脾阳虚证大鼠能量代谢和神经内分泌改变的实验研究

【作者】 唐汉庆；

【导师】 李国彰；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2010， 博士

【摘要】 脾阳虚证存在两症状群：①阳虚症状群,表现是畏寒喜温、形寒肢冷、蜷缩倦怠等。中医理论基础是脾失温煦,而生物学基础是能量代谢低下、产热不足；②脾(气)虚症状群,表现是纳少、消瘦、乏力、脘腹胀痛、便溏及腹泻等。中医理论基础是脾失健运,而生物学基础是胃肠消化和吸收功能减退。简而言之,该两大症状群可概括为“不温”和“不运”症状群。一、本研究首先系统综述了脾阳虚证理论研究进展、脾阳虚证实验研究进展和脾阳虚证神经内分泌研究进展。重点介绍了脾阳虚证动物模型制备的现状、缺陷与发展方向,系统概述了解偶联蛋白家族(uncoupling proteins, UCPs)在能量代谢(特别是产热)中的作用特点,概括了新的脑肠肽胃生长素(ghrelin)对胃肠消化吸收功能和摄食、能量代谢双重调控的特点。这些进展为本研究寻找创新点提供了前提条件。二、实验研究设计的基本思路以研创“新型脾阳虚模型”为基点,瞄准脾阳虚证能量代谢改变特征,探索脾阳虚证中最为关键的“阳虚”机制；以现代系统生物学“整体论”思想为理论基础,探索脾阳虚证中桥联“脾失健运”(“不运”)和“脾失温煦”(“不温”)两大板块(症状群)的神经内分泌调控因素。(一)建立新型脾阳虚证动物模型本研究采用肩胛间棕色脂肪组织(brown adipose tissue, BAT)切除术+高脂饲料喂养+隔日寒冷环境刺激的复合因素造模方法,建立了可靠的脾阳虚证动物模型。其主要特色是加强了“伤阳”设计,通过切除BAT,大幅度减少大鼠产热来源,衰其阳气,使脾阳虚证动物模型造模的阳虚环节坚实,该方法具有创新性。(二)脾阳虚证的能量代谢研究脾阳虚证患者有形寒肢冷、畏寒喜温等症状和体征,和现代医学理论中的基础代谢率和能量代谢水平下降而导致的产热不足表现相似,从现代生物化学观点看,细胞的物质代谢活动都伴随有能量转化,而三磷酸腺苷(ATP)是机体储能和直接供能的物质,因此,了解脾阳虚证大鼠荷能物质(糖和脂肪)水平、能荷和ATP酶的改变特征对于阐释脾阳虚证阳虚的本质具有重要的意义。线粒体是细胞能量代谢和生物氧化的主要场所,UCP约占线粒体内膜蛋白总量的6%-14%,与机体产热作用密切相关。现代生物学研究显示其生理作用是使线粒体膜电位降低,使物质氧化磷酸化和ATP合成解偶联,从而抑制ATP的合成,使能量以热能形式散失,使呼吸过程中由质子梯度形成的电化学势能转变为热能而不产生ATP,即产热而不产能,由于UCP对能量代谢的潜在影响,脾阳虚既然有产热低下的表现,理论上应该和UCP的表达异常有关。本课题拟检测血糖、血脂、线粒体能荷、线粒体UCP和骨骼肌肌球蛋白ATP酶活性等能量代谢相关指标,研究脾阳虚证大鼠能量代谢的特征,探讨脾阳虚证阳虚的细胞分子机制。(三)脾阳虚证的神经内分泌研究现代生物学的最新研究进展表明脑肠肽、胃肠激素和肠神经系统递质对胃肠进行着整合控制。脾阳虚证主要是以消化系统为主的多系统、多器官的功能低下,并与相关神经内分泌调控的改变有着密切的联系,特别是近年新发现的脑肠肽Ghrelin,与胆囊收缩素相似,具有广泛的胃肠功能和摄食、能量代谢双重调控作用。有望成为连接脾阳虚消化吸收功能状态和能量代谢状况的桥梁之一因此,本课题拟检测脾阳虚Ghrelin水平,了解脾阳虚证大鼠Ghrelin改变的特征,以探讨Ghrelin作为神经内分泌物质在脾阳虚证发病机制中的作用。三、实验研究结果(一)异丙肾上腺素致脾阳虚证大鼠产热量效关系的实验研究1目的采用肩胛间BAT切除术+高脂饲料喂养(高脂饮食)+隔日寒冷环境刺激的多因素造模模式制备脾阳虚证动物模型,然后以异丙肾上腺素作为外部刺激源,观察脾阳虚证动物对异丙肾上腺素适应性产热的反应性和剂量效应相关性。2方法参考有关对脾阳虚证动物模型的评价,对大鼠序贯施行BAT切除、高脂饮食喂养、隔日寒冷环境刺激的措施,模拟中医学脾阳虚的发生发展过程,观察大鼠曲行为学表现,建立脾阳虚证大鼠模型,在大鼠适宜环境(28℃)下,以异丙肾上腺素对大鼠进行尾静脉注射,测定大鼠肛温、胸部体表温度、肢温,并探索异丙肾上腺素剂量和各部温度变化之间的关系。3结果异丙肾上腺素刺激大鼠产热的量效关系如下：肛温峰值和曲线下面积EDso分别为0.28 mg/kg和0.106 mg/kg,胸部体表温度峰值以及曲线下面积ED50分别为0.066 mg/kg和0.070 mg/kg,肢温峰值以及曲线下面积ED50分别为0.071mg/kg和0.172 mg/kg,肛温、肢温与胸部体表温度三部分温度之间变化趋势呈正相关。4结论(1)脾阳虚证大鼠整体上产热能力下降,可能是脾阳虚动物畏冷寒战的原因。采用药物干预可以改善适应性产热低下状况,为主要以温热药组成的附子理中汤干预脾阳虚动物的实验奠定了基础。(2)异丙肾上腺素刺激脾阳虚证大鼠产热效能,量效关系正相关,但最佳剂量受环境(如外界温度、噪音和光线等)因素影响而难以确定。(二)脾阳虚证大鼠BAT和UCP1关联性的实验研究1目的探讨BAT、解偶联蛋白1(uncoupling protein 1,UCP1)、高脂饮食和中药干预在实验动物能量代谢中的关联性和机制。2方法动物随机分为4组。每组16只。(1)对照组：普通饲料喂养；d70-d98生理盐水10ml/kg/d灌胃。d98检测刺激性产热,观察2h肛温变化曲线和温度峰值,计算温度曲线下面积；(2)阳虚组：d42手术切除肩胛间BAT,余同(1)组；(3)脾阳虚组：d49-d98高脂饲料(83%普通饲料,15%甘油三酯,2%胆固醇)喂养；隔日19℃环境喂养,余同(2)组；(4)中药组：d70-d98附子理中汤5ml/kg/d灌胃。余同(3)组。每周测体重一次。以效能体重增长率、效能产热温度峰值、产热温度曲线下面积为参考指标。取BAT测UCP1表达量。3结果(1)效能体重增长率阳虚组与对照组相比体重增长减慢(P＜0.01)；脾阳虚组与阳虚组相比体重增长减慢(P＜0.01)；中药组与脾阳虚组相比,体重增长加快(P＜0.01)。(2)效能产热温度峰值阳虚组与对照组相比效能产热温度峰值降低(P＜0.01)；脾阳虚组与阳虚组相比效能产热温度峰值升高(P＜0.01)；中药组与脾阳虚组相比,效能产热温度峰值升高(P＜0.01)；中药组与脾阳虚组相比,效能体重增长率与效能产热温度峰值变化程度呈正相关。(3)产热温度曲线下面积阳虚组与对照组相比产热曲线下面积减少(P＜0.01)；脾阳虚组与阳虚组相比产热曲线下面积增加(P＜0.05)；中药组与脾阳虚组相比,产热曲线下面积增加(P＜0.01)。效能体重增长率与产热温度曲线下面积变化程度呈正相关。中药组上调UCP1表达量,和阳虚组以及脾阳虚组相比,差异均具有高度显著性(P＜0.01)。4结论(1)温阳健脾中药促使高脂饮食转化为BAT并提高其产热活性。(2)温阳健脾中药上调UCP1在BAT中的表达,通过UCP1的解偶联作用,使BAT分解产热而改善阳虚畏寒的症状。(三)脾阳虚证大鼠骨骼肌线粒体能荷和UCP3的关联性研究1目的研究附子理中汤对脾阳虚证大鼠骨骼肌解偶联蛋白3(uncoupling protein3,UCP3)含量以及骨骼肌能荷的影响。2方法动物随机分为四组,每组18只,四组动物均饲以普通饲料和饮水喂养至(142,然后取三组造模,剩下一组作为对照组,对照组依然是普通饲料和饮水,对造模的三组采取以下措施：(1)d42,随机取其中一组施行肩胛间BAT切除术,术后饲以普通饮食、饮水,为阳虚组；(2)d42,余下的两组,也施行肩胛间BAT切除术,但术后饲以高脂饲料(83%基本饲料,15%甘油三酯,2%胆固醇),隔日19℃环境喂养；至d82随机从两组中取一组,每只大鼠按照5ml/kg体重每天灌饲附子理中汤,为中药组；另一组为脾阳虚组,全部四组d98取左侧腓肠肌检测指标。3结果(1)中药组肌能荷值显著低于其他三组,说明合成ATP的量低于其他三组,(和对照组相比P＜0.01；和阳虚组比,P＜0.05；和脾阳虚组比,P<0.05)。中药组UCP3表达量和ATP酶活性均较脾阳虚组升高(分别为P＜0.01,P＜0.05)。(2)脾阳虚组和对照组相比,能荷值降低(P＜0.05)；和阳虚组相比,能荷值也降低,但差异没有显著性(P＞0.05)。