An Experimental Study On The Actions Of RAS And CTGF In Diabetic Myocardial Lesion Of Rats And The Prevention And Treatment

Diabetic cardiomyopathy (DCM) is a common long-term complication of diabetes mellitus as well as a special cardiomyopathy. The morphological development of diabetic cardiomyopathy is characterized by myocardial extracellular matrix(ECM)accumulation and myocardial interstitial remodeling. The onset diabetic cardiomyopathy is the result of combined action of multiple factors, including glucose metabolism disturbance, production, release of cytokine factors and growth factors. Among these factors,the enhanced activity of rennin angiotensin system(RAS)in myocardium tissue was considered as an important factor in pathogenesis of diabetic cardiomyopathy. The potential mechanism of how the activity enhancing RAS cause myocardiac pathological change is not clear. Such a deep research did not appear in the world so far. In the developing process of DCM, transforming growth factor-β(TGF-β), which serves as an antifibrotic target, was also considered as potent profibrotic growth factor. A series of new approaches toward interference with TGF-βexpression has been developed in the past decades. However, TGF-βalso has anti-inflammatory and anti-proliferative effects in its normal expression. Taking account of the safety of long-term and chronic inhibition of TGF-β, more specific antifibrotic target should be searched. Indirect and direct evidences have indicated that connective tissue growth factor (CTGF), a recently identified new growth factor, acts as a downstream mediator of TGF-βprofibrotic effects. CTGF only mediates the profibrotic effects of TGF-βand does not affect the anti-inflammatory and anti-proliferative effects of TGF-β. Therefore, CTGF may serve as a new therapeutic target for diabetic cardiomyopathy. However,the expressive condition of CTGF in the myocardium of diabetic rats induced by streptozotocin (STZ),and the effects of CTGF on ECM accumulation as well as the expressive impact of RAS towards CTGF has not been determined. It has been demonstrated that the occurrence and progression of DCM is the result of the imbalance between synthesis and degradation of myocardiac ECM components. Therefore, the research only focusing on the synthesis of ECM can not account for the whole pathologic process and mechanism of the ECM accumulation. The decreased ECM degradation is also a key link leading to ECM accumulation. The deposition of ECM may be due to increased synthesis and/or decreased degradation of ECM. Being one of the major proteolytic enzymes, matrix metalloproteinase (MMPs) play an important role in myocardiac ECM accumulation. The decreased activity of MMPs is an important mechanism of myocardiac ECM accumulation and myocardiac interstitial sclerosis. The activity of MMPs is mainly arranged by tissue inhibitor of metalloproteinase (TIMPs). The decreased activity of MMPs is due to decreased production and release of MMPs and/or increased activity of TIMPs. It has been demonstrated that the activity of MMPs and TIMPs determines degradation of ECM. MMP-1 has the quantities advantage among the myocardial cells and its main inhibitor is TIMP-1. The expressive condition of MMP-1 and TIMP-1 in myocardium of diabetic rats, and the relationship between MMP-1 / TIMP-1 with RAS have not been reported. There are no effective therapies for DCM till now in the modern medicine. In recent years, the clinical and experimental study on preventing and treating DCM of traditional Chinese medicine has shown the promising prospecct. JieDuTongLuo capsule (JDTL capsule) is composition of traditional Chinesemedicine, and it can obviously inhibit ECM accumulation of DN. However, the study of JDTL capsule acting on myocardium of diabetic rats and the relative molecular mechanism has not been covered. The aim of this study is to determine the RAS effects on MMP-1 /TIMP-1 protein expression and TGF-β1, CTGF gene expression of myocardium of diabetic rats, as well as the relation of these cytokine factors on ECM accumulation. We anticipate determining the new effects of RAS on CTGF mRNA expression and definituding the relation of CTGF mRNA expression on ECM accumulation and clarifying the proceeding mechanism of the activity enhancing RAS with ECM accumulation and providing experimental basis for determining a new therapeutic target for diabetic cardiomyopathy. Besides, with Benazepril being positive control group, we preliminarily determine the effects of RAS on myocardium of diabetic rats and mechanism, and anticipate