肝郁—脾虚—肝郁脾虚证与疏肝健脾方关系的肝脏蛋白组学研究
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摘要
“方证相关”是中医辨证论治科学原理和现代方剂学的核心研究领域之一,方剂的疗效不仅与方药的药量、药味、剂型、服法等要素相关,还与其所主治的对象-病证之间具有高度的针对或适配性,因而方剂的功效大小体现在方中药味的配伍与所主病证病机之间的吻合或针对程度,表现为临床同一或不同方剂治疗多个或某个特定病证显示不同的疗效,即方与证之间存在关联度高低的问题。“方证相关”蕴含的科学问题是方证之间的关联性大小及其现代生物学基础,开展方证相关的研究对于揭示中医辨证论治的科学内涵具有重要的意义。
本研究以与中医经验中具有一定方证关联关系的方和证为切入点,从“同方异证”的角度,以中医病因病机学说和现代疾病的病生理变化为背景,运用同步条件控制下的相关病因交叉模型复制方法,即采用束缚、饮食失节+过度疲劳及其复合因素造模方法复制出肝郁证、脾虚证及肝郁脾虚证大鼠模型,在对三个证候模型整体评价的基础上,运用蛋白质组学技术,探查肝郁、脾虚及肝郁脾虚三证模型肝脏的差异蛋白谱；运用生物信息学分析方法,比较各证模型的肝脏蛋白组学特征及其异同,探讨其生物学含义；同时考察疏肝健脾代表方—柴疏四君子汤对三证模型动物肝脏蛋白组学影响及其异同,试图从分子网络调控的角度探查三证的肝脏分子表征和柴疏四君子汤对三证作用的分子机制,论证柴疏四君子汤与肝郁证、脾虚证及肝郁脾虚证间的关联性及探讨其关联的现代生物学基础。
论文分为文献综述和实验研究两大部分。文献综述主要包括中医“方证相关”的研究进展和基于组学的中医证候研究进展两个部分。正文实验研究包括模型复制和蛋白组学分析二个部分,第一部分涉及肝郁证、脾虚证及肝郁脾虚证三证动物模型复制及其评价,第二部分则基于蛋白组学研究技术与方法,分析关联的三证动物模型肝脏的蛋白组学特征和疏肝健脾方(柴疏四君子汤)对三证模型肝脏蛋白组学的干预效应比较,探讨三证模型肝脏所涉及的信号通路及其调控网络变化和柴疏四君子汤对三证模型生物网络的不同作用。
研究一、肝郁-脾虚-肝郁脾虚三证大鼠模型的复制
方法：两批大鼠随机分为正常对照组、肝郁证模型组、脾虚证模型组、肝郁脾虚证模型组4组,每组10-13只。肝郁证模型组、脾虚证模型组、肝郁脾虚证模型组大鼠分别采用慢性束缚、过度疲劳+饮食失节、慢性束缚+过度疲劳+饮食失节法造模,正常对照组不予处理。每周末观察并记录各组大鼠体重。于实验第14和28天,采用旷场实验观察各组大鼠的行为活动特征,记录爬格数、直立次数、中央格停留时间、修饰时间及测算活动里程数。实验第29天,分别取血和下丘脑及垂体,分离血浆,试剂盒放免法测定血浆ACTH、COR1、CRH、下丘脑CRH及垂体ACTH。SPSS17.0软件处理各组数据,各组指标数据均以(x±s)表示,采用单因素方差分析(one-way ANOVA)后,行多组间比较,采用LSD检验。
结果：造模四周,与正常组相比,肝郁证大鼠体重降低,中央格停留时间显著升高(P<0.05或P<0.01),爬格数、直立次数、修饰时间、里程数均显著降低(P<0.05或P<0.01),血浆CRH、CORT、下丘脑CRH均显著升高(P<0.05),垂体ACTH无显著变化(P>0.05)；脾虚证大鼠体重降低,修饰时间和直立次数均显著升高(P<0.01),爬格数、里程数、中央格停留时间、血浆ACTH、CORT、垂体ACTH显著降低(P<0.05或P<0.01),血浆和下丘脑CRH无显著变化(P>0.05)；肝郁脾虚证大鼠体重降低明显,中央格停留时间显著升高(P<0.01),爬格数、直立次数、修饰时间、里程数显著降低(P<0.01),血浆CRH、ACTH、CORT、下丘脑CRH、垂体ACTH显著升高(P<0.05)。
结论：肝郁证、脾虚证及肝郁脾虚证模型大鼠均存在不同程度的外观表征和实验室指标的异常改变,但三证模型各有特点：1)体重变化上,随着造模时间的延长,各模型大鼠体重呈缓慢增长趋势,其中以脾虚证和肝郁脾虚证模型大鼠体重减低较为明显。2)行为学变化上,肝郁证初期活动探究性加强,而随造模时间的不断延长,从早期的活跃逐渐转为抑制,呈现出兴奋性由强转弱的特点,最终表现为中枢兴奋性减弱；脾虚证从早期的中枢兴奋到后期中枢神经系统反应失调；肝郁脾虚证从早期活动度降低,且随造模时间的延长进一步降低,呈现出中枢抑制性渐进加重的趋势。3)实验室指标变化上,在神经内分泌方面,肝郁证和肝郁脾虚证大鼠的HPA功能轴均呈亢进状态,脾虚证HPA功能轴则呈低下状态；肝郁证和脾虚证大鼠HPA轴异常的主要调点分别涉及下丘脑和垂体,肝郁脾虚证则同时伴有下丘脑及垂体反馈调节功能损伤。三个模型的上述结果分别与中医肝郁证、脾虚证及肝郁脾虚证三证的症状描述类同,与之前所了解到的三证的部分现代病生理内涵基本一致。结果表明本次复制的三个大鼠证候模型对中医肝郁证、脾虚证及肝郁脾虚证具有较好的模拟性。
研究二、肝郁-脾虚-肝郁脾虚三证模型大鼠肝脏蛋白组学特征及柴疏四君子汤作用
方法：大鼠随机分为正常对照组、肝郁证模型组、脾虚证模型组、肝郁脾虚证模型组、肝郁证干预组、脾虚证干预组、肝郁脾虚证干预组7组,每组10只。肝郁证模型组、脾虚证模型组、肝郁脾虚证模型组及其各证干预组大鼠分别采用上述各法造模,正常对照组不予处理；3个模型干预组分别于造模第2周末(即造模第15天起),按3.57g.kg-1体重给予大鼠柴疏四君子汤颗粒灌胃,连续2周；各模型组和正常对照组给予等量蒸馏水。实验第29天,无菌条件下剖取肝脏,取出肝脏相同部位的部分组,经生理盐水冲洗。随机从每组取出6个样品,进行蛋白质的提取后等量混合,对组织蛋白进行酶切后应用ITRAQ试剂标记,高效液相色谱分离后；应用液相色谱与质谱联用方法对蛋白质组样品进行分析,对质谱检测结果应用反相数据库检索方法评估数据的假阳性率；根据所鉴定出蛋白质的ITRAQ相对定量值,确定各模型组与正常对照组之间的差异蛋白。运用IPA软件分析各证模型及相应干预组的差异蛋白,获得各组差异蛋白涉及的部分信号传导通路及关联蛋白网络。
结果：1)三证模型肝脏的差异表达蛋白及其相关信号传导通路与功能网络：肝证模型组的差异蛋白共有67个,其中上调46个,下调21个；蛋白质信号传导通路显变化的有7条,包括EIF2pathway、Nicotine Degradation pathway、Androg Biothesis Thombin signaling、Regulation of actin-based mortility by Rho Chemokine Signaling和tRNA charging;主要的关联蛋白相互作用网络1个。脾虚模型组差异蛋白共有123个,其中上调52个,下调71个；蛋白质信号传导通路明显化的有11条,包括Alanine Degradation and synthesis、Geranyldiphospha Biosynthesis、Xenobiotic metabolism signaling、Glutathione Redox Glutathione-mediated Detoxification、Thyroid Hormone Metabolism Ⅱ、 Retin biosynthesis、LPS/IL-1mediated inhibition of RXR Function、FcR Pathway Serotonin degradation和Retinotate biosynthesis I;主要的蛋白相互作用网络个。肝郁脾虚证模型组差异蛋白共有296个,其中上调51个,下调245个；蛋白质号传导通路明显变化的有11条,包括Xenobiotic metabolism, LPS/IL-1mediat inhibition of RXR Function、Nicotine degradation Ⅱ、nicotine degradation II Serotonin degradation、Medatonin Degradation pathway. PXR/RXR pathway、Extrog biothethesis、Superpathway of metalon in degradation、Thyoid Hormone Metaboli Ⅱ、Phenylalanine degradation IV,主要的蛋白相互作用网络network4个。2)证模型肝脏差异表达蛋白的异同：肝郁证、脾虚证、肝郁脾虚证三个模型组共有差异白12个,其表达趋势一致；蛋白质信号传导通路明显变化的有3条,包括Retinota Biosynthesis I、GM-CSF signaling、nNOS signaling in neurons。其中肝郁证和虚证模型组共有差异蛋白20个,其表达趋势一致；涉及差异高通量蛋白质信号传导路明显变化的有3条,包括Retinotate Biothenthesis I、Thombin signaling和tR charging。肝郁证和肝郁脾虚证模型组共有差异蛋白22个,其中4个蛋白表达趋势反；涉及差异高通量蛋白质信号传导通路明显变化的有3条,包括Chemokine signal in Thrombin pathway、Nicotine degradation。脾虚证和肝郁脾虚证模型组共有差异蛋69个,其中1个蛋白表达趋势相反；蛋白质信号传导通路明显变化的有7条,包Xenobiotic singaling、serotonin degradation、Retinol and ret inotate biothesi Thyroid Hormone Metabolism、glutathione redox reaction、glutathione-mediat detoxif ication、Melatonin degradation。3)柴疏四君子汤对三证模型肝脏蛋白的响：①肝郁证模型组差异蛋白67个,肝郁证干预组的差异蛋白167个,其中经柴疏君子汤干预后恢复的差异蛋白22个,新增差异蛋白122个；干预后恢复的差异蛋白要涉及3条信号传导通路,包括LPS/IL-1mediated inhibition of RXR Function Xenobiotic metabolism pathway和Triacylglycerol Biothethesis,主要的蛋白相作用网络network1个；新增差异蛋白涉及4条信号传导通路,包括EIF2pathway Epithelial Adherens Junction Signaling、Cysteine BioSynthesis Homocy steidegradation、Co-enzyme Biothethesis,主要的蛋白相互作用网络network1个。②脾虚证模型组差异蛋白123个,脾虚证干预组差异蛋白243个,其中经柴疏四君子汤干预后恢复的差异蛋白49个,新增差异蛋白169个；干预后恢复的差异蛋白主要涉及5条信号传导通路,包括Alanine Biosynthesis/Degradation、 Geranylgeranyldiphosphate Biosynthesis、LPS/IL-1mediated inhibition of RXR Function、Trans-farnesyl diphosphate biosynthesis和Seenocysteine BiosynthesisⅡ,主要涉及的相关蛋白网络功能network2个；新增差异蛋白涉及7条信号传导通路,包括melatonin degradation、Nicotine degradation Ⅱ/Ⅲ, Estrogen biosynthesis. Serotonin degradation、Brupropion degradation和Acetone Degradation。③肝郁脾虚证模型组差异蛋白296个,肝郁脾虚证干预组差异蛋白403个,其中经柴疏四君子汤干预后恢复的差异蛋白68个,新增差异蛋白175个；干预后恢复的差异蛋白主要涉及6条信号传导通路,包括EIF2pathway、Clathrin-mediated endocytosis signaling。 LPS/IL-1mediated inhibition of RXR Function、Taurine Biothesis、Ceramide biothesis和Remodeling of Epithelial Adheres functions,主要涉及的相关蛋白网络功能network2个；新增差异蛋白涉及5条信号传导通路,包括Mitochondrial dysfunction、Bruprion degradation、Acetone degradation、Melatonin degradation. Estrogen Biothesis,主要涉及的相关蛋白网络功能network4个。
