黄芪散对糖尿病心肌病变的干预作用及其机制研究
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摘要
糖尿病是一种由多种原因引起的糖、脂肪、蛋白质代谢紊乱综合征,进而导致多系统、多脏器损害,是常见的慢性全身进行性内分泌代谢疾病。文献报道糖尿病致残率、死亡率仅次于肿瘤和心血管疾病,而且其中95%左右为2型糖尿病。糖尿病的根本危害在于其各种慢性并发症(心、脑、肾和血管),这些并发症不仅严重危害人类的生命和健康,而且其昂贵的医药费用还给患者带来沉重的经济负担,已成为当今世界范围的重大公共健康问题之一。因此,糖尿病的防治不是单纯的降糖问题,其关键问题是长期控制高血糖和高血脂等,预防和延缓并发症的发生,从而降低病死率。在各种并发症中,其中心脏病变是糖尿病最严重而突出的问题,因此,“从降糖治疗到全面防治心血管危险因素”被称为“2型糖尿病防治策略的革命”。
糖尿病心肌病是糖尿病重要的慢性心脏并发症之一,是导致糖尿病晚期心功能衰竭和死亡的主要原因,其病理表现主要为心肌细胞肥大、凋亡和心肌间质纤维化等。糖尿病心肌病发病机制尚未完全明了,目前认为这些特征性的病理改变主要与心肌细胞的能量代谢障碍、氧化应激、细胞因子异常、胰岛素抵抗等多种因素有关。由于其病因复杂,其确切的病理机制尚未彻底阐明,临床治疗尚无特效药物,西药主要以对症治疗为主,疗效欠佳。
中医治疗糖尿病的历史源远流长,尤其是在防治糖尿病并发症的整体调节上具有很大的优势,往往起到标本兼顾的治疗作用。因此,寻求安全而有效的中医药疗法对糖尿病及其并发症的治疗有着重要的社会意义和经济意义。
糖尿病在中医属于“消渴”范畴,综合历代医家对消渴病的病因病机和辨证论治的论述发现,气阴不足,燥热内盛是消渴病的主要病机。糖尿病心肌病则属于“消渴”“心悸”“胸痹”“胸痛”“消渴病心病”范畴,本病主要由消渴病日久迁延所致,现代医家认为阴虚燥热是本病基本病机,为标;正气不足、心脾两虚是其病机的关键,为本。为此提出益气养阴为基本治疗原则,本研究所用的方剂就是基于这种治疗原则建立的。
古方黄芪散方源自北宋《圣济总录》,全方由葛根、黄芪、桑白皮三味药组成。葛根生津止渴,升脾中清阳,输津液以溉五脏而滋阴清热,为方中君药;黄芪益气健脾,取其气复津还之意,用为臣药,君臣相配,既可益脾气,又可滋脾阴,清中焦之热而生津止渴以治中消之症;佐以桑白皮甘寒泻肺中伏火,滋阴润燥以疗上消之症,兼调下消,且其甘寒之性以制黄芪其气稍热之性。三药相伍,共奏益气健脾,滋阴清热,生津止渴,三消并治,标本兼顾之效。临床观察和文献研究发现三药单用或与它药联合配伍应用对糖尿病及并发症均有良好疗效,但关于整方的实验研究几近空白。
前期我们通过系列小鼠实验考证了黄芪散的降血糖药效:分别采用链脲佐菌素糖尿病模型、肾上腺素高血糖模型研究黄芪散对病理状态下高血糖和糖耐量的影响;采用正常小鼠研究黄芪散对正常状态下血糖和糖耐量的影响。实验结果表明,黄芪散能明显降低糖尿病小鼠的空腹血糖和改善糖耐量;对肾上腺素性高血糖小鼠体现一定的降血糖作用;黄芪散连续给药3周对正常小鼠空腹血糖和糖耐量没有明显的影响。
在这种基础上,我们通过复合因素建立2型糖尿病实验动物模型,观察一定周期内出现的心肌病变,研究黄芪散对糖尿病心肌病变的干预作用及其保护机制。
目的
系统研究古方黄芪散对糖尿病及其心肌病变的防治作用及其机理,利用现代科学技术阐释传统中医药的治疗机理,同时为古方的临床应用和糖尿病新药开发提供实验依掘。
方法
采用高脂喂养+链脲佐菌素的方法建立具备胰岛素抵抗特征的2型糖尿病大鼠模型,在此基础上观察糖尿病大鼠的心肌病变及药物的干预作用。同时,实验还比较了不同病程的2型糖尿病大鼠心肌病变差异。
黄芪散的干预作用及其机制研究:大鼠高脂饲料喂养4周继而腹腔注射链脲佐菌素,选择链脲佐菌素成模后大鼠随机分为五组,糖尿病心肌病变模型组和黄芪散高、中、低治疗组、罗格列酮阳性对照组。另设正常对照组平行观察。除正常对照组和模型对照组外,各组大鼠分别予以不同药物干预治疗8周,同时维持高脂饲料造模至实验结束。
实验中,定期记录大鼠的体重、进食量、饮水量、尿量等一般体征,检测大鼠空腹血糖、餐后血糖和葡萄糖耐量。实验全部结束后处死大鼠,采血分离血清,采用生化法检测大鼠空腹血糖、糖化血清蛋白、血脂常规、游离脂肪酸等指标,采用放射免疫方法测定空腹胰岛素、肿瘤坏死因子(TNF-α)等指标;取部分心肌组织制备组织匀浆液,测定心肌组织超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、心肌羟脯氨酸的含量变化;对各组大鼠心脏进行HE、MASSON染色,观察糖尿病大鼠心肌组织的病理改变;应用透射电镜观察心肌组织的超微结构改变;应用原位术端标记法(TUNEL)观察心肌细胞凋亡情况;采用RT-PCR方法检测大鼠心肌组织葡萄糖转运蛋白4(GLU-T4)、核转录因子(NF-κB)、转化生长因子-β1(TGF-β1)及其信号蛋白SAMD3的mRNA表达情况;采用免疫组织化学方法观察TGF-β1和SAMD3蛋白在心肌组织的表达情况。
结果
1.高脂喂养+链脲佐菌素方法可以建立2型糖尿病大鼠心肌病变模型:首先,该模型具有多饮、多食、多尿、肥胖、胰岛素抵抗以及糖、脂肪、蛋白质代谢紊乱等特征,与临床上2型糖尿病患者的特征比较接近;成模8周后可出现典型糖尿病心肌病变:心肌细胞肥大、变性、凋亡、间质胶原纤维增生,心肌细胞超微结构损伤性变化(肌原纤维排列紊乱,线粒体明显增多,线粒体嵴断裂、溶解,细胞核核膜不完整,异染色质凝聚等)。随着时间的延长,心肌纤维化和凋亡现象加重,成模12周以后大鼠心肌出现大片胶原纤维增生,心肌明显纤维化病变。两个病程的模型组大鼠在体重、血糖、血脂、胰岛素方面的变化没有明显区别,都高于正常组。
2.在糖代谢方面,与模型组相比,黄芪散高、中剂量均可降低糖尿病大鼠的空腹血糖、糖化血清蛋白(P<0.05),改善葡萄糖耐量,黄芪散高剂量具有显著降低空腹胰岛素以及改善胰岛素抵抗的作用(P<0.01),黄芪散中剂量也具有明显改善胰岛素抵抗的作用(P<0.01)。在脂代谢方面,黄芪散各剂量均可显著降低模型大鼠的血清总胆固醇和低密度脂蛋白水平,降低游离脂肪酸(P<0.01～P<0.05);黄芪散高、中剂量还可显著降低大鼠的甘油三酯水平(P<0.05),黄芪散中剂量明显升高大鼠的高密度脂蛋白水平(P<0.05);而且在血清总胆固醇和低密度脂蛋白水平,游离脂肪酸的改善方面,作用要略优于罗格列酮组。此外,黄芪散各剂量还可明显减少肥胖的2型糖尿病大鼠体重的增加(P<0.01～P<0.05),减少糖尿病大鼠的饮水量和进食量,明显减少24h尿量(P<0.01～P<0.05),这些都有利于糖尿病临床症状的改善。进一步对与心肌细胞糖代谢相关的关键葡萄糖转运蛋白GLU-T4表达的研究结果发现,与模型组相比,黄芪散高剂量能明显抑制糖尿病大鼠心肌GLU-T4 mRNA表达下降(P<0.05),提示黄芪散可能通过调节GLU-T4改善心肌细胞的能量代谢从而防止心肌病变。
