基于二维液相色谱技术的元胡活性成分筛选及其药效和毒性机制的蛋白组学研究
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摘要
“中药研究中最困难的,也是最重要的一点,就是找出它的有效成分,然后进行作用机制研究,并将其推荐于临床应用,或进一步进行构效关系研究,以求新的发展。”(陈宜张于《中药延胡索研究中的新发现》序三)。尽管中药的应用历史可以追溯几千年,但是中药发挥疗效的物质基础是什么?其作用的靶点是什么?作用机制是什么?这些问题一直未被世人所彻底揭示。近年来,随着中药不良反应的增加,许多传统认为没有毒性的中药也出现不良反应,对中药不良反应的认识,不能再局限于传统文献记载的那些有毒中药上,必须利用现代科技开展药物安全性评价工作并对中药的毒理机制进行系统研究。另外,如何在中医药基本理论的指导下,结合现代科研手段,在细胞和分子水平明确中药的作用靶点和分子机制,也是目前中药研究亟需解决的问题。
系统生物学是研究一个生物系统中所有组成成分(基因、mRNA、蛋白质、代谢物等)的构成,以及在特定条件下这些组分间相互关系的学科。系统生物学的研究方法与中医治疗疾病的整体观念是一致的。蛋白组学是系统生物学的重要组成部分。蛋白组学认为中药有效部位或有效成分进入细胞或组织发挥作用,必然会引起分子、细胞、器官、整体多个层面的结构与功能状态的改变。蛋白质是生物体内功能的执行者,生物体结构和功能状态的改变必然在蛋白组水平上表现出来。因此,蛋白组学以蛋白质的丰度或翻译后修饰为指标,对中药活性成分的作用靶点和效应蛋白进行识别,建立起相应的作用网络,探讨其药理、毒副作用机制。化学蛋白组学主要用于研究在复杂蛋白质体系中,能与活性小分子相互作用的蛋白组。通过对该活性小分子进行标记或固定化,可以实现与其相互作用的蛋白组的选择性富集和鉴定。与传统的差异蛋白组学技术相比,只有与被标记或固定化小分子相互作用的蛋白质才能够被富集并检测,因而可以大大降低特定蛋白组的复杂度,从而提高对中、低丰度蛋白的检测能力。此外,通过质谱鉴定可以获得关于活性小分子靶蛋白的信息,并进一步阐述该分子的可能作用机理。
本课题以传统中药元胡为研究对象,以二维液相色谱—串联质谱为检测手段,借助血清药理学的研究理念,筛选中药元胡的活性镇痛成分;基于二维液相的筛选结果及相关文献报道,我们发现左旋延胡索乙素(l-THP)是元胡的主要镇痛活性成分之一,并对其进行进一步研究。首先,我们应用液相色谱-串联质谱法结合微透析取样方法研究l-THP在大鼠纹状体的药代动力学;针对l-THP临床应用中出现的肝脏毒副作用,本文应用2D-nano-LC-MS/MS技术,研究其肝脏毒性的分子机制;l-THP能明显抑制福尔马林诱导的疼痛行为,本文联用差异蛋白组学和化学蛋白组学技术,研究l-THP的作用靶点和镇痛机制,具体内容包括:
1.本文首先建立了福尔马林镇痛模型以评价元胡总生物碱(TAC)的镇痛效果,发现灌胃给药元胡总生物碱150 mg/kg后能够有效抑制福尔马林诱导的疼痛行为。接下来我们利用HSA生物色谱柱和ODS整体柱建立全二维液相色谱-串联质谱系统,通过比对元胡总生物碱、元胡总生物碱入血和入纹状体成分之间的差异,初步筛选元胡生物碱的镇痛活性成分。在系统优化全二维液相色谱串联质谱法条件后,我们将其应用于TAC的原成分、入血成分及入纹状体成分的分析。在原成分分析时,我们共检测到100多个化学成分,并对其中13种成分进行结构鉴定;我们共检测到40余种入血成分并鉴定了其中7种主要成分,分别是原阿片碱、海罂粟碱、四氢巴马亭、紫堇碱、小檗碱、四氢小檗碱和蓝堇辛;在进入纹状体的约20种化合物中,4种原形化合物(原阿片碱、海罂粟碱、四氢巴马亭、紫堇碱)的浓度较高。通过文献查阅,我们发现这4种化学物具有潜在的镇痛作用。尽管对元胡总生物碱的镇痛机制研究还存在很多不确定的因素,需要进一步探讨和验证,但是我们推测TAC的镇痛作用与原阿片碱、海罂粟碱、四氢巴马亭、紫堇碱的协同作用有关。本文的研究结果显示全二维液相色谱串联质谱技术是中药活性成分筛选的有效工具,能为中药的作用机制研究提供有效的信息。
2. L-THP是元胡的主要活性成分之一,具有显著的镇痛作用,作为镇痛药在临床上已应用多年。L-THP是多巴胺受体的阻滞剂,通过阻滞纹状体的D2多巴胺受体,抑制痛觉信息在脊髓水平的传递,实现镇痛作用。本研究首先建立了大鼠脑部纹状体的微透析取样模型,通过反透析法测定l-THP在纹状体内的回收率,并考察了l-THP浓度、取样时间对体内回收率的影响,发现微透析采样技术能够用于l-THP在脑部纹状体组织的药代动力学研究。另外,我们建立了高通量、高灵敏度的LC-MS/MS分析方法,并进行了完整的方法学验证。本文的研究证实微透析技术结合LC-MS/MS分析方法,能够成功的用于l-THP在纹状体的药代动力学研究。