脾阳虚组UCP3表达量升高(与对照组相比P＜0.01)。脾阳虚组骨骼肌肌球蛋白ATP酶活性升高(与对照组相比P＜0.01)。(3)阳虚组和对照组相比,能荷值降低但差异没有显著性(P＞0.05)。4结论本实验结果表明,附子理中汤通过上调骨骼肌UCP3表达和骨骼肌肌球蛋白ATP酶活性以及抑制机体合成ATP(产能),通过UCP3解偶联作用提高产热而发挥温阳作用。(四)脾阳虚证大鼠UCP3和血糖、甘油三酯以及总胆固醇关联性实验研究1目的分析附子理中汤对脾阳虚证大鼠UCP3的影响以及UCP3和血糖、甘油三酯、总胆固醇能量物质之间的内在联系。2方法实验动物分为4组,每组16只。(1)对照组：普通饲料喂养,饮水；d70-d98灌胃生理盐水10ml/kg/d;(2)阳虚组：d42手术切除BAT,余同(1)组；(3)脾阳虚组：d49-d98高脂饲料(83%普通饲料,15%甘油三酯,2%胆固醇)在28℃和19℃环境隔日交替喂养；余同(2)组；(4)中药组：d70-d98附子理中汤5ml/kg/d灌胃；余同(3)组。全部四组动物d98左颈总动脉插管取血检测血糖、血清甘油三酯、血清总胆固醇含量,取骨骼肌检测UCP3。3结果(1)各组血糖含量的比较阳虚组与对照组相比血糖降低(P＜0.01)；脾阳虚组与阳虚组相比血糖升高(P＜0.01)；中药组与阳虚组相比,血糖升高(P＜0.01)；中药组与脾阳虚组相比,血糖血糖有下降趋势(P＞0.05)。(2)各组血清甘油三酯含量的比较阳虚组与对照组相比血清甘油三酯降低(P＜0.01)；脾阳虚组与阳虚组相比血清甘油三酯增加(P＜0.01)；中药组与阳虚组相比,血清甘油三酯升高(P＜0.01),中药组与脾阳虚组相比,血清甘油三酯有下降趋势(P＞0.05)。(3)各组血清总胆固醇含量的比较脾阳虚组、中药组与对照组相比血清总胆固醇均升高(P＜0.01)。(4)各组UCP3表达细胞形态观察①对照组UCP3表达细胞形态镜下对照组骨骼肌肌原纤维排列整齐,肌原纤维有较多阳性颗粒表达。②阳虚组UCP3表达细胞形态镜下阳虚组骨骼肌肌原纤维阳性颗粒表达较对照组少。③脾阳虚组UCP3表达细胞形态镜下脾阳虚组骨骼肌肌原纤维阳性颗粒表达较对照组少。④中药组UCP3表达细胞形态镜下中药组骨骼肌肌原纤维阳性颗粒表达较脾阳虚组和阳虚组多。(5)各组骨骼肌UCP3表达量的比较中药组、脾阳虚组、阳虚组与对照组相比,UCP3表达上调(P＜0.01)；中药组经过中药干预后,UCP3表达上调(P＜0.01)。4结论(1)附子理中汤促使骨骼肌内UCP3增加,UCP3主要促进脂肪酸代谢,使血清甘油三酯降低；增加对葡萄糖的利用及加强其代谢,使血糖水平降低。(2)脾阳虚组给于高脂饮食,血清甘油三酯升高,则游离脂肪酸(free fatty acid, FFA)也增多,FFA和葡萄糖作为参与能量代谢物质,相互间存在竞争,FFA水平升高使葡萄糖氧化受抑和摄取减少,表现为血糖升高。(3)由于UCP1作用的组织是BAT, UCP3主要作用于骨骼肌,均有高度的专一性,对胆固醇代谢的作用不明显；另外,由于高脂饲料中含有胆固醇,这增加了实验动物胆固醇摄入量,以上两点是脾阳虚组和中药组血清总胆固醇水平比对照组高的原因。(4)附子理中汤上调UCP3表达量,通过UCP3对骨骼肌的解偶联作用、促进产热是引起血糖、血清甘油三酯、血清总胆固醇水平变化的核心机制。(五)附子理中汤对脾阳虚证大鼠血清胃生长素影响的实验研究1目的探讨脾阳虚证大鼠血清Ghrelin的变化特征、生理作用机制以及附子理中汤对脾阳虚证大鼠血清Ghrelin的影响。2方法实验动物体重均衡随机分为4组,每组16只。①对照组：普通饲料喂养,饮水；d70-d98灌胃生理盐水10 ml/kg/d。d98禁食12h后,尾静脉取血,：EDTA-Na2及抑肽酶抗凝,检测血清Ghrelin水平；②阳虚组：d42手术切除BAT,余同①组；③脾阳虚组：d49-d98高脂饲料(83%普通饲料,15%甘油三酯,2%胆固醇)在28℃和19℃环境交替喂养；余同②组；④中药组：d70-d98附子理中汤5ml/kg/d体重灌胃；余同③组。3结果(1)从各组体重增长率看,阳虚组与对照组相比体重增长减慢(P＜0.01)；脾阳虚组与对照组相比体重增长减慢(P＜0.01)；中药组与脾阳虚组相比,体重增长加快(P＜0.01)。(2)从各组血清Ghrelin含量看,脾阳虚组和对照组相比,血清Ghrelin含量明显降低(P＜0.01)；中药组与脾阳虚组相比,血清Ghrelin含量明显上升(P＜0.01)。4结论附子理中汤上调脾阳虚证大鼠血清Ghrelin水平,提高Ghrelin的双重调控的效能,从而改善脾阳虚症状：(1)附子理中汤上调Ghrelin,加强胃肠消化和吸收功能,这可能是附子理中汤“健脾”功效的神经内分泌基础之一。(2)附子理中汤上调Ghrelin.水平,提高食欲促进摄食、增加体重,这可能是附子理中汤“温阳”功效的神经内分泌基础之一。四、实验结论(一)脾阳虚证中能量代谢改变的特征依据上述脾阳虚证大鼠能量代谢的实验结果,进行多因素综合分析可以作出如下推断：1脾阳虚证大鼠能量代谢水平下降,荷能物质(血糖和甘油三酯)减少,能量分子(ATP)减少,体温下降。2机体在脾阳虚证时,面对能量代谢水平下降,通过整体、器官系统和细胞分子三个水平进行代偿性调节,不断进行自调节和自适应。在机体神经-内分泌-免疫网络的调控下,骨骼肌通过寒战前肌紧张、寒战产热、BAT的非寒战产热等措施,代偿能量代谢的下降。在细胞分子水平的表现是,BAT细胞线粒体UCP1和骨骼肌线粒体UCP3的代偿性产热；骨骼肌肌球蛋白ATP酶活性代偿性升高。(二)附子理中汤干预脾阳虚的生物学基础包括两个方面：1提高机体能量代谢水平,以达到温煦脾阳的目的。主要根据是附子理中汤干预后,与脾阳虚组相比,BAT的UCP1表达量升高、骨骼肌线粒体的UCP3表达量升高、骨骼肌肌球蛋白ATP酶活性升高。2提高脑肠肽Ghrelin水平,发挥对胃肠消化吸收功能和摄食、能量代谢的双重正调节效应,以恢复“脾主运化”和“脾主温煦”的功能。五、本课题研究的创新点(一)立足中医脾阳虚的传统理论,既考虑脾阳虚能量代谢失调方面,也考虑脾阳虚胃肠功能失调方面。采取“肩胛间BAT切除术+高脂饲料喂养+隔日寒冷环境刺激”的多因素序贯干预措施,时间较长,从整体上模拟人体脾阳虚的发生发展过程,建立的“脾阳虚证”证候模型,与人体脾阳虚证的表现吻合度较高,模型可靠。(二)以UCPs和能量代谢的关系为主要研究内容,重点观察UCP1、UCP3、能荷以及肌球蛋白ATP酶活性的改变特征,探讨脾阳虚证大鼠能量代谢低下、产热不足的机制。(三)选取Ghrelin为研究对象,从神经内分泌角度阐释其对脾阳虚证大鼠的作用及机制、调节方式,角度选取具有新颖性。更多还原

【Abstract】 There are two types of syndromes about spleen yang deficiency syndrome, one is yang deficiency syndrome whose symptoms include chill, cold limbs, low body temperature, the other is spleen Qi deficiency syndrome which involves with symptoms as weakness, anorexia, weight loss, abdominal pain, diarrhea, lassitude,etc. the above are based on the theory of traditional Chinese medicine while its biological base is disorder of digestion and absorption system, in a word, spleen yang deficiency syndrome include two types of syndromes, one is low ability of heat generating, the other is disorder of digestion and absorption system.1 This