The Role And Signaling Pathogenesis Of Tribble 3 On Cardiac Fibrosis In Diabetic Mouse

BackgroundDuring the past three decades epidemiological, clinical, and laboratory studies have confirmed the existence of a cardiomyopathy associated with diabetes mellitus (DM). The diagnosis of a diabetic cardiomyopathy is made in patients in whom no other known etiological factors, such as coronary artery disease, alcoholism, or hypertension, are present . In these patients, a higher incidence of congestive heart failure is observed. Pathological alterations include those relevant to muscle tissue and cells, such as the presence of myocardial hypertrophy and impaired contraction, those associated with extracellular matrix (ECM) proteins, such as excess deposition of collagens, abnormal glycosylation/crosslinking, and alterations in diastolic compliance. However, the machanisms of cardiac fibrosis in diabetes were still unclear.Tribbles is a family of Drosophila protein including TRBl,TRB2,TRB3,which shows to regulate String activity and hence mitosis during ventral furrow formation,. Recently, TRB3 has been found to block the activation of Akt/PKB and TRB3 expression increases in fasted mice and functions to promote glucose output. The prevalent TRB3 missense Q84R polymorphism is significantly associated with several insulin resistance-related abnormalities and with the presence of a cluster of insulin resistance-related cardiovascular risk factors in diabetic patients.TRB3 can selectively regulate the activation of MAPKs by interacting with MAP kinase kinases (MAPKKs) in a specific manner that regulates their activity, and inhibit NF-KB -dependent transcription by interacting with p65.Both MAPKs and NF-KB play an important role in cell proliferation and apoptosis.Therfore,TRB3 might participate in cardiovascular complication of diabetes. objective1 .To investigate the mRNA expression of TRBs in the left ventricular of diabetic rats.2.To evaluate the correlation between the mRNA level of TRBs and collagen content in left ventricular of rats. Methods1.The animal model induction 36 male Wistar rats were randomly divided into 2 groups: the control group (n=18) and the diabetic (DM) group (n=18). The rats of the control group were fed a normal chow diet. The rats in DM group rats were injected with streptozotocin (STZ) and fed with a high-fat and high-calorie diet.2.Measurement of body weight and biochemical indexes. The rats in two groups were weighted every week and the contents of fast blood-glucose were detected every two weeks. The contents of fast blood-glucose,insulin, triglyceride, cholesterol 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 and at the end of the experiment. Electrocardiograms of animals were recorded and hemodynamics indexes were measured by cardiac catheterization.4. Myocardial ultrastructral and histopathological changes were observed at the end of the experiment. Ultrastructral characters were observed by transmission electron microscopy and the content of collagen were quantified by Masson-staining.5.The mRNA expression of TRBs including TRB1,TRB2 and TRB3 were measured by quantification real-time RT PCR. Results1 .Body weight and biochemical indices measurement:Before STZ injection, body weight values of the DM groups was significantly higher than that of the control group (P<0.05). the level of fast blood-glucose and insulin were significantly higher than that of the control group (P<0.05), but insulin sensitive index(ISI) of the DM group was slightly lower than that of the control group(P<0.01).At the 20^th weeks of the experiment, Fast blood-glucose of the DM group was significantly higher than that of the control group (P<0.01 )but the content of fast insulin heve no diference in the two groups (P>0.05). ISI of the DM group was significantly lower than that of the control group (P<0.01).2. Echocardiographic detectionAt the end of the experiment, compared with the control group,left atrial dimension(LAD), left ventricular dimension (LVD) and right ventricular dimension (RVD) of the DM group were enlarged obviously (P<0.05). left ventricular ejection fraction(LVEF) of the DM group was decreased (P<0.05).3. Hemodynamic indices determinationAt the end of the experiment, compared with the control group, left ventricular systolic pressure (LVSP) of the DM group was decreased (P<0.01),left ventricular end-diastolic pressure (LVEDP) increased significantly(P<0.01), the peak left ventricular pressure descending rate (-dp/dtmaX) and the peak left ventricular pressure ascending rate (dp/dtmaX) were all decreased significantly (P<0.01),while the relaxation time constant (T) of the DCM group was longer than that of the control group (P<0.01).4. Ultrastructural change observation by transmission electron microscopy:The left ventricular myocytes from the control group arranged regularly. A little fibroblast and collagenous fibers distributed in extra-cellular matrix.The left ventricular myocytes from the DM group arranged irregularly. The pericellular membrane