四妙勇安汤抗糖尿病心肌病的效应成分及分子机制研究

【作者】 李琳；

【导师】 郭淑贞；

【作者基本信息】 北京中医药大学 ， 中西医结合药理学， 2021， 博士

【摘要】 糖尿病心肌病是糖尿病患者发生心衰的主要原因之一,19%的心衰患者合并有糖尿病,目前尚无特效的治疗方法。四妙勇安汤由金银花、玄参、当归、甘草四味药组成,具有活血通络、清热解毒兼具滋阴之效。中医理论认为,糖尿病心肌病的病机主要是燥热内伤,久病致瘀,心血瘀阻,蕴久成毒,瘀毒互结,心体心用俱损。四妙勇安汤切中糖尿病心肌病瘀毒损心病机,临床经辨证加减后常用于糖尿病心肌病治疗。基础研究证明,心肌底物代谢异常诱发的心肌脂毒性在糖尿病心肌病发生和发展中发挥着重要作用,胰高血糖素是介导心肌脂毒性的关键介质。心肌脂毒性可能是中医瘀毒的生物学基础之一。本课题组前期研究已证明四妙勇安汤能抑制异丙肾上腺素及主动脉弓缩窄术诱导的心衰。然而,有关四妙勇安汤抗糖尿病心肌病的作用、机制及其效应成分,目前尚不清楚。本研究以胰高血糖素脂毒性通路为靶标,通过体内外实验研究四妙勇安汤抗糖尿病心肌病的作用机制及物质基础。另外,通过筛选出四妙勇安汤干预以胰高血糖素为核心的脂毒性信号传导的最优效应成分,部分揭示四妙勇安汤干预糖尿病心肌病的物质基础及作用靶点,为抗糖尿病心肌病的创新药物研发提供实验依据。第一部分 文献综述对胰高血糖素在糖尿病心肌病中的研究进展进行系统探索,梳理胰高血糖素与心衰相关疾病的关系及潜在的信号通路,分析了目前研究的不足之处,为实验研究思路构建提供依据。对四妙勇安汤及其主要成分改善代谢综合征的作用机制进行系统归纳,为四妙勇安汤改善糖尿病心肌病作用及其机制研究提供实验基础。第二部分四妙勇安汤防治糖尿病心肌病的作用及机制研究通过链脲佐菌素构建糖尿病模型小鼠,通过测定给药后血糖血脂、口服葡萄糖耐量、血清胰岛素、胰高血糖素水平、胰腺及肝脏病理形态、胰岛α和β细胞分布,阐明四妙勇安汤对糖尿病小鼠糖脂代谢、胰岛素敏感性及高胰高血糖素血症的改善作用。通过超声影像确定在第18周糖尿病心肌病成模,结合心肌组织HE、WGA、Masson、油红O、TUNEL、CD45及透射电镜染色结果表明:四妙勇安汤能改善糖尿病小鼠心功能,抑制心肌细胞肥大、心肌纤维化、心肌细胞凋亡、心肌细胞脂质积聚、心肌炎细胞浸润及心肌细胞线粒体形态,其中,降脂、抑制心肌细胞凋亡及维持线粒体形态作用明显,说明四妙勇安汤抗糖尿病心肌病的作用与抑制脂毒性相关。随后通过WB检测心肌组织中胰高血糖素通路相关蛋白表达,发现四妙勇安汤能抑制糖尿病小鼠心肌组织中胰高血糖素(GLC)、胰高血糖素受体(GCGR)、过氧化物酶体增殖物激活受体α(PPARα)、过氧化物酶体增殖物激活受体辅激活因子1α(PGC-1α)及核因子活化B细胞κ轻链增强子(NF-κB)的表达或激活,能促进糖尿病小鼠心肌组织中AMP依赖的蛋白激酶(AMPK)的激活,提示四妙勇安汤抗糖尿病心肌病作用与调节GLC/AMPK/NF-kB 和 GLC/PPARα/PGC-1α 通路相关。第三部分四妙勇安汤干预心肌脂毒性的效应成分研究250μM棕榈酸(PA)作用于H9C2心肌细胞,建立心肌细胞脂毒性模型。通过分析20个入血原型成分对PA诱导的H9C2心肌细胞活力及凋亡的影响,结果显示绿原酸、新绿原酸、甘草素、异甘草苷、新异甘草苷、芹糖甘草苷、芹糖异甘草苷、木犀草素和阿魏酸能够显著抑制PA诱导的心肌细胞活力的降低及凋亡的增加,提示这9个成分是该复方中改善心肌细胞脂毒性的有效成分。其中,绿原酸和阿魏酸作用最强,用于后续研究。第四部分四妙勇安汤效应成分干预心肌脂毒性的作用机制研究流式细胞仪检测Annexin V/PI双染结果证明,绿原酸和阿魏酸可抑制PA诱导的心肌细胞凋亡。油红O染色及RT-PCR检测脂代谢相关基因表达,结果表明:绿原酸和阿魏酸能降低PA诱导的心肌细胞脂质积聚及脂代谢相关基因PPARα,CD36,UCP3的表达。通过激光共聚焦对棕榈酸和葡萄糖吸收分析证明,绿原酸和阿魏酸可抑制PA诱导的心肌细胞底物利用失常。通过Mitotracker线粒体染色及Seahorse线粒体功能分析,证明绿原酸和阿魏酸能改善PA诱导的线粒体分裂及呼吸功能损伤。初步证明绿原酸和阿魏酸抑制心肌细胞脂毒性作用与心肌细胞能量底物代谢调节及线粒体功能改善相关。GCGR抑制剂(Adomeglivant)干预PA刺激的心肌细胞能抑制PA诱导的心肌细胞凋亡,发现PA通过激活GCGR诱导心肌细胞脂毒性。WB检测GLC通路关键蛋白表达,结果显示:绿原酸和阿魏酸能下调PA诱导的心肌细胞中GCGR、PPARα和PGC-1α的表达,增加p-AMPK的激活,说明绿原酸和阿魏酸通过抑制GCGR信号通路改善PA诱导的H9C2心肌细胞凋亡。综上,体内和体外研究结合阐释了四妙勇安汤及其活性成分绿原酸和阿魏酸能通过调节GLC/PPARα和GLC/AMPK通路,从而改善糖尿病心肌糖脂代谢,抑制心肌脂毒性,发挥防治糖尿病心肌病的作用。特色与创新1.理论创新:将活血解毒治法及其经典方——四妙勇安汤用于糖尿病心肌病的干预,丰富并发展了糖尿病心肌病的瘀毒病机理论。2.技术创新:整合了四妙勇安汤给药后入血原型成分库及体外药效筛选,构建四妙勇安汤干预糖尿病心肌脂毒性的效应成分优选方法,为创新药物的发现提供了新策略。3.机制创新:以GLC/AMPK/NF-kB和GLC/PPARα/PGC-1α通路为切入点,通过在体实验与离体实验相结合,首次深入探讨四妙勇安汤及其效应成分绿原酸、阿魏酸对心肌细胞脂代谢的改善作用,为四妙勇安汤的进一步研究及其临床糖尿病心肌病治疗提供可靠的实验依据。更多还原

