生脉解毒通络胶囊对实验性糖尿病大鼠心肌组织微炎症干预研究

【作者】 王聃红；

【导师】 南征；

【作者基本信息】 长春中医药大学 ， 中医内科学， 2008， 博士

【摘要】 糖尿病心肌病(DCM)属中医消渴心病,是糖尿病(DM)重要的心脏并发症,是导致慢性心功能不全的主要原因,本论文从文献综述、理论探讨、实验研究等方面对生脉解毒通络胶囊防治DCM进行较系统的研究。重点探讨毒损心络病机理论与DCM炎症发病机制的相关性,论述生脉解毒通络胶囊通过干预DCM炎症机制发挥治疗上的优势,进一步从不同的角度阐述生脉解毒通络法的科学性,实验研究通过运用中药抑制炎症因子表达途径,进一步阐明DCM的发病机理,有较强的创新性和实用性。1文献综述1.1中医药研究糖尿病心肌病进展从古代和现代中医对DCM病名的认识、病因病机的探索、辨证论治进展情况及中医药防治DCM作用机制进展等方面进行了综述,并对当前在诊断及疗效评定标准、辨证分型和科研等方面存在的缺乏规范性研究等问题进行了述评和展望。1.2糖尿病心肌病的现代研究进展就近年来国内外在DCM的发病机理即心肌组织糖、脂代谢紊乱,血管紧张素Ⅱ(AngⅡ)、单核细胞趋化蛋白-1(MCP-1)等相关炎症因子作用机制与心肌细胞外基质转运失衡之间的最新研究成果及它们之间的关系,并包括了糖尿病心肌病的其他病因、发病机理、临床诊断、治疗及实验研究的最新进展做以综述。2理论探讨运用中医理论引经据典,并结合现代研究成果,对消渴心病的病机理论、治则治法等进行了探讨,提出并完善毒损心络是消渴心病的发病关键,阐明毒损心络理论与DCM炎症发病机制的相关性,论述了益气养阴,解毒通络法通过干预DCM炎症发病机制,为消渴心病的研究思路及治法提供理论依据。3实验研究3.1生脉解毒通络胶囊对实验性糖尿病大鼠糖、脂代谢的影响采用链脲佐菌素(STZ)诱导大鼠实验性DM模型,设正常对照组、DM模型组、中药实验组(DM+生脉解毒通络胶囊高、中、低剂量组)和阳性对照组(DM+依那普利)实验观察各项指标。结果表明,生脉解毒通络胶囊能明显改善DM大鼠的一般状态,具有一定的降血糖作用,并能够降低血清胆固醇(TC)、甘油三酯(TG)、升高密度脂蛋白(HDL-C)而调节脂代谢,与DM模型组比较有显著性统计学意义(P＜0.01,P＜0.05)。3.2生脉解毒通络胶囊对实验性糖尿病大鼠心肌超微结构的保护本实验通过光镜HE及PAS染色观察各组实验性DM大鼠心肌组织形态学改变,利用电镜观察生脉解毒通络胶囊对DM大鼠心肌超微结构的变化,结果表明:DM模型组光镜主要表现为心肌间质纤维细胞、成纤维细胞增生,PAS阳性物质沉着明显增多,透射电镜下心肌细胞肌丝排列紊乱,心肌细胞线粒体间质内胶原纤维增多,线粒体水肿,数量增多,呈凝聚状,肌原纤维溶解,肌浆网扩张。经生脉解毒通络胶囊治疗的中药实验组上述病理改变较DM模型组显著减轻。说明生脉解毒通络胶囊对糖尿病心肌组织结构及超微结构损害起到保护作用。3.3生脉解毒通络胶囊对实验性糖尿病大鼠AngⅡ及ECM积聚的影响用放射免疫法测各组实验动物血清及心肌组织AngⅡ含量,结果表明DM模型组血清及心肌组织AngⅡ含量原均明显升高,与正常对照组比较有显著性统计学意义(P＜0.01),依那普利治疗的阳性对照组及中药实验组心肌组织上述指标均显著下降,与DM模型组比较有显著性统计学意义(P＜0.05、P＜0.01),免疫组织化学法检测心肌Ⅰ、Ⅲ(ColⅠ、ColⅢ)胶原的蛋白表达,DM组从强度到面积都明显增强,与正常对照组比较有显著性统计学意义(P＜0.01),依那普利治疗的阳性对照组及中药实验组心肌组织上述指标均显著下降,与DM模型组比较有显著性统计学意义(P＜0.05、P＜0.01)。本实验说明生脉解毒通络胶囊至少在部分程度上是可能通过减血清及心肌组织中AngⅡ含量,进而阻断ColⅠ、ColⅢ过度表达,促进ECM降解。AngⅡ作为炎性因子,在抑制DCM炎症机制的过程中起调节作用,是生脉解毒通络胶囊防治DCM作用机制之一。3.4生脉解毒通络胶囊对实验性糖尿病大鼠心肌MCP-1蛋白及基因表达途径的影响采用免疫组化检测各组实验大鼠心肌MCP-1,逆转录聚合酶链式反应(RT-PCR)检测各组实验大鼠心肌MCP-1mRNA表达,观察生脉解毒通络胶囊对实验性DM大鼠心肌MCP-1表达的影响,目的是探讨MCP-1途径在DCM发生发展中的作用机制及与AngⅡ、心肌ECM转运失衡在DCM发生发展中的相关性。三者在DCM发病机制中的作用可能是AngⅡ诱导MCP-1表达增加,导致ECM积聚,加重DCM。结果表明:DM模型组大鼠心肌MCP-1蛋白及基因表达明显高于正常对照组(P＜0.01);中药实验组和阳性对照组MCP-1、表达较DM模型组均明显下降(P＜0.01,P＜0.05),提示生脉解毒通络胶囊有较强的抑制DM大鼠心肌MCP-1过度表达的作用,其防治DCM的作用可能部分通过抑制MCP-1途径,发挥干预DCM炎症机制的作用。更多还原

