitative indicators of poor standard, quantitative in dicators lack individual patient clinically important change assessment.Ultimately affect the results of the sub group meta-analysis of clinical significance explanation.From the liver to confirm the clinical effectiveness of the treatment of insomnia, and still need to carry out more high-quality multi-center, large randomized controlled clinical trials.
2Experimental Study
From the liver side by enhancing GABAA receptor affinity for GABA; slow GABAA recept or in activation; enhance GABAA receptor expression on the cell membrane, enhance GABAA receptor-mediated mlPSP, played neuronal inhibition, there by may improve insomnia. From the liver side play sedative and hypnotic effects by increasing the total protein in neurons αl subunit content; through increased neuronal membrane proteina2, a3subunit content to play the role of anti-abnormal mood; neurons by reducing the total protein and membrane proteins the α5subunit content to avoid memory impairment. But away from the cell body functional status and mode of administration and differences insomatic cells, with the overall results still need to refer to each other confirmed.
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