【Abstract】 Diabetic cardiomyopathy is one of the main causes of heart failure in diabetic patients,and 19%of patients with heart failure have diabetes,but there is no effective treatment.Si-Miao-Yong-An decoction is composed of Lonicerae Japonicae Flos,Scrophulariae Radix,Angelicae Sinensis Radix,and Glycyrrhizae Radix et Rhizoma,which has the function of improving blood circulation,alleviating fever and detoxification and nourishing Yin.According to the theory of traditional Chinese medicine,the pathogenesis of diabetic cardiomyopathy is mainly hot and dry internal injury,blood stasis caused by long-term disease,heart blood stasis,accumulated for a long time into poison,mutual stagnation of blood stasis and toxin,and heart body and heart function all damaged.Si-Miao-Yong-An decoction targets the pathogenesis of the stasis toxin injuring heart in diabetic cardiomyopathy and has been frequently used in the treatment of diabetic cardiomyopathy after syndrome differentiation.Basic research has proved that myocardial lipotoxicity induced by abnormal myocardial substrate metabolism plays an important role in the occurrence and development of diabetic cardiomyopathy and glucagon is a key mediator in mediating cardiolipid toxicity.Cardiac lipid toxicity may be the biological basis of stasis toxicity in traditional Chinese medicine.Our previous studies have shown that Si-Miao-Yong-An decoction can improve the cardiac function of mice with heart failure induced by isoproterenol or aortic arch constriction.However,there has been no report on the effect of Si-Miao-Yong-An decoction against diabetic cardiomyopathy,its mechanism and effective components.In this study,the glucagon lipid toxicity pathway was used as the target to study the mechanism and material basis of Si-Miao-Yong-An decoction in improving diabetic cardiomyopathy through in vivo and in vitro experiments.This study will select the optimal effective components of Si-Miao-Yong-An decoction for intervention of lipotoxic signal transduction with glucagon as the core,and partially reveal the effective components and targets of Si-Miao-Yong-An decoction for intervention of diabetic cardiomyopathy.Provide support for the development of innovative drugs against diabetic cardiomyopathy.Part one:literature reviewThis paper reviews the research progress on the role of glucagon in diabetic cardiomyopathy,comb out the relationship between glucagon and heart failure related diseases and potential signaling pathways,and analyze the shortcomings of the current research,so as to provide a basis for the construction of experimental research ideas.To systematically discuss the mechanism of Si-Miao-Yong-An decoction and its main components in improving metabolic syndrome,and to provide experimental reference for the study on the effect and mechanism of Si-Miao-Yong-An decoction in improving diabetic cardiomyopathy.Part two:Study on the effect and mechanism of Si-Miao-Yong-An decoction in the prevention and treatment of diabetic cardiomyopathyThe diabetic mice were constructed by streptozotocin.It has been demonstrated that Si-Miao-Yong-An decoction improves glucolipid metabolism,insulin