3. L-THP是临床上应用多年的镇痛药,但是近年来,l-THP在临床应用中出现了严重的肝脏副作用。在本文中,我们发现l-THP能够诱导BALB/c小鼠肝细胞和human liver L-02细胞凋亡。通过Western Blots实验,我们证实一些凋亡指标性蛋白(caspase-3、Bcl-2、Bax)的表达发生了显著变化。我们以human liver L-02 (L-02)细胞为研究对象,应用基于2D-nano-LC-MS/MS的高通量蛋白组学研究技术,研究L-02细胞在毒性剂量的l-THP处理后,在蛋白表达层次上呈现的差异,从而探索l-THP致肝损伤可能的信号途径。我们发现经过l-THP处理后,L-02细胞共有156个蛋白的表达呈现明显差异。这些蛋白涵盖了复杂的功能领域,包括能量代谢、细胞骨架、核酸代谢和细胞凋亡等。我们选取了跟凋亡相关的蛋白进行功能探讨,并且选取两个重要蛋白(mTOR and MEK2),对其含量表达进行Western Blots验证。最后我们发现,l-THP主要通过改变凋亡途径中发挥重要作用的一些蛋白的表达,诱导细胞凋亡。并且从本实验可以看出,基于纳升级二维液相色谱—串联质谱技术的蛋白组学是研究药源性肝毒性机制的有效手段。
4. L-THP能明显抑制福尔马林诱导的疼痛行为,本文联用差异蛋白组学和化学蛋白组学技术,研究l-THP的作用靶点和镇痛机制。口服40 mg/kg的l-THP能够显著抑制福尔马林产生的疼痛行为。基于文献报道,纹状体是l-THP的主要镇痛部位。我们以纹状体蛋白组为研究对象,应用基于2D-nano-LC-MS/MS的高通量蛋白组学研究技术,研究福尔马林致痛大鼠在l-THP处理后,其纹状体蛋白组的表达差异。我们设定了严格的标准,对鉴定到的上千种蛋白进行筛选。最终我们选取得到17种具有显著表达差异的蛋白。我们通过对随机抽取的两种蛋白(Neurabin-1和Calcium-dependent secretion activator 1)的含量进行Western Blots验证,验证蛋白组学结果的可靠性,发现Western Blots实验结果能与蛋白组学结果较好吻合。另外,我们合成了基于l-THP的分子探针,通过化学蛋白组学技术,研究能与l-THP相互作用的蛋白组。我们发现差异蛋白组学和化学蛋白组学的结果能够较好互补的。综合化学蛋白组学和差异蛋白组学的研究结果,我们发现与离子通道,神经递质释放以及信号传导途径等相关蛋白在l-THP的镇痛机制中发挥重要作用。并且,联用差异蛋白组学和化学蛋白组学技术能够有效加深我们对l-THP的镇痛机制的了解
“The most difficult and most important challenges for Traditional Chinese medicine (TCM) research in order to gain further development is to screen the active ingredients, and illustrate the target proteins and related recognition mechanisms, and ultimately recommend it for clinical application and/or for further structure-activity relationship study”said Chen, Yizhang, the member of the Chinese Academy of Sciences. Despite TCMs’existence and continued use over many centuries and its popularity and extensive use during the last decade, substantial gaps remain both in our understanding of the“real”specific bioactive compounds, and the target proteins and related recognition mechanisms for these compounds. The knowledge on safety and efficacy is lacking because few TCMs have been evaluated by rigorous scientifically-designed trials. Herbal toxicity is increasingly recognized as the use of these medications has increased, and toxicity from TCMs seems to be a serious problem all over world now. We should no longer confine the adverse reactions of TCM to traditional Chinese medicine literature and expansion of basic research into mechanisms of herbal toxicity is warranted. To illustrate the target proteins and related recognition mechanisms of TCM under the guidance of TCM theory is another urgent task.