3.黄芪散可以防止或延缓糖尿病大鼠心肌的形态学病理改变:黄芪散各剂量可以不同程度防止大鼠心肌出现心肌细胞排列紊乱,疏松,心肌细胞肥大、变性,细胞核固缩、裂解,成纤维细胞增生,胶原纤维增生的病理变化。用药后大部分大鼠心肌细胞基本排列整齐,部分病变区有心肌细胞肥大、变性。黄芪散高、中剂量可明显防止大鼠心肌的超微结构改变:高、中剂量组肌原纤维排列基本整齐,线粒体和细胞核形态基本正常,糖原颗粒存在状况与正常组相似。黄芪散高、中剂量还可明显降低心肌细胞凋亡指数(P<0.01～P<0.05),提示黄芪散可以干扰心肌细胞凋亡程序。
4.黄芪散可以抑制氧化应激-炎症反应:糖尿病心肌病变模型组大鼠心肌SOD、GSH-Px活性均显著低于正常组(P<0.01),脂质过氧化产物MDA含量则显著高于正常组(P<0.01)。黄芪散高、中剂量组大鼠心肌SOD、GSH-P_X活性显著高于模型组(P<0.01～P<0.05),MDA含量显著低于模型组(P<0.05),提示黄芪散能提高大鼠心肌的抗氧化能力,减轻氧化应激反应。过激的氧化应激反应可以进一步激活NF-κB,启动或加重炎症反应。研究结果发现,糖尿病心肌病变模型组大鼠NF-κB和TNF-α表达增强,而黄芪散高、中剂量能抑制NF-κB和TNF-α的过度表达,从而抑制心肌和全身的炎症反应。
5.黄芪散可以通过调控TGF-β1/SMAD3通路显著抑制心肌纤维化病变:糖尿病心肌病变大鼠心脏指数增加(P<0.01),MASSON图像分析发现心肌间质胶原纤维增生(P<0.01),生化检测也证实心肌组织羟脯氨酸含量增加(P<0.05),提示糖尿病大鼠存在心肌纤维化从而导致心肌肥厚病变。研究发现,模型组大鼠TGF-β1及其信号蛋白SMAD3的mRNA和蛋白表达均明显增强(P<0.01)。而黄芪散各剂量具有不同程度抗心肌纤维化的作用,可明显降低心脏指数,减少心肌羟脯氨酸含量,抑制胶原纤维增生从而抑制心肌肥厚病变,并显著抑制大鼠心肌TGF-β1/SMAD3 mRNA和蛋白的强表达,提示黄芪散抑制心肌纤维化的作用可能主要是与抑制TGF-β1/SMAD3信号通路有关。
结论
糖尿病心肌病的发生发展存在高血糖/氧化应激/NF-κB/TGFβ1信号传导途径,表现为糖尿病由于持续的高血糖和脂肪、蛋白质代谢紊乱以及胰岛素抵抗,导致心肌细胞能量代谢障碍,引起心肌细胞结构与功能受损,氧化应激反应逐渐加重,可以激活NF-κB,启动下游基因的转录,诱导全身性和心血管反应加重心肌细胞的损伤,进一步可促使TGF-β1/SMADs通路活化。上述各种因素最终可导致心肌细胞肥大、凋亡、坏死、间质胶原纤维增生,并发展成为糖尿病心肌间质纤维化,即糖尿病性心肌特征性病变。本研究结果证明,以益气养阴为主要治则的古方黄芪散能有效防治糖尿病心肌病变,作用与干扰高血糖/氧化应激/NF-κB/TGF-β1信号传导途径有关,具体表现为减少尿量、饮食量、进水量,以及改善胰岛素抵抗、降低血糖和改善糖耐量、调节血脂、上调GLUT-4 mRNA表达,改善心肌能量代谢;增强心脏抗氧化能力,抑制NF-κB和TNF-α的表达,减轻心脏和全身炎症反应;降低TGF-β1和SMAD3 mRNA及蛋白表达等多方面的作用,从而整体调节和防治糖尿病及其心肌病变。
Diabetes Melliuts(DM) is a multi-cause of endocrine and metabolic disease, which caused by defects of insulin secretion and / or activity also it is a common and chronic life-long disease.It is reported that the disability and mortality rate of diabetes is only after those of tumor and cardiovascular diseases(ranking No.3),of which about 95%for type 2 diabetes.The cardinal harm of DM is determined by various chronic complications(ex heart,brain, kidney and blood vessels complications),which not only cause significant harm to human life and health,and also cause the expensive medical costs that bring heavy financial burden to patients.Therefore DM has been become one of the major public health problems all of the world.That is why prevention and treatment of diabetes is not a simple problem of lowering blood glucose,the key issues is the long-term control of high blood glucose,high blood lipids and high blood pressure,then preventing and delaying the incidence of complications,thereby reducing the mortality of DM.In variety of complications,heart disease one of the most serious and outstanding problems,including diabetic coronary heart disease,diabetic cardiomyopathy and diabetic cardiac autonomic neuropathy.Therefore,now people refer to "the full treatment for lowering blood glucose convert to controlling of cardiovascular risk factors" as " the revolution of prevention and treatment strategies of type 2 diabetes."