结果表明：肝郁、脾虚、肝郁脾虚三证均存在肝脏蛋白的异常表达,涉及到血液(与造血前体细胞生长有关的GM-CSF signaling通路)、甲状腺激素代谢和视物障碍(与视黄醇合成相关的Retinotate Biosynthesis I通路)等方面的异常,但各有不同。其中肝郁证以肝脏蛋白质的合成和翻译功能加强,尼古丁降解增强和雄激素合成加速,内分泌代谢亢进,免疫机能低下、神经发育功能低下,肝细胞再生修复功能下降,凝血等循环功能障碍为主要特点；脾虚证以乳酸肌糖酵解激活,异物清除及代谢能力下降,氧化还原、解毒能力低下,视黄醇等合成减少、视网膜合成障碍,甲状腺激素代谢水平低下,炎性反应和清除细菌能力下降、脂肪酸代谢水平低下为主要特点；肝郁脾虚证以清除异物毒物能力低下,胆固醇、胆汁酸和脂肪酸代谢水平低下,褪黑素降解抑制,雌激素合成减少、甲状腺激素代谢低下,尼古丁降解能力低下,免疫机能低下为主要特点；肝郁证和脾虚证均存在神经功能和心血管功能的低下,但肝郁证存在肝脏蛋白合成和翻译功能亢进,而在脾虚证中未见变化；脾虚证存在视黄醇合成减少、脂肪酸代谢水平低下,而在肝郁证中未见变化；肝郁脾虚证涵括了肝郁证和脾虚证的部分机制,其中肝郁和肝郁脾虚两证在肝细胞修复及凝血酶原导致的血管障碍等方面的生理功能异常表现有相同之处；脾虚和肝郁脾虚两证在血清素降解,视黄醇合成抑制,甲状腺激素代谢抑制,褪黑素降解等方面的生理功能异常表现有相同之处；但在糖代谢、异物清除、神经调节功能方面则与两证有所不同,较为突出的表现为肝郁脾虚证肝脏糖代谢处于抑制状态,而脾虚证中糖代谢机能表现亢进；肝郁脾虚证存在异物清除和尼古丁降解功能抑制,与肝郁证异物清除和尼古丁降解功能激活趋势有所不同。
柴疏四君子汤对肝郁证、脾虚证、肝郁脾虚证模型的肝脏差异蛋白表达均有不同程度的调节或逆转恢复作用,其中对肝郁证的效应主要是促进其免疫和异物清除力的恢复,对脾虚证主要是促进其免疫和脂代谢水平的恢复,对肝郁脾虚证主要是促进其免疫和神经调节、脂代谢、牛磺酸及上皮细胞功能的恢复。疏肝健脾方对肝郁脾虚证的肝脏显示了更为广泛的生物学调节作用。
本课题基于中医病因病机学说和中医证候病生理学内涵为背景,采用同步条件控制下的相关病因交叉叠加的模型复制方法,成功复制出中医肝郁证、脾虚证、肝郁脾虚证大鼠模型。在此基础上运用蛋白质组学分析技术,比较观察了肝郁、脾虚、肝郁脾虚三证模型动物肝脏的蛋白组学变化及体现中医疏肝健脾法的柴疏四君子汤对其模型肝脏蛋白组学的影响。结果获得肝郁、脾虚、肝郁脾虚三证模型的肝脏差异表达蛋白谱及柴疏四君子汤对三证模型作用后的肝脏差异蛋白谱。生物信息学分析表明,此三证肝脏蛋白组学变化涉及到神经与血管功能、三大物质及激素代谢、解毒及清除异物等多方面的生物学功能；三证之间具有较为复杂的交互异同关系并各具特点；柴疏四君子汤对此三证模型肝脏蛋白组学变化及其相关信号传导通路具有不同程度的选择性作用表现出不同的生物效应。
课题运用系统生物学方法,首次从肝脏蛋白组学的角度探查中医肝郁、脾虚、肝郁脾虚三证生物学内涵,比较疏肝健脾方对三证的作用异同,并获得疏肝健脾方与三证关联的部分分子机制,为在分子调控水平上揭示中医证候和方证关联的现代内涵提供重要的科学依据。
"Correlation between Formula and Syndrome" is one of the core content of research in TCM of the diagnosis and treatment based on differentiation of symptoms and signs and modern formula-ology. The function and usage of formula not only have correlation with medical elements by dose, taste, form, method, but also depends on complete matching with the main syndrome. So the main effect of the formula reflects to the role of targets of medical compatibility and a considerable extent on the pathology. The different functions give expression to the same or different formula treating one or more diseases,that is a problem of extent of correlation between the formula and syndrome. Until now the important scientific problems of correlation between formula and syndrome mainly study on the extent of correlation between the formula and syndrome and the foundation of modern biology. Based on the background, the research is began with the associating formula with diseases and cross etiology by different relating diseases and same formula actioning on different models. We use comparative analysis and proteomics researching methods to reveal scientific connotation of correlation between formula and syndrome,that will totally improve TCM of the diagnosis and treatment based on differentiation of symptoms and signs.
According to the TCM's etiology and pathogenesis theory and clinical manifestation and modern physiological and pathological characteristics, we take chronic restraint; excess fatigue plus out of constant diet and chronic restraint-fatigue-diet disorder methods to establish models of the liver-stagnation syndrome; the spleen-deficiency syndrome and combining the liver-stagnation and spleen-deficiency syndrome. Depending on liver proteomics, we study on the foundation of modern biology for rat models of GY; PX and GYPX from the view of the same formula to different syndromes. Compared with relating etiology under the same controling step to reveal TCM's essence on molecular level. Based on those factors, we choose the same medicine of Chaishu Sijunzi decoction to observe action of similarities and differences to models of GY; PX and GYPX. By the view of regulating molecular network,we try to discuss mechanism for the liver of the three models relationship and effects of CSJUZ decoction to those models in molecular level.