【Abstract】 diabetic cardiomyopathy(DCM)is an important microvascular complication of diabetic mellitus(DM) as well as the common reason resulting in chronic heart failure.Systematic studies on the prevention and treatment for DCM by ShengMaiJieDuTongLuo capsule(SJT capsule) had been performed from the aspects of reference reviews,theory and experimental study in this essay.It layed stress on exploring the relation between the inflammatory pathogenesis of DCM and the pathogenesis theory of the damage of heart by poison and discussing remedial advantages of SJT capsule from intervening inflammatory pathogenesis of DCM."Method of Removing toxicity,removing obstruction and supplementing kiDCMey" being scientific is clarified from different aspects ulteriorly.The experimental study clarifies pathological mechanism of DCM ulteriorly from herb restraining expressed pathway of inflammatory factor,which is practical and innovative.1 Reference review1.1 The skeleton research of traditional Chinese medicine(TCM) on DCMThis essay reviewed from the advances of such fields as followed:the source and name of TCM for DCM,the etiology and pathogenesis for DCM,selection of treatment based on the differential syndrome diagnosis,pathogenesis of TCM on DCM,which is in ancient and modern times,moreover,included the evaluations andprospects on the criterions of diagnosis and curative effect as well as the problems lying in the selection of treatment based on the differential syndrome diagnosis or scientific study.1.2 The latest study of pathogenesis of DCMThis essay summarized the experimental study results and advances on the principle of preventing and treating DCM,according to the pathologenesis of DCM,such as glucoregulation tubulence,fat matabolism tubulence,monocyte chemoattractant protein-1 (MCP-1),angiotensinⅡ(AngⅡ)involving the inflammatory pathogenesis of DCM,et cl.the losing balance of extracellular matrix(ECM),and the relations between them.other etiology, pathogenesis,clinical diagnosis,laboratory examination were also included in recent years years. 2 Theory StudyBy discussing the pathogenesis theory,principle and method of treatment of Xiao ke heart desease with Chinese medicine theories quoting from classics works and combining modern research result,we put forward and perfect the theory that the main mechanism of Xiao ke heart desease is the damnification of heart by poison and we also clarify the relation between the inflammatory pathogenesis of DCM and the pathogenesis theory of the damage of heart by poison.Theory gist of treating Xiao ke heart desease is provided by us from discussing the effective way of "Method of Removing toxicity,removing obstruction and supplementing heart" intervening inflammatory pathogenesis of DCM.3 Experimental study3.1 The influence about SJT capsule on glucose and lipids in blood of diabetic cardiomyopathy ratsSTZ-induced diabetic rats were divided randomly into normal control group,diabetic model group,positive control group(DM+Enalapril),experimental group(DM+SJT capsule of large,median and small dose respectively) in Chinese herb.The various marks were observed.The results showed JDTLBS capsule obviously improve the rats’ general condition and had a certain effect on reducing blood sugar,serum cholesterol(TC) and triglyceride(TG), low density lipoprotein(LDL),which had a significant statistic sense in comparing with diabetic model group(P＜0.01,P＜0.05).The results indicated that SJT capsule was beneficial for regulating glucose and lipid metabolism.3.2 Effect of shengmai jiedu tongluo capsule(SJT capsule) on myocardial ultrastructure in Diabetic RatsThe changes of myocardial tissue morphology in experimental diabetic rats were observed by optical microscope and electric microscope.What were seen in model group by optical microscope were that the number of myocardiac interstitial fibrocyte increased, protein substances with positive marked by PAS,Under the transmission electric microscope, the myofilament of myocardial cell disarranged,the number of mitochondrion increased and was agglomerate,the content of the collagenous fiber in myocardiac interstitium increased, Myocardium hypertrophy,a focal loss,disorganization of myofibril,mitochondril swelling, and lysis of cistae were observed。