疏肝—健脾—疏肝健脾方与肝郁脾虚证相关的研究

【作者】 王玉杰；

【导师】 谢鸣；

【作者基本信息】 北京中医药大学 ， 中医方剂学， 2010， 博士

【摘要】 方证相关是中医辨证论治和方剂学的的核心内容。方剂的疗效不仅与药物的组成有关,还与其所主病证紧密相关。方与证之间关联程度的大小直接影响方剂的效用。虽然近年围绕方证相关的研究取得一些进展,但是鲜有研究能为方证相关提供系统的实证依据。本研究基于上述背景,以不同方剂作用于同一病证模型为切入点,通过观察比较药物干预模型后的效应差异,揭示方证相关的科学内涵。本研究首先以肝郁脾虚证的中医病因学说、临床研究及与其相关的现代疾病的生理病理变化为背景,采用慢性束缚+过度疲劳+饮食失节法复制肝郁脾虚证动物模型,通过观察神经-免疫-内分泌系统的多个指标,探查肝郁脾虚证动物模型的生物学基础。在此基础上,选择疏肝、健脾、疏肝健脾的代表方—柴胡疏肝散、四君子汤、柴疏四君汤作用于肝郁脾虚证动物模型,比较三方的效应异同,探讨三方与肝郁脾虚证动物模型之间的关联性及生物学基础。最后运用基因芯片技术探查肝郁脾虚证模型及柴疏四君汤干预模型后的大鼠肝脏全基因表达,从分子层面为科学理解方证相关提供依据。全文分为文献综述和实验研究两大部分,文献综述主要包括肝郁脾虚证的临床及实验研究,柴胡疏肝散、四君子汤、柴疏四君汤的临床及实验研究。实验研究主要从免疫系统、甲状腺轴、HPA轴、消化系统、血液系统、肝脏差异基因表达等方面探查肝郁脾虚证大鼠模型的变化,比较观察柴胡疏肝散、四君子汤、柴疏四君汤对肝郁脾虚证大鼠模型作用后的效应异同。研究一、肝郁脾虚证大鼠模型的免疫系统变化及相关方剂的作用方法：将大鼠随机分为正常组模型组,柴胡疏肝散组、四君子汤组、柴疏四君汤组,每组12只。模型组大鼠采用慢性束缚应激+过度疲劳+饮食失节法造模,将大鼠于每天上午8：00置于束缚盒中限制3小时,下午2：00置于盛有温水(22±1℃)的大塑料桶中游泳10分钟。隔日喂食(隔日禁食,隔日足量给食),连续4周.正常对照组不加任何刺激,自然饲养。于造模第15d,中药各组大鼠分别按柴胡疏肝散4.2g／kg·d、四君子汤4.53g／kg·d、柴疏四君汤4.27g／kg·d,灌胃,1次／d,连续14d；模型组和对照组分别给予等量蒸馏水。于第29d取材,用放免试剂盒检测IL-2和L-6,用MTT法检测T淋巴细胞增殖率。结果：模型组大鼠血清IL-2、IL-6及均T淋巴细胞增殖率显著降低。柴胡疏肝散、四君子汤、柴疏四君汤三组大鼠的血清IL-2均呈显著性升高；柴胡疏肝散、四君子汤、柴疏四君汤三组大鼠血清IL-6均见不同程度升高,其中柴胡疏肝散和柴疏四君汤组呈显著性升高；柴胡疏肝散、四君子汤和柴疏四君汤三组T淋巴细胞增殖率均呈不同程度升高,其中柴疏四君汤组呈显著性升高。结论：肝郁脾虚证模型大鼠免疫功能低下；柴疏四君汤在提高肝郁脾虚证模型大鼠免疫功能方面优于柴胡疏肝散和四君子汤。研究二、肝郁脾虚证大鼠模型甲状腺变化及相关方剂的作用方法：造模方法及药物干预同前。用放免试剂盒检测T3、T4、TRH、TSH。结果：模型组大鼠血清TRH、TSH、T3、T4均见显著降低。柴胡疏肝散、四君子汤、柴疏四君汤三组大鼠的血清T3、T4均呈显著性升高；柴胡疏肝散、四君子汤、柴疏四君汤三组大鼠血清TRH、TSH均见不同程度升高,其中柴疏四君汤组呈显著性升高。结论：肝郁脾虚证模型大鼠甲状腺功能低下；柴疏四君汤在提高肝郁脾虚证模型大鼠甲状腺功能方面优于柴胡疏肝散和四君子汤。研究三、肝郁脾虚证大鼠模型HPA轴变化及相关方剂的作用方法：造模方法及药物干预同前。用放免试剂盒检测浆CRH、ACTH、COR和下丘脑CRH结果：模型组大鼠血浆CRH、COR及下丘脑CRH均见显著升高,血浆ACTH显著降低。柴胡疏肝散组、四君子汤组、柴疏四君汤组大鼠血浆中CRH含量均有不同程度降低,但差异均无显著性意义；柴胡疏肝散组、四君子汤组、柴疏四君汤组大血浆中ACTH含量均显著增加；柴胡疏肝散组、四君子汤组、柴疏四君汤组大鼠血浆中COR含量均不同程度降低,其中柴疏四君汤组显著降低；柴胡疏肝散组、四君子汤组、柴疏四君汤组大鼠下丘脑CRH含量均不同程度的降低,其中柴疏四君汤组和柴胡疏肝散组显著降低。结论：肝郁脾虚证模型大鼠HPA轴功能异常；柴疏四君汤在恢复肝郁脾虚证模型大鼠HPA轴功能异常方面优于柴胡疏肝散和四君子汤。研究四、肝郁脾虚证大鼠模型消化系统的变化及相关方剂的作用方法：造模方法及药物干预同前。用放免试剂盒检测MTL、SS,对溴苯胺法检测D-木糖排泄率,分光光度法检测胃蛋白酶,用酶组织化学法观测肝脏SDH和G-6-PD。结果：模型组大鼠血浆MTL、SS及胃蛋白酶均见显著升高,D-木糖排泄率显著降低,肝脏SDH、G-6-PD均见显著升高。柴胡疏肝散、四君子汤、柴疏四君汤三组大鼠的血清MTL均呈显著性降低；柴胡疏肝散、柴疏四君汤二组大鼠血清SS均见不同程度降低,四君子汤组显著升高；柴胡疏肝散、四君子汤、柴疏四君汤三组大鼠的D-木糖排泄率均呈不同程度升高,其中柴疏四君汤组和四君子汤组均呈显著升高；柴胡疏肝散、四君子汤、柴疏四君汤三组大鼠的胃蛋白酶均呈不同程度降低,其中柴疏四君汤组显著降低。柴胡疏肝散、四君子汤、柴疏四君汤三组大鼠SDH均呈显著性降低；柴胡疏肝散、四君子汤、柴疏四君汤三组大鼠G-6-PD均见不同程度升高,其中柴疏四君汤组呈显著性升高。结论：肝郁脾虚证模型大鼠消化功能及肝酶表达异常；柴疏四君汤在恢复消化功能及肝酶方面优于柴胡疏肝散和四君子汤。研究五、肝郁脾虚证大鼠模型血液系统的变化及相关方剂的作用方法：造模方法及药物干预同前。用MVIS-2035全自动血液流变分析仪检测全血流变,血液全自动分析仪检测血常规,CA—500全自动血凝分析仪检测凝血功能。结果：模型组大鼠的红细胞压积指数显著升高,红细胞聚集指数显著降低(P<0.001),红细胞变形指数无显著性差异,150/s、38/s、10/s、5/s切变率下的全血粘度均显著升高,150/s、38/s、10/s、5/s切变率下的还原粘度无显著变化,血常规(WBC、RBC、HGB、PLT)、凝血功能(APTT、PT、PTA、Fib)均无显著变化。柴胡疏肝散组、四君子汤组和柴疏四君汤组的红细胞压积、聚集指数、变形指数、全血还原粘度、WBC、RBC、HGB、PLT、凝血功能均无显著变化；四君子汤可显著降低各切变率下全血粘度,柴胡疏肝散能显著降低高切下全血粘度,其余均无显著性变化。结论：肝郁脾虚证模型大鼠全血流变异常,血常规及凝血功能指标均无明显改变；三方对血液常规和凝血功能指标无明显影响,四君子汤可改善肝郁脾虚模型大鼠血流变的异常,柴胡疏肝散次之,柴疏四君汤对其无明显作用。研究六肝郁脾虚证大鼠模型肝脏的差异基因探查及柴疏四君汤的作用方法：造模方法及药物干预同前。无菌条件下剖取肝脏,进行脾脏RNA的提取、样品RNA荧光标记、杂交与清洗、芯片扫描、图像采集与数据分析；主要利用http://www. genome.ad. jp/kegg/中的基因库对差异基因进行功能检索。结果：模型组的差异表达基因有59条,其中上调基因有27条,下调基因有32条；其中涉及到已知功能基因有Scdl、Dbp、Upp2、Slc28a2、Sds、Hpn、Gldc、Tm7sf2、Gcat、Btg3、Lin7a、Zfp354a、Rgs2、Duspl1、Ldha。柴疏四君汤作用肝郁脾虚证模型差异表达基因25条,其中上调基因有13条,下调基因有12条；涉及到已知功能基因有Hhex、Myc、Hmgcr、Ccl7、Bubl。结论：肝郁脾虚证存在基因异常表达,涉及脂类代谢、糖代谢等多个方面。柴疏四君汤作用于肝郁脾虚证模型的差异基因主要涉及细胞周期和糖代谢方面。提示多个基因的异常表达可能是中医肝郁脾虚证的分子基础,而柴疏四君汤对肝郁脾虚证多个相关基因发挥调节作用。本课题采用慢性束缚+过度疲劳+饮食失节法复制肝郁脾虚证动物模型,比较观察了柴胡疏肝散、四君子汤和柴疏四君汤三方对该肝郁脾虚证模型的作用异同。结果发现,肝郁脾虚证大鼠模型存在胃肠功能、神经内分泌,血流变及肝酶等多系统的异常改变；柴疏四君汤在调节肝郁脾虚模型免疫系统、甲状腺轴、HPA轴、消化系统、肝酶方面优于柴胡疏肝散和四君子汤,并且影响模型的多个差异基因的表达。结果表明,柴疏四君汤与肝郁脾虚证具有较高的相关性。该课题为揭示中医肝郁脾虚证及方证相关的现代内涵提供了一定的客观依据。更多还原