This thesis is divided into two main parts:literature review and experimental research. Literature review include research progress of TCM's theory of'Correlation between Formula and Syndrome" and using proteomics to study TCM's syndromes. Experimental research include model replication and proteomics analysis.For the first part,it involves rat models of GY; PX and GYPX replication and evaluation. For the second part,we discuss the change of liver protein expression in those rat models and comparing to the same medicine of Chaishu Sijunzi decoction to similarities and differences to those models effectiveness, and then we analysis differences proteins through different groups and understand the function of signal pathway and protein network to reveal foundation of modern biology with those models.
Research1:Establishing the different rat models of liver-stagnation syndrome, spleen-deficiency syndrome and both liver-stagnation and spleen-deficiency syndrome
Methods:The rats are randomly assigned into the normal group, the liver-stagnation group, the spleen-deficiency group, the liver-stagnation and spleen-deficiency group with ten rats for each group. The models were established in the liver-stagnation group, the spleen-deficiency group, the liver-stagnation and spleen-deficiency group by chronic restraint, excess fatigue plus out of constant diet or chronic restraint-fatigue-diet disorder method for4weeks, while the normal group is not with intervention. On the14th and28th day, using open-field-test to observe all rats included with the crossed squares, number of rearing,the stay period in the central zone, decorating time and the total distance of movement. On the29th day, all rats of serum ACTH. CORT. CRH are detected. SPSS17.0analysis different group data and take method of one-way ANOVA and LSD to compare for different groups.
Results:After building rat models4weeks,compared with normal group, the liver-stagnation group of the stay period in the central zone. CRH、CORT、the hypothalamus of CRH shown increased (P<0.05), weight, the crossed squares、number of rearing、 decorating time. the total distance of movement shown decreased (P<0.05);the spleen-deficiency group of number of rearing、decorating time shown increased (P<0.05), weight、the crossed squares、the stay period in the central zone、the total distance of movement、ACTH. CORT. the pituitary gland of ACTH shown decreased (P Conclusion:Rats models of the liver-stagnation, the spleen-deficiency, the liver-stagnation and spleen-deficiency are changed to apperant syndromes and laboratory indicators. In weight changing aspect, by the extension of time, the weight of rat models show a trend of slow growth and the weight significantly decreased in GY and GYPX group. In the autonomic activities, there are abnormal at different degrees in rat models of GY. PX and GYPX group. GY is involved in central excitatory from strong to weak. PX is involved in reaction of central nervous system disorder. GYPX is involved in central excitatory restraining deeply. In metabolic endocrine aspect, both of the spleen-deficiency and the liver-stagnation and spleen-deficiency are overactive to its function. The liver-stagnation and spleen-deficiency is also involved in impairment of feedback changing with hypothalamus and pituitary.The results of models are similar to the syndrome of GY,PX and GYPX. Replication of three rat models has a better simulation.
Research2:Study on effectiveness of the same formula of Chaishu Sijunzi decoction to similarities and differences in GY, PX and GYPX groups with liver protein expression changing
Methods:The rats are randomly assigned into the normal group, the liver-stagnation group, the spleen-deficiency group, the liver-stagnation and spleen-deficiency group, the drug treatment to liver-stagnation group, the drug treatment to spleen-deficiency group and the drug treatment to liver-stagnation and spleen-deficiency group with ten rats for each group. The models are established by chronic restraint method in GY and GY treatment groups, by excess fatigue plus out of constant diet method in PX and PX treatment groups, by chronic restraint-fatigue-diet disorder method in GYPX and GYPX treatment groups for4weeks. On the15th day, rats of all treatment groups have been fed with medicine of Chaishu Sijun decoction3.57g· kg-1for two weeks. The ones of the normal group and model groups are fed with equivalent physiological saline for two weeks. On the29th day, taking the liver outside in aseptic condition, then we eliminate hemoglobin by repeat washing. Randomly taking6sample for each group and extracting protein to mixing with equal amount. After enzyme digestion in microwave condition, we use ITRAQ reagent to mark, and then through liquid chromatographic separation we use mass spectrometry method to identify and quantitative analysis proteins. We take reverse database to estimate its false positive rate. According to identificating proteins ITRAQ relative quantitative values to ensure differential proteins for groups.At last, the differences in protein are analysis by IPA database and get partial signaling pathways and networks.
