糖尿病心肌病发病机制、早期诊断和治疗的实验研究
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摘要
背景
心血管病变是糖尿病患者的主要死亡原因,约80%死于心血管病。糖尿病心肌病(diabetic cardiomyopathy,DCM)作为一种独立的糖尿病心脏并发症日益引起重视,诊断DCM的三个要素是:(1)明确的长期糖尿病病史可能引起的心脏损伤。(2)存在心脏收缩、舒张功能异常或其他结构功能损伤的证据。(3)可以排除其他已知的心脏疾病或其他有关疾病引起的心脏损害。DCM发生后,随着心功能的进行性恶化,死亡率很高。近20余年来,国内外学者在DCM方面进行了大量的基础和临床研究,虽然取得了阶段性成果,但DCM的发生机制、早期诊断和治疗措施,仍处在探索中,深入研究DCM的发生机制,探讨阻断DCM发病途径无疑对DCM的诊治具有重要意义。
DCM的发生涉及多种影响因素。临床研究表明,DCM通常以舒张性心力衰竭为早期表现,即心肌松弛性减低和僵硬度增大,心肌间质纤维化在舒张性心力衰竭的发生发展中起重要作用。无高血压和冠心病的糖尿病患者心肌活检也证实有心肌间质纤维化,因此,心肌间质纤维化在DCM发病中的作用逐渐引起人们的重视,但其发生机制尚不明确。在糖尿病肾脏纤维化和细胞培养实验中证实,血糖可以刺激TSP-1表达升高,而TSP-1是非活性型TGFβ_1重要的激活因子,随后激活型的A-TGFβ_1可以通过其细胞膜上的受体,将促纤维化信号传递到细胞内,进一步激活Smads蛋白家族,Smads相关蛋白的激活可以调控纤维基质和相关因子的表达。以上这些信号分子与糖尿病肾病的关系,国外可见零散的研究报道,但这一系列因子与DCM心肌间质纤维化的关系,国内外均未见系统的研究报道。
BackgroundThe main death reason of patients with diabetes mellitus is cardiovasculardiseases and 80% of diabetic patients died for these complications. Diabetic cardiomyopathy (DCM), as an independent diabetic cardiac complication, has been paid more attention to. The three gist of DCM diagnosis was brought forward: (1) A definitive long diabetic duration, which might result in cardiac impairment; (2) The evidence of systolic, diastolic dysfunction damages and structure changes in heart; (3) To exclude the cardiac damage of other known heart diseases and concerned diseases. The mortality of DCM is very high along with the progressed cardiac function deterioration. For recent two decades, a number of basical and clinical studies on DCM have been finished. The phasic achievements were obstained, but the machanism, early diagnosis and therapeutic measures of DCM were still explored. It is important for treatment of DCM to explore deeply the machanism and the methods to block the pathway of DCM development.The development of DCM is multifactorial. In clinical study, DCM was characterized by diastolic heart failure, decreased relaxation and increased stiffness at the early stage. The myocardial interstitial fibrosis is the main mechanism of DCM development and was observed in myocardial biopsy of diabetes mellitus without hypertension and coronary heart disease. The effects of myocardial interstitial fibrosis on DCM were gradually paid attention to, but the mechanism is still unclear. In a foreign study about interstitial fibrosis of diabetic nephropathy and cell culture, it was
reported that high glucose levels could stimulate TSP-1 expression, which is an important activator of latent-TGFPj (L-TGFp,). Active-TGFpi (A-TGFp,) can propagate the fibrosis-inducing signal into cells by its cell-membrane receptor to activate intracellular Smads family members. Activatory Smads can regulate and control expression of collagenous fibers and related factors. There were a few of reports about the relationship of these molecules and diabetic nephropathy, but the relationship between these molecules and DCM was still not reported systematically. According to the comprehensive analysis on the studies of cell signal pathway on DCM, we supposed that "Glucose/TSP-l/TGFP)/Smads" signal transduction pathway (GTTS pathway) might exert important effects on DCM. We studied the effects of GTTS pathway on occurrence and progress of DCM by molecular biology, cytobiology and echocardiography. In addition, some studies reported that RAS was activated in hyperglycemic environment. As an important factor to stimulate TSP-1 expression, Angll can induce activation of GTTS fibrosis pathway by its ATI receptor. By using valsartan, the ATI receptor antagonist, to treat diabetic animals, we want to unravel the effect of valsartan and its molecular-biological mechanism. It is anticipated to establish new target point and method for early diagnosis and treatment of DCM. Objective(1) To establish a DCM animal model of type 2 diabetes mellitus.(2) To observe the damages of myocardial ultrastructure and cardiac function.(3) To prove the existence of GTTS pathway in the deterioration of DCM. To explore the effects of GTTS pathway in DCM by the molecular biological techniques.(4) To evaluate the effects of valsartan on GTTS pathway and the feasibility of clinical treatment on DCM. To offer new target molecules for DCM treatment. Methods40 male Wistar rats were randomly divided into 3 groups: a control group (n=8), a DCM group (n=16) and a valsartan group (n=16). The control group rats were fed a normal chow diet. The DCM group and valsartan group rats were fed a high-fat and high-calorie diet. After 5 weeks on either diet, the DCM and valsartan groups were
given with STZ (30mg/kg) by intraperitoneal injection, but the control group animals were injected with equal citrate acid/sodium citrate buffer solution after 12 hours fast. The standards of diabetic animals are the concentrations of fasting blood glucose ^ ll.lmmol/1 by 2 consecutive analyses and the insulin sensitivity index (ISI) is lower than that of the control group. The animals, which did not reach the above standard, were excluded. After that, the valsartan group animals were dosed orally with valsartan (30mg/kg) and the other two groups animal were given with 0.9% saline every day for 16 weeks duration after glucose was heightened. The detection contents were listed below. (1) The animals were weighted every week and the contents of fast blood-glucose were detected every two weeks. (2) The contents of fast blood-glucose, insulin, triglyceride (TG), cholesterol (Choi) and ISI were analyzed before STZ injection, one week after STZ injection and at the end of the experiment. (3) The common echocardiographic indexes were detected before the experiment, the 12th week after hyperglycemia and at the end of the experiment. (4) Electrocardiograms of animals were recorded and hemodynamics indexes were measured by cardiac catheterization. (5) Myocardial ultrastructral and histopathological changes were observed. (6) The content of collagen were quantified by Masson-staining. (7) The mRNA expression of TSP-1, TGFpi, TGFpR II, Smad2, Smad3 and Smad7 of GTTS pathway were detected by quantification real-time RT-PCR. (8) The protein levels of TSP-1, A-TGFpj, L-TGFPi, TGFpR II, P-Smad2, P-Smad3 and Smad7 were detected by Western-blot and immunohistochemistry. Results1. Body weight and biochemical indices measurementBefore STZ injection, body weight values of the DCM and valsartan groups were significantly higher than that of the control group (PO.05). Fast blood-glucose was insignificantly different between the three groups (P>0.05). The content of fast insulin of the other two groups were significantly higher than that of the control group (P<0.05), but ISI of the other two groups were significantly lower than that of the control group (P<0.01). Serum TG and Choi of the other two groups were significantly higher than those of the control group (P<0.05).
