摘要
第一部分文献研究
糖尿病(Diabetes mellitus, DM)与心血管疾病密切相关,合并糖尿病更易发生冠状动脉粥样硬化性心脏病、心力衰竭、动脉粥样硬化、高血压等心血管疾病。糖尿病心脏病(Diabetic Cardiopathy, DC)(?)旨糖尿病并发或伴发的心脏病,是糖尿病患者的主要死亡原因之一。糖尿病心肌病(Diabetic Cardiomyopathy, DCM)是糖尿病心脏病的其中一个类型,是糖尿病引起心脏微血管病变和心肌代谢紊乱所致的心肌广泛局灶性坏死。流行病学研究发现,糖尿病病人70%以上死于心血管系统疾病,是非糖尿病人群心血管系统疾病病死率的2-3倍。因此,更好地了解和采取积极的预防措施,减缓疾病的进程是治疗的关键。
糖尿病心脏病的发病机制复杂,到目前未有统一的说法,可能涉及心肌细胞代谢障碍、心肌纤维化、自主神经病变和干细胞等多种因素。DC的临床表现早期通常为心肌顺应性降低和舒张期充盈受阻为主的舒张功能不全,晚期以收缩功能不全为主,易发生充血性心力衰竭及心律失常。目前西医的防治方法无特效的治疗方案,一旦发现心脏的舒张功能障碍,应及早治疗糖尿病心肌病。控制血糖、血压、血脂,预防动脉粥样硬化,减轻心脏负荷,是防治糖尿病心脏病变必不可少的手段。
近研究发现胰高血糖素样肽-1(glucagon like peptide-1, GLP-1)及类似物可能具有心血管保护作用,尤其是对2型糖尿病患者心血管系统的影响已成为关注的焦点.GLP-1是一种主要由肠L细胞分泌的肠促胰素,具有葡萄糖浓度依赖性降糖作用,刺激胰岛素分泌、保护胰岛β细胞、抑制胰高血糖素分泌、抑制胃排空、降低食欲和食物摄入量的作用。其类似物已用于2型糖尿病治疗。
糖尿病心脏病在祖国医学中,属于消渴并发心悸、胸痹心痛等范畴,现代中医命名为“消渴病心病”。通过古代文献的整理,总结本病病因为禀赋虚弱、饮食不节、过服丹石、情志失调、劳逸过度。对于消渴病心病的病机,历代医家提出许多不同观点,大致可归纳为以下几种学说：阴虚燥热论、肾虚论、火热论、气虚论、脾胃论、瘀血论、情志致病论。糖尿病中医防治指南认为本病以气血阴阳两虚为本,气滞、痰浊、血瘀、寒凝为标,将糖尿病心脏病分为六个证型,即气阴两虚证、痰浊阻滞证、心脉瘀阻证、阴阳两虚证、心肾阳虚证及水气凌心证。但总的来说本病离不开“瘀”,所以从瘀论治是目前防治糖尿病心脏病的主要研究方向。中医中药对防治本病的研究具有广阔的前景。
心胃相关理论是在邓铁涛教授“五脏相关”理论的基础上产生的。中医学对胃的概念,还包括了大肠和小肠。心胃相关的理论是根据心胃的经络关系和五行理论的母子相生关系。现代研究也证明心与胃肠的生理相关、病理互相影响。因此,以心脏病为主要威胁的糖尿病心脏病也可以采用“心病治胃”的思路进行施治。
广州中医药大学第一附属医院熊曼琪教授为首的研究课题组在上世纪80年代,围绕气阴两虚、瘀血阻滞为病机,以通腑泻热逐瘀为主的治法,由《伤寒论》经方桃核承气汤加味研制而成中药降糖三黄片。在临床及机理研究方面发现中药降糖三黄片具有保护心脏的作用。本研究通过文献记载来探讨降糖三黄片对糖尿病心脏病的保护作用存在“心胃相关”理论,结合实验研究进一步证明其理论科学性。
第二部分实验研究
目的：
通过观察降糖三黄片对糖尿病性心肌病大鼠的血糖、糖化血红蛋白、血浆中GLP-1的浓度、GLP-1受体基因表达量及心肌组织病理改变的影响,初步探讨其保护心脏的作用机制,验证“心胃相关”理论的科学性。
方法：
SPF级雄性49只SD大鼠普通饲料适应性喂养5天后,随机分为2组,正常组(9只)和造摸组。正常组继续以普通饲料喂养,造摸组改用高糖高脂饲料喂养2周。造摸前禁食12h,造摸组按40mg/kg剂量腹腔内注射STZ,正常组腹腔内注射等量柠檬酸缓冲液。1W后,测定空腹血糖。连续两次空腹血糖≥11.1mmol/L,且有多饮、多尿、多食现象确认为2型糖尿病模型,共35只大鼠完成实验。糖尿病模型成模后,造摸组进一步分层,随机分为模型组、降糖三黄片高剂量组、降糖三黄片低剂量组、二甲双胍组及DPP-4高剂量组,每组7只,余5只不符合条件不列入统计分析。降糖三黄片高剂量组按787.5mg/kg/d体重的剂量给药,降糖三黄片低剂量组按393.75mg/kg/d的剂量给药,二甲双胍按52.5mg/kg/d的剂量给予,DPP-4抑制剂组按10.5mg/kg/d的剂量给予,正常组及模型组灌服等量蒸馏水。给药5周后,经股静脉注射垂体后叶素(Pit)1U/kg,制造急性心肌缺血模型,以Ⅱ导联心电图记录为证。30分钟后取材。①每日观察饮水量、进食量及排泄量,每周观测体重及空腹血糖；使用糖化血红蛋白测定试剂盒进行手工检测糖化血红蛋白。②酶联免疫吸附分析法(ELISA)检测血浆中GLP-1浓度；实时定量-聚合酶链式反应(RT-PCR)检测大肠组织及下丘脑组织的GLP-1受体基因表达量。③以光学显微镜下观察心肌细胞的病理形态学改变并测定心肌缺血面积。