Diabetic cardiomyopathy is one of the important chronic complications of diabetes,which often lead to later-period heart failure and death.The pathogenesis of diabetic cardiomyopathy have not yet been fully understood, now it is considered that the reasons such as the encumbrance of myocardial cells energy metabolism,oxidative stress,abnormality of cytokines and insulin resistance induce the myocardial lesions,resulted in hypertrophy and apoptosis of myocardial cells,fibrosis of myocardial interstitium and other pathological changes.Because of its complex etiology,and the non-clear pathological mechanism,there is no effective drugs in clinical,mainly treat symptoms by western medicine which only play poor efficacy.
Traditional Chinese medicine have a long history of treating diabetes, especially is good at the prevention and treatment of diabetic complications on the overall regulation,and it often treat symptoms and essence in the same time.Therefore,it has important social and economic significance in searching safe and effective therapy of Chinese medicine for diabetes and its complications.
In tradtional Chinese medicine,diabetes belongs to " Xiaoke ",by synthesizing theroy of etiology,pathogenesis and treatment according to syndrome differentiation of medicals in past dynasties,we could find deficiency of Qi and Yin,excessive dry and heat are the major pathogenesis of Xiaoke diseases,thus tonify qi,nourish yin,and eliminating heat become the main treatment principles of Chinese medicine.Prescriptions used in this study is based on this principle.
The ancient prescription i.e Huangqisan Originated from "Shengji zonglu" in Northern Song Dynasty,the entire prescription is made up of three herbs: Pueraria,Radix Astragali,Morus alba.Sweet and cool Pueraria can produce body fluid to stop thirst,Promote Qing-yang in the spleen,transport body fluid to irrigate five internal organs,nourish yin and eliminate hot,so it plays as a prime-drug in the prescription;Sweet and warm radix Astragali can increase Qi and tonify spleen,which means to body fluid recovering as soon as Qi regaining,so it plays as a official-drug in the prescription,which can treat middle-xiao symptoms combining with the prime-drug by eliminating hot of Zhongjiao accordingly producing body fluid to stop thirst;Sweet and cold Morus alba can clear away fiery in the lung,nourish yin and moisture dry so as to treat up-xiao and down-xiao symptoms,futhermore the Sweet and cold nature can constrain the warm nature of Radix Astragali.The effects of total prescription are increasing Qi and tonifying spleen,nourishing yin and eliminating heat,producing body fluid to stop thirst,treating for three-xiao symptoms,and treating symptoms and essence in the same time.Clinical observation and literature study have found that it have a good effect on diabetes and complications,whether three drugs alone or combined with other drugs.However,the research of the whole prescription(Huangqisan) is vacant.
First of all,we carried out some pre-experimental studies in mice,which used to testing and verifying effects of lowing glucose of Huangqisan.Applying two high blood sugar models to study the effects on pathological high glucose and impaired glucose tolerance,such as diabetic model induced by streptozotocin(STZ),and hyperglycemia model caused by adrenergic;In addition,we used the normal mice to study effects on blood sugar and glucose tolerance of normal state.At last,the experimental results showed that Huangqisan decreased FBG and ameliorated glucose tolerance of diabetic mice induced by STZ;Huangqisan also showed lowing glucose effect on adrenergic model;However,Huangqisan play no evdient influence on normal fasting glucose and glucose tolerance of normal mice after three weeks'administration.
On this basis,we established experimental animal models of type 2 diabetes melliuts(T2-DM)by imitating complex factors,observed cardiac complications during a certain period,and to study the intervention effect and its protection mechanisms of Huangqisan on diabetic cardiomyopathy.
Objective
To study the intervention effect and its protection mechanisms of Huangqisan on diabetic cardiomyopathy,using modern science and technology to explain the mechanism of traditional Chinese medicine theraphy,as well as to provide experimental basis for popularization and application of ancient prescription and clinical practice.
Methods
Applying the methods which combined high fat diet with STZ to establish T2-DM rats model with characteristics of insulin resistance,on the basis of these T2-DM rats we observed the pathological changes of myocardial and the intervention effects of drugs.
The intervention effect of and its protection mechanisms of Huangqisan on diabetic cardiomyopathy:The successful model rats were selected,and randomly divided into five groups:diabetic cardiomyopathy model group,high,medium and low treatment group of Huangqisan,rosiglitazone positive control group.Furthermore,a normal control group was set up for parallel observing.In addition to the normal control group and the model control group,other rats in each group were treated by eight weeks of drugs intervention,the high fat diet were maintained until the end of the experiment.
During the experiment,we regularly recorded general condition of all the rats such as weight,food intake,water intake,urine output,and detected fasting blood glucose,random blood glucose and glucose tolerance of the rats. After the end of the experiment,all rats were killed,blood were collected for serum separating,then the fasting blood glucose,glycosylated serum protein,blood lipid,free fatty acids were detected by biochemical methods, fasting insulin and tumor necrosis factor(TNF-α) were detected by radioimmunoassay method;Cut part of cardiac tissue for tissue fluid, myocardial superoxide dismutase(SOD),malondialdehyde(MDA),glutathione peroxidase(GSH-Px) of content were detected by kits;The pathological changes in heart tissue of the diabetic rats were observed by HE and MASSON staining methods;The ultrastructural changes of myocardial tissue were detected by transmission electron microscopy;Apoptosis condition of myocardial cells observed by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL) method;The mRNA expression of transporter 4(GLU-T4), nuclear transcription factor(NF-κB),transforming growth factor-β1(TGF-β1) and its SAMD3 signal protein were detected by RT-PCR method;The protein expression of TGF-β1 and SAMD3 in cardiac tissue were detected by immunohistochemical method.