One week after STZ injection, body weight values of the three groups were insignificant (P>0.05). Fast blood-glucose of the other two groups was obviously increased, compared with that of the control group (P<0.01). The content of fast insulin was insignificantly different between the three groups (P>0.05). ISI of the other two groups were still significantly lower than that of the control group (P<0.01). Serum TG and Choi of the other two groups were still significantly higher than those of the control group (PO.01).At the end of the experiment, body weight values of the DCM and valsartan groups were obviously decreased, compared with that of the control group (PO.01). Fast blood-glucose of the other two groups was still significantly higher than that of the control group (P<0.01). The content of fast insulin was still insignificantly different between the three groups (P>0.05). ISI of the other two groups were still significantly lower than that of the control group (PO.01). Serum TG and Choi of the other two groups were still significantly higher than those of the control group (PO.01).2. Echocardiographic detectionMitral and tricuspid valvular regurgition detection: None of 8 animals in the control group showed mitral and tricuspid valvular regurgitation. In 11 animals of the DCM group, mild mitral valvular regurgitation was occurred in 3 rats; moderate mitral valvular regurgitation was occurred in one rat; severe mitral valvular regurgitation was occurred in one rat; mild tricuspid valvular regurgitation was occurred in one rat; moderate tricuspid valvular regurgitation was occurred in 2 rats; severe tricuspid valvular regurgitation was occurred in one rat; 2 rats of the DCM group showed both mitral and tricuspid valvular simultaneous regurgition; 4 rats of the DCM group did not show valvular regurgition. In 13 animals of the valsartan group, mild mitral valvular regurgitation was occurred in 2 rats; moderate mitral valvular regurgitation was occurred in one rat; mild tricuspid valvular regurgitation was occurred in 2 rats; moderate tricuspid valvular regurgitation was occurred in one rat; there was no rats, which showed severe mitral or tricuspid valvular regurgitation and the two valvular simultaneous regurgitation; 7 rats of the valsartan group did not
show valvular regurgition. There were no rats with valvular regurgitation in the control group. The incidence of valvular regurgitation in the DCM group was 63.64% and 46.15% in the valsartan group. Fisher analysis: Compared with that of the control group, the incidence of valvular regurgitation in the DCM group and the valsartan group was significantly increased (PO.05). Compared with that of the DCM group, the incidence of valvular regurgitation in the valsartan group was decreased, but which was insignificant (P>0.05).The common echocardiographic indices: 12 weeks after hyperglycemia, Compared with the indices of the control group, left atrial dimension (LAD) of the DCM group enlarged obviously (P<0.01), left ventricular ejection fraction (LVEF) of the DCM group was obviously decreased (P<0.05). LAD of the valsartan group was significantly smaller than that of the DCM group (PO.05).At the end of the experiment, compared with the indices of the control group, LAD, left ventricular dimension (LVD) and right ventricular dimension (RVD) of the DCM group enlarged obviously (P<0.05~0.01). LVEF of the DCM group was obviously decreased (P<0.05). LAD and LVD of the valsartan group were significantly smaller than those of the DCM group (P<0.05). LVEF of the valsartan group was significantly higher than that of the DCM group (PO.05). LAD of the valsartan group was significantly larger than that of the control group (P<0.05).3. Electrocardiogram recordationElectrocardiograms (ECG) of 8 animals in the control group were all normal and ECGs of 11 rats in the DCM group were all abnormal. 9 rats of the DCM group showed characteristic QRS wave interchanges and 2 rats of the DCM group showed complex arrhythmia. Change of the valsartan group ECG was mild. 6 rats of the valsartan group showed normal ECG, 7 rats showed QRS wave interchanges ECG, and none of the valsartan group showed complex arrhythmia. There were no rats with arrhythmia in the control group. The incidence of arrhythmia was 100% in the DCM group and 53.85% in the valsartan group. Fisher analysis: Compared with that of the control group, the incidence of arrhythmia in the DCM group and the valsartan group was significantly increased (PO.05~0.01). Compared with that of the DCM group,
the incidence of arrhythmia in the valsartan group was significantly decreased (PO.05).4. Hemodynamic indices determinationAt the end of the experiment, compared with the indices of the control group, left ventricular systolic pressure (LVSP) of the DCM group decreased obviously (P<0.01), left ventricular end-diastolic pressure (LVEDP) increased significantly (PO.01), the peak left ventricular pressure descending rate (-dp/dtmax) and the peak left ventricular pressure ascending rate (dp/dtmax) decreased significantly (PO.01), while the relaxation time constant (T) of the DCM group was longer than that of the control group (P<0.01). Compared with the indices of the DCM group, LVSP of the valsartan group increased obviously (P<0.05), LVEDP decreased significantly (P<0.05), -dp/dtmax was increased obviously (P<0.05). The T of the valsartan group was shorter than that of the DCM group (P<0.05). LVEDP of the valsartan group was higher than that of the control group (P<0.05). The -dp/dtmax of the valsartan group was lower than that of the control group (P<0.05).5. Ultrastructural change observation by transmission electron microscopy The left ventricular myocytes from the control group arranged regularly. Thepericellular membrane was uninterrupted and intact. The thick and thin myofilament arranged regularly. The sarcomere and light dark band were clear. The uniformly sized mitochondrial was abundant and showed round or oval shape. The nuclear membrane was smooth and intact. A typical distribution of heterochromatin in the form of clusters at the nuclear membrane was present in all cardiomyocyte nuclei. Intercalated disk was clear, intact and normally structured. A little fibroblast and collagenous fibers distributed in extra-cellular matrix. The microvessel lumen was normal and the structure of endothelial cell was normal.The left ventricular myocytes from the DCM group arranged irregularly. The pericellular membrane was interrupted and unclear. The local myofibril was disintegrated. The myofilament was distorted and interrupted. The sarcomere was in a bad apposition. The swelling mitochondrial increased and accumulated. The finger print mitochondrial could be found. The nuclear shape was abnormal with deep notch.