The proteomic approach is widely applied nowadays in the development of novel biomarker candidates for early detection of disease and identification of new targets for therapeutics, mainly by delineation of protein expression changes depending on factors such as the organism's physiological state and the stage of development of disease. However, traditional proteomics methodology provides information for abundant proteins but only provides limited information for proteins with low abundance. Chemical proteomics which used small molecules as baits to fish for interacting proteins, has emerged as a powerful way to investigate the interacting proteome. By chemical proteomic approach, only a manageable fraction of the proteome interacting with the fixed small molecules will be collected and analyzed and this approach greatly reduces the complexity of a certain proteome and thus enhances the ability to detect and to characterize low-abundance proteins. Furthermore, chemical proteomics becomes a direct readout for the characterization of target proteins and related recognition mechanisms of many drugs whose targets are still unclear, especially for compounds of natural origin.
Corydalis yanhusuo W.T. Wang has been widely used to treat spastic pain, abdominal pain and pain due to injury. In this paper, two-dimensional LC was applied to screen the bioactive compounds of Corydalis yanhusuo. L-THP, one of its main active ingredients was selected for further study. Microdialysis combined with LC-MS/MS was applied in the pharmacokinetic studies of l-THP in the straitum, the traget sites, and shotgun-based proteomics was applied to illustrate the detailed mechanisms involved in the l-THP-induced-antinociception and -hepatotoxicology. The detailed results were presented as follows:
1. In the present study, an animal mode of nociception based on the formalin injection into the hinder paw of rats was applied to evaluate the anti-nociceptive effect of total alkaloids of Corydalis yanhusuo (TAC), and the results of the formalin test indicated that formalin-evoked spontaneously nociceptive responses (licking behavior) could be inhibited by given (i.g.) TAC at a single dose of 150 mg/kg. Subsequently, an online comprehensive two-dimensional biochromatography method with a silica-bonded human serum albumin (HSA) column in the first dimension and a monolithic ODS column in the second was developed, and the absorbed bioactive components were screened by comparing and contrasting the components detected in the plasma and striatum with those in TAC. More than 100 compounds were separated and detected in the TAC, among which 13 compounds were identified. About 40 compounds (7 compounds identified) were absorbed into the plasma with appropriate concentrations, and about 20 compounds (4 compounds identified) passed through the blood-brain barrier into the striatum. Of interest, four compounds (protopine, glaucine, tetrahydropalmatine and corydaline) which were reported to possess profound anti-nociceptive effects, exhibited high concentrations in the striatum, therefore, it appears that they participated synergistically in regulating the formalin-induced nociception. The results indicated that the developed comprehensive two-dimensional biochromatography method is capable of screening the bioactive components in Corydalis yanhusuo, and providing valuable information for understanding the mechanisms by which Corydalis yanhusuo alleviates nociception.
2. Levo-tetrahydropalmatine (l-THP), which is officially listed in the Chinese pharmacopoeia was demonstrated to have excellent analgesic effects and has been in use in clinical practice for years in China. L-THP was recently found to elicit profound effects on the dopaminergic system in the straitum to produce anti-nociception. In the present study, the factors affecting the in vivo recovery from microdialysis probe (drug concentrations and within-day stability) were investigated, and the results indicated that microdialysis was an excellent method for in vivo sampling to measure the concentration of l-THP in the striatum. We developed and validated a sensitive, specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the quantitative determination of l-THP using diphenhydramine as the internal standard. The method combined LC-MS/MS with microdialysis was successfully applied in the pharmacokinetic studies of l-THP in the straitum.