was interrupted and unclear. The local myofibrillar was disintegrated. The myofilament was distorted and interrupted. A lot of collagenous fibers distributed in extra-cellular matrix. The microvessel lumen was narrow.5. The content of collagen detection by Masson-stainingThe collagen tissue was appropriate arranged among cardiomyocytes in the control group. However, collagen tissue increased markedly, and disrupted in some area in theDM group. Quantitative analysis results: The content of collagen in the DM groupwas higher significantly than that of the control group (P<0.01).There was a significantly positive correlation of the collagen content with the fastblood-glucose in the DM group (r=0.746, P<0.01).6.The mRNA expression of TRBs in left ventricular of the ratsThe mRNA of TRB1,TRB2 and TRB3 were all expressed in laft ventricular of therats. Thereare no obvious change of the mRNA level of TRB1 and TRB2 in the twogroups,However,The mRNA level of TRB3 in DM group is 5.6 folds of the controlgroup(P<0.01).Conclusions1. A rat model of type 2 diabetes mellitus was established by high-calorie diet, STZ injection of small dose, detection of echocardiography and histopathology.2. The main histopathologic changes were collagen deposition and myocardial interstitial fibrosis.3.A11 the mRNA of TRBs are expressed in the cardiac tissue of the rat.4.The mRNA level of TRB3 is overexpressed in cardiac tissue of diabetic rats, which is correlated with cardiac fibrosis,which suggest that TRB3 might participate in the pathogenesis of cardiac fibrosis in diabetic states. The mRNA expression of TRB1 and TRB2 have no manifestive difference between the two groups.Whichsuggest that TRB3 might be related with cardiac fibrosis in diabetes. BackgroundChronic hyperglycemia in humans is the main feature of diabetes mellitus and is responsible for pathologic changes in the cardiovascular system. Hyperglycemia may promote the progression of heart failure mainly by excessive interstitial myocardial collagen accumulation, which could result in impaired diastolic and systolic function. The composition of the ECM, a complex network of structural proteins, mainly collagen types I and III in the myocardium, provides architectural support for the muscle cells and plays an important role in diastolic dysfunction in diabetic cardiomyopathy.Prolonged activation of cardiac fibroblasts, defined by increased proliferation and subsequent increased ECM, leads to cardiac fibrosis. A few studies reported that high glucose up-regulate collagen produced by cardiac fibroblasts, but the expression of the subtype of collagens in cardiac fibroblasts under high extracellular glucose is still unclear.One potential mechanism that has been defined for high glucose is the stimulation of lipogenic cytokine production by activation of the mitogen activated protein kinase (MAPK) signaling pathway. Both extracellular signal kinase 1/2 (ERK1/2) and p38 MAPK are involved in the regulation of collagens production by fibroblasts. Our previous study showed that TRB3 mRNA is overexpressed in cardiac tissue of diabetic rats associated with cardiac fibrosis, which suggest that TRB3 might participate in the pathogenesis of cardiac fibrosis in diabetic states. TRB3 can selectively regulate the activation of MAPKs in a specific manner.Therefore,we hypothesized that high glucose may lead to TRB3 overexpression and thus up-regulate the production of collagen types I and III through MAPKs signaling pathway. Objective1. To investigate the effect of high glucose on collagen types I and III expressed by rat cardiac fibroblasts2. To explore whether MAPKs signaling pathway is responsible for collagen metabolism in cardiac fibroblasts.3. To explore whether TRB3 affect high glucose- induced collagens expression through MAPKs signaling pathwayMetholds1. Neonatal rat cardiac fibroblasts were treated with normal glucose (5.5 mmol/L), OC (mannose, 25mmol/L) and high glucose (25mmol/L) respectively for Oh, 4h, 8h, 12h, 24h, 48h, 72h, Cells were collected, and both the mRNA and protein level of collagen types I and III by real time PCR and enzyme linked immunosorbent assay (ELISA), the mRNA level of TRB3.2.After the cardiac fibroblasts were incubated with by normal glucose, OC and high glucose for 0min,30min,60min and 120min ,the activity of MAPKs including p38,ERK1/2 and JNK were measured respectively.3. According to the effect of high glucose on the activity of MAPKs, MAPKs inhibitor was choosed to block reciprocal signaling pathway, the change of mRNA and protein level of collagen types I and III by glucose were measured .4. After blockade of TRB3 expression by TRB3 siRNA,the cardiac fibroblasts were incubated with high glucose, mannose and normal glucose,respectively. The mRNA level of collagen types I and III by glucose for 12hours were measured,and the protein level of collagen types I and III in the medium aere measurd at 72h. 5. After blockade of TRB3 expression by TRB3 siRNA,the cardiac fibroblasts were incubated with high glucose, mannose and normal glucose, respectivly. The activity of MAPKs of cardiac fibroblasts at 0min and 120min were measured. Results1. Our data showed an increase in both the mRNA and protein level of collagen types I and III by cardiac fibroblast following incubation with high glucose(P<0.01) . These effects were not due to the changes in osmotic pressure.2. Extracellular signal regulated kinase 1/2 (ERK1/2) was activated by high glucose (P<0.01) .After ERK phosphorylation was blocked by PD98059, the mRNA and protein expression of collagen types I and III were significantly decreased (K<0. 