There was accumulative euchromatin. The unclear Intercalated disk was distorted and interrupted. A lot of collagenous fibers distributed in extra-cellular matrix. The microvessel lumen was narrow. The endothelial cell was swelling obviously and protruded to the lumen.Compared with the DCM group, the ultrastructural change of the valsartan group was obviously improved. The myocytes from the valsartan group arranged more regularly than the DCM group. The phenomenon that the local myofibril was disintegrated was decreased. The T tubule was moderately dilated. The mitochondrial was more regular than the DCM group and the finger print mitochondrial was not found. The nuclear shape was more regular than the DCM group. Intercalated disk was improved. The collagenous fibers in extra-cellular matrix decreased obviously and the swelling endothelial cell of microvessel was alleviated.6. Pathological detectionThe myocytes from the control group arranged regularly. The size of the nuclear was uniform. The staining cytoplasm was homogeneous. The myocytes from the DCM group arranged irregularly. The nuclear was irregular and the interrupted myofibril arranged irregularly. The valsartan group was obviously improved. The myocytes from the valsartan group arranged more regularly than the DCM group. The interrupted myofibril was not common. The nuclear shape was more regular than the DCM group.7. The content of collagen detection by Masson-stainingThe myocyte was red or yellow and the collagen was green or blue. The collagen tissue was appropriate arranged among cardiomyocytes in the control group. However, collagen tissue increased markedly, and disrupted in some area in the DCM group. The collagen tissue decreased and arranged regularly in the valsartan group, compared with the DCM group. Quantitative analysis results: The content of collagen in the DCM group was higher significantly than that of the control group (P<0.01). The content of collagen in the valsartan group was lower significantly than that of the DCM group (P<0.05). There was a significantly positive correlation of the collagen content with the fast blood-glucose in the DCM group (r = 0.746, PO.01).
8. Immunohistochemistry detectionThe positive reaction of TSP-1, A-TGFp, and TGFpRlI protein were stained brown and absent in myocardial cytoplasm. There was uniform distribution of weak brown granule in the control group. There was thick brown granule in the DCM group. The granule of the valsartan group was weaker than the DCM group.There was uniform distribution of weak brown granule on L-TGFpi in the control group. The granule of the DCM group and the valsartan group was thicker than the DCM group.It is a rare case to observe the expression of P-Smad2/3 in the control group. There was obvious brown granule of P-Smad2/3 in the DCM group. The granule of P-smad2/3 in the valsartan group was weaker than the DCM group.There was uniform distribution of weak brown granule on Smad7 in the control group. There was thick brown granule on Smad7 in the DCM group. The granule of the valsartan group was even thicker than the DCM group.9. The mRNA expression of several genes on GTTS pathwayThe expression of TSP-1 mRNA in the DCM group was higher significantly than that of the control group (PO.05). The expression of TSP-1 mRNA in the valsartan group was lower significantly than that of the DCM group (P<0.05).The expression of TGFpi mRNA in the DCM group was higher significantly than that of the control group (PO.01). The expression of TGFPi mRNA in the valsartan group was lower significantly than that of the DCM group (P<0.05) and higher than that of the control group (PO.05).The expression of TGFPIIR mRNA in the DCM group was higher significantly than that of the control group (PO.05). The expression of TGFp IIR mRNA in the valsartan group was lower significantly than that of the DCM group (PO.05).The expression of Smad2 mRNA in the DCM group was higher significantly than that of the control group (PO.01). The expression of Smad2 mRNA in the valsartan group was lower significantly than that of the DCM group (PO.05) and higher than that of the control group (PO.05).The expression of Smad3 mRNA in the DCM group was higher significantly
than that of the control group (P<0.05). The expression of Smad3 mRNA in the valsartan group was lower significantly than that of the DCM group (P<0.05).The expression of Smad7 mRNA in the valsartan group was higher significantly than those of the other two groups (PO.05). The expression of Smad7 mRNA in the DCM group was higher significantly than that of the control group (P<0.05).The Smad2/Smad7 in the DCM group was higher significantly than that of the control group (P<0.05). The smad2/smad7 in the valsartan group was lower significantly than that of the DCM group (P<0.05).The Smad3/Smad7 in the DCM group was higher significantly than that of the control group (P<0.05). The Smad3/Smad7 in the valsartan group was lower significantly than that of the DCM group (PO.05).In the DCM group, the fast blood-glucose showed a significantly positive correlation with the expression of TSP-1, TGFp), Smad2 mRNA (r=0.762, 0.714, 0.686, PO.05~0.01).In the DCM group, the collagen content showed a significantly positive correlation with TSP-1, TGFp,, TGFp IIR, Smad2 mRNA (r=0.717, 0.632, 0.675, 0.703, PO.05).In the