【Abstract】 "Correlation between Formula and Syndrome" is the core content of in the diagnosis and treatment based on differentiation of symptoms and signs and formula-ology. The function and usage of formula not only havecorrelation with medical compatibility but also compatible with the syndrome which it will have effect on, that is the main effect of the formula depends on a considerable extent on the pathology and the role of targets of medicalcompatibility. Although the progress has been made about "Correlation between Formula and Syndrome",less scientific evidence is provided for it. Based on the background, the research is began with different formulae’s action on the same model.The scientific meaning of Correlation between Formula and Syndrome is revealed through comparing and observing the effect of three formulae’s action on the same model.The dissertation was to explore the rats’syndrome model of stagnation of live anddeficiency of spleen (GYPX) which was made by the method of compound causes of diseaseaccording to the TCM’s theory of attack, clinical manifestation and some physiological and pathological characteristics of some modern diseases about this syndrome and to acquire the Chinese syndrome and some biological characteristics of this model through observing manytargets about the nerve-endocrine-immunity system; On these achievements, we observed and compared similarities and differences of action of, SJZT and CSSJT which had therespective function of soothing liver, invigorating spleen andsoothing liver and invigorating spleen on GYPX model and probed into the associatedcharacter and partial modern biological basis of these associate between the effectiveness ofthe three prescriptions and the model of disease and syndrome on the condition of different prescription on the same syndrome. Finally, we explored genes expression of the model and CSSJT influencing it by applying microarray technology, and deeply approachedmolecular mechanism of the model and cssjt influencing it.This thesis is divided into two main parts:literature review and experimental research. literature review include clinnic and experimental research of GYPX, CHSGS, SJZT and CSSJT. The change of immune system, HPTA, HPA, digest system and blood circulation made by CHSGS, SJZT and CSSJT and Gene expression profiles associated with preliminary exploration of the syndromes of GYPX deficiency of rat model and study of the effect of CSSJT on the model. Research 1:the change of immune system of the model of GYPX syndrome and the effect of CHSGS, SJZT and CSSJT on the model of GYPX syndromeMethods:The rats were assigned to five groups randomly:normal group, model group of stagnation of liver and deficiency of spleen, CHSGS group, SJZT group and CSSJT group,12 rats in each group. The rats of latter four groups were administrated by chronic restricting and excess fatigue with out constant diet for four weeks and