01) .3. Compared with the control, the TRB3 mRNA increased 5.7 folds following 12h-incubation with high glucose (P<0.01) ,OC and normal glucose can not affect the TRB3 mRNA.4. The mRNA and protein expression of collagen types I and III induced by high glucose were all down-regulated after TRB3 was blocked by TRB3 siRNA (P<0.01).5.The activity of ERK1/2 at 120min stimulated by high glucose was significantly repressed after TRB3 had been inhibited. Conclusions1. High glucose increase the synthesis of collagen types I and III through activation of ERK1/2 .2. High glucose increase the the mRNA level of TRB3, and TRB3 play an role in collagens expression induced by high glucose in cardiac fibroblasts through ERK1/2 signaling pathway. BackgroundThe formation of advanced glycation end products (AGEs) is an important biochemical abnormality that accompanies diabetes mellitus in general. It has been recognized as an important pathophysiological mechanism in the development of complications in diabetic patients. Recently AGEs has been found to be related with cardiac fibrosis in aged and diabetic heart. In the heart of diabetic rat, accompanied with increased AGEs, there is an increase in total collagen,the serum level of AGEs is related with diastolic function in both diabetic patients and diabetic animals.AGEs can not only structurally stiffen structural collagen backbones by suppressing collagen turnover in diabetes states, but also act as agonists to AGE receptors (RAGE) on various cell types, which stimulate the release of profibrotic growth factors, promote collagen deposition, increase inflammation, and ultimately lead to tissue fibrosis.In addition to direct effects such as formation of protein cross-links, AGEs can exert their effects via cell activation. In many different cell Studies such as in human dermal fibroblasts and Mesangium cells have shown that AGEs are able to activate MAP kinases and encourage collagens expression.Our previous study showed that TRB3 mRNA level was associated with collagen content in the heart of diabetic rat .We also found that TRB3 up-regulated collagen production through activation of ERK1/2 .Besides, evidence proved that TRB3 interacte with MAPKs specically and extensively, which suggest that TRB3 may participate in cardiac fibrosis through MAPKs signaling pathway. Cardiac fibroblasts is the origin of collagens in heart, however, the role of TRB3 on collagen production by cardiac fibroblasts induced by AGEs are not fully understood.Excessive advanced glycation end products (AGEs) as a result of chronic hyperglycemia, in tissues or in the circulation may critically affect the progression of diabetic cardiomyopathy. Cardiac fibrosis is a typical pathologic feature characterized by the increase of the extracellular matrix (ECM) in diabetic cardiomyopathy. It has been reported previously that collagen types I and III are the major components of ECM mainly origin from cardiac fibroblasts in the heart, which are down-regulated by AGEs breaker. We have reported that myocardial collagens were accumulated along with significantly increased TRB3 mRNA expression in the heart of streptozotocin-induced diabetic rats. Recently TRB3 have been proved to affect MAPKs specifically and extensively, and MAPKs have been demonstrated to participate in collagens expressions by fibroblasts and be an important mediator of the fibrotic process. We hypothesis that TRB3 regulate collagens expression induced by AGEs in cardiac fibroblasts through MAPKs signaling pathway. Objective(1)To investigate collagens production induced by AGEs in cardiac fibroblasts.(2)To investigate the role of MAPKs in AGEs-induced collagens production.(3)To investigate the mRNA expression of TRB3 in cardiac fibroblasts induced by AGEs.(4)To explore the role of TRB3 in AGE-induced collagens production and wether MAPKs pathway was involved in this process. Metholds(1)Neonatal rat cardiac fibroblasts were treated with AGEs, BSA (200ug/ml) respectively from 0h-72h,then collect the cells and measure the mRNA expression of TRB3,collagen types I and III by real time RT-PCR, the protein level of collagen types I and III in medium were measured by enzyme linked immunosorbent assay (ELISA).(2)The activity of MAPKs including p38,ERKl/2 and JNK in cardiac fibroblasts was measured at 0min, 15 min, 30min,60min induced by AGEs(200ug/ml) and BSA(200ug/ml) respectively.(3)Inhibitors were used to block MAPKs signaling pathway, then