结果：
1.造摸后,造摸组均出现“三多一少”的症状,其饮水量、进食量、排出量及血糖明显高于正常组(P<0.05或P<0.01),而体重明显减轻,与正常组比较有显著差异(P<0.01),提示DM造模成功。经给药5周后,结果显示：降糖三黄片能改善多饮症状(与模型组比较,分别为高剂量组P<0.01,低剂量组P<0.05),但未能改善多食的症状(P>0.05)；降糖三黄片高剂量组及低剂量组的平均排出量均明显增加(P<0.01),提示降糖三黄片的“通下”结果；可以增加糖尿病大鼠的体重(P<0.01),与二甲双胍组及DPP-4抑制剂组比较无显著差异；同时能够降低糖尿病大鼠的空腹血糖(P<0.05或P<0.01),其降糖作用均从给药第2周开始显效,给药期间有血糖波动,提示降糖作用仍不稳定；然而,降糖三黄片高、低剂量组于实验末的糖化血红蛋白与模型组比较无显著性差异(P>0.05),考虑因为实验时间较短。从血糖控制程度来分析,降糖效果由优至劣依次为DPP-4抑制剂组、二甲双胍组、降糖三黄片高剂量组、降糖三黄片低剂量组。
2.酶联免疫吸附分析法(ELISA)检测血浆中GLP-1浓度；实时定量-聚合酶链式反应(RT-PCR)检测大肠组织及下丘脑组织的GLP-1受体基因表达量。结果显示：降糖三黄片高剂量组及降糖三黄片低剂量组血浆中的GLP-1浓度高于模型组、二甲双胍组及DPP-4抑制剂组,但无统计学意义。经过药物干预5周后,与模型组为对照(相对表达量为1),则降糖三黄片高剂量组大肠中GLP-1mRNA表达水平为模型组的2.48倍(P>0.05),而下丘脑中GLP-1mRNA表达水平为模型组的6.46倍(P<0.05)；而降糖三黄片低剂量组大肠中GLP-1mRNA表达水平为模型组的1.14倍(P>0.05),而下丘脑中GLP-1mRNA表达水平为模型组的2.87倍(P>0.05),但两组的GLP-1受体基因表达量仍低于二甲双胍组及DPP-4抑制剂组,提示降糖三黄片具有一定的提高GLP-1的作用。然而,降糖三黄片高、低剂量组大鼠血清中GLP-1浓度与模型组无统计学意义,可能降糖三黄片的靶点不在血清中GLP-1,而直接影响GLP-1受体mRNA表达。
3.以光学显微镜下观察心肌细胞的病理形态学改变并测定心肌缺血面积。结果显示：降糖三黄片低剂量组内7只大鼠都见心肌缺血区局灶性心肌细胞变性、坏死,胞浆嗜伊红染,横纹不清,细胞核固缩或消失,心肌细胞间间隙增大,间质散在炎细胞浸润,未见纤维组织增生。而降糖三黄片高剂量组只见6只与降糖三黄片低剂量组病理改变相似,余1只未见明显变性、坏死,间质未见出血、水肿、炎细胞浸润及纤维组织增生,且其心肌缺血面积小于降糖三黄片低剂量组。总体来说,降糖三黄片高剂量组及降糖三黄片低剂量组的心肌缺血面积小于模型组,但无统计学意义,其面积大于二甲双胍组及DPP-4抑制剂组。
结论：
1.降糖三黄片具有改善糖代谢作用,能提升血浆中GLP-1浓度、大肠及下丘脑中GLP-1受体基因表达量,减少心肌缺血面积。但作用不及二甲双胍和DPP-4抑制剂。
2.降糖三黄片高剂量组对糖尿病大鼠的心脏保护作用及对GLP-1的影响优于降糖三黄片低剂量组。
Part One Literature research
Diabetes is closely related to cardiovascular disease. Diabetic patiens are more likely to suffer from coronary atherosclerosis, heart disease, heart failure, atherosclerosis, hypertension and other cardiovascular diseases. Diabetic cardiopathy (DC) is a decline in heart function associated with diabetes, is one of the leading cause of mortality within the diabetic population. Diabetic cardiomyopathy is one of its types. The pathogenic performance mainly exists in the heart capillaries, cardiac muscle metabolism and fibrosis. Therefore, early prevention is the key of DC treatment.