sensitivity and hyperglucagonemia through measuring blood sugar,blood fat,oral glucose tolerance test,serum insulin,glucagon,pancreas and liver pathological morphology,alpha and beta cell distribution.The diabetic cardiomyopathy model is established in the 18th week,which is confirmed by cardiac ultrasound.Si-Miao-Yong-An decoction improves cardiac function,inhibits myocyte hypertrophy,myocardial fibrosis,myocardial apoptosis,lipid accumulation,inflammatory cell infiltration,and mitochondrial morphology through myocardial tissue HE,WGA,Masson,oil red O,TUNEL,CD45 and transmission electron mcroscopy staining,among which,the obvious lipid-lowering,inhibiting myocardial cell apoptosis and maintaining mitochondrial morphology effects indicate that the improvement of diabetic cardiomyopathy by Si-Miao-Yong-An decoction was related to the inhibition of lipid toxicity.Then,it is found that Si-Miao-Yong-An decoction can downregulate the expression or activation of glucagon(GLC),glucagon receptor(GCGR),Peroxisome proliferators-activated receptor α(PPARα),Peroxisome proliferator-activated receptor coactivator 1α(PGC-1α),and Nuclear transcription factor κB(NF-kB),and upregulate the activation of AMP-activated protein kinase(AMPK)by WB showing that the improvement of diabetic cardiomyopathy by Si-Miao-Yong-An decoction is related to the regulation of GLC/AMPK/NF-κB and GLC/PPARα/PGC-1α pathway.Part three:Study on the effective components of Si-Miao-Yong-An decoction against myocardial lipid toxicityThe lipid toxicity model of H9C2 cardiomyocytes was established.The activity of H9C2 cardiomyocytes was significantly decreased after treated with 250 μM palmitic acid(PA)for 24h.Chlorogenic acid,Neochlorogenic acid,Liquiritigenin,Isoliquiritin,Neoisoliquiritin,Liquiritin apioside,Isoliquiritin apioside,luteolin,and Ferulic acid were shown to significantly inhibit the decrease of myocardial cells activity and the increase of myocardial cells apoptosis induced by PA through analyzing the effects of 20 prototype components on the PA-induced myocardial cells activity or apoptosis,which indicates that the 9 ingredients are the active components of Si-Miao-Yong-An Decoction to improve myocardial cell lipid toxicity.Among 9 active ingredients,chlorogenic acid and ferulic acid show the strongest effect,which are used in the follow-up study.Part four:Study on the effect mechanism of active ingredients of Si-Miao-Yong-An decoction against myocardial lipid toxicityThe results of Annexin V/PI double staining by flow cytometry showed that chlorogenic acid and ferulic acid could inhibit PA-induced apoptosis of cardiomyocytes.The expression of lipid metabolism-related genes was detected by RT-PCR.The results of oil red O and RT-PCR showed that chlorogenic acid and