Results
The methods combining high fat diet with streptozotocin and then continue high fat diet can create a model of T2-DM in rats,which has characteristic of excessive drink,excessive food,excessive urine,fat,insulin resistance,and metabolic disorders of sugar,fat,protein,etc,and those characteristics were close to T2-DM patients in clinical;The rats showed typical diabetic cardiomyopathy after models having been established for eight weeks:such as cardiac myocyte hypertrophy,degeneration,increasing of apoptotic cells interstitial collagen fibrosis,and myocardial cell injury changes in ultrastructure(disorder of myofibril,increasing of mitochondria, fragmentation and dissolution of mitochondrial cristae,incomplete nuclear membrane and eterochromatin condensation,etc).Accompanied by time prolonging, myocardial fibrosis aggravated after 12 weeks,myocardial of the rats showed large areas of interstitial collagen fibers had proliferated.Two course of model rats had no apparent distinction in the weight,blood sugar,and blood lipid,those were significantly higher than the normal group.
In the glucose metabolism area,when compared with the model control group,high and middle doses of Huangqisan decreased fasting blood glucose, glycosylated serum protein of T2-DM rats(P<0.05),ameliorated glucose tolerance(P<0.05),high doses of Huangqisan significantly decreased fasting insulin,improved insulin sensitivity index and insulin resistance index(P <0.01),middle dose also improved insulin sensitivity index and insulin resistance index(P<0.01).In the lipid metabolism area,the three doses of Huangqisan significantly reduced serum total cholesterol and low-density lipoprotein levels(LDL-L),lowered free fatty acid(P<0.01-P<0.05);High and middle doses also significantly reduced triglyceride levels in rats,increased levels of high density lipoprotein(HDL-L);and in controlling of serum total cholesterol,LDL-L,and free fatty acids levels,the action of Huangqisan was better than the rosiglitazone group.In addition,each dose of Huangqisan decreased weight gain in these obesity rats(P<0.01-P<0.05), reduced water intake and food intake,significantly reduced urinary excretion in 24 hours(P<0.01-P<0.05),and all those indicators were in favor of the improvement of symptoms of T2-DM rats.By analysing expression of GLU-T4 which gene Closely related with glucose metabolism in myocardial cells,we found that high dose of Huangqisan significantly increased cardiac GLU-T4 mRNA expression (FKO.05),when compared with the model group,this results implys Huangqisan may improve the energy metabolism of myocardial cells by adjusting GLU-T4 thus prevent myocardial lesions.
Huangqisan can prevent or delay the pathological and ultrastructure changes of T2-DM rats'myocardial.Each dose of Huangqisan prevented the pathological changes in varying degrees:preventing myocardial disorders, osteoporosis,myocardial hypertrophy,degeneration,cell shrinkage,cracking, fibroblasts hyperplasia.After treatment by Huangqisan,most rats showed arranging regularly cardiac cells,only myocardial hypertrophy,degeneration in some areas.High and middle doses of Huangqisan showed that in ultrastructure:myofibril basic neatly arranged,mitochondrial and nuclear morphology were normal,the existence of glycogen granules similar with the normal group.Furthermore,High and middle doses of Huangqisan significantly reduced myocardial apoptosis index(P<0.01 - P<0.05),which means Huangqisan can interfere the process of cardiomyocyte apoptosis.
Compared with the normal group,SOD and GSH-Px activity of myocardial in diabetic cardiomyopathy rats were significantly decreased(P<0.01),while content of lipid peroxidation product MDA was significantly increased(P <0.01).High and medium dose of Huangqisan significantly increased SOD,GSH-PX activity(P<0.01 -P<0.05)and decreased MDA content(P<0.05),when Compared with the model group,that implied Huangqisan could improve antioxidant capacity of rats' myocardial then reduce oxidative stress reaction.Excessive oxidative stress can further activate NF-κB,then start or increase the inflammatory response.The results found that NF-κB and TNF-αexpression in diabetic cardiomyopathy rats were increased Obviously,but high and medium dose of Huangqisan inhibited over-expression of NF-κB and TNF-α,thereby inhibited myocardial inflammatory response and cardiovascular response of the whole body.
Diabetic cardiomyopathy rats had those characteristicrcardiac index increased(P<0.01),myocardial interstitial collagen fiber proliferated by analyzing MASSON image(P<0.01),content of hydroxyproline of myocardial tissue also increased detected by biological and chemical method (P<0.05),these results indicated the existence of myocardial fibrosis in diabetic rats which indued cardiac hypertrophy lesions.The study also found that compared with the normal group,mRNA and protein expression of TGF-β1 and its signaling protein SMAD3 in model rats were significantly enhanced (P<0.01).However,each dose of Huangqisan had different degree effects on myocardial fibrosis,significantly reduced the cardiac index,reduced myocardial hydroxyproline content,and inhibited collagen fibers thus prevented cardiac hypertrophy hyperplasia lesions,Huangqisan also significantly inhibited over-expression of TGF-β1 / SMAD3 mRNA and protein in diabetic rats cardiac,which suggested that prevention effects of Huangqisan on myocardial fibrosis maybe mainly related to the inhibition effect of TGF-β1 / SMAD3 signaling pathway.
Conelusion
Because of sustained high blood sugar,disorder of fat and protein metabolism,which induce dysfunction of myocardial cell energy metabolism,resulting in the structure and function damage of myocardial cell, oxidative stress reaction increase then activate NF-κB,which induce systemic and cardiovascular responses thus aggravate cardiac cell injury,resulting in myocardial hypertrophy,degeneration,apoptosis,necrosis and other Pathological changes,all the above-mentioned factors can activate TGF-β1/SMAD3 pathway,eventually leading to proliferation of Myocardial interstitial collagen fibers,and then developed into diabetic myocardial fibrosis,that is diabetic myocardial characteristic disease.Experiment proved that the ancient prescription Huangqisan prevented and treated diabetic cardiomyopathy effectively,mainly through the interference of high blood glucose / oxidative stress / NF-κB / TGF-β1 signal transduction pathway, these effects displayed as that reduce blood glucose,blood lipids and ameliorate insulin resistance,up-regulate GLUT -4 gene expression in cardiac cell,thereby improving myocardial energy metabolism(including glucose metabolism and lipid metabolism);and that enhance cardiac antioxidant capacity,inhibit NF-κB and TNF-αover-expression,reduce heart inflammation, and further control TGF-β1 and SMAD3 gene transcription and protein expression levels,thereby reducing oxidative stress - inflammation - myocardial fibrosis reaction.