The pathogenesis of DC is very complex, may involve many factors such as myocardial metabolic abnormalities, myocardial fibrosis, autonomic neuropathy, stem cells, etc. DC is characterized by reduced myocardial compliance and diastolic filling blocked in the presence of a diastolic dysfunction as early clinical manifestations, followed by systolic dysfunction in later period which can lead to heart failure or arrhythmia. At present, there is not a single clinically effective treatment for DC. Once diastolic dysfunction has occurred, DC should be treated as soon as possible. Controlling blood glucose, blood pressure and blood lipids, preventing atherosclerosis, reducing cardiac stress are the prevention and treatment important steps of DC.
Recent studies found that glucagon-like-peptide-1(GLP-1) and its analogs may have cardiovascular protective effects, especially for the cardiovascular system in patients with type2diabetes, what makes it become the focus of attention. GLP-1is an incretin hormone secreted by intestinal L cells, has glucose concentration-dependent of the hypoglycemic effect, that stimulates insulin secretion and protects β-cells, inhibits glucagon secretion and gadtric emptying, andreduces appetite and food intake. Recently, its analogs have been used for the treatment of type2diabetes.
In Traditional Chinese Medicine(TCM) theory, diabetic cardiomyopathy belong to the scope of "XiaoKe","heart palpitations","chestpain", etc., Modern Chinese Medicine named it "Xiao Ke Heart Disease". Through the collation of ancient literatures, the pathogenesis of this disease is summarized to the weak endowment, improper diet, over clothing bladder stone, emotional disorders, and excessive works. There are many different points of view about pathogenesis of this disease, that can be broadly summarized as: Yin hot theory, deficiency of kidney theory, fiery theory, deficiency of Qi theory, the spleen and stomach theory, stasis theory, emotions pathogenic theory. TCM Diabetes Prevention Guide consider that DC is caused by deficiency of Qi, Xue, Yin, Yang as the primary causes, and Qi stagnation, phlegm, blood stasis coagulated cold as the secondary causes. The guide put DC into6syndromes:syndrome of deficiency both Qi and Yin, syndrome of blockade of phlegm-turbidity, syndrome of blood stasis blocking heart vessel, syndrome of deficiency both Yin and Yang, syndrome of Yang deficiency of heart and kidney, syndrome of pathogenic water attacking heart. But in general, the disease can not be separated from "stasis" of blood stasis, therefore, nowadays it become the main research direction of prevention and treatment of diabetic cardiopathy. Chinese medicine has broad prospects for research on the prevention and treatment of this disease.
"Relevance of Heart and Stomach" theory is generated on the basis of the "Five Visceras" theory of Professor Deng Tietao. The concept of the stomach in traditional chinese medicine includes small and large intestine."Relevance of Heart and Stomach" theory is based on the meridian relations between heart and stomach, and relationship between mother and child according to the Five Elements theory. Recent researches had also proved that the physiological between heart and gastrointestine are related each other, so as their pathology which affecting each other. Therefore, as the main threat to diabetic cardiopathy, the treatment for heart disease can also adopt the idea of "heart disease take the cure from stomach".
The first affiliated hospital of Guangzhou University of Traditional Chinese Medicine Xiongmanqi's research team invented Chinese patent drug-Jiangtangsanhuang Tablet in the1980's, originating from Taohe chengqi decoction from Shanghan Lun, based on the the pathogenesis of deficiency in qi and yin, stagnation of blood stasis, with tonificating qi and yin, also eliminating stagnation of blood stasis as the therapeutic method. Many clinical and mechanism studies founded that Jiangtangsanhuang tablet has a cardioprotective effect. Based on the former research, these literature and experiments are meant to explore the cardioprotective effect of JTSH to further prove "Relevance of Heart and Stomach" theoretical science.