Results:1)Three rat models'liver differential proteins and pathways and network:There are67differentially expressed proteins in GY group, among them,46proteins are upregulation and21downregulation. There are7changing pathways, include EIF2pathway、Nicotine Degradation pathway Androgen Bio thesi、Thomb in signaling、Regulation of actin-based mortility by Rho、Chemokine Signaling、tRNA charging and there is1network. There are123differentially expressed proteins in PX group, among them,52proteins are upregulation and71down regulation. There are7changing pathways, include Alanine Degradation and synthesis、 Geranyldiphosphate Biosynthesis、Xenobiotic metabolism signaling、Glutathione Redox、Glutathione-mediated Detoxification、Thyroid Hormone Metabolism Ⅱ、Retinal biosynthesis、LPS/IL-1mediated inhibition of RXR Function、FcR Pathway、Serotonin degradation、Retinotate biosynthesis I and there are2networks. There are296differentially expressed proteins in GYPX group, among them,51proteins are upregulation and245down regulation. There are11changing pathways, include Xenobiotic metabolism. LPS/IL-1mediated inhibition of RXR Function. Nicotine degradation II. nicotine degradation III. Serotonin degradation. Medatonin Degradation pathway、PXR/RXR pathway. Extrogen biothethesis. Superpathway of metalonin degradation、Thyoid Hormone Metabolism II. Phenylalanine degradation IV and there are4networks.2) Similarities and differences between three rat models'liver differential proteins:There are12differentially expressed proteins in GY, PX and GYPX groups with the same expression trend. There are3changing pathways, include Retinotate Biosynthesis I、GM-CSF signaling、nNOS signaling in neurons.There are20differentially expressed proteins in GY and PX groups with the same expression trend.There are3changing pathways, include Retinotate Biothenthesis I. Thombin signaling and tRNA charging.There are22differentially expressed proteins in GY and GYPX groups, among them,4proteins are upregulation in GY and downregulation in GYPX groups. There are3changing pathways, include Chemokine signaling、Thrombin pathway、Nicotine degradation.There are69differentially expressed proteins in PX and GYPX groups, among them,1protein is upregulation in PX and downregulation in GYPX groups. There are7changing pathways, include Xenobiotic singaling、serotonin degradation、Retinol and retinotate biothesis、Thyroid Hormone Metabolism、glutathione redox reaction、glutathione-mediated detoxification. Melatonin degradation.3) Influence of CSSJZ Decoction to three rat models'liver differential proteins:①There are67differentially expressed proteins in GY group and167differentially expressed proteins in GY treatment group, among them,22differentially expressed proteins turn to normal and122new differentially expressed proteins adding by taking CSSJZT. There are3changing pathways, include LPS/IL-1mediated inhibition of RXR Function、Xenobiotic metabolism pathway. Triacylglycerol Biothethesis and there is1network for differentially expressing proteins turned to normal. There are4changing pathways, include EIF2pathway、Epithelial Adherens Junction Signaling、Cysteine BioSynthesis Homocy steine degradation、Co-enzyme Biothethesis and there is1network for new differentially expressed proteins adding by taking CSSJZT in GY group.②There are123differentially expressed proteins in PX group and243differentially expressed proteins in PX treatment group, among them,49differentially expressed proteins turn to normal and169new differentially expressed proteins adding by taking CSSJZT. There are5changing pathways, include Alanine Biosynthesis/Degradation、Geranylgeranyldiphosphate Biosynthesis、LPS/IL-1mediated inhibition of RXR Function、Trans-farnesyl diphosphate biosynthesis、Seenocysteine Biosynthesis II and there are2networks for differentially expressing proteins turned to normal. There are7changing pathways, include melatonin degradation、Nicotine degradationⅡ/Ⅲ、Estrogen biosynthesis. Serotonin degradation、Brupropion degradation、Acetone Degradation for new differentially expressed proteins adding by taking CSSJZT in PX group.③There are296differentially expressed proteins in GYPX group and403differentially expressed proteins in GYPX treatment group, among them,68differentially expressed proteins turn to normal and175new differentially expressed proteins adding by taking CSSJZT. There are6changing pathways, include EIF2pathway、Clathrin-mediated endocytosis signaling. LPS/IL-1mediated inhibition of RXR Function、Taurine Biothesis、Ceramide biothesis、Remodeling of Epithelial Adheres functions and there are2networks for differentially expressing proteins turned to normal. There are5changing pathways, include Mitochondrial dysfunction、 Bruprion degradation、Acetone degradation、Melatonin degradation、Estrogen Biothesis and there are4networks for new differentially expressed proteins adding by taking CSSJZT in GYPX group.
Conclusion:The liver proteins are differentially expressed in GY, PX and GYPX groups, that connects with aspects of blood, thyroid hormone metabolism and visual impairment. In GY group, the characteristics include the activating functions of liver protein synthesis and translation、nicotine degradation、androgen synthesis、endocrine metabolism and the restraining functions of immune、neural development、repairment of liver cell regeneration and disorder function of blood coagulation. In PX group, the characteristics include the activating functions of muscle lactate glycolysis and the restraining functions of foreign matter removal、redox、detoxification、retinol and retina synthesis、thyroid hormone metabolism、inflammatory reaction、fatty acid metabolism. In GYPX group, the characteristics include the restraining functions of foreign matter removal、cholesterol、bile acid and fatty acids metabolism、melatonin degradation、estrogen synthesis、thyroid hormone metabolism、nicotine degradation. In GY and PX groups, the characteristics include the restraining functions of nerve and cardiovascular. The characteristics of activating functions of liver protein synthesis and translation are only found in GY group and restraining functions of retinol and retina synthesis、fatty acid metabolism are only found in PX group. GYPX partly include GY and PX characteristic, while those which are different from aspects of glucose metabolism, foreign matter removal capacity and neural. Among them, the hyperfunction of glucose metabolism in PX opposite to depressing in GYPX. The hyperfunction of foreign matter removal and nicotine degradation capacity in G Y opposite to depressing in GYPX.
The formula of soothing liver qi and tonifying spleen qi can regulate the function of GY, PX and GYPX with liver differentially expressed proteins. The function of GY is regulated by immunity and foreign matter removal capacity. The function of PX is regulated by immunity and lipid metabolism. The function of GYPX is regulated by immunity, neural, lipid metabolism melatonin and estrogen synthesis. Based on those conclusions, it prompts that CSSJZT can improve GYPX in a wide range.