The pathological changes of experimental group and positive control group treated by SJT capsule and Enalapri for 12 weeks were alleviated more obviously than diabetic group.The results showed SJT Capsule may decrease the myocardial injury of diabetic rats significantly.3.3 The study about SJT capsule to decrease of content of AngⅡand decomposite the experimental diabetic rat’s myocardial ECMStudying the content of AngⅡin the experimental animals’ serum and local myocardial tissue with radioimmunoassay,the results showed:the content of AngⅡin DM modal group’ s serum and local myocardial tissue obviously increased,which have remarkable difference from the normal control group(P＜0.01);the content of AngⅡin the serum and local myocardial tissue of experimental group that cured by SJT capsule and postive control group that cured by Enalapril obviously decreased,which have remarkable difference from the DM model group(P＜0.01);So SJT capsule can decrease the content of AngⅡin the serum and local myocardial tissue.Studying the protein expression of ColⅠ、Ⅲin the experimental animals’ myocardial tissue with immunohistochemical method and observing the effect of SJT capsule on the protein expression of ColⅠ、Ⅲin the experimental animal’s myocardial tissue are to investigate the molecular mechanism of SJT capsule preventing and treating DCM.The results showed:the protein expression of ColⅠ、Ⅲin the modal group’s myocardial tissue was in high level,immunostaining of it was increased from intention and area which was more 8 times than that in normal control group.The number of ColⅠ、Ⅲpositive cells in the myocardial tissue of experimental group that cured by SJT capsule and postive control group that cured by Enalapril is remarkably fewer than that in the DM modal group(P＜0.01、P＜0.01).So SJT capsule can inhibit the protein expression of ColⅠ、Ⅲin the experimental animals’ myocardial tissue markedly.It indicate that the decrease of content of AngⅡis one of the function mechanisms that SJT capsule prevent and treat DCM though promote the decomposition of ECM in myocardial tissue,which maybe partly had correlation with its ability to interdict the expressed way of inflammatory factors and then have a regular effect on inhibiting the inflammatory pathogenesis of DCM.3.4 The study about effect of SJT capsule on the protein and gene expression pathway of MCP-1 in experimental diabetic rat’s myocardial tissueIt was done that assaying the protein expression of MCP-1 in experimental rats’ myocardial tissue of each group by immunohistochemical method and the expressive content of MCP-1 mRNA in experimental rats’ myocardial tissue of each group by RT-PCR method and observing SJT capsule’s effect on MCP-1 in diabetic rats’ myocardial tissue,so that we could probe the mechanism of and progress of diabetic glomerular sclerosis.The action of the three factors on the pathogenesis of DCM maybe have concerned with the following:AngⅡcan stimulate the expression of induce the expression of MCP-1 and consequently MCP-1 activate the ColⅠ,Ⅲ,,at last ECM accumulate,DCM is worse.The results showed:the protein expression of MCP-1 and the level of MCP-1 mRNA in modal group obviously increased more than normal control group(P＜0.01);the protein expression of MCP-1 in the myocardial tissue of experimental group that cured by SJT capsule and postive control group that cured by Enalapril obviously decreased,which have remarkable difference from the DM model group(P＜0.01,P＜0.05);It indicated that SJT capsule could obviously inhibit the excess expression of MCP-1 in diabetic rats’ myocardial tissue,which maybe partly had correlation with its ability to prevent and treat DCM by inhibiting the way of MCP-1 and intervening the inflammatory pathogenesis of DCM.更多还原