normal group rats were fed as usual.The rats of three medical groups were orally administrated with CHSGS, SJZT, CSSJT (lml·100g-1·d-1) respectively from the 15th day to the end and rats of normal and model groups were given equal volume of distilled water. Interleukin-2 (IL-2) and interleukin-6(IL-6) in serum and spleen T lymphocyte proliferation rate of groups were observed by the method of RIA.Results:IL-2, IL-6, spleen T lymphocyte proliferation rate of model group decreased significantly. IL-2 of CHSGS,SJZT,CSSJT increased significa-ntly. IL-6 of CHSGS, SJZT, CSSJT increased in varying degrees, and CHSGS and CSSJT were ignificant. Spleen T lymphocyte proliferation rate of CHS-GS, SJZT, CSSJT increased in varying degrees,and CSSJT was significant. Conclusion:The abnormal changes turned up in immune system. CSSJTwas better than CHSGS and SJZT in adjusting these abnormal changes.Research 2:the change of throid system of the model of GYPX syndrome and the effect of CHSGS, SJZT and CSSJT on the model of GYPX syndromeMethods:The rats were assigned to five groups randomly:normal group, model group of stagnation of liver and deficiency of spleen, CHSGS group, SJZT group and CSSJT group,12 rats in each group. The rats of latter four groups were administrated by chronic restricting and excess fatigue with out constant diet for four weeks and normal group rats were fed as usual.The rats of three medical groups were orally administrated with CHSGS, SJZT, CSSJT (lml·100g-1·d-1) respectively from the 15th day to the end and rats of normal and model groups were given equal volume of distilled water. T3, T4, TRH and TSH of groups were determined by the method of RIA.Results:T3,T4,TRH and TSH of model group were significantly decreased. T3 and T4 in serum of CHSGS, SJZT and CSSJT groups were significantly increased. TRH and TSH in serum of CSSJT group markedly increased.Conclusion:The abnormal changes turned up in thyroid system. CSSJT was better than CHSGS and SJZT in adjusting these abnormal changes.Research 3:the change of HPA of the model of GYPX syndrome and the effect of CHSGS, SJZT and CSSJT on the model of GYPX syndromeMethods:The rats were assigned to five groups randomly:normal group, model group of stagnation of liver and deficiency of spleen, CHSGS group, SJZT group and CSSJT group,12 rats in each group. The rats of latter four groups were administrated by chronic restricting and excess fatigue with out constant diet for four weeks and normal group rats were fed as usual. The rats of three medical groups were orally administrated with CHSGS, SJZT, CSSJT (1ml·100g-1·d-1) respectively from the 15th day to the end and rats of normal and model groups were given equal volume of distilled water. CRH, ACTH, COR, CRH in the hypothalamus were determined.by the method of RIA.Results:CRH,COR in plasma and CRH in the hypothalamus of model group increased significantly and ACTH in plasma of model group decreased significantly. CRH in plasma of CHSGS, SJZT, CSSJT decreased in varying degrees,not significantly. ACTH in plasma of CHSGS, SJZT, CSSJT increased significantly. COR in plasma of CHSGS, SJZT, CSSJT decreased in varying degrees,and chsjt was significantly. CRH in the hypothalamus of