thesis systemically discusses the development of spleen yang deficiency syndrome.At first, the research shows the development of the theory, experimental research and neuroendocrine research of spleen yang deficiency syndrome, and focuses on the present status, deficiency and future to systemically indicate the traits on energy metabolism especially on heat generating about UCPs; meanwhile, summarizes the regulating features of digestion, absorption,feed and metabolism about Ghrelin, which are the foundations for this research.2 The original consider about designing the researchBased on creating a new spleen yang deficiency syndrome model, aiming at the traits during the energy metabolism and exploring the key mechanism in spleen yang deficiency syndrome. To discuss the main factors of disorder of spleen function and low heat generating based on modern systemic biological integration theory.2.1 Setting up rat model with spleen yang deficiency syndromeIncising BAT, high fatty feeding and cold stimulating every other day to create a reliable spleen yang deficiency syndrome rat model, which emphasizing the traits of reinforce " destroying yang" by incising BAT to decrease the resource of heat generating, which is an innovation in model creating field.2.2 Research on energy metabolism in spleen yang deficiency syndromeSpleen yang deficiency syndrome includes chill, low body temperature symptoms, which is similar with the symptoms caused by low energy metabolism and basal metabolism rate. In modern biochemistry, during the process of energy metabolism always companied with ATP change which can directly supply energy. Many biologic mole and substance take part in this process of energy metabolism.Mitochondrion is main site of cell energy metabolism. the content of UCP is about 6%-14%of total content of inner membrane of mitochondrion which has close relationship with heat generating.Uncoupling proteins(UCPs) is a protein family which are involved with heat generating function.Physiologic research has shown it can decrease mitochondrial membrane potential and uncouple the process of oxidation and phosphorylation, in this way, it can inhibit synthesis of ADP even ATP and the result is to make energy to emit by way of heat. During the process, UCPs generates heat but not energy, and it also has close relationship with energy metabolism and is key to maintain body temperature. Since spleen yang deficiency syndrome is involved with low ability of heat generating, then, it should have a relationship with UCPs expression. This thesis will explain the relationship between UCP and spleen yang deficiency syndrome.2.3 Research on neuroendocrine in rats with spleen yang deficiency syndromeThe latest modern biology research shows brain-intestinal, gastric-intestinal hormone and intestinal nervous system controlling the stomach and intestine. Spleen yang deficiency syndrome has a close relationship with neuroendocrine based on main digestion system and multi organs’disorder. Ghrelin and CCK is familiar with regulating the food in-intake and energy metabolism and hopefully to become a bridge between function of digestive system and energy metabolism status.3 Conclusions about experimental researches3.1 Research on isoproterenol (ISO) inducing heat generating in rats with spleen yang deficiency syndrome3.1.1 Objective:Creating spleen yang deficiency syndrome rat model by removing interscapular brown adipose tissue, high fatty diet, and coldness stimulating every other day, then, injecting