3. L-THP, received much attention as an analgesic agent, has been associated with acute or chronic hepatitis in clinical practice. We found that l-THP can induce apoptosis in the hepatocytes of BALB/c mice and human normal liver L-02 (L-02) cells. Several key molecules, including caspase-3, Bcl-2 and Bax, were modulated by l-THP treatment. A novel high-throughput proteomic approach based on shotgun approach was applied to simultaneously evaluate the alterations of global protein expression involved in the response of l-THP treatment in L-02 cells. A total of 156 proteins were differentially expressed, among which 12 proteins play regulatory or constitutive roles in apoptosis pathways. Further analysis of two proteins by Western Blots, mTOR and MEK2, confirmed that these proteins were expressed at lower levels in l-THP treated L-02 cells compared with those of control. The current study provided detailed evidence to support that l-THP is capable of inducing apoptosis in mammalian liver cells and improve the understanding of mechanisms mediating the hepatotoxity of l-THP.
4. This study investigated the mechanisms involved in the antinociceptive action induced by l-THP in the formalin test by combined comparative and chemical proteomics. Rats were pre-treated with l-THP by the oral route (40 mg/kg) 1 h before formalin injection. The antinociceptive effect of l-THP was shown in the first and second phases of the formalin test. To address the mechanisms by which l-THP inhibits formalin-induced nociception in rats, the combined comparative and chemical proteomics were applied. A novel high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS was applied to simultaneously evaluate the deregulated proteins involved in the response of l-THP treatment in formalin-induced pain rats. Thousands of proteins were identified, among which 17 proteins survive the stringent filter criteria were further included for functional discussion. Two proteins (Neurabin-1 and Calcium-dependent secretion activator 1) were randomly selected and their expression levels were further confirmed by Western Blots. The results matched well with those of proteomics. In the present study, we also described the development and application of l-THP immobilized beads to bind the targets. Following incubation with cellular lysates, the proteome interacting with the fixed l-THP was identified. The results of comparative and chemical proteomics are quite complementary. Although the precise roles of these identified moleculars in l-THP-induced antinociception need further study, the combined results indicated that proteins associated with signal transduction, vesicular trafficking and neurotransmitter release, energy metabolism, and ion transport play important roles in l-THP-induced antinociception in formalin test,
