| Diabetic mellitus(DM),an endocrine and metabolic disease, is rapidly becoming one of the most serious chronic diseases that threaten human health globally.Insulin resistance is central to the pathophysiology of DM. In our country, a rapid increase in the prevalence of DM has been reported. A recent study suggested that China has overtaken India and has become the global epicentre of the diabetes epidemic, with92.4million adults(9.7%) affected. Cardiovascular diseases(CVD) induced by DM has become the primary cause of death in Diabetic patients,which accounts for nearly70%of the total cause of death.The morbidity of CVD in diabetic patients is2to4times higher than the non-diabetic patients,which seriously threaten the quality of life in diabetic patients and results in a huge economic burden for society and patients.The prevention and treatment of diabetes on CVD is imminent.Therefore,to study the pathogenesis of diabetic myocardial damage and find the new molecular pharmacal target point to prevent and treat DM and its complication will have markedly significance in improving the the quality of life and decreasing the mortality of diabetic patients. G protein-coupled receptors(GPCR) comprise a large protein family of transmembrane receptors related to GTP binding regulatory proteins. They have the same structural features which are composed with7transmembrane domains,3intracellular loops and3extracellular loops. They mediate signal not only be involve in regulating blood pressure and blood glucose,but also regulating immunological response.GPCR has been a hot area of research in cardiovascular disease at present,including M2-muscarinic receptor, β1-adrenergic receptor, al-adrenergic receptor, AT1-receptor,etc. Their autoantibodies all have pathological agonistic activity, since they can represent an important risk factor for future cardiovascular disease. We have discovered that the autoantibodies against al-adrenergic receptor(α1-AA) increased significantly in sera of patients with diabetic cardiomyopathy(DCM), accompanied by neuroendocrine activation, further developped into cardiomegaly cardiac dysfunction, which demonstrated that immune dysfunction might contribute to the pathogenesis of DCM. α1-AA is an IgM or IgG immunoglobulin, which corresponds to amino acids192-218of the second extracellular loop of the human α1-adrenergic receptor. Its function is similar to norepinephrine.The activity of α1-AA could be blocked by α1-receptor inhibitor,which would be the important measure to alleviate the damage of target organs.Now, the research about α1-AA mostly focus on hypertension and preeclampsia. The characteristic change of myocardial ultra-structure and the mechanism of correlated signal transduction in diabetic myocardial damage mediated by α1-AA is still unclear.α1-AA is the ligand of G protein-coupled receptor and the binding specificity with its receptor is very high.This characteristic contributes to the new target of pharmacotherapy. This experiment research the immunologic mechanism of diabetic myocardial damage through the translational medicine. Established the diabetes model with α1-AA, then explored the pathogenesis of myocardial fibrosis in diabetic rats mediated by α1-AA and find the new target of the prevention and treatment for DCM.Transforming growth factor-β1(TGF-β1) is a pleiotropic cytokine, which plays a major role in the regulation of myocardial fibrosis and the transcription and expression of target gene. TGF-β1mediates progressive myocardial fibrosis by stimulating extracellular matrix (ECM) production while inhibiting its degradation. Smads is a key mediator in transferring the TGF-βsignal from extracellular to nucleus, which plays a major role in the regulation of myocardial cells proliferation, growth and apoptosis,while smad2/3is the positive regulating factor of cadiovascular remodeling.It is reported that activated GPCR could activate the TGF-βsignal which participates the activation of various proinflammatory factors and inflammatory factors such as nuclear factor κB(NF-κB), TNF-a and IL-1,which result in the effect of inflammatory cascade amplification.This experiment studies the immunologic mechanism of diabetic myocardial damage mediated by α1-AA through the TGF-P signal and explores the interventional effect of α1-receptor inhibitor on this signaling pathway and the protective mechanism of al-receptor inhibitor on cardiomyopathy of diabetic rats.In this animal experiment, we detect the serumal α1-AA by ELISA after establishment of diabetes model with streptozocin,and then observe the expression of TGF-β1and collagen fiber on myocardium in diabetic rats by strept avidin-biotin