糖尿病心肌病是糖尿病重要的慢性心脏并发症之一,是导致糖尿病晚期心功能衰竭和死亡的主要原因,其病理表现主要为心肌细胞肥大、凋亡和心肌间质纤维化等。糖尿病心肌病发病机制尚未完全明了,目前认为这些特征性的病理改变主要与心肌细胞的能量代谢障碍、氧化应激、细胞因子异常、胰岛素抵抗等多种因素有关。由于其病因复杂,其确切的病理机制尚未彻底阐明,临床治疗尚无特效药物,西药主要以对症治疗为主,疗效欠佳。
中医治疗糖尿病的历史源远流长,尤其是在防治糖尿病并发症的整体调节上具有很大的优势,往往起到标本兼顾的治疗作用。因此,寻求安全而有效的中医药疗法对糖尿病及其并发症的治疗有着重要的社会意义和经济意义。
糖尿病在中医属于“消渴”范畴,综合历代医家对消渴病的病因病机和辨证论治的论述发现,气阴不足,燥热内盛是消渴病的主要病机。糖尿病心肌病则属于“消渴”“心悸”“胸痹”“胸痛”“消渴病心病”范畴,本病主要由消渴病日久迁延所致,现代医家认为阴虚燥热是本病基本病机,为标;正气不足、心脾两虚是其病机的关键,为本。为此提出益气养阴为基本治疗原则,本研究所用的方剂就是基于这种治疗原则建立的。
古方黄芪散方源自北宋《圣济总录》,全方由葛根、黄芪、桑白皮三味药组成。葛根生津止渴,升脾中清阳,输津液以溉五脏而滋阴清热,为方中君药;黄芪益气健脾,取其气复津还之意,用为臣药,君臣相配,既可益脾气,又可滋脾阴,清中焦之热而生津止渴以治中消之症;佐以桑白皮甘寒泻肺中伏火,滋阴润燥以疗上消之症,兼调下消,且其甘寒之性以制黄芪其气稍热之性。三药相伍,共奏益气健脾,滋阴清热,生津止渴,三消并治,标本兼顾之效。临床观察和文献研究发现三药单用或与它药联合配伍应用对糖尿病及并发症均有良好疗效,但关于整方的实验研究几近空白。
前期我们通过系列小鼠实验考证了黄芪散的降血糖药效:分别采用链脲佐菌素糖尿病模型、肾上腺素高血糖模型研究黄芪散对病理状态下高血糖和糖耐量的影响;采用正常小鼠研究黄芪散对正常状态下血糖和糖耐量的影响。实验结果表明,黄芪散能明显降低糖尿病小鼠的空腹血糖和改善糖耐量;对肾上腺素性高血糖小鼠体现一定的降血糖作用;黄芪散连续给药3周对正常小鼠空腹血糖和糖耐量没有明显的影响。
在这种基础上,我们通过复合因素建立2型糖尿病实验动物模型,观察一定周期内出现的心肌病变,研究黄芪散对糖尿病心肌病变的干预作用及其保护机制。
目的
系统研究古方黄芪散对糖尿病及其心肌病变的防治作用及其机理,利用现代科学技术阐释传统中医药的治疗机理,同时为古方的临床应用和糖尿病新药开发提供实验依掘。
方法
采用高脂喂养+链脲佐菌素的方法建立具备胰岛素抵抗特征的2型糖尿病大鼠模型,在此基础上观察糖尿病大鼠的心肌病变及药物的干预作用。同时,实验还比较了不同病程的2型糖尿病大鼠心肌病变差异。
黄芪散的干预作用及其机制研究:大鼠高脂饲料喂养4周继而腹腔注射链脲佐菌素,选择链脲佐菌素成模后大鼠随机分为五组,糖尿病心肌病变模型组和黄芪散高、中、低治疗组、罗格列酮阳性对照组。另设正常对照组平行观察。除正常对照组和模型对照组外,各组大鼠分别予以不同药物干预治疗8周,同时维持高脂饲料造模至实验结束。
实验中,定期记录大鼠的体重、进食量、饮水量、尿量等一般体征,检测大鼠空腹血糖、餐后血糖和葡萄糖耐量。实验全部结束后处死大鼠,采血分离血清,采用生化法检测大鼠空腹血糖、糖化血清蛋白、血脂常规、游离脂肪酸等指标,采用放射免疫方法测定空腹胰岛素、肿瘤坏死因子(TNF-α)等指标;取部分心肌组织制备组织匀浆液,测定心肌组织超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、心肌羟脯氨酸的含量变化;对各组大鼠心脏进行HE、MASSON染色,观察糖尿病大鼠心肌组织的病理改变;应用透射电镜观察心肌组织的超微结构改变;应用原位术端标记法(TUNEL)观察心肌细胞凋亡情况;采用RT-PCR方法检测大鼠心肌组织葡萄糖转运蛋白4(GLU-T4)、核转录因子(NF-κB)、转化生长因子-β1(TGF-β1)及其信号蛋白SAMD3的mRNA表达情况;采用免疫组织化学方法观察TGF-β1和SAMD3蛋白在心肌组织的表达情况。
结果
1.高脂喂养+链脲佐菌素方法可以建立2型糖尿病大鼠心肌病变模型:首先,该模型具有多饮、多食、多尿、肥胖、胰岛素抵抗以及糖、脂肪、蛋白质代谢紊乱等特征,与临床上2型糖尿病患者的特征比较接近;成模8周后可出现典型糖尿病心肌病变:心肌细胞肥大、变性、凋亡、间质胶原纤维增生,心肌细胞超微结构损伤性变化(肌原纤维排列紊乱,线粒体明显增多,线粒体嵴断裂、溶解,细胞核核膜不完整,异染色质凝聚等)。随着时间的延长,心肌纤维化和凋亡现象加重,成模12周以后大鼠心肌出现大片胶原纤维增生,心肌明显纤维化病变。两个病程的模型组大鼠在体重、血糖、血脂、胰岛素方面的变化没有明显区别,都高于正常组。
2.在糖代谢方面,与模型组相比,黄芪散高、中剂量均可降低糖尿病大鼠的空腹血糖、糖化血清蛋白(P<0.05),改善葡萄糖耐量,黄芪散高剂量具有显著降低空腹胰岛素以及改善胰岛素抵抗的作用(P<0.01),黄芪散中剂量也具有明显改善胰岛素抵抗的作用(P<0.01)。在脂代谢方面,黄芪散各剂量均可显著降低模型大鼠的血清总胆固醇和低密度脂蛋白水平,降低游离脂肪酸(P<0.01～P<0.05);黄芪散高、中剂量还可显著降低大鼠的甘油三酯水平(P<0.05),黄芪散中剂量明显升高大鼠的高密度脂蛋白水平(P<0.05);而且在血清总胆固醇和低密度脂蛋白水平,游离脂肪酸的改善方面,作用要略优于罗格列酮组。此外,黄芪散各剂量还可明显减少肥胖的2型糖尿病大鼠体重的增加(P<0.01～P<0.05),减少糖尿病大鼠的饮水量和进食量,明显减少24h尿量(P<0.01～P<0.05),这些都有利于糖尿病临床症状的改善。进一步对与心肌细胞糖代谢相关的关键葡萄糖转运蛋白GLU-T4表达的研究结果发现,与模型组相比,黄芪散高剂量能明显抑制糖尿病大鼠心肌GLU-T4 mRNA表达下降(P<0.05),提示黄芪散可能通过调节GLU-T4改善心肌细胞的能量代谢从而防止心肌病变。