系统生物学是研究一个生物系统中所有组成成分(基因、mRNA、蛋白质、代谢物等)的构成,以及在特定条件下这些组分间相互关系的学科。系统生物学的研究方法与中医治疗疾病的整体观念是一致的。蛋白组学是系统生物学的重要组成部分。蛋白组学认为中药有效部位或有效成分进入细胞或组织发挥作用,必然会引起分子、细胞、器官、整体多个层面的结构与功能状态的改变。蛋白质是生物体内功能的执行者,生物体结构和功能状态的改变必然在蛋白组水平上表现出来。因此,蛋白组学以蛋白质的丰度或翻译后修饰为指标,对中药活性成分的作用靶点和效应蛋白进行识别,建立起相应的作用网络,探讨其药理、毒副作用机制。化学蛋白组学主要用于研究在复杂蛋白质体系中,能与活性小分子相互作用的蛋白组。通过对该活性小分子进行标记或固定化,可以实现与其相互作用的蛋白组的选择性富集和鉴定。与传统的差异蛋白组学技术相比,只有与被标记或固定化小分子相互作用的蛋白质才能够被富集并检测,因而可以大大降低特定蛋白组的复杂度,从而提高对中、低丰度蛋白的检测能力。此外,通过质谱鉴定可以获得关于活性小分子靶蛋白的信息,并进一步阐述该分子的可能作用机理。
本课题以传统中药元胡为研究对象,以二维液相色谱—串联质谱为检测手段,借助血清药理学的研究理念,筛选中药元胡的活性镇痛成分;基于二维液相的筛选结果及相关文献报道,我们发现左旋延胡索乙素(l-THP)是元胡的主要镇痛活性成分之一,并对其进行进一步研究。首先,我们应用液相色谱-串联质谱法结合微透析取样方法研究l-THP在大鼠纹状体的药代动力学;针对l-THP临床应用中出现的肝脏毒副作用,本文应用2D-nano-LC-MS/MS技术,研究其肝脏毒性的分子机制;l-THP能明显抑制福尔马林诱导的疼痛行为,本文联用差异蛋白组学和化学蛋白组学技术,研究l-THP的作用靶点和镇痛机制,具体内容包括:
1.本文首先建立了福尔马林镇痛模型以评价元胡总生物碱(TAC)的镇痛效果,发现灌胃给药元胡总生物碱150 mg/kg后能够有效抑制福尔马林诱导的疼痛行为。接下来我们利用HSA生物色谱柱和ODS整体柱建立全二维液相色谱-串联质谱系统,通过比对元胡总生物碱、元胡总生物碱入血和入纹状体成分之间的差异,初步筛选元胡生物碱的镇痛活性成分。在系统优化全二维液相色谱串联质谱法条件后,我们将其应用于TAC的原成分、入血成分及入纹状体成分的分析。在原成分分析时,我们共检测到100多个化学成分,并对其中13种成分进行结构鉴定;我们共检测到40余种入血成分并鉴定了其中7种主要成分,分别是原阿片碱、海罂粟碱、四氢巴马亭、紫堇碱、小檗碱、四氢小檗碱和蓝堇辛;在进入纹状体的约20种化合物中,4种原形化合物(原阿片碱、海罂粟碱、四氢巴马亭、紫堇碱)的浓度较高。通过文献查阅,我们发现这4种化学物具有潜在的镇痛作用。尽管对元胡总生物碱的镇痛机制研究还存在很多不确定的因素,需要进一步探讨和验证,但是我们推测TAC的镇痛作用与原阿片碱、海罂粟碱、四氢巴马亭、紫堇碱的协同作用有关。本文的研究结果显示全二维液相色谱串联质谱技术是中药活性成分筛选的有效工具,能为中药的作用机制研究提供有效的信息。
2. L-THP是元胡的主要活性成分之一,具有显著的镇痛作用,作为镇痛药在临床上已应用多年。L-THP是多巴胺受体的阻滞剂,通过阻滞纹状体的D2多巴胺受体,抑制痛觉信息在脊髓水平的传递,实现镇痛作用。本研究首先建立了大鼠脑部纹状体的微透析取样模型,通过反透析法测定l-THP在纹状体内的回收率,并考察了l-THP浓度、取样时间对体内回收率的影响,发现微透析采样技术能够用于l-THP在脑部纹状体组织的药代动力学研究。另外,我们建立了高通量、高灵敏度的LC-MS/MS分析方法,并进行了完整的方法学验证。本文的研究证实微透析技术结合LC-MS/MS分析方法,能够成功的用于l-THP在纹状体的药代动力学研究。
3. L-THP是临床上应用多年的镇痛药,但是近年来,l-THP在临床应用中出现了严重的肝脏副作用。在本文中,我们发现l-THP能够诱导BALB/c小鼠肝细胞和human liver L-02细胞凋亡。通过Western Blots实验,我们证实一些凋亡指标性蛋白(caspase-3、Bcl-2、Bax)的表达发生了显著变化。我们以human liver L-02 (L-02)细胞为研究对象,应用基于2D-nano-LC-MS/MS的高通量蛋白组学研究技术,研究L-02细胞在毒性剂量的l-THP处理后,在蛋白表达层次上呈现的差异,从而探索l-THP致肝损伤可能的信号途径。我们发现经过l-THP处理后,L-02细胞共有156个蛋白的表达呈现明显差异。这些蛋白涵盖了复杂的功能领域,包括能量代谢、细胞骨架、核酸代谢和细胞凋亡等。我们选取了跟凋亡相关的蛋白进行功能探讨,并且选取两个重要蛋白(mTOR and MEK2),对其含量表达进行Western Blots验证。最后我们发现,l-THP主要通过改变凋亡途径中发挥重要作用的一些蛋白的表达,诱导细胞凋亡。并且从本实验可以看出,基于纳升级二维液相色谱—串联质谱技术的蛋白组学是研究药源性肝毒性机制的有效手段。
4. L-THP能明显抑制福尔马林诱导的疼痛行为,本文联用差异蛋白组学和化学蛋白组学技术,研究l-THP的作用靶点和镇痛机制。口服40 mg/kg的l-THP能够显著抑制福尔马林产生的疼痛行为。基于文献报道,纹状体是l-THP的主要镇痛部位。我们以纹状体蛋白组为研究对象,应用基于2D-nano-LC-MS/MS的高通量蛋白组学研究技术,研究福尔马林致痛大鼠在l-THP处理后,其纹状体蛋白组的表达差异。我们设定了严格的标准,对鉴定到的上千种蛋白进行筛选。最终我们选取得到17种具有显著表达差异的蛋白。我们通过对随机抽取的两种蛋白(Neurabin-1和Calcium-dependent secretion activator 1)的含量进行Western Blots验证,验证蛋白组学结果的可靠性,发现Western Blots实验结果能与蛋白组学结果较好吻合。另外,我们合成了基于l-THP的分子探针,通过化学蛋白组学技术,研究能与l-THP相互作用的蛋白组。我们发现差异蛋白组学和化学蛋白组学的结果能够较好互补的。综合化学蛋白组学和差异蛋白组学的研究结果,我们发现与离子通道,神经递质释放以及信号传导途径等相关蛋白在l-THP的镇痛机制中发挥重要作用。并且,联用差异蛋白组学和化学蛋白组学技术能够有效加深我们对l-THP的镇痛机制的了解