complex (SABC) and VG staining.To study the process that activated GPCR could enhance NF-κB activation mediated by α1-AA, thereby promoting the expression of TGF-β1, and result in the effect of inflammatory cascade amplification.Then explored the relationship between α1-AA and the pathogenetic of diabetic myocardial damage.With the confirmation of our hypothesis that inflammation and immunolesion induced by α1-AA could lead to diabetic myocardial damage. Our research priorities go into investigation that how to interrupt this criticality link in the DM cadiovascular complication, predict DM Cadiovascular event and protect the function of the heart early.Based on the experiment as mentioned above, we treated DM rats mediated by α1-AA with α1-receptor inhibitor(Doxazosin, Cardura XL), further to study the possible protective mechanism of Doxazosin on cardiomyopathy of diabetic rats,in order to offer the new theory for the treatment of DM whose serumal α1-AA is positive and expand the new idea of diabetes cardiomyopathy prevention.Chart I The effect of autoantibodies against α1-adrenergic receptor on the expression of TGF-β1and smad2/3in the myocardium of diabetic ratsObject:To study the pathogenesis of autoantibodies against α1-adrenergic receptors (α1-AA) on cardiomyopathy of diabetic rats through observe the expression of TGF-β1, and smad2/3in diabetic rats mediated by α1-AA.Methods:1.Induction of diabetes:Diabetes was induced in male Wistar rats (160-170g body weight) by intraperitoneal administration of STZ (single dose of0.5mL/100g b.w.) dissolved in freshly prepared0.1mmol/L citrate buffer, pH4.3after feeding with high-sugar and high-lipid fodder (contained10%lard,10%sucrose and2.5%cholesterol in the general fodder) for four weeks. After72h rats with marked hyperglycemia (FBG>16.7mmol/L) were selected and used for the study. Rats were fed with the general fodder after the establishment of diabetes.2. Group of experiments:Before completion of the experiment, we detect the serumal α1-AA by ELISA in diabetic rats after fasting12h gotten blood from caudal vein.α1-AA positive diabetic rats were used as model (α1-AA positive group),with α1-AA negative diabetic rats (α1-AA negative group) and normal Wistar rats (normal group)of the same age as controls.All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in20-24℃, relative humidity50%-70%. 3. Observation index and laboratory testing①general condition:Observed the change of fur skin, mental status, food consumption, the volume of urine and in-take water and body weight.②ELISA Assay of α1-AA:The epitopes of the second extracellular loop of α1-receptor was synthesized and used as antigens to detect the α1-receptor by ELISA, then calculated the positive rate.③pathologic changes in myocardium:Obtain the myocardial tissue of left ventricular in rats. Pathological changes in the myocardium were observed by electron microscope.④The expression of TGF-β1and smad2/3:The expression of TGF-β1and smad2/3in myocardium of left ventricle were detected by immunohistochemical staining and observed by light microscope,and calculated the value of MOD.Results:1.30rats were used to established the DM models. The rate of successful models was80%(24/30). And the death rate of DM model rats was zero(0/32).2. The positive rate of α1-AA:There was a rat whose α1-AA was positive in normal group, the positive rate is8.33%(1/12); there were eleven rats in DM model group, the positive rate is45.83%(11/24).3.Change of the body weight:At the end of this experiment,the weight in α1-AA positive group and negative group were lower than before(both P<0.01),but normal group was higher than before(P<0.01).4. Pathologic changes in myocardium:Myocardial pathological changes in α1-AA positive group were the most serious,mitochondrial reduce,arrangement disorder,show vacuolar degeneration,interstitial collagen hyperplasia, microvascular basement membrane thickening.There was on obviously abnormal in normal group.5.The expression of TGF-β1:In α1-AA positive group,the expression of TGF-β1 in cardiac muscle cell and interstitial cell were higher than α1-AA negative group and normal group(respectively,P<0.05, P<0.01).6. The expression of smad2/3:In α1-AA positive group,the expression of smad2/3in subendocardial myocardium and nucleus were higher than α1-AA negative group and normal group(respectively,P<0.05, P<0.01).Conclusions:1. The α1-AA highly express in diabetic rats, which demonstrates that immune dysfunction might contribute to the pathogenesis of DM.2. The α1-AA could effect the expression of TGF-β1and smad2/3in the myocardium of diabetic rats, resulting in the aggratate of cardiomyopathy