We take methods of chronic restraint, excess fatigue plus out of constant diet, chronic restraint-fatigue-diet disorder to establish rats models of GY, PX and GYPX at the same lever and conditions. On this basis,we take proteomic analysis technology and observe effectives of the same CSSJZT to GY, PX and GYPX similarities and differences with the method of liver proteomics.We get the differentially expressed proteins for the same CSSJZT decoction to the syndrome of GY, PX and GYPX. According to the bio informatics analysis,three syndromes of GY, PX and GYPX involve in the function changing of nerves and blood vessels、three materials and hormone metabolism、detoxify and foreign matter removal capacity.Those syndromes have complex interactions and characteristics and CSSJZ decoction select to show different biological effects to the different model liver proteins with pathway and network analysis.
Using biology system method,we discuss the biology connotation of TCM's GY、PX、 GYPX in Liver proteomics at first.Comparing the different effects of CSSJZ decoction to three syndromes,we get partial molecular mechanisms to the formula for soothing liver qi and tonifying spleen qi and three syndromes.The discussion reveal essence and connotation of correlation between formula and syndrome to offer objective evidence.
本研究以与中医经验中具有一定方证关联关系的方和证为切入点,从“同方异证”的角度,以中医病因病机学说和现代疾病的病生理变化为背景,运用同步条件控制下的相关病因交叉模型复制方法,即采用束缚、饮食失节+过度疲劳及其复合因素造模方法复制出肝郁证、脾虚证及肝郁脾虚证大鼠模型,在对三个证候模型整体评价的基础上,运用蛋白质组学技术,探查肝郁、脾虚及肝郁脾虚三证模型肝脏的差异蛋白谱；运用生物信息学分析方法,比较各证模型的肝脏蛋白组学特征及其异同,探讨其生物学含义；同时考察疏肝健脾代表方—柴疏四君子汤对三证模型动物肝脏蛋白组学影响及其异同,试图从分子网络调控的角度探查三证的肝脏分子表征和柴疏四君子汤对三证作用的分子机制,论证柴疏四君子汤与肝郁证、脾虚证及肝郁脾虚证间的关联性及探讨其关联的现代生物学基础。
论文分为文献综述和实验研究两大部分。文献综述主要包括中医“方证相关”的研究进展和基于组学的中医证候研究进展两个部分。正文实验研究包括模型复制和蛋白组学分析二个部分,第一部分涉及肝郁证、脾虚证及肝郁脾虚证三证动物模型复制及其评价,第二部分则基于蛋白组学研究技术与方法,分析关联的三证动物模型肝脏的蛋白组学特征和疏肝健脾方(柴疏四君子汤)对三证模型肝脏蛋白组学的干预效应比较,探讨三证模型肝脏所涉及的信号通路及其调控网络变化和柴疏四君子汤对三证模型生物网络的不同作用。
研究一、肝郁-脾虚-肝郁脾虚三证大鼠模型的复制
方法：两批大鼠随机分为正常对照组、肝郁证模型组、脾虚证模型组、肝郁脾虚证模型组4组,每组10-13只。肝郁证模型组、脾虚证模型组、肝郁脾虚证模型组大鼠分别采用慢性束缚、过度疲劳+饮食失节、慢性束缚+过度疲劳+饮食失节法造模,正常对照组不予处理。每周末观察并记录各组大鼠体重。于实验第14和28天,采用旷场实验观察各组大鼠的行为活动特征,记录爬格数、直立次数、中央格停留时间、修饰时间及测算活动里程数。实验第29天,分别取血和下丘脑及垂体,分离血浆,试剂盒放免法测定血浆ACTH、COR1、CRH、下丘脑CRH及垂体ACTH。SPSS17.0软件处理各组数据,各组指标数据均以(x±s)表示,采用单因素方差分析(one-way ANOVA)后,行多组间比较,采用LSD检验。
结果：造模四周,与正常组相比,肝郁证大鼠体重降低,中央格停留时间显著升高(P<0.05或P<0.01),爬格数、直立次数、修饰时间、里程数均显著降低(P<0.05或P<0.01),血浆CRH、CORT、下丘脑CRH均显著升高(P<0.05),垂体ACTH无显著变化(P>0.05)；脾虚证大鼠体重降低,修饰时间和直立次数均显著升高(P<0.01),爬格数、里程数、中央格停留时间、血浆ACTH、CORT、垂体ACTH显著降低(P<0.05或P<0.01),血浆和下丘脑CRH无显著变化(P>0.05)；肝郁脾虚证大鼠体重降低明显,中央格停留时间显著升高(P<0.01),爬格数、直立次数、修饰时间、里程数显著降低(P<0.01),血浆CRH、ACTH、CORT、下丘脑CRH、垂体ACTH显著升高(P<0.05)。
结论：肝郁证、脾虚证及肝郁脾虚证模型大鼠均存在不同程度的外观表征和实验室指标的异常改变,但三证模型各有特点：1)体重变化上,随着造模时间的延长,各模型大鼠体重呈缓慢增长趋势,其中以脾虚证和肝郁脾虚证模型大鼠体重减低较为明显。2)行为学变化上,肝郁证初期活动探究性加强,而随造模时间的不断延长,从早期的活跃逐渐转为抑制,呈现出兴奋性由强转弱的特点,最终表现为中枢兴奋性减弱；脾虚证从早期的中枢兴奋到后期中枢神经系统反应失调；肝郁脾虚证从早期活动度降低,且随造模时间的延长进一步降低,呈现出中枢抑制性渐进加重的趋势。3)实验室指标变化上,在神经内分泌方面,肝郁证和肝郁脾虚证大鼠的HPA功能轴均呈亢进状态,脾虚证HPA功能轴则呈低下状态；肝郁证和脾虚证大鼠HPA轴异常的主要调点分别涉及下丘脑和垂体,肝郁脾虚证则同时伴有下丘脑及垂体反馈调节功能损伤。三个模型的上述结果分别与中医肝郁证、脾虚证及肝郁脾虚证三证的症状描述类同,与之前所了解到的三证的部分现代病生理内涵基本一致。结果表明本次复制的三个大鼠证候模型对中医肝郁证、脾虚证及肝郁脾虚证具有较好的模拟性。
研究二、肝郁-脾虚-肝郁脾虚三证模型大鼠肝脏蛋白组学特征及柴疏四君子汤作用
方法：大鼠随机分为正常对照组、肝郁证模型组、脾虚证模型组、肝郁脾虚证模型组、肝郁证干预组、脾虚证干预组、肝郁脾虚证干预组7组,每组10只。肝郁证模型组、脾虚证模型组、肝郁脾虚证模型组及其各证干预组大鼠分别采用上述各法造模,正常对照组不予处理；3个模型干预组分别于造模第2周末(即造模第15天起),按3.57g.kg-1体重给予大鼠柴疏四君子汤颗粒灌胃,连续2周；各模型组和正常对照组给予等量蒸馏水。实验第29天,无菌条件下剖取肝脏,取出肝脏相同部位的部分组,经生理盐水冲洗。随机从每组取出6个样品,进行蛋白质的提取后等量混合,对组织蛋白进行酶切后应用ITRAQ试剂标记,高效液相色谱分离后；应用液相色谱与质谱联用方法对蛋白质组样品进行分析,对质谱检测结果应用反相数据库检索方法评估数据的假阳性率；根据所鉴定出蛋白质的ITRAQ相对定量值,确定各模型组与正常对照组之间的差异蛋白。运用IPA软件分析各证模型及相应干预组的差异蛋白,获得各组差异蛋白涉及的部分信号传导通路及关联蛋白网络。
结果：1)三证模型肝脏的差异表达蛋白及其相关信号传导通路与功能网络：肝证模型组的差异蛋白共有67个,其中上调46个,下调21个；蛋白质信号传导通路显变化的有7条,包括EIF2pathway、Nicotine Degradation pathway、Androg Biothesis Thombin signaling、Regulation of actin-based mortility by Rho Chemokine Signaling和tRNA charging;主要的关联蛋白相互作用网络1个。脾虚模型组差异蛋白共有123个,其中上调52个,下调71个；蛋白质信号传导通路明显化的有11条,包括Alanine Degradation and synthesis、Geranyldiphospha Biosynthesis、Xenobiotic metabolism signaling、Glutathione Redox Glutathione-mediated Detoxification、Thyroid Hormone Metabolism Ⅱ、 Retin biosynthesis、LPS/IL-1mediated inhibition of RXR Function、FcR Pathway Serotonin degradation和Retinotate biosynthesis I;主要的蛋白相互作用网络个。肝郁脾虚证模型组差异蛋白共有296个,其中上调51个,下调245个；蛋白质号传导通路明显变化的有11条,包括Xenobiotic metabolism, LPS/IL-1mediat inhibition of RXR Function、Nicotine degradation Ⅱ、nicotine degradation II Serotonin degradation、Medatonin Degradation pathway. PXR/RXR pathway、Extrog biothethesis、Superpathway of metalon in degradation、Thyoid Hormone Metaboli Ⅱ、Phenylalanine degradation IV,主要的蛋白相互作用网络network4个。2)证模型肝脏差异表达蛋白的异同：肝郁证、脾虚证、肝郁脾虚证三个模型组共有差异白12个,其表达趋势一致；蛋白质信号传导通路明显变化的有3条,包括Retinota Biosynthesis I、GM-CSF signaling、nNOS signaling in neurons。其中肝郁证和虚证模型组共有差异蛋白20个,其表达趋势一致；涉及差异高通量蛋白质信号传导路明显变化的有3条,包括Retinotate Biothenthesis I、Thombin signaling和tR charging。