“The most difficult and most important challenges for Traditional Chinese medicine (TCM) research in order to gain further development is to screen the active ingredients, and illustrate the target proteins and related recognition mechanisms, and ultimately recommend it for clinical application and/or for further structure-activity relationship study”said Chen, Yizhang, the member of the Chinese Academy of Sciences. Despite TCMs’existence and continued use over many centuries and its popularity and extensive use during the last decade, substantial gaps remain both in our understanding of the“real”specific bioactive compounds, and the target proteins and related recognition mechanisms for these compounds. The knowledge on safety and efficacy is lacking because few TCMs have been evaluated by rigorous scientifically-designed trials. Herbal toxicity is increasingly recognized as the use of these medications has increased, and toxicity from TCMs seems to be a serious problem all over world now. We should no longer confine the adverse reactions of TCM to traditional Chinese medicine literature and expansion of basic research into mechanisms of herbal toxicity is warranted. To illustrate the target proteins and related recognition mechanisms of TCM under the guidance of TCM theory is another urgent task.
The proteomic approach is widely applied nowadays in the development of novel biomarker candidates for early detection of disease and identification of new targets for therapeutics, mainly by delineation of protein expression changes depending on factors such as the organism's physiological state and the stage of development of disease. However, traditional proteomics methodology provides information for abundant proteins but only provides limited information for proteins with low abundance. Chemical proteomics which used small molecules as baits to fish for interacting proteins, has emerged as a powerful way to investigate the interacting proteome. By chemical proteomic approach, only a manageable fraction of the proteome interacting with the fixed small molecules will be collected and analyzed and this approach greatly reduces the complexity of a certain proteome and thus enhances the ability to detect and to characterize low-abundance proteins. Furthermore, chemical proteomics becomes a direct readout for the characterization of target proteins and related recognition mechanisms of many drugs whose targets are still unclear, especially for compounds of natural origin.