DCM group, there was a significantly positive correlation of TSP-1 mRNA with LVEDP (r = 0.658, PO.05), in contrast, there was a significantly negative correlation of TSP-1 mRNA with LVSP and -dp/dtmax (r = -0.605,-0.694, PO.05). There was a significantly positive correlation of TGFPi mRNA with LVEDP (r = 0.689, PO.05), in contrast, there was a significantly negative correlation of TGFpi mRNA with -dp/dtmax and LVEF (r = -0.634, -0.647, PO.05). There was a significantly negative correlation of TGFP IIR mRNA with dp/dtmax and -dp/dtmax (r = -0.628, -0.729, PO.05).There was a significantly positive correlation of Smad2 mRNA with LVEDP (r = 0.669, PO.05), in contrast, there was a significantly negative correlation of Smad2 mRNA with -dp/dtmax (r = -0.738, PO.05). There was a significantly positive correlation of Smad3 mRNA with LVEDP (r = 0.746, PO.05), in contrast, there was a significantly negative correlation of Smad3 mRNA with
-dp/dtmax (r = -0.639, PO.05). There was no insignificant correlation of Smad7 mRNA with the other indices.10. The protein expression of several factors on GTTS pathwayThe expression of TSP-1 protein in the DCM group was higher significantly than that of the control group (P<0.05). The expression of TSP-1 protein in the valsartan group was lower significantly than that of the DCM group (P<0.05).The expression of A-TGFPi protein in the DCM group was higher significantly than that of the control group (PO.01). The expression of A-TGFPi protein in the valsartan group was lower significantly than that of the DCM group (PO.05).The expression of L-TGFpi protein in the DCM group and the valsartan group were both higher significantly than that of the control group (P<0.05). There was no difference of the expression of L-TGF|3i protein between the DCM group and the valsartan group (P>0.05).The expression of P-Smad2 protein in the DCM group was higher significantly than that of the control group (PO.01). The expression of P-Smad2 protein in the valsartan group was lower significantly than that of the DCM group (P<0.01) and higher than that of the control group (P<0.01).The expression of P-Smad3 protein in the DCM group was higher significantly than that of the control group (P<0.01). The expression of P-Smad3 protein in the valsartan group was lower significantly than that of the DCM group (PO.05) and higher than that of the control group (PO.05).The expression of Smad7 protein in the DCM group was higher significantly than that of the control group (PO.05). The expression of Smad7 protein in the valsartan group was higher significantly than those of the other two groups (PO.05~0.01).The P-Smad2/Smad7 in the DCM group was higher significantly than that of the control group (PO.05). The P-Smad2/Smad7 in the valsartan group was lower significantly than that of the DCM group (PO.05).The P-Smad3/Smad7 in the DCM group was higher significantly than that of the control group (PO.05). The P-Smad3/Smad7 in the valsartan group was lower
significantly than that of the DCM group (PO.05).In the DCM group, the fast blood-glucose showed a significantly positive correlation with the expression of TSP-1, A-TGFp,, P-Smad2, P-Smad3 protein (r=0.735, 0.726, 0.807, 0.764, PO.05~0.01).In the DCM group, the collagen content showed a significantly positive correlation with the expression of TSP-1, A-TGFpi, P-Smad2, P-Smad3 protein (r=0.750, 0.820, 0.689, 0.744, PO.05~0.01).In the DCM group, there was a significantly positive correlation of TSP-1 protein with LVEDP (r = 0.716, PO.05), in contrast, there was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r = -0.633, -0.669, PO.05). There was a significantly positive correlation of A-TGFPi protein with LVEDP (r = 0.697, PO.05), in contrast, there was a significantly negative correlation of A- TGFpi protein with -dp/dtmax, dp/dtmax and LVEF (r = -0.717, -0.637, -0.683, PO.05). There was a significantly positive correlation of P-Smad2 protein with LVEDP (r = 0.776, PO.01), in contrast, there was a significantly negative correlation of P-Smad2 protein with -dp/dtmax and LVEF (r = -0.705, -0.637, PO.05). There was a significantly positive correlation of P-Smad3 protein with LVEDP (r = 0.656, PO.05), in contrast, there was a significantly negative correlation of P-Smad3 protein with -dp/dtmax (r = -0.696, PO.05). There was no insignificant correlation of L-TGFpi n Smad7 protein with the other indices. Conclusion(1) A DCM animal model of type 2 diabetes mellitus was established by high-calorie diet, STZ injection of small dose, detection of echocardiography and histopathology. The animal model was valuable for the study of the mechanism in DCM.(2) The main histopathologic changes were collagen deposition and myocardial interstitial fibrosis.(3) The early change was the diastolic dysfunction and the mixed dysfunction (diastolic and systolic dysfunction) ocurred at the late stage of disease.(4) The pathyway of Glucose/TSP-1/ TGFpVSmads was proved in type 2
diabetes mellitus by quantification real-time RT-PCR and western blot. GTTS pathway showed obviously correlation with the changes of cardiac function and myocardial interstitial fibrosis. The main mechanism of DCM was GTTS pathway.(5) Valsartan can prevent cardiac from damage in DCM. Blocking the effects of Ang II on the factors of GTTS pathway was an important mechanism.