3.黄芪散可以防止或延缓糖尿病大鼠心肌的形态学病理改变:黄芪散各剂量可以不同程度防止大鼠心肌出现心肌细胞排列紊乱,疏松,心肌细胞肥大、变性,细胞核固缩、裂解,成纤维细胞增生,胶原纤维增生的病理变化。用药后大部分大鼠心肌细胞基本排列整齐,部分病变区有心肌细胞肥大、变性。黄芪散高、中剂量可明显防止大鼠心肌的超微结构改变:高、中剂量组肌原纤维排列基本整齐,线粒体和细胞核形态基本正常,糖原颗粒存在状况与正常组相似。黄芪散高、中剂量还可明显降低心肌细胞凋亡指数(P<0.01～P<0.05),提示黄芪散可以干扰心肌细胞凋亡程序。
4.黄芪散可以抑制氧化应激-炎症反应:糖尿病心肌病变模型组大鼠心肌SOD、GSH-Px活性均显著低于正常组(P<0.01),脂质过氧化产物MDA含量则显著高于正常组(P<0.01)。黄芪散高、中剂量组大鼠心肌SOD、GSH-P_X活性显著高于模型组(P<0.01～P<0.05),MDA含量显著低于模型组(P<0.05),提示黄芪散能提高大鼠心肌的抗氧化能力,减轻氧化应激反应。过激的氧化应激反应可以进一步激活NF-κB,启动或加重炎症反应。研究结果发现,糖尿病心肌病变模型组大鼠NF-κB和TNF-α表达增强,而黄芪散高、中剂量能抑制NF-κB和TNF-α的过度表达,从而抑制心肌和全身的炎症反应。
5.黄芪散可以通过调控TGF-β1/SMAD3通路显著抑制心肌纤维化病变:糖尿病心肌病变大鼠心脏指数增加(P<0.01),MASSON图像分析发现心肌间质胶原纤维增生(P<0.01),生化检测也证实心肌组织羟脯氨酸含量增加(P<0.05),提示糖尿病大鼠存在心肌纤维化从而导致心肌肥厚病变。研究发现,模型组大鼠TGF-β1及其信号蛋白SMAD3的mRNA和蛋白表达均明显增强(P<0.01)。而黄芪散各剂量具有不同程度抗心肌纤维化的作用,可明显降低心脏指数,减少心肌羟脯氨酸含量,抑制胶原纤维增生从而抑制心肌肥厚病变,并显著抑制大鼠心肌TGF-β1/SMAD3 mRNA和蛋白的强表达,提示黄芪散抑制心肌纤维化的作用可能主要是与抑制TGF-β1/SMAD3信号通路有关。
结论
糖尿病心肌病的发生发展存在高血糖/氧化应激/NF-κB/TGFβ1信号传导途径,表现为糖尿病由于持续的高血糖和脂肪、蛋白质代谢紊乱以及胰岛素抵抗,导致心肌细胞能量代谢障碍,引起心肌细胞结构与功能受损,氧化应激反应逐渐加重,可以激活NF-κB,启动下游基因的转录,诱导全身性和心血管反应加重心肌细胞的损伤,进一步可促使TGF-β1/SMADs通路活化。上述各种因素最终可导致心肌细胞肥大、凋亡、坏死、间质胶原纤维增生,并发展成为糖尿病心肌间质纤维化,即糖尿病性心肌特征性病变。本研究结果证明,以益气养阴为主要治则的古方黄芪散能有效防治糖尿病心肌病变,作用与干扰高血糖/氧化应激/NF-κB/TGF-β1信号传导途径有关,具体表现为减少尿量、饮食量、进水量,以及改善胰岛素抵抗、降低血糖和改善糖耐量、调节血脂、上调GLUT-4 mRNA表达,改善心肌能量代谢;增强心脏抗氧化能力,抑制NF-κB和TNF-α的表达,减轻心脏和全身炎症反应;降低TGF-β1和SMAD3 mRNA及蛋白表达等多方面的作用,从而整体调节和防治糖尿病及其心肌病变。
Diabetes Melliuts(DM) is a multi-cause of endocrine and metabolic disease, which caused by defects of insulin secretion and / or activity also it is a common and chronic life-long disease.It is reported that the disability and mortality rate of diabetes is only after those of tumor and cardiovascular diseases(ranking No.3),of which about 95%for type 2 diabetes.The cardinal harm of DM is determined by various chronic complications(ex heart,brain, kidney and blood vessels complications),which not only cause significant harm to human life and health,and also cause the expensive medical costs that bring heavy financial burden to patients.Therefore DM has been become one of the major public health problems all of the world.That is why prevention and treatment of diabetes is not a simple problem of lowering blood glucose,the key issues is the long-term control of high blood glucose,high blood lipids and high blood pressure,then preventing and delaying the incidence of complications,thereby reducing the mortality of DM.In variety of complications,heart disease one of the most serious and outstanding problems,including diabetic coronary heart disease,diabetic cardiomyopathy and diabetic cardiac autonomic neuropathy.Therefore,now people refer to "the full treatment for lowering blood glucose convert to controlling of cardiovascular risk factors" as " the revolution of prevention and treatment strategies of type 2 diabetes."