ferulic acid could reduce the lipid accumulation and the expression of lipid metabolism-related genes PPARα,CD36 and UCP3 in PA-induced cardiomyocytes.The results of palmitic acid and glucose absorption indicated that chlorogenic acid and ferulic acid could inhibit PA-induced substrate utilization disorders in H9C2 cardiomyocytes.Mitotracker staining and Seahorse function analysis of mitochondria showed that chlorogenic acid and ferulic acid could improve mitochondrial division and respiratory function injury induced by PA.The results showed that the inhibition of chlorogenic acid and ferulic acid for the cardiomyocyte lipid toxicity was related to the regulation of cardiomyocyte glucolipid metabolism and mitochondrial function.GCGR inhibitor(Adomelivant)can inhibit PA-induced apoptosis of cardiomyocytes,and it has been preliminarily proved that PA induces lipotoxicity of cardiomyocytes by activating GCGR.The expression of GLC pathway key proteins was detected by WB and the results demonstrated that chlorogenic acid and ferulic acid could down-regulate the expressions of GCGR,PPARa and PGC-1α in PA-induced cardiomyocytes,and increase the activation of p-AMPK,suggesting that chlorogenic acid and ferulic acid could improve PA-induced apoptosis of H9C2 cardiomyocytes by inhibiting GCGR signaling pathway.In conclusion,in vivo and in vitro study demonstrated the improvement of Si-Miao-Yong-An decoction and its active components(chlorogenic acid and ferulic acid)could improve the myocardial glucose and lipid metabolism,inhibit myocardial lipotoxicity,and prevent diabetic cardiomyopathy through regulating GLC/PPARa and GLC/AMPK pathways.Characteristics and innovation1.Theoretical innovation:the activating blood and detoxifying therapy and its classic prescription Si-Miao-Yong-An decoction are used in the intervention of diabetic cardiomyopathy,which enriches and develops the blood stasis toxin pathogenesis of diabetic cardiomyopathy.2.Technological innovation:integrating plasma prototype library after oral administration of Si-Miao-Yong-An decoction and in vitro efficacy screening,establishing the optimization method of effective components of Si-Miao-Yong-An decoction in intervening diabetic cardiolipid toxicity,and improving the efficiency of innovative drug discovery.3.Mechanism innovation:regarding GLC/AMPK/NF-κB and GLC/PPARα/PGC-1α as the breakthrough point,through in vivo and in vitro experiment,firstly in-depth discuss the intervention for myocardial cell lipid metabolism of Si-Miao-Yong-An decoction and the effective components(chlorogenic acid and ferulic acid)during the development of diabetic cardiomyopathy,and provide reliable experimental basis for clinical treatment of diabetic cardiomyopathy and further investigation.更多还原