心血管病变是糖尿病患者的主要死亡原因,约80%死于心血管病。糖尿病心肌病(diabetic cardiomyopathy,DCM)作为一种独立的糖尿病心脏并发症日益引起重视,诊断DCM的三个要素是:(1)明确的长期糖尿病病史可能引起的心脏损伤。(2)存在心脏收缩、舒张功能异常或其他结构功能损伤的证据。(3)可以排除其他已知的心脏疾病或其他有关疾病引起的心脏损害。DCM发生后,随着心功能的进行性恶化,死亡率很高。近20余年来,国内外学者在DCM方面进行了大量的基础和临床研究,虽然取得了阶段性成果,但DCM的发生机制、早期诊断和治疗措施,仍处在探索中,深入研究DCM的发生机制,探讨阻断DCM发病途径无疑对DCM的诊治具有重要意义。
DCM的发生涉及多种影响因素。临床研究表明,DCM通常以舒张性心力衰竭为早期表现,即心肌松弛性减低和僵硬度增大,心肌间质纤维化在舒张性心力衰竭的发生发展中起重要作用。无高血压和冠心病的糖尿病患者心肌活检也证实有心肌间质纤维化,因此,心肌间质纤维化在DCM发病中的作用逐渐引起人们的重视,但其发生机制尚不明确。在糖尿病肾脏纤维化和细胞培养实验中证实,血糖可以刺激TSP-1表达升高,而TSP-1是非活性型TGFβ_1重要的激活因子,随后激活型的A-TGFβ_1可以通过其细胞膜上的受体,将促纤维化信号传递到细胞内,进一步激活Smads蛋白家族,Smads相关蛋白的激活可以调控纤维基质和相关因子的表达。以上这些信号分子与糖尿病肾病的关系,国外可见零散的研究报道,但这一系列因子与DCM心肌间质纤维化的关系,国内外均未见系统的研究报道。
BackgroundThe main death reason of patients with diabetes mellitus is cardiovasculardiseases and 80% of diabetic patients died for these complications. Diabetic cardiomyopathy (DCM), as an independent diabetic cardiac complication, has been paid more attention to. The three gist of DCM diagnosis was brought forward: (1) A definitive long diabetic duration, which might result in cardiac impairment; (2) The evidence of systolic, diastolic dysfunction damages and structure changes in heart; (3) To exclude the cardiac damage of other known heart diseases and concerned diseases. The mortality of DCM is very high along with the progressed cardiac function deterioration. For recent two decades, a number of basical and clinical studies on DCM have been finished. The phasic achievements were obstained, but the machanism, early diagnosis and therapeutic measures of DCM were still explored. It is important for treatment of DCM to explore deeply the machanism and the methods to block the pathway of DCM development.The development of DCM is multifactorial. In clinical study, DCM was characterized by diastolic heart failure, decreased relaxation and increased stiffness at the early stage. The myocardial interstitial fibrosis is the main mechanism of DCM development and was observed in myocardial biopsy of diabetes mellitus without hypertension and coronary heart disease. The effects of myocardial interstitial fibrosis on DCM were gradually paid attention to, but the mechanism is still unclear. In a foreign study about interstitial fibrosis of diabetic nephropathy and cell culture, it was
reported that high glucose levels could stimulate TSP-1 expression, which is an important activator of latent-TGFPj (L-TGFp,). Active-TGFpi (A-TGFp,) can propagate the fibrosis-inducing signal into cells by its cell-membrane receptor to activate intracellular Smads family members. Activatory Smads can regulate and control expression of collagenous fibers and related factors. There were a few of reports about the relationship of these molecules and diabetic nephropathy, but the relationship between these molecules and DCM was still not reported systematically. According to the comprehensive analysis on the studies of cell signal pathway on DCM, we supposed that "Glucose/TSP-l/TGFP)/Smads" signal transduction pathway (GTTS pathway) might exert important effects on DCM. We studied the effects of GTTS pathway on occurrence and progress of DCM by molecular biology, cytobiology and echocardiography. In addition, some studies reported that RAS was activated in hyperglycemic environment. As an important factor to stimulate TSP-1 expression, Angll can induce activation of GTTS fibrosis pathway by its ATI receptor. By using valsartan, the ATI receptor antagonist, to treat diabetic animals, we want to unravel the effect of valsartan and its molecular-biological mechanism. It is anticipated to establish new target point and method for early diagnosis and treatment of DCM. Objective(1) To establish a DCM animal model of type 2 diabetes mellitus.(2) To observe the damages of myocardial ultrastructure and cardiac function.(3) To prove the existence of GTTS pathway in the deterioration of DCM. To explore the effects of GTTS pathway in DCM by the molecular biological techniques.(4) To evaluate the effects of valsartan on GTTS pathway and the feasibility of clinical treatment on DCM. To offer new target molecules for DCM treatment. Methods40 male Wistar rats were randomly divided into 3 groups: a control group (n=8), a DCM group (n=16) and a valsartan group (n=16). The control group rats were fed a normal chow diet. The DCM group and valsartan group rats were fed a high-fat and high-calorie diet. After 5 weeks on either diet, the DCM and valsartan groups were
given with STZ (30mg/kg) by intraperitoneal injection, but the control group animals were injected with equal citrate acid/sodium citrate buffer solution after 12 hours fast. The standards of diabetic animals are the concentrations of fasting blood glucose ^ ll.lmmol/1 by 2 consecutive analyses and the insulin sensitivity index (ISI) is lower than that of the control group. The animals, which did not reach the above standard, were excluded. After that, the valsartan group animals were dosed orally with valsartan (30mg/kg) and the other two groups animal were given with 0.9% saline every day for 16 weeks duration after glucose was heightened. The detection contents were listed below. (1) The animals were weighted every week and the contents of fast blood-glucose were detected every two weeks. (2) The contents of fast blood-glucose, insulin, triglyceride (TG), cholesterol (Choi) and ISI were analyzed before STZ injection, one week after STZ injection and at the end of the experiment. (3) The common echocardiographic indexes were detected before the experiment, the 12th week after hyperglycemia and at the end of the experiment. (4) Electrocardiograms of animals were recorded and hemodynamics indexes were measured by cardiac catheterization. (5) Myocardial ultrastructral and histopathological changes were observed. (6) The content of collagen were quantified by Masson-staining. (7) The mRNA expression of TSP-1, TGFpi, TGFpR II, Smad2, Smad3 and Smad7 of GTTS pathway were detected by quantification real-time RT-PCR. (8) The protein levels of TSP-1, A-TGFpj, L-TGFPi, TGFpR II, P-Smad2, P-Smad3 and Smad7 were detected by Western-blot and immunohistochemistry. Results1. Body weight and biochemical indices measurementBefore STZ injection, body weight values of the DCM and valsartan groups were significantly higher than that of the control group (PO.05). Fast blood-glucose was insignificantly different between the three groups (P>0.05). The content of fast insulin of the other two groups were significantly higher than that of the control group (P<0.05), but ISI of the other two groups were significantly lower than that of the control group (P<0.01). Serum TG and Choi of the other two groups were significantly higher than those of the control group (P<0.05).