providing a new therapeutic way to prevent and treat diabetic cardiomyopathy. The research methods applied in this study include the myocardiac morphological changes, immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The parameters evaluation includes: ①the myocardium of diabetic rats structure and the protective domino effects of JDTL capsule; ②MMP-1 /TIMP-1 protein expression; ③TGF-β1 mRNA expression; ④CTGF mRNA expression; ⑤the impact of JDTL capsule on RAS; ⑥the impact of JDTL capsule on the degraded mechanism of ECM components; ⑦the impact of JDTL capsule on the synthetic mechanism of ECM components. The main results are as follows: 1. Diabetic rats induced by STZ were observed by optical microscope and electric microscope. Their myocardial structures were obviously altered at the end of 12 weeks. The results showed the number of myocardiac interstitialfibrocyte increased, protein substances with positive marked by PAS increased and the content of fibronectin (FN), collagenⅠ(ColⅠ) and collagen Ⅲ(Col Ⅲ)as well as the index of ColⅠ/Col Ⅲall increased (P<0.01, P<0.05). Under the transmission electric microscope, the myofilament of myocardial cell disarranged, the number of mitochondrion increased and was agglomerate, the content of the collagenous fiber in myocardiac interstitium increased. The pathological changes of experimental group and positive control group treated by JDTL capsule and Benazepril for 12 weeks were alleviated more obviously than diabetic group (P<0.01, P<0.05). Besides JDTL capsule has appreciable effect in regulating blood glucose and lipid concentration. 2. At the end of 12 weeks, compared with normal control group, MMP-1 protein expression of myocardium of diabetic rats decreased, and TIMP-1 protein expression increased 2 times (P<0.01). Compared with normal control group, MMP-1 protein expression of positive control group and experimental group increased, and TIMP-1 protein expression of positive control group and experimental group decreased (P<0.01). MMP-1 protein expression of experimental group obviously increased than positive control group, and TIMP-1 protein expression of experimental group decreased 12% than positive control group (P<0.01). 3. At the end of 12 weeks, TGF-β1 and CTGF mRNA expression of myocardium of diabetic rats all increased, differentially increased 1 times and 0.5 times than normal control group (P<0.01). TGF-β1 mRNA expression decreased 24% and CTGF mRNA expression decreased 14% (P<0.01) after being treated by Benazepril for 12 weeks. TGF-β1 and CTGF gene expression shows parallel. After being treated by JDTL capsule for 12 weeks, TGF-β1 and CTGF mRNA expression of experimental group differentially decreased 32% and 20% than diabetic control group (P<0.01), and TGF-β1 and CTGFmRNA expression of experimental group differentially decreased 11% and 6.9% than positive control group (P<0.01). 4. After being treated by JDTL capsule for 12 weeks, the activity of RA of experimental group increased than normal control group (P<0.01), but it obviously decreased than positive control group (P<0.01). The activity of ACE and the content of Ang Ⅱof experimental group differentially decreased 40% and 16% than diabetic control group (P<0.01, P<0.05), and the activity of ACE and the content of AngⅡof experimental group all increased than positive control group (P>0.05). The main conclusions are: 1. AT the end of 12 weeks, the pathological changes of myocardium of diabetic rats mainly included that FN multiplying and ColⅠand Col Ⅲdisproportionably depositing as well as the alteration of ultrastructure. JDTL capsule can alleviate the pathological changes of myocardiac interstitial remodelling. 2. AT the end of 12 weeks, the activity of ACE and the content of AngⅡand RA of myocardium of diabetic rats significantly increase, and they are parallel to the pathological changes of myocardium of diabetic rats. JDTL capsule can significantly decrease the activity of ACE and the content of AngⅡof myocardium of diabetic rats and protect the myocardium. 3. The decreasing of the content of AngⅡand melioration of the pathological changes of myocardium of diabetic rats can be achieved through blockading the activity of ACE. JDTL capsule can decrease the activity of ACE and the content of AngⅡof myocardium of diabetic rats. 4. The decreasing of the activity of MMP-1 and the increasing of the activity of TIMP-1 of myocardium of diabetic rats suggest the impe-diment of degradation of ECM components. JDTL capsule can meliorate the abnormal activity of MMP-1 and TIMP-1 of myocardium of diabetic rats.