ISO as external stimulating resource and observing the sensibility to heat generating and ISO dose-effect relationship with spleen yang deficient rats.3.1.2 Methods:Removing interscapular brown adipose tissue, high fatty diet, and coldness stimulating every other day in rats with reference to evaluation on spleen yang deficiency syndrome rat model referencing the process of emergence and development of spleen yang deficiency syndrome according to the theory of traditional Chinese medicine, observing the rats’ behavior, setting up spleen yang deficiency syndrome rat model. then, under 28℃circumstance, injecting ISO and recording the rectal temperature,the temperature of chest and rear leg in spleen yang deficiency syndrome rats, and exploring the relationship between ISO dose and the temperature of each part.3.1.3 Results:the relationship between ISO dose and effect:effective dose 50(EDso) of the rectal temperature peak and the area under curve is 0.28 mg/kg and 0.106 mg/kg, ED50 of the temperature peak of chest and the area under curve is 0.066 mg/kg and 0.070 mg/kg, ED50 of the temperature of real leg peak and the area under curve is 0.071 mg/kg and 0.172 mg/kg. The changing tendency of the rectal temperature, the temperature of rear leg and chest is direct ratio.3.1.4 Conclusions:①totally, spleen yang deficiency syndrome model rats is not that sensible to external heat generating stimulating resource. Low ability of heat generating is probably one of the reasons that rats’slivering and chilling.drugs can improve symptoms of low ability of heat generating which makes an example for how Acotinum Lizhong decoction acts on rats with spleen yang deficiency syndrome.②the tendency of ISO dose-effect of heat generating is direct ratio, but the appropriate dose is not easy to fix due to the change of temperature of circumstance,noise, and light factors etc.3.2 Experimental research on the relationship between brown adipose tissue and UCP1 in spleen yang deficiency syndrome3.2.1 Objective:Exploring the relationship and mechanism about brown adipose tissue, (uncoupling protein 1,UCP1),high fatty diet and Chinese herbal decoction during the process of energy metabolism.3.2.2 Methods:the rats are divided into four groups, and each 16 cases.①control group:fed with ordinary food, d70-d98 ig. With 0.9% NaCl 10 ml/kg/d. d98 testing the ability of ISO deducing heat generating and observing variety curve of the rectal temperature and its peak within two hours and recording the area under the curve.②yang deficiency syndrome group:the same as the control group’s except removing brown adipose tissue on d42③spleen yang deficiency syndrome group:the same as the yang deficiency syndrome group’s except of being fed with high fatty diet (including 83% ordinary diet,15% triglycerides,2% cholesterol)from d49 to d98 and placed in 19℃circumstance every other day.④Chinese herbal decoction group:the same as the spleen yang deficiency syndrome group except of ig.5ml/kg/d with Aconitum Lizhong decoction and recording weight every week. Recording the index of the weight growth rate, the peak of temperature and the area under the curve of temperature. Using brown adipose tissue to test the content of UCP1.3.2.3 Results:①weight growth rate compared with control group, in yang deficiency syndrome group weight growth rate got lower (P<0.01), compared with yang deficiency syndrome group, weight growth rate in spleen yang deficiency syndrome group got lower(P<0.01), compared with spleen yang deficiency syndrome group, weight growth rate got higher in Chinese herbal decoction group (P<0.01).