肝郁证和肝郁脾虚证模型组共有差异蛋白22个,其中4个蛋白表达趋势反；涉及差异高通量蛋白质信号传导通路明显变化的有3条,包括Chemokine signal in Thrombin pathway、Nicotine degradation。脾虚证和肝郁脾虚证模型组共有差异蛋69个,其中1个蛋白表达趋势相反；蛋白质信号传导通路明显变化的有7条,包Xenobiotic singaling、serotonin degradation、Retinol and ret inotate biothesi Thyroid Hormone Metabolism、glutathione redox reaction、glutathione-mediat detoxif ication、Melatonin degradation。3)柴疏四君子汤对三证模型肝脏蛋白的响：①肝郁证模型组差异蛋白67个,肝郁证干预组的差异蛋白167个,其中经柴疏君子汤干预后恢复的差异蛋白22个,新增差异蛋白122个；干预后恢复的差异蛋白要涉及3条信号传导通路,包括LPS/IL-1mediated inhibition of RXR Function Xenobiotic metabolism pathway和Triacylglycerol Biothethesis,主要的蛋白相作用网络network1个；新增差异蛋白涉及4条信号传导通路,包括EIF2pathway Epithelial Adherens Junction Signaling、Cysteine BioSynthesis Homocy steidegradation、Co-enzyme Biothethesis,主要的蛋白相互作用网络network1个。②脾虚证模型组差异蛋白123个,脾虚证干预组差异蛋白243个,其中经柴疏四君子汤干预后恢复的差异蛋白49个,新增差异蛋白169个；干预后恢复的差异蛋白主要涉及5条信号传导通路,包括Alanine Biosynthesis/Degradation、 Geranylgeranyldiphosphate Biosynthesis、LPS/IL-1mediated inhibition of RXR Function、Trans-farnesyl diphosphate biosynthesis和Seenocysteine BiosynthesisⅡ,主要涉及的相关蛋白网络功能network2个；新增差异蛋白涉及7条信号传导通路,包括melatonin degradation、Nicotine degradation Ⅱ/Ⅲ, Estrogen biosynthesis. Serotonin degradation、Brupropion degradation和Acetone Degradation。③肝郁脾虚证模型组差异蛋白296个,肝郁脾虚证干预组差异蛋白403个,其中经柴疏四君子汤干预后恢复的差异蛋白68个,新增差异蛋白175个；干预后恢复的差异蛋白主要涉及6条信号传导通路,包括EIF2pathway、Clathrin-mediated endocytosis signaling。 LPS/IL-1mediated inhibition of RXR Function、Taurine Biothesis、Ceramide biothesis和Remodeling of Epithelial Adheres functions,主要涉及的相关蛋白网络功能network2个；新增差异蛋白涉及5条信号传导通路,包括Mitochondrial dysfunction、Bruprion degradation、Acetone degradation、Melatonin degradation. Estrogen Biothesis,主要涉及的相关蛋白网络功能network4个。
结果表明：肝郁、脾虚、肝郁脾虚三证均存在肝脏蛋白的异常表达,涉及到血液(与造血前体细胞生长有关的GM-CSF signaling通路)、甲状腺激素代谢和视物障碍(与视黄醇合成相关的Retinotate Biosynthesis I通路)等方面的异常,但各有不同。其中肝郁证以肝脏蛋白质的合成和翻译功能加强,尼古丁降解增强和雄激素合成加速,内分泌代谢亢进,免疫机能低下、神经发育功能低下,肝细胞再生修复功能下降,凝血等循环功能障碍为主要特点；脾虚证以乳酸肌糖酵解激活,异物清除及代谢能力下降,氧化还原、解毒能力低下,视黄醇等合成减少、视网膜合成障碍,甲状腺激素代谢水平低下,炎性反应和清除细菌能力下降、脂肪酸代谢水平低下为主要特点；肝郁脾虚证以清除异物毒物能力低下,胆固醇、胆汁酸和脂肪酸代谢水平低下,褪黑素降解抑制,雌激素合成减少、甲状腺激素代谢低下,尼古丁降解能力低下,免疫机能低下为主要特点；肝郁证和脾虚证均存在神经功能和心血管功能的低下,但肝郁证存在肝脏蛋白合成和翻译功能亢进,而在脾虚证中未见变化；脾虚证存在视黄醇合成减少、脂肪酸代谢水平低下,而在肝郁证中未见变化；肝郁脾虚证涵括了肝郁证和脾虚证的部分机制,其中肝郁和肝郁脾虚两证在肝细胞修复及凝血酶原导致的血管障碍等方面的生理功能异常表现有相同之处；脾虚和肝郁脾虚两证在血清素降解,视黄醇合成抑制,甲状腺激素代谢抑制,褪黑素降解等方面的生理功能异常表现有相同之处；但在糖代谢、异物清除、神经调节功能方面则与两证有所不同,较为突出的表现为肝郁脾虚证肝脏糖代谢处于抑制状态,而脾虚证中糖代谢机能表现亢进；肝郁脾虚证存在异物清除和尼古丁降解功能抑制,与肝郁证异物清除和尼古丁降解功能激活趋势有所不同。
柴疏四君子汤对肝郁证、脾虚证、肝郁脾虚证模型的肝脏差异蛋白表达均有不同程度的调节或逆转恢复作用,其中对肝郁证的效应主要是促进其免疫和异物清除力的恢复,对脾虚证主要是促进其免疫和脂代谢水平的恢复,对肝郁脾虚证主要是促进其免疫和神经调节、脂代谢、牛磺酸及上皮细胞功能的恢复。疏肝健脾方对肝郁脾虚证的肝脏显示了更为广泛的生物学调节作用。
本课题基于中医病因病机学说和中医证候病生理学内涵为背景,采用同步条件控制下的相关病因交叉叠加的模型复制方法,成功复制出中医肝郁证、脾虚证、肝郁脾虚证大鼠模型。在此基础上运用蛋白质组学分析技术,比较观察了肝郁、脾虚、肝郁脾虚三证模型动物肝脏的蛋白组学变化及体现中医疏肝健脾法的柴疏四君子汤对其模型肝脏蛋白组学的影响。结果获得肝郁、脾虚、肝郁脾虚三证模型的肝脏差异表达蛋白谱及柴疏四君子汤对三证模型作用后的肝脏差异蛋白谱。生物信息学分析表明,此三证肝脏蛋白组学变化涉及到神经与血管功能、三大物质及激素代谢、解毒及清除异物等多方面的生物学功能；三证之间具有较为复杂的交互异同关系并各具特点；柴疏四君子汤对此三证模型肝脏蛋白组学变化及其相关信号传导通路具有不同程度的选择性作用表现出不同的生物效应。
课题运用系统生物学方法,首次从肝脏蛋白组学的角度探查中医肝郁、脾虚、肝郁脾虚三证生物学内涵,比较疏肝健脾方对三证的作用异同,并获得疏肝健脾方与三证关联的部分分子机制,为在分子调控水平上揭示中医证候和方证关联的现代内涵提供重要的科学依据。
"Correlation between Formula and Syndrome" is one of the core content of research in TCM of the diagnosis and treatment based on differentiation of symptoms and signs and modern formula-ology. The function and usage of formula not only have correlation with medical elements by dose, taste, form, method, but also depends on complete matching with the main syndrome. So the main effect of the formula reflects to the role of targets of medical compatibility and a considerable extent on the pathology. The different functions give expression to the same or different formula treating one or more diseases,that is a problem of extent of correlation between the formula and syndrome. Until now the important scientific problems of correlation between formula and syndrome mainly study on the extent of correlation between the formula and syndrome and the foundation of modern biology. Based on the background, the research is began with the associating formula with diseases and cross etiology by different relating diseases and same formula actioning on different models. We use comparative analysis and proteomics researching methods to reveal scientific connotation of correlation between formula and syndrome,that will totally improve TCM of the diagnosis and treatment based on differentiation of symptoms and signs.