Diabetic cardiomyopathy is one of the important chronic complications of diabetes,which often lead to later-period heart failure and death.The pathogenesis of diabetic cardiomyopathy have not yet been fully understood, now it is considered that the reasons such as the encumbrance of myocardial cells energy metabolism,oxidative stress,abnormality of cytokines and insulin resistance induce the myocardial lesions,resulted in hypertrophy and apoptosis of myocardial cells,fibrosis of myocardial interstitium and other pathological changes.Because of its complex etiology,and the non-clear pathological mechanism,there is no effective drugs in clinical,mainly treat symptoms by western medicine which only play poor efficacy.
Traditional Chinese medicine have a long history of treating diabetes, especially is good at the prevention and treatment of diabetic complications on the overall regulation,and it often treat symptoms and essence in the same time.Therefore,it has important social and economic significance in searching safe and effective therapy of Chinese medicine for diabetes and its complications.
In tradtional Chinese medicine,diabetes belongs to " Xiaoke ",by synthesizing theroy of etiology,pathogenesis and treatment according to syndrome differentiation of medicals in past dynasties,we could find deficiency of Qi and Yin,excessive dry and heat are the major pathogenesis of Xiaoke diseases,thus tonify qi,nourish yin,and eliminating heat become the main treatment principles of Chinese medicine.Prescriptions used in this study is based on this principle.
The ancient prescription i.e Huangqisan Originated from "Shengji zonglu" in Northern Song Dynasty,the entire prescription is made up of three herbs: Pueraria,Radix Astragali,Morus alba.Sweet and cool Pueraria can produce body fluid to stop thirst,Promote Qing-yang in the spleen,transport body fluid to irrigate five internal organs,nourish yin and eliminate hot,so it plays as a prime-drug in the prescription;Sweet and warm radix Astragali can increase Qi and tonify spleen,which means to body fluid recovering as soon as Qi regaining,so it plays as a official-drug in the prescription,which can treat middle-xiao symptoms combining with the prime-drug by eliminating hot of Zhongjiao accordingly producing body fluid to stop thirst;Sweet and cold Morus alba can clear away fiery in the lung,nourish yin and moisture dry so as to treat up-xiao and down-xiao symptoms,futhermore the Sweet and cold nature can constrain the warm nature of Radix Astragali.The effects of total prescription are increasing Qi and tonifying spleen,nourishing yin and eliminating heat,producing body fluid to stop thirst,treating for three-xiao symptoms,and treating symptoms and essence in the same time.Clinical observation and literature study have found that it have a good effect on diabetes and complications,whether three drugs alone or combined with other drugs.However,the research of the whole prescription(Huangqisan) is vacant.
First of all,we carried out some pre-experimental studies in mice,which used to testing and verifying effects of lowing glucose of Huangqisan.Applying two high blood sugar models to study the effects on pathological high glucose and impaired glucose tolerance,such as diabetic model induced by streptozotocin(STZ),and hyperglycemia model caused by adrenergic;In addition,we used the normal mice to study effects on blood sugar and glucose tolerance of normal state.At last,the experimental results showed that Huangqisan decreased FBG and ameliorated glucose tolerance of diabetic mice induced by STZ;Huangqisan also showed lowing glucose effect on adrenergic model;However,Huangqisan play no evdient influence on normal fasting glucose and glucose tolerance of normal mice after three weeks'administration.
On this basis,we established experimental animal models of type 2 diabetes melliuts(T2-DM)by imitating complex factors,observed cardiac complications during a certain period,and to study the intervention effect and its protection mechanisms of Huangqisan on diabetic cardiomyopathy.
Objective
To study the intervention effect and its protection mechanisms of Huangqisan on diabetic cardiomyopathy,using modern science and technology to explain the mechanism of traditional Chinese medicine theraphy,as well as to provide experimental basis for popularization and application of ancient prescription and clinical practice.
Methods
Applying the methods which combined high fat diet with STZ to establish T2-DM rats model with characteristics of insulin resistance,on the basis of these T2-DM rats we observed the pathological changes of myocardial and the intervention effects of drugs.
The intervention effect of and its protection mechanisms of Huangqisan on diabetic cardiomyopathy:The successful model rats were selected,and randomly divided into five groups:diabetic cardiomyopathy model group,high,medium and low treatment group of Huangqisan,rosiglitazone positive control group.Furthermore,a normal control group was set up for parallel observing.In addition to the normal control group and the model control group,other rats in each group were treated by eight weeks of drugs intervention,the high fat diet were maintained until the end of the experiment.
During the experiment,we regularly recorded general condition of all the rats such as weight,food intake,water intake,urine output,and detected fasting blood glucose,random blood glucose and glucose tolerance of the rats. After the end of the experiment,all rats were killed,blood were collected for serum separating,then the fasting blood glucose,glycosylated serum protein,blood lipid,free fatty acids were detected by biochemical methods, fasting insulin and tumor necrosis factor(TNF-α) were detected by radioimmunoassay method;Cut part of cardiac tissue for tissue fluid, myocardial superoxide dismutase(SOD),malondialdehyde(MDA),glutathione peroxidase(GSH-Px) of content were detected by kits;The pathological changes in heart tissue of the diabetic rats were observed by HE and MASSON staining methods;The ultrastructural changes of myocardial tissue were detected by transmission electron microscopy;Apoptosis condition of myocardial cells observed by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL) method;The mRNA expression of transporter 4(GLU-T4), nuclear transcription factor(NF-κB),transforming growth factor-β1(TGF-β1) and its SAMD3 signal protein were detected by RT-PCR method;The protein expression of TGF-β1 and SAMD3 in cardiac tissue were detected by immunohistochemical method.