One week after STZ injection, body weight values of the three groups were insignificant (P>0.05). Fast blood-glucose of the other two groups was obviously increased, compared with that of the control group (P<0.01). The content of fast insulin was insignificantly different between the three groups (P>0.05). ISI of the other two groups were still significantly lower than that of the control group (P<0.01). Serum TG and Choi of the other two groups were still significantly higher than those of the control group (PO.01).At the end of the experiment, body weight values of the DCM and valsartan groups were obviously decreased, compared with that of the control group (PO.01). Fast blood-glucose of the other two groups was still significantly higher than that of the control group (P<0.01). The content of fast insulin was still insignificantly different between the three groups (P>0.05). ISI of the other two groups were still significantly lower than that of the control group (PO.01). Serum TG and Choi of the other two groups were still significantly higher than those of the control group (PO.01).2. Echocardiographic detectionMitral and tricuspid valvular regurgition detection: None of 8 animals in the control group showed mitral and tricuspid valvular regurgitation. In 11 animals of the DCM group, mild mitral valvular regurgitation was occurred in 3 rats; moderate mitral valvular regurgitation was occurred in one rat; severe mitral valvular regurgitation was occurred in one rat; mild tricuspid valvular regurgitation was occurred in one rat; moderate tricuspid valvular regurgitation was occurred in 2 rats; severe tricuspid valvular regurgitation was occurred in one rat; 2 rats of the DCM group showed both mitral and tricuspid valvular simultaneous regurgition; 4 rats of the DCM group did not show valvular regurgition. In 13 animals of the valsartan group, mild mitral valvular regurgitation was occurred in 2 rats; moderate mitral valvular regurgitation was occurred in one rat; mild tricuspid valvular regurgitation was occurred in 2 rats; moderate tricuspid valvular regurgitation was occurred in one rat; there was no rats, which showed severe mitral or tricuspid valvular regurgitation and the two valvular simultaneous regurgitation; 7 rats of the valsartan group did not
show valvular regurgition. There were no rats with valvular regurgitation in the control group. The incidence of valvular regurgitation in the DCM group was 63.64% and 46.15% in the valsartan group. Fisher analysis: Compared with that of the control group, the incidence of valvular regurgitation in the DCM group and the valsartan group was significantly increased (PO.05). Compared with that of the DCM group, the incidence of valvular regurgitation in the valsartan group was decreased, but which was insignificant (P>0.05).The common echocardiographic indices: 12 weeks after hyperglycemia, Compared with the indices of the control group, left atrial dimension (LAD) of the DCM group enlarged obviously (P<0.01), left ventricular ejection fraction (LVEF) of the DCM group was obviously decreased (P<0.05). LAD of the valsartan group was significantly smaller than that of the DCM group (PO.05).At the end of the experiment, compared with the indices of the control group, LAD, left ventricular dimension (LVD) and right ventricular dimension (RVD) of the DCM group enlarged obviously (P<0.05~0.01). LVEF of the DCM group was obviously decreased (P<0.05). LAD and LVD of the valsartan group were significantly smaller than those of the DCM group (P<0.05). LVEF of the valsartan group was significantly higher than that of the DCM group (PO.05). LAD of the valsartan group was significantly larger than that of the control group (P<0.05).3. Electrocardiogram recordationElectrocardiograms (ECG) of 8 animals in the control group were all normal and ECGs of 11 rats in the DCM group were all abnormal. 9 rats of the DCM group showed characteristic QRS wave interchanges and 2 rats of the DCM group showed complex arrhythmia. Change of the valsartan group ECG was mild. 6 rats of the valsartan group showed normal ECG, 7 rats showed QRS wave interchanges ECG, and none of the valsartan group showed complex arrhythmia. There were no rats with arrhythmia in the control group. The incidence of arrhythmia was 100% in the DCM group and 53.85% in the valsartan group. Fisher analysis: Compared with that of the control group, the incidence of arrhythmia in the DCM group and the valsartan group was significantly increased (PO.05~0.01). Compared with that of the DCM group,
the incidence of arrhythmia in the valsartan group was significantly decreased (PO.05).4. Hemodynamic indices determinationAt the end of the experiment, compared with the indices of the control group, left ventricular systolic pressure (LVSP) of the DCM group decreased obviously (P<0.01), left ventricular end-diastolic pressure (LVEDP) increased significantly (PO.01), the peak left ventricular pressure descending rate (-dp/dtmax) and the peak left ventricular pressure ascending rate (dp/dtmax) decreased significantly (PO.01), while the relaxation time constant (T) of the DCM group was longer than that of the control group (P<0.01). Compared with the indices of the DCM group, LVSP of the valsartan group increased obviously (P<0.05), LVEDP decreased significantly (P<0.05), -dp/dtmax was increased obviously (P<0.05). The T of the valsartan group was shorter than that of the DCM group (P<0.05). LVEDP of the valsartan group was higher than that of the control group (P<0.05). The -dp/dtmax of the valsartan group was lower than that of the control group (P<0.05).5. Ultrastructural change observation by transmission electron microscopy The left ventricular myocytes from the control group arranged regularly. Thepericellular membrane was uninterrupted and intact. The thick and thin myofilament arranged regularly. The sarcomere and light dark band were clear. The uniformly sized mitochondrial was abundant and showed round or oval shape. The nuclear membrane was smooth and intact. A typical distribution of heterochromatin in the form of clusters at the nuclear membrane was present in all cardiomyocyte nuclei. Intercalated disk was clear, intact and normally structured. A little fibroblast and collagenous fibers distributed in extra-cellular matrix. The microvessel lumen was normal and the structure of endothelial cell was normal.The left ventricular myocytes from the DCM group arranged irregularly. The pericellular membrane was interrupted and unclear. The local myofibril was disintegrated. The myofilament was distorted and interrupted. The sarcomere was in a bad apposition. The swelling mitochondrial increased and accumulated. The finger print mitochondrial could be found. The nuclear shape was abnormal with deep notch.