CHSGS, SJZT, CSSJT decreased in varying degrees,and CSSJT and chsgs were sig-nificantly.Conclusion:The abnormal changes turned up in HPA. CSSJT was better than CHSGS and SJZT in adjusting these abnormal changes.Research 4:the change of digest system of the model of GYPX syndrome and the effect of CHSGS, SJZT and CSSJT on the model of GYPX syndromeMethods:The rats were assigned to five groups randomly:normal group, model group of stagnation of liver and deficiency of spleen, CHSGS group, SJZT group and CSSJT group,12 rats in each group. The rats of latter four groups were administrated by chronic restricting and excess fatigue with out constant diet for four weeks and normal group rats were fed as usual.The rats of three medical groups were orally administrated with CHSGS, SJZT, CSSJT (1ml·100g-1·d-1) respectively from the 15th day to the end and rats of normal and model groups were given equal volume of distilled water. MTL and SS in plasma by the method of RIA, D-xyloseexcretion rate in urine by the method of adjacent aminotoluene, pepsin by the method of spectrophotometer,and SDH and G-6-PD in liver by the method of NBT were determined.Results:MTL and SS in plasma, pepsin, and SDH and G-6-PD in liver of model group increased significantly. D-xyloseexcretion rate in urine of model group decreased significantly. MTL in plasma of CHSGS, SJZT, CSSJT group decreased significantly. SS in plasma of CHSGS and CSSJT group decreased in varying degrees,and SJZT group increased significantly. D-xyloseexc-retion rate in urine of CHSGS, SJZT, CSSJT group increased in varying degrees, and SJZT and CSSJT were significant. Pepsin of CHSGS, SJZT, CSSJT group decreased in varying degrees, and CSSJT was significant. SDH in liver of CHSGS, SJZT, CSSJT group decreased significantly. G-6-PD in liver of CHS-GS, SJZT, CSSJT group increased significantly,and CSSJT was significant. Conclusion:The abnormal changes turned up in digest system and SDH and G-6-PD in liver. CSSJTwas better than CHSGS and SJZT in adjusting these abnormal changes.Research 5:the change of blood circulation in liver of the model of GYPX syndrome and the effect of CHSGS, SJZT and CSSJT on the model of GYPX syndromeMethods:The rats were assigned to five groups randomly:normal group, model group of stagnation of liver and deficiency of spleen, CHSGS group, SJZT group and CSSJT group,12 rats in each group. The rats of latter four groups were administrated by chronic restricting and excess fatigue with out constant diet for four weeks and normal group rats were fed as usual.The rats of three medical groups were orally administrated with CHSGS, SJZT, CSSJT (1ml·100g-1·d-1) respectively from the 15th day to the end and rats of normal and model groups were given equal volume of distilled water. Hemorheology measured by blood viscosimeter, blood routine test by automatic blood cell counter, blood coagulation function by Automatic coagulation analyzer were determined.Results:The index of erythrocyte aggregation decreased, hematocrit increased significantly and the index of erythrocyte deformability had no change. The whole blood viscosity under different shear rates of model group’s rats increased significantly and reduced viscosity had no change. Blood routine test and blood coagulation function had no change. The index of erythrocyte aggregation, hematocrit, index of erythrocyte deformabi-lity, reduced viscosity under different shear rates, blood routine test (WBC、RBC、HGB、PLT) and blood coagulation (APTT、PT、PTA、Fib) of CHSGS,SJZT,CSSJT group had no change. The whole blood viscosity under different shear rates of SJZT group decreased significantly. The whole blood viscosity under higher shear rates of CHSGS group decreased sign-ificantly.Conclusion:The abnormal changes turned up in blood circulation. CSSJTwas better than CHSGS and SJZT in adjusting these abnormal changes. Research 6:Exploration of gene expression on the syndromes of GYPX and the effects of cssjt.Methods:The rats were assigned to 3 groups randomly:normal group, model group of stagnation of liver and deficiency of spleen, CSSJT group,12 rats in each group. The rats of CSSJT group were administrated by chronic restricting and excess fatigue with out constant diet for four weeks and normal group rats were fed as usual. The rats of CSSJT group were orally administrated with CSSJT (lml·100g-1·d-1) respectively from the 15th day to the end and rats of normal and model groups were given equal volume of distilled water. We extract RNA from liver, and tag RNA by fluores-cence, hybridizate and cleanse, scan chip, collect picture and analyze data; At last we inquest functions of obstained different genes by gene bank of http://www. genome.ad. jp/kegg/.Results:There are 59 differentially expressed genes in model group, genes, among them,27 genes are upregulation and 32 downregulation, referring to inquest known functional genes as followed:Scdl、Dbp、Upp2、 Slc28a2、Sds、Hpn、Gldc、Tm7sf2、Gcat、Btg3、Lin7a、Zfp354a、Rgs2、Duspll、Ldha; CSSJT group has 25 differentially expressed genes, among them 13 are upregulation and 12 aredownregulation; referring to inquest known functional genes as followed:Hhex、Myc、Hmgcr、Ccl7、Bubl.Conclusion:The model of the syndromes of GYPX exists abnormal expression of part genes, known functional genes of involved in metabolism of glucose and metabolism of fatty; CSSJT has generally regulate to genes expression of GYPX, the main known functional genes mainly influence metabolism of glucose and cell circle. The results cue that abnormal expression of several genes is possibly molecular foundation of the syndromes of GYPX, however related formula of treating the syndromes of GYPX have complicate regulate to many genes including abnormal expression genes of the synd-romes of GYPX.The change of the function of stomach and intestine, NEI network, blood circulation, SDH and G-6-PD in liver had been observed in GYPX model made bythe method of chronic astricting, excess fatigue and out of constant diet on ratsBased on the result, we observed the different action of CHSGS, SJZT, and CSSJT on the GYPX model made by the method of chronic astricting, excess fatigue and out of constant diet on rats and discovered that the function of CSSJT was better than that of CHSGS and SJZT in the way of adjusting abnormal change of immune system, HPTA, HPA,digest system and blood circulation. The syudy had provided objective evidence for cor-relation between prescription and syndrome.更多还原