Corydalis yanhusuo W.T. Wang has been widely used to treat spastic pain, abdominal pain and pain due to injury. In this paper, two-dimensional LC was applied to screen the bioactive compounds of Corydalis yanhusuo. L-THP, one of its main active ingredients was selected for further study. Microdialysis combined with LC-MS/MS was applied in the pharmacokinetic studies of l-THP in the straitum, the traget sites, and shotgun-based proteomics was applied to illustrate the detailed mechanisms involved in the l-THP-induced-antinociception and -hepatotoxicology. The detailed results were presented as follows:
1. In the present study, an animal mode of nociception based on the formalin injection into the hinder paw of rats was applied to evaluate the anti-nociceptive effect of total alkaloids of Corydalis yanhusuo (TAC), and the results of the formalin test indicated that formalin-evoked spontaneously nociceptive responses (licking behavior) could be inhibited by given (i.g.) TAC at a single dose of 150 mg/kg. Subsequently, an online comprehensive two-dimensional biochromatography method with a silica-bonded human serum albumin (HSA) column in the first dimension and a monolithic ODS column in the second was developed, and the absorbed bioactive components were screened by comparing and contrasting the components detected in the plasma and striatum with those in TAC. More than 100 compounds were separated and detected in the TAC, among which 13 compounds were identified. About 40 compounds (7 compounds identified) were absorbed into the plasma with appropriate concentrations, and about 20 compounds (4 compounds identified) passed through the blood-brain barrier into the striatum. Of interest, four compounds (protopine, glaucine, tetrahydropalmatine and corydaline) which were reported to possess profound anti-nociceptive effects, exhibited high concentrations in the striatum, therefore, it appears that they participated synergistically in regulating the formalin-induced nociception. The results indicated that the developed comprehensive two-dimensional biochromatography method is capable of screening the bioactive components in Corydalis yanhusuo, and providing valuable information for understanding the mechanisms by which Corydalis yanhusuo alleviates nociception.
2. Levo-tetrahydropalmatine (l-THP), which is officially listed in the Chinese pharmacopoeia was demonstrated to have excellent analgesic effects and has been in use in clinical practice for years in China. L-THP was recently found to elicit profound effects on the dopaminergic system in the straitum to produce anti-nociception. In the present study, the factors affecting the in vivo recovery from microdialysis probe (drug concentrations and within-day stability) were investigated, and the results indicated that microdialysis was an excellent method for in vivo sampling to measure the concentration of l-THP in the striatum. We developed and validated a sensitive, specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the quantitative determination of l-THP using diphenhydramine as the internal standard. The method combined LC-MS/MS with microdialysis was successfully applied in the pharmacokinetic studies of l-THP in the straitum.
3. L-THP, received much attention as an analgesic agent, has been associated with acute or chronic hepatitis in clinical practice. We found that l-THP can induce apoptosis in the hepatocytes of BALB/c mice and human normal liver L-02 (L-02) cells. Several key molecules, including caspase-3, Bcl-2 and Bax, were modulated by l-THP treatment. A novel high-throughput proteomic approach based on shotgun approach was applied to simultaneously evaluate the alterations of global protein expression involved in the response of l-THP treatment in L-02 cells. A total of 156 proteins were differentially expressed, among which 12 proteins play regulatory or constitutive roles in apoptosis pathways. Further analysis of two proteins by Western Blots, mTOR and MEK2, confirmed that these proteins were expressed at lower levels in l-THP treated L-02 cells compared with those of control. The current study provided detailed evidence to support that l-THP is capable of inducing apoptosis in mammalian liver cells and improve the understanding of mechanisms mediating the hepatotoxity of l-THP.
4. This study investigated the mechanisms involved in the antinociceptive action induced by l-THP in the formalin test by combined comparative and chemical proteomics. Rats were pre-treated with l-THP by the oral route (40 mg/kg) 1 h before formalin injection. The antinociceptive effect of l-THP was shown in the first and second phases of the formalin test. To address the mechanisms by which l-THP inhibits formalin-induced nociception in rats, the combined comparative and chemical proteomics were applied. A novel high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS was applied to simultaneously evaluate the deregulated proteins involved in the response of l-THP treatment in formalin-induced pain rats. Thousands of proteins were identified, among which 17 proteins survive the stringent filter criteria were further included for functional discussion. Two proteins (Neurabin-1 and Calcium-dependent secretion activator 1) were randomly selected and their expression levels were further confirmed by Western Blots. The results matched well with those of proteomics. In the present study, we also described the development and application of l-THP immobilized beads to bind the targets. Following incubation with cellular lysates, the proteome interacting with the fixed l-THP was identified. The results of comparative and chemical proteomics are quite complementary. Although the precise roles of these identified moleculars in l-THP-induced antinociception need further study, the combined results indicated that proteins associated with signal transduction, vesicular trafficking and neurotransmitter release, energy metabolism, and ion transport play important roles in l-THP-induced antinociception in formalin test,
引文
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