②temperature peak of heat generating compared with control group, in yang deficiency syndrome group temperature peak of heat generating got lower (P<0.01), compared with yang deficiency syndrome group, temperature peak of heat generating in spleen yang deficiency syndrome group got higher(P<0.01), compared with spleen yang deficiency syndrome group, temperature peak of heat generating got higher in Chinese herbal decoction group (P<0.01). Variety between weight growth rate and temperature peak of heat generating shows direct ratio.③area under the tempreature curve of heat generatingCompared with control group, in yang deficiency syndrome group, area under the tempreature curve of heat generating decreasing (P<0.01), compared with yang deficiency syndrome group, area under the tempreature curve of heat generating in spleen yang deficiency syndrome group increasing (P＜0.01), compared with spleen yang deficiency syndrome group, area under the tempreature curve of heat generating increasing in Chinese herbal decoction group (P<0.01). Variety between weight growth rate and area under the tempreature curve of heat generating is direct ratio.Compared with yang deficiency syndrom group and spleen yang deficiency syndrome group, Chinese herbal decoction group upregulates content of UCP1 and difference has high significance (P<0.01).3.2.4 Conclusions:①Chinese herbal decoction with function of heat generating and tonifying spleen is inclined to make high fatty diet turn into brown adipose tissue and advance its activity.②Chinese herbal decoction with function of warming middle energizer and tonifying spleen can upgrade level and expression of UCP1 in brown adipose tissue, and then make brown adipose tissue decomposed by the mechanism of uncoupling with UCP1, finally dispelling coldness from the bodies in the rats with yang deficiency syndrome.3.3 Interrelationship between energy charge and UCP3 in mitochondrion of skeletal muscle in rats with spleen yang deficiency syndrome3.3.1 Objective:Exploring effect of UCP3 and muscle energy charge after administrated Aconitum Lizhong decoction in rats with spleen yang deficiency syndrome.3.3.2 Methods:Rats are divided into four groups, and each 18 cases. All rats in four groups fed with ordinary food and water for 42 days, then chosen randomly three groups to mock the spleen yang deficiency syndrome, then the left as control group, to the control group always ordinary diet and water. For the three groups:①to choose randomly one group to remove brown adipose tissue(BAT) on d42 and then fed with ordinary diet and water, this group called yang deficiency syndrome group.②the other two groups, also, removed BAT on d42 but both of them are fed with high fatty diet (including 83% ordinary diet,15% triglycerides,2% cholesterol) and placed in the circumstance of 19℃every other day. When on d82, to choose randomly one of groups to ig. Aconitum Lizhong decoction according to the standard of 5ml/kg/d each rat, so this group called Chinese herbal decoction group while the other is called spleen yang deficiency syndrome group. On d98 using the rats blood and skeletal muscle to test indications.3.3.3 Results:①energy charge in Chinese herbal decoction group is lower than those in other three groups, which indicates content of ATP is also lower than those in other three groups and compared with control group, difference has highly significance (P<0.01); compared with yang deficiency syndrome group, difference has significance (P<0.05);compared with spleen yang deficiency syndrome group,difference has significance (P<0.05).compared with spleen yang deficiency group, chinese herbal decoction group upgrade the expresson of UCP3 (P<0.01) and acitivity of ATPase (P<0.05)②compared with control group, energy charge in spleen yang deficiency syndrome is lower and difference has significance (P<0.05); compared with yang deficiency syndrome group, energy charge is also lower but there is no significance (P>0.05).compared with control group, activity of ATPase of myosin raises in spleen yang dificency group (P<0.01)③compared with control group,energy charge in yang deficiency syndrome group is lower but there is no significance (P>0.05)3.3.4 Conclusions:The research shows that the mechanism of dysfunctional heat generating lies in the unbalance between heat generating and energy generating, Aconitum Lizhong decoction increases the content of muscle UCP3 and activity of ATPase of myosin,then inhibits synthesis of ATP (energy generating) to maintain the body temperature by the mechanism of uncoupling of UCP3 finally.3.4 Experimental research on the relationship between UCP3 and blood sugar, triglyceride, total cholesterol in rats with spleen yang deficiency syndrome3.4.1 Objective:Exploring the relationship between UCP3 and blood sugar, triglyceride, total cholesterol after administrated Aconitum Lizhong decoction in rats with spleen yang deficiency syndrome.3.4.2 Methods:The rats are divided into four groups, and each 16 cases.①control group:fed with ordinary food and water, d70-d98 ig. with 0.9% NaCl 10 ml/kg/d.②yang deficiency syndrome group:the same as the control group’s except removing brown adipose tissue on d42③pleen yang deficiency syndrome group:the same as the yang deficiency syndrome group’s except that fed with high fatty diet (including 83% ordinary diet,15% triglycerides,2% cholesterol)from d49 to d98 and placed in circumstance of 28℃and 19℃in turn.④Chinese herbal decoction group:the same as the spleen yang deficiency syndrome group’s except of ig.5ml/kg/d with Aconitum Lizhong decoction. To draw blood from left common carotid artery in all the rats in the four groups on d98 and then to test the content of blood sugar, triglyceride, total cholesterol. Finally to test the content of UCP3 of skeletal muscle by Immunohistochemistry technology.3.4.3 Results:①content of blood sugar compared with control group,content of blood sugar is lower than that in yang deficiency syndrome group (P<0.01); compared with yang deficiency syndrome group, content of blood sugar in spleen yang deficiency syndrome group is higher (P<0.01);compared with yang deficiency syndrome group, content of blood sugar in Chinese herbal decoction group is higher (P<0.01).②content of triglyceride compared with control group,content of triglyceride is lower than that in yang deficiency syndrome group (P<0.01); compared with yang deficiency syndrome group, content of triglyceride in spleen yang deficiency syndrome group is higher (P<0.01); compared with yang deficiency syndrome group, content of triglyceride in Chinese herbal decoction group is higher (P＜0.01).③content of total cholesterol compared with control group, content of total cholesterol in both spleen yang deficiency syndrome group and Chinese herbal decoction group is higher (P<0.01).