There was accumulative euchromatin. The unclear Intercalated disk was distorted and interrupted. A lot of collagenous fibers distributed in extra-cellular matrix. The microvessel lumen was narrow. The endothelial cell was swelling obviously and protruded to the lumen.Compared with the DCM group, the ultrastructural change of the valsartan group was obviously improved. The myocytes from the valsartan group arranged more regularly than the DCM group. The phenomenon that the local myofibril was disintegrated was decreased. The T tubule was moderately dilated. The mitochondrial was more regular than the DCM group and the finger print mitochondrial was not found. The nuclear shape was more regular than the DCM group. Intercalated disk was improved. The collagenous fibers in extra-cellular matrix decreased obviously and the swelling endothelial cell of microvessel was alleviated.6. Pathological detectionThe myocytes from the control group arranged regularly. The size of the nuclear was uniform. The staining cytoplasm was homogeneous. The myocytes from the DCM group arranged irregularly. The nuclear was irregular and the interrupted myofibril arranged irregularly. The valsartan group was obviously improved. The myocytes from the valsartan group arranged more regularly than the DCM group. The interrupted myofibril was not common. The nuclear shape was more regular than the DCM group.7. The content of collagen detection by Masson-stainingThe myocyte was red or yellow and the collagen was green or blue. The collagen tissue was appropriate arranged among cardiomyocytes in the control group. However, collagen tissue increased markedly, and disrupted in some area in the DCM group. The collagen tissue decreased and arranged regularly in the valsartan group, compared with the DCM group. Quantitative analysis results: The content of collagen in the DCM group was higher significantly than that of the control group (P<0.01). The content of collagen in the valsartan group was lower significantly than that of the DCM group (P<0.05). There was a significantly positive correlation of the collagen content with the fast blood-glucose in the DCM group (r = 0.746, PO.01).
8. Immunohistochemistry detectionThe positive reaction of TSP-1, A-TGFp, and TGFpRlI protein were stained brown and absent in myocardial cytoplasm. There was uniform distribution of weak brown granule in the control group. There was thick brown granule in the DCM group. The granule of the valsartan group was weaker than the DCM group.There was uniform distribution of weak brown granule on L-TGFpi in the control group. The granule of the DCM group and the valsartan group was thicker than the DCM group.It is a rare case to observe the expression of P-Smad2/3 in the control group. There was obvious brown granule of P-Smad2/3 in the DCM group. The granule of P-smad2/3 in the valsartan group was weaker than the DCM group.There was uniform distribution of weak brown granule on Smad7 in the control group. There was thick brown granule on Smad7 in the DCM group. The granule of the valsartan group was even thicker than the DCM group.9. The mRNA expression of several genes on GTTS pathwayThe expression of TSP-1 mRNA in the DCM group was higher significantly than that of the control group (PO.05). The expression of TSP-1 mRNA in the valsartan group was lower significantly than that of the DCM group (P<0.05).The expression of TGFpi mRNA in the DCM group was higher significantly than that of the control group (PO.01). The expression of TGFPi mRNA in the valsartan group was lower significantly than that of the DCM group (P<0.05) and higher than that of the control group (PO.05).The expression of TGFPIIR mRNA in the DCM group was higher significantly than that of the control group (PO.05). The expression of TGFp IIR mRNA in the valsartan group was lower significantly than that of the DCM group (PO.05).The expression of Smad2 mRNA in the DCM group was higher significantly than that of the control group (PO.01). The expression of Smad2 mRNA in the valsartan group was lower significantly than that of the DCM group (PO.05) and higher than that of the control group (PO.05).The expression of Smad3 mRNA in the DCM group was higher significantly
than that of the control group (P<0.05). The expression of Smad3 mRNA in the valsartan group was lower significantly than that of the DCM group (P<0.05).The expression of Smad7 mRNA in the valsartan group was higher significantly than those of the other two groups (PO.05). The expression of Smad7 mRNA in the DCM group was higher significantly than that of the control group (P<0.05).The Smad2/Smad7 in the DCM group was higher significantly than that of the control group (P<0.05). The smad2/smad7 in the valsartan group was lower significantly than that of the DCM group (P<0.05).The Smad3/Smad7 in the DCM group was higher significantly than that of the control group (P<0.05). The Smad3/Smad7 in the valsartan group was lower significantly than that of the DCM group (PO.05).In the DCM group, the fast blood-glucose showed a significantly positive correlation with the expression of TSP-1, TGFp), Smad2 mRNA (r=0.762, 0.714, 0.686, PO.05~0.01).In the DCM group, the collagen content showed a significantly positive correlation with TSP-1, TGFp,, TGFp IIR, Smad2 mRNA (r=0.717, 0.632, 0.675, 0.703, PO.05).In the DCM group, there was a