through increasing the expression of TGF-β1.That is the damage of myocardium in diabetic rats is closely related to autoimmunity reaction mediated by α1-AA.Chart Ⅱ The relative investigation of autoantibodies against α1-adrenergic receptor and the expression of type I collagen fiber and type Ⅲ collagen fiber, in myocardium of DM ratsObject:To study the correlation of α1-AA and myocardial fibrosis and and its possible pathogenic mechanism in diabetic rats through observe the expression of type I collagen fiber and type Ⅲ collagen fiber in diabetic rats mediated by α1-AA.Methods:1.Induction of diabetes:The same with chart I.2.Group of Experiments:The same with chart I.3. Observation index and laboratory testing: ①general condition,②ELISA Assay of α1-AA,③pathologic changes in myocardium,④The expression of type I collagen fiber and type Ⅲ collagen fiber: The same with chart I.⑤Changes of collagen metabolism in myocardium:Collagen fiber change in myocardium were detected by Van Gieson staining(VG) and observed by light microscope.Results:The result of SABC:In α1-AA positive group.the expression of type I collagen fiber and type Ⅲ collagen fiber in interstitial cell were higher than α1-AA negative group and normal group(respectively,P<0.05, P<0.01); The result of VG staining: Myocardial collagen fiber in α1-AA positive group with VG staining significantly increased and were malalignmen. Compared with α1-AA negative group and normal group, myocardial collagen deposition in α1-AA positive group obviously increased. The remaining results same with above.Conclusions:1. The α1-AA highly express in diabetic rats, which demonstrates that immune dysfunction might contribute to the pathogenesis of DM.2. The α1-AA could effect the expression of type I collagen fiber and type Ⅲ collagen fiber in the myocardial interstitial cell of diabetic rats. The progress of myocardial fibrosis in diabetic rats is closely related to α1-AA. The immunolesion participates the progress of DM myocardial damage.Chart Ⅲ The study on protective mechanism of the treatment of doxazosin on myocardium in α1-adrenergic receptors antibody mediated diabeticrats Object:To observe the effects of α1-receptor inhibitor (Doxazosin) on the expression of transforming growth factor β1(TGF-β1) and collagen fiber in α1-AA positive diabetic rats, and investigate the protective mechanism of Doxazosin on cardiomyopathy of diabetic rats whether it could reduce the expressing of TGF-β1to alleviate myocardial fibrosis in DM rats.Methods:1. Production of intragastric administration drug:Doxazosin (4mg/slice), dispensing the drug according to0.36mg/kg dosage and administer10ml/kg per rat capacity after grinding into powder.2. Induction of diabetes:The same with chart I.3. Group of Experiments:Wistar rats were divided into five groups:Group A(diabetic non-mediated group), Group B(diabetic doxazosin intervention group), Group C(diabetic α1R-Ab mediated group), Group D(diabetic both aiR-Ab mediated and doxazosin intervention group).After establishment of diabetes model with Streptozocin, Group C and Group D were syringed α1-AA (100μg/100g) by tails vein at0,4,8,12and16weeks. Doxazosin (0.36mg/kg) was administered for16weeks per day in Group B and Group D. Other groups were given the same volume of saline every day. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in20-24℃, relative humidity50%-70%.4. Observation index and laboratory testing:①General condition,②Pathologic changes in myocardium,③The expression of TGF-β1and type I collagen fiber:The same with chart I.Results: 1.40rats were used to established the DM models. The rate of successful models was90%(36/40). And the death rate of DM model rats was zero(0/36).2. By the end of the experiment, DM rats’weight were significantly lower than before (P<0.01).3. Expression of TGF-β1and type I collagen fiber in Group B were lower than Group A (P<0.05). Expression of TGF-β1and type I collagen fiber on myocardium in Group D were lower than Group C (P<0.05).4. Myocardial pathological changes in Group C were the most serious, mitochondrial reduce, arrangement disorder, show vacuolar degeneration, interstitial collagen hyperplasia, microvascular basement membrane thickening. Compared with Group C, the cardiomyopathy of Group D showed a decreased damage. Compared with Group A, the cardiomyopathy of Group B showed a decreased damage as well.Conclusions:Doxazosin can suppress expressions of TGF-β1and type I collagen fiber in myocardium of diabetic rats with α1-AA mediated, thus resulting in the alleviate of myocardial fibrosis and protective effect of myocardium in diabetic rats. |
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