④expression of UCP3 cytomorphology in each groupⅠ. expression of UCP3 cytomorphology in control group under microscope observing muscle cell fiber arranging regular and in muscle cell fiber is full of yellow (means positive) parts.Ⅱ. expression of UCP3 cytomorphology in yang deficiency syndrome group under microscope observing muscle cell fiber with yellow parts of muscle cell fiber decreases when compared with that in control group.Ⅲ. expression of UCP3 cytomorphology in spleen yang deficiency syndrome group under microscope observing muscle cell fiber with yellow parts of muscle cell fiber decreases when compared with that in control group.Ⅳ. expression of UCP3 cytomorphology in Chinese herbal decoction group under microscope observing muscle cell fiber with yellow parts of muscle cell fiber are more than that in both spleen yang deficiency syndrom and yang deficiency syndrom group.⑤compare of relative content of UCP3 in each group compared with control group, relative content of UCP3 is higher in Chinese herbal decoction group, spleen yang deficiency syndrom group and yang deficiency syndrom group,difference has high significance(P<0.01); after administrating Chinese herbal decoction, relative content of UCP3 increases in Chinese herbal decoction group and difference has high significance when compared with yang deficiency syndrome group and spleen yang deficiency syndrome group(P<0.01).3.4.4 Conclusions:①Chinese herbal decoction increase content of UCP3, and UCP3 alleviates metabolism of fatty acid, which lower content of triglyceride and increase use of glucose and alleviates its metabolism and then cause its levels lower.②fed with high fatty diet in spleen yang deficiency syndrome group cause content of triglyceride rise, which lead to increase free fatty acid (FFA), but due to competitive with glucose, level of FFA can inhibit use of glucose,which cause level of blood sugar to rise.③UCP1 mainly takes effect in brown adipose tissue and UCP3 act in muscle cell fiber, both of them have high specificity and have slight effect on metabolism of cholesterol, besides, high fatty diet increases level of cholesterol, which cause level of cholesterol to rise,and it is the reason that level of cholesterol in control group is higher than that in both spleen yang deficiency syndrome group and Chinese herbal decoction group.④Chinese herbal decoction raises content of UCP3 and then by the mechanism of uncoupling of UCP3 to improv heat generating, which in turn causing content of blood sugar, triglyceride and total cholesterol to rise, so the mechanism of uncoupling of UCP3 is main and key in this process of metabolism.3.5 Experimental research on effects on Ghrelin after administrated Acotinum Lizhong decoction in rats with spleen yang deficiency syndrome3.5.1 Objective:Exploring effects on Ghrelin after administrated Chinese herbal decoction in rats with spleen yang deficiency syndrome and its physiologic mechanism.3.5.2 Methods:The rats are divided into four groups, and each 16 cases.①control group:fed with ordinary food and water, d70-d98 ig. with 0.9% NaCl 10 ml/kg/d. On d98 prohibited to take in food for 12 hours, and then to draw blood from caudal vein and to add EDTA-Na2 and Aprotinin Protein inhibitor to the blood, then to test level of serum Ghrelin. ②yang deficiency syndrome group:the same as the control group’s except removing brown adipose tissue on d42..③spleen yang deficiency syndrome group:the same as the yang deficiency syndrome group’s except that fed with high fatty diet (including 83% ordinary diet,15% triglycerides,2% cholesterol)from d49 to d98 and placed in circumstance of 28℃and 19℃in turn.