Results
The methods combining high fat diet with streptozotocin and then continue high fat diet can create a model of T2-DM in rats,which has characteristic of excessive drink,excessive food,excessive urine,fat,insulin resistance,and metabolic disorders of sugar,fat,protein,etc,and those characteristics were close to T2-DM patients in clinical;The rats showed typical diabetic cardiomyopathy after models having been established for eight weeks:such as cardiac myocyte hypertrophy,degeneration,increasing of apoptotic cells interstitial collagen fibrosis,and myocardial cell injury changes in ultrastructure(disorder of myofibril,increasing of mitochondria, fragmentation and dissolution of mitochondrial cristae,incomplete nuclear membrane and eterochromatin condensation,etc).Accompanied by time prolonging, myocardial fibrosis aggravated after 12 weeks,myocardial of the rats showed large areas of interstitial collagen fibers had proliferated.Two course of model rats had no apparent distinction in the weight,blood sugar,and blood lipid,those were significantly higher than the normal group.
In the glucose metabolism area,when compared with the model control group,high and middle doses of Huangqisan decreased fasting blood glucose, glycosylated serum protein of T2-DM rats(P<0.05),ameliorated glucose tolerance(P<0.05),high doses of Huangqisan significantly decreased fasting insulin,improved insulin sensitivity index and insulin resistance index(P <0.01),middle dose also improved insulin sensitivity index and insulin resistance index(P<0.01).In the lipid metabolism area,the three doses of Huangqisan significantly reduced serum total cholesterol and low-density lipoprotein levels(LDL-L),lowered free fatty acid(P<0.01-P<0.05);High and middle doses also significantly reduced triglyceride levels in rats,increased levels of high density lipoprotein(HDL-L);and in controlling of serum total cholesterol,LDL-L,and free fatty acids levels,the action of Huangqisan was better than the rosiglitazone group.In addition,each dose of Huangqisan decreased weight gain in these obesity rats(P<0.01-P<0.05), reduced water intake and food intake,significantly reduced urinary excretion in 24 hours(P<0.01-P<0.05),and all those indicators were in favor of the improvement of symptoms of T2-DM rats.By analysing expression of GLU-T4 which gene Closely related with glucose metabolism in myocardial cells,we found that high dose of Huangqisan significantly increased cardiac GLU-T4 mRNA expression (FKO.05),when compared with the model group,this results implys Huangqisan may improve the energy metabolism of myocardial cells by adjusting GLU-T4 thus prevent myocardial lesions.
Huangqisan can prevent or delay the pathological and ultrastructure changes of T2-DM rats'myocardial.Each dose of Huangqisan prevented the pathological changes in varying degrees:preventing myocardial disorders, osteoporosis,myocardial hypertrophy,degeneration,cell shrinkage,cracking, fibroblasts hyperplasia.After treatment by Huangqisan,most rats showed arranging regularly cardiac cells,only myocardial hypertrophy,degeneration in some areas.High and middle doses of Huangqisan showed that in ultrastructure:myofibril basic neatly arranged,mitochondrial and nuclear morphology were normal,the existence of glycogen granules similar with the normal group.Furthermore,High and middle doses of Huangqisan significantly reduced myocardial apoptosis index(P<0.01 - P<0.05),which means Huangqisan can interfere the process of cardiomyocyte apoptosis.
Compared with the normal group,SOD and GSH-Px activity of myocardial in diabetic cardiomyopathy rats were significantly decreased(P<0.01),while content of lipid peroxidation product MDA was significantly increased(P <0.01).High and medium dose of Huangqisan significantly increased SOD,GSH-PX activity(P<0.01 -P<0.05)and decreased MDA content(P<0.05),when Compared with the model group,that implied Huangqisan could improve antioxidant capacity of rats' myocardial then reduce oxidative stress reaction.Excessive oxidative stress can further activate NF-κB,then start or increase the inflammatory response.The results found that NF-κB and TNF-αexpression in diabetic cardiomyopathy rats were increased Obviously,but high and medium dose of Huangqisan inhibited over-expression of NF-κB and TNF-α,thereby inhibited myocardial inflammatory response and cardiovascular response of the whole body.
Diabetic cardiomyopathy rats had those characteristicrcardiac index increased(P<0.01),myocardial interstitial collagen fiber proliferated by analyzing MASSON image(P<0.01),content of hydroxyproline of myocardial tissue also increased detected by biological and chemical method (P<0.05),these results indicated the existence of myocardial fibrosis in diabetic rats which indued cardiac hypertrophy lesions.The study also found that compared with the normal group,mRNA and protein expression of TGF-β1 and its signaling protein SMAD3 in model rats were significantly enhanced (P<0.01).However,each dose of Huangqisan had different degree effects on myocardial fibrosis,significantly reduced the cardiac index,reduced myocardial hydroxyproline content,and inhibited collagen fibers thus prevented cardiac hypertrophy hyperplasia lesions,Huangqisan also significantly inhibited over-expression of TGF-β1 / SMAD3 mRNA and protein in diabetic rats cardiac,which suggested that prevention effects of Huangqisan on myocardial fibrosis maybe mainly related to the inhibition effect of TGF-β1 / SMAD3 signaling pathway.
Conelusion
Because of sustained high blood sugar,disorder of fat and protein metabolism,which induce dysfunction of myocardial cell energy metabolism,resulting in the structure and function damage of myocardial cell, oxidative stress reaction increase then activate NF-κB,which induce systemic and cardiovascular responses thus aggravate cardiac cell injury,resulting in myocardial hypertrophy,degeneration,apoptosis,necrosis and other Pathological changes,all the above-mentioned factors can activate TGF-β1/SMAD3 pathway,eventually leading to proliferation of Myocardial interstitial collagen fibers,and then developed into diabetic myocardial fibrosis,that is diabetic myocardial characteristic disease.Experiment proved that the ancient prescription Huangqisan prevented and treated diabetic cardiomyopathy effectively,mainly through the interference of high blood glucose / oxidative stress / NF-κB / TGF-β1 signal transduction pathway, these effects displayed as that reduce blood glucose,blood lipids and ameliorate insulin resistance,up-regulate GLUT -4 gene expression in cardiac cell,thereby improving myocardial energy metabolism(including glucose metabolism and lipid metabolism);and that enhance cardiac antioxidant capacity,inhibit NF-κB and TNF-αover-expression,reduce heart inflammation, and further control TGF-β1 and SMAD3 gene transcription and protein expression levels,thereby reducing oxidative stress - inflammation - myocardial fibrosis reaction.
引文
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