Part Two experimental research
Objective:
To observe the cardio-protective effect and the expression of GLP-1of JTSH tablet against the experimental diabetic cardiopathy rats,to reveal the "relevance of heart and stomach" theory.
Methods:
Healthy SD rats were randomly divided into normal group(n=9) with regular feed and diabetic model group with high cholesterol diet (HCD) throughout the study period. The model group induced by low-dosage streptozocin (STZ)(40mg/kg) after2weeks of HCD and were randomly divided into four group with fasting blood glucose>11.1mmol/L:model group, high-dose of JTSH group (787.5mg/kg/d), low-dose of JTSH group (393.75mg/kg/d), metformin group (52.5mg/kg/d), DPP-4inhibitor group (10.5mg/kg/d),7rats each group. After5weeks treatment, acute myocardial ischemia model were established on all groups induced by Pituitrin injection.①The general status (food and water intake, excretion) were measured everyday, body weight and fasting blood glucose were measured every week during the treatment. The glycosylated hemoglobin were measured after acute myocardial ischemia model were established.②GLP-1amide in plasma were measured using ELISA. The large intestine and hypothalamic were detected for the expression of GLP-1mRNA Real Time PCR.③The myocardial tissue pathological changes were observed by optics microscope and ischemia area were measured by Image-Proα Express5.1.
Results:
1. After modeling, polydipsia, polyphagia, polyuria and weight loss symptoms appear on model group; their water intake, food intake, excretion and fasting blood glucose were significantly higher than normal group, indicates that diabetic modeling was succeed. After5weeks treatment, JTSH can improve the symptomps of polydipsia(P<0.05or P<0.01), but can't yet improve the symptoms of polyphagia. The excretion of JTSH both high-dose and low-dose are more than before treatment, which is the effect of JTSH. JTSH also can increase the body weight of diabetic rats and reduced their blood glucose significantly (P<0.05or P<0.01). However, HbAlC level on high and low-dose of JTSH groups were not significantly lower than model groups, probably due to shorter experimental period. DPP-4inhibitor group has the best effect of glycemic control, followed by metformin group, high-dose of JTSH group and low-dose of JTSH group respectively.
2. GLP-1amide in plasma were measured using ELISA. The large intestine and hypothalamic were detected for the expression of GLP-1mRNA Real Time PCR. The result indicated that the GLP-1amide in plasma on high and low-dose of JTSH group were higher than the model group, the metformin group and the DPP-4inhibitor group, but it hadn't statistically significance. In high-dose of JTSH group, the expression of GLP-1mRNA on its large intestine were2.48times higher than the model group (P>0.05), while the expression of GLP-1mRNA on its hypothalamic were6.46times higher than the model group(P<0.05); in low-dose of JTSH group, the expression of GLP-1mRNA on its large intestine were1.14times higher than the model group(P>0.05), while the expression of GLP-1mRNA on its hypothalamic were2.87times higher than the model group(P>0.05). But still, GLP-1mRNA expression level on JTSH groups were lower than the metformin group and the DPP-4inhibitor group. These results indicated that JTSH has a certain role in improving GLP-1, by directly effecting on the gene.
3. The myocardial tissue pathological changes were observed by optics microscope and ischemia area were measured by Image-Proα Express5.1. Pathological changes such as focal myocardial cell degeneration or necrosis, eosinophilic cytoplasm stained, stripes unclear, karyopyknosis or nucleus disappear, gap increases between myocardial cells, interstitial scattered infiltration of inflammatory cells, but no fibrosis, could be found at myocardial ischemia area of all rats in low-dose of JTSH group. While similar pathological changes could also be found at6rats in high-dose of JTSH group, but there was1rat which its myocardial tissue was no obvious degeneration nor necrosis, no interstitial hemorrhage nor edema, no inflammatory cell infiltration nor fibrosis. The result indicated that the ischemia area of high and low-dose of JTSH group were smaller than the model group, but bigger than the metformin group and the DPP-4inhibitor group.
Conclusion:
1. Jiangtangsanhuang Tablet can improve glucose metabolism, elevate GLP-1in amide plasma and its mRNA expression in large intestine and hypothalamic. But the efficacy of metformin and DPP-4inhibitor were better than JTSH tablet.
2. The cardio-protective effect and the GLP-1expressions at high-dose of JTSH group were more obvious than low-dose of JTSH group.
引文
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