The Intervention Effect And Mechanism Of Huangqisan On Diabetic Cardiomyopathy

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.ObjectiveTo 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.MethodsApplying 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.ResultsThe 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.ConelusionBecause 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.