significantly positive correlation of TSP-1 mRNA with LVEDP (r = 0.658, PO.05), in contrast, there was a significantly negative correlation of TSP-1 mRNA with LVSP and -dp/dtmax (r = -0.605,-0.694, PO.05). There was a significantly positive correlation of TGFPi mRNA with LVEDP (r = 0.689, PO.05), in contrast, there was a significantly negative correlation of TGFpi mRNA with -dp/dtmax and LVEF (r = -0.634, -0.647, PO.05). There was a significantly negative correlation of TGFP IIR mRNA with dp/dtmax and -dp/dtmax (r = -0.628, -0.729, PO.05).There was a significantly positive correlation of Smad2 mRNA with LVEDP (r = 0.669, PO.05), in contrast, there was a significantly negative correlation of Smad2 mRNA with -dp/dtmax (r = -0.738, PO.05). There was a significantly positive correlation of Smad3 mRNA with LVEDP (r = 0.746, PO.05), in contrast, there was a significantly negative correlation of Smad3 mRNA with
-dp/dtmax (r = -0.639, PO.05). There was no insignificant correlation of Smad7 mRNA with the other indices.10. The protein expression of several factors on GTTS pathwayThe expression of TSP-1 protein in the DCM group was higher significantly than that of the control group (P<0.05). The expression of TSP-1 protein in the valsartan group was lower significantly than that of the DCM group (P<0.05).The expression of A-TGFPi protein in the DCM group was higher significantly than that of the control group (PO.01). The expression of A-TGFPi protein in the valsartan group was lower significantly than that of the DCM group (PO.05).The expression of L-TGFpi protein in the DCM group and the valsartan group were both higher significantly than that of the control group (P<0.05). There was no difference of the expression of L-TGF|3i protein between the DCM group and the valsartan group (P>0.05).The expression of P-Smad2 protein in the DCM group was higher significantly than that of the control group (PO.01). The expression of P-Smad2 protein in the valsartan group was lower significantly than that of the DCM group (P<0.01) and higher than that of the control group (P<0.01).The expression of P-Smad3 protein in the DCM group was higher significantly than that of the control group (P<0.01). The expression of P-Smad3 protein in the valsartan group was lower significantly than that of the DCM group (PO.05) and higher than that of the control group (PO.05).The expression of Smad7 protein in the DCM group was higher significantly than that of the control group (PO.05). The expression of Smad7 protein in the valsartan group was higher significantly than those of the other two groups (PO.05~0.01).The P-Smad2/Smad7 in the DCM group was higher significantly than that of the control group (PO.05). The P-Smad2/Smad7 in the valsartan group was lower significantly than that of the DCM group (PO.05).The P-Smad3/Smad7 in the DCM group was higher significantly than that of the control group (PO.05). The P-Smad3/Smad7 in the valsartan group was lower
significantly than that of the DCM group (PO.05).In the DCM group, the fast blood-glucose showed a significantly positive correlation with the expression of TSP-1, A-TGFp,, P-Smad2, P-Smad3 protein (r=0.735, 0.726, 0.807, 0.764, PO.05~0.01).In the DCM group, the collagen content showed a significantly positive correlation with the expression of TSP-1, A-TGFpi, P-Smad2, P-Smad3 protein (r=0.750, 0.820, 0.689, 0.744, PO.05~0.01).In the DCM group, there was a significantly positive correlation of TSP-1 protein with LVEDP (r = 0.716, PO.05), in contrast, there was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r = -0.633, -0.669, PO.05). There was a significantly positive correlation of A-TGFPi protein with LVEDP (r = 0.697, PO.05), in contrast, there was a significantly negative correlation of A- TGFpi protein with -dp/dtmax, dp/dtmax and LVEF (r = -0.717, -0.637, -0.683, PO.05). There was a significantly positive correlation of P-Smad2 protein with LVEDP (r = 0.776, PO.01), in contrast, there was a significantly negative correlation of P-Smad2 protein with -dp/dtmax and LVEF (r = -0.705, -0.637, PO.05). There was a significantly positive correlation of P-Smad3 protein with LVEDP (r = 0.656, PO.05), in contrast, there was a significantly negative correlation of P-Smad3 protein with -dp/dtmax (r = -0.696, PO.05). There was no insignificant correlation of L-TGFpi n Smad7 protein with the other indices. Conclusion(1) A DCM animal model of type 2 diabetes mellitus was established by high-calorie diet, STZ injection of small dose, detection of echocardiography and histopathology. The animal model was valuable for the study of the mechanism in DCM.(2) The main histopathologic changes were collagen deposition and myocardial interstitial fibrosis.(3) The early change was the diastolic dysfunction and the mixed dysfunction (diastolic and systolic dysfunction) ocurred at the late stage of disease.(4) The pathyway of Glucose/TSP-1/ TGFpVSmads was proved in type 2
diabetes mellitus by quantification real-time RT-PCR and western blot. GTTS pathway showed obviously correlation with the changes of cardiac function and myocardial interstitial fibrosis. The main mechanism of DCM was GTTS pathway.(5) Valsartan can prevent cardiac from damage in DCM. Blocking the effects of Ang II on the factors of GTTS pathway was an important mechanism.
引文
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