④Chinese herbal decoction group:the same as the spleen yang deficiency syndrome group’s except of ig.5ml/kg/d with Aconitum Lizhong decoction.3.5.3 Results:①compared with control group, weight growth rate in yang deficiency syndrome group is lower (P<0.01); compared with spleen yang deficiency syndrom group,weith growth rate in Chinese herbal decoction group is higher (P<0.01) difference has high significance.②compared with control group, in spleen yang deficiency syndrome group, the content of Ghrelin decreases, and difference has high significance (P<0.01); while compared with spleen yang deficiency syndrome group, the content of Ghrelin in Chinese herbal decoction group rises, difference high significance (P<0.01). and is close to that of control group (P>0.05).3.5.4 Conclusions:①mechanismⅠ:Aconitum Lizhong decoction can raise level of Ghrelin, and then Ghrelin regulates energy metabolism,alleviates gastric acid secretion,raises food intake and growth,which thus can alleviates the symptoms of anorexia, weight loss of spleen yang deficiency syndrome.②mechanismⅡ:Aconitum Lizhong decoction raises level of Ghrelin, and the latter can alleviate disorder of gastrointestinal motility, which also alleviates the symptoms of lack of appetite, weight loss of spleen yang deficiency syndrome.4 Summarization for the experimental researches4.1 The traits about energy metabolism in spleen yang deficiency syndromeWith the results of above experiences and by multi-factors analysis, here are some infers:(1) level of energy metabolism lowering, and energy substance such as glucose lowering, ATP and body temperature are also decreasing. (2) Regulating in integration, organ and cell or mole to adapt the changing circumstance in the status of low energy metabolism under the control of neuroendocrineimmune network. And by pre-shivering tone, shivering generating heat and non-shivering generating heat with UCP1 in BAT or UCP3 in skeletal muscle.4.2 Two aspects about how Aconitum Lizhong decoction interfering spleen yang deficiency syndrome. (1) elevating the level of energy metabolism to warm spleen yang since it can raise expression of UCP1 of BAT,UCP3 of skeletal muscle and activity of ATPase in myosin. (2) the expression of Ghrelin can regulate digestive and absorption function and energy metabolism to return the normal function of spleen.5 Innovations of the research5.1 Based on traditional theory of spleen yang deficiency syndrome, and considering both disorder of energy metabolism and disorder of gastric and intestinal function. Firstly remodeling spleen yang deficiency syndrome rat model with removing brown adipose tissue, feeding high fatty food. Coldness stimulating every other day with multi-factors. It takes a long time to create the model and totally considers the course of happening and development of spleen yang deficiency syndrome, for above reasons, the model is reliable and practicable.5.2 Ghrelin, UCPs and ATPase are chosen as main indexes, for Ghrelin which is discussed about its physiologic mechanism and the way of regulation from angle of theory of neuroendocrine, which is a new angle deserving further research. For UCPs, CUP1 and CUP3 are related with synthesis of ATP and energy charge, which are viewed as innovations in this research too.5.3 Ghrelin is chosen to be research on its mechanism and the methods on spleen yang deficiency syndrome, which is also a creative way or angle for the research.更多还原