According to the TCM's etiology and pathogenesis theory and clinical manifestation and modern physiological and pathological characteristics, we take chronic restraint; excess fatigue plus out of constant diet and chronic restraint-fatigue-diet disorder methods to establish models of the liver-stagnation syndrome; the spleen-deficiency syndrome and combining the liver-stagnation and spleen-deficiency syndrome. Depending on liver proteomics, we study on the foundation of modern biology for rat models of GY; PX and GYPX from the view of the same formula to different syndromes. Compared with relating etiology under the same controling step to reveal TCM's essence on molecular level. Based on those factors, we choose the same medicine of Chaishu Sijunzi decoction to observe action of similarities and differences to models of GY; PX and GYPX. By the view of regulating molecular network,we try to discuss mechanism for the liver of the three models relationship and effects of CSJUZ decoction to those models in molecular level.
This thesis is divided into two main parts:literature review and experimental research. Literature review include research progress of TCM's theory of'Correlation between Formula and Syndrome" and using proteomics to study TCM's syndromes. Experimental research include model replication and proteomics analysis.For the first part,it involves rat models of GY; PX and GYPX replication and evaluation. For the second part,we discuss the change of liver protein expression in those rat models and comparing to the same medicine of Chaishu Sijunzi decoction to similarities and differences to those models effectiveness, and then we analysis differences proteins through different groups and understand the function of signal pathway and protein network to reveal foundation of modern biology with those models.
Research1:Establishing the different rat models of liver-stagnation syndrome, spleen-deficiency syndrome and both liver-stagnation and spleen-deficiency syndrome
Methods:The rats are randomly assigned into the normal group, the liver-stagnation group, the spleen-deficiency group, the liver-stagnation and spleen-deficiency group with ten rats for each group. The models were established in the liver-stagnation group, the spleen-deficiency group, the liver-stagnation and spleen-deficiency group by chronic restraint, excess fatigue plus out of constant diet or chronic restraint-fatigue-diet disorder method for4weeks, while the normal group is not with intervention. On the14th and28th day, using open-field-test to observe all rats included with the crossed squares, number of rearing,the stay period in the central zone, decorating time and the total distance of movement. On the29th day, all rats of serum ACTH. CORT. CRH are detected. SPSS17.0analysis different group data and take method of one-way ANOVA and LSD to compare for different groups.
Results:After building rat models4weeks,compared with normal group, the liver-stagnation group of the stay period in the central zone. CRH、CORT、the hypothalamus of CRH shown increased (P<0.05), weight, the crossed squares、number of rearing、 decorating time. the total distance of movement shown decreased (P<0.05);the spleen-deficiency group of number of rearing、decorating time shown increased (P<0.05), weight、the crossed squares、the stay period in the central zone、the total distance of movement、ACTH. CORT. the pituitary gland of ACTH shown decreased (P
Research2:Study on effectiveness of the same formula of Chaishu Sijunzi decoction to similarities and differences in GY, PX and GYPX groups with liver protein expression changing
Methods:The rats are randomly assigned into the normal group, the liver-stagnation group, the spleen-deficiency group, the liver-stagnation and spleen-deficiency group, the drug treatment to liver-stagnation group, the drug treatment to spleen-deficiency group and the drug treatment to liver-stagnation and spleen-deficiency group with ten rats for each group. The models are established by chronic restraint method in GY and GY treatment groups, by excess fatigue plus out of constant diet method in PX and PX treatment groups, by chronic restraint-fatigue-diet disorder method in GYPX and GYPX treatment groups for4weeks. On the15th day, rats of all treatment groups have been fed with medicine of Chaishu Sijun decoction3.57g· kg-1for two weeks. The ones of the normal group and model groups are fed with equivalent physiological saline for two weeks. On the29th day, taking the liver outside in aseptic condition, then we eliminate hemoglobin by repeat washing. Randomly taking6sample for each group and extracting protein to mixing with equal amount. After enzyme digestion in microwave condition, we use ITRAQ reagent to mark, and then through liquid chromatographic separation we use mass spectrometry method to identify and quantitative analysis proteins. We take reverse database to estimate its false positive rate. According to identificating proteins ITRAQ relative quantitative values to ensure differential proteins for groups.At last, the differences in protein are analysis by IPA database and get partial signaling pathways and networks.
Results:1)Three rat models'liver differential proteins and pathways and network:There are67differentially expressed proteins in GY group, among them,46proteins are upregulation and21downregulation. There are7changing pathways, include EIF2pathway、Nicotine Degradation pathway Androgen Bio thesi、Thomb in signaling、Regulation of actin-based mortility by Rho、Chemokine Signaling、tRNA charging and there is1network. There are123differentially expressed proteins in PX group, among them,52proteins are upregulation and71down regulation. There are7changing pathways, include Alanine Degradation and synthesis、 Geranyldiphosphate Biosynthesis、Xenobiotic metabolism signaling、Glutathione Redox、Glutathione-mediated Detoxification、Thyroid Hormone Metabolism Ⅱ、Retinal biosynthesis、LPS/IL-1mediated inhibition of RXR Function、FcR Pathway、Serotonin degradation、Retinotate biosynthesis I and there are2networks. There are296differentially expressed proteins in GYPX group, among them,51proteins are upregulation and245down regulation. There are11changing pathways, include Xenobiotic metabolism. LPS/IL-1mediated inhibition of RXR Function. Nicotine degradation II. nicotine degradation III. Serotonin degradation. Medatonin Degradation pathway、PXR/RXR pathway. Extrogen biothethesis. Superpathway of metalonin degradation、Thyoid Hormone Metabolism II. Phenylalanine degradation IV and there are4networks.2) Similarities and differences between three rat models'liver differential proteins:There are12differentially expressed proteins in GY, PX and GYPX groups with the same expression trend. There are3changing pathways, include Retinotate Biosynthesis I、GM-CSF signaling、nNOS signaling in neurons.There are20differentially expressed proteins in GY and PX groups with the same expression trend.There are3changing pathways, include Retinotate Biothenthesis I. Thombin signaling and tRNA charging.There are22differentially expressed proteins in GY and GYPX groups, among them,4proteins are upregulation in GY and downregulation in GYPX groups. There are3changing pathways, include Chemokine signaling、Thrombin pathway、Nicotine degradation.There are69differentially expressed proteins in PX and GYPX groups, among them,1protein is upregulation in PX and downregulation in GYPX groups. There are7changing pathways, include Xenobiotic singaling、serotonin degradation、Retinol and retinotate biothesis、Thyroid Hormone Metabolism、glutathione redox reaction、glutathione-mediated detoxification. Melatonin degradation.3) Influence of CSSJZ Decoction to three rat models'liver differential proteins:①There are67differentially expressed proteins in GY group and167differentially expressed proteins in GY treatment group, among them,22differentially expressed proteins turn to normal and122new differentially expressed proteins adding by taking CSSJZT. There are3changing pathways, include LPS/IL-1mediated inhibition of RXR Function、Xenobiotic metabolism pathway. Triacylglycerol Biothethesis and there is1network for differentially expressing proteins turned to normal. There are4changing pathways, include EIF2pathway、Epithelial Adherens Junction Signaling、Cysteine BioSynthesis Homocy steine degradation、Co-enzyme Biothethesis and there is1network for new differentially expressed proteins adding by taking CSSJZT in GY group.②There are123differentially expressed proteins in PX group and243differentially expressed proteins in PX treatment group, among them,49differentially expressed proteins turn to normal and169new differentially expressed proteins adding by taking CSSJZT. There are5changing pathways, include Alanine Biosynthesis/Degradation、Geranylgeranyldiphosphate Biosynthesis、LPS/IL-1mediated inhibition of RXR Function、Trans-farnesyl diphosphate biosynthesis、Seenocysteine Biosynthesis II and there are2networks for differentially expressing proteins turned to normal. There are7changing pathways, include melatonin degradation、Nicotine degradationⅡ/Ⅲ、Estrogen biosynthesis. Serotonin degradation、Brupropion degradation、Acetone Degradation for new differentially expressed proteins adding by taking CSSJZT in PX group.③There are296differentially expressed proteins in GYPX group and403differentially expressed proteins in GYPX treatment group, among them,68differentially expressed proteins turn to normal and175new differentially expressed proteins adding by taking CSSJZT. There are6changing pathways, include EIF2pathway、Clathrin-mediated endocytosis signaling. LPS/IL-1mediated inhibition of RXR Function、Taurine Biothesis、Ceramide biothesis、Remodeling of Epithelial Adheres functions and there are2networks for differentially expressing proteins turned to normal. There are5changing pathways, include Mitochondrial dysfunction、 Bruprion degradation、Acetone degradation、Melatonin degradation、Estrogen Biothesis and there are4networks for new differentially expressed proteins adding by taking CSSJZT in GYPX group.
Conclusion:The liver proteins are differentially expressed in GY, PX and GYPX groups, that connects with aspects of blood, thyroid hormone metabolism and visual impairment. In GY group, the characteristics include the activating functions of liver protein synthesis and translation、nicotine degradation、androgen synthesis、endocrine metabolism and the restraining functions of immune、neural development、repairment of liver cell regeneration and disorder function of blood coagulation. In PX group, the characteristics include the activating functions of muscle lactate glycolysis and the restraining functions of foreign matter removal、redox、detoxification、retinol and retina synthesis、thyroid hormone metabolism、inflammatory reaction、fatty acid metabolism. In GYPX group, the characteristics include the restraining functions of foreign matter removal、cholesterol、bile acid and fatty acids metabolism、melatonin degradation、estrogen synthesis、thyroid hormone metabolism、nicotine degradation. In GY and PX groups, the characteristics include the restraining functions of nerve and cardiovascular. The characteristics of activating functions of liver protein synthesis and translation are only found in GY group and restraining functions of retinol and retina synthesis、fatty acid metabolism are only found in PX group. GYPX partly include GY and PX characteristic, while those which are different from aspects of glucose metabolism, foreign matter removal capacity and neural. Among them, the hyperfunction of glucose metabolism in PX opposite to depressing in GYPX. The hyperfunction of foreign matter removal and nicotine degradation capacity in G Y opposite to depressing in GYPX.
The formula of soothing liver qi and tonifying spleen qi can regulate the function of GY, PX and GYPX with liver differentially expressed proteins. The function of GY is regulated by immunity and foreign matter removal capacity. The function of PX is regulated by immunity and lipid metabolism. The function of GYPX is regulated by immunity, neural, lipid metabolism melatonin and estrogen synthesis. Based on those conclusions, it prompts that CSSJZT can improve GYPX in a wide range.
We take methods of chronic restraint, excess fatigue plus out of constant diet, chronic restraint-fatigue-diet disorder to establish rats models of GY, PX and GYPX at the same lever and conditions. On this basis,we take proteomic analysis technology and observe effectives of the same CSSJZT to GY, PX and GYPX similarities and differences with the method of liver proteomics.We get the differentially expressed proteins for the same CSSJZT decoction to the syndrome of GY, PX and GYPX. According to the bio informatics analysis,three syndromes of GY, PX and GYPX involve in the function changing of nerves and blood vessels、three materials and hormone metabolism、detoxify and foreign matter removal capacity.Those syndromes have complex interactions and characteristics and CSSJZ decoction select to show different biological effects to the different model liver proteins with pathway and network analysis.
Using biology system method,we discuss the biology connotation of TCM's GY、PX、 GYPX in Liver proteomics at first.Comparing the different effects of CSSJZ decoction to three syndromes,we get partial molecular mechanisms to the formula for soothing liver qi and tonifying spleen qi and three syndromes.The discussion reveal essence and connotation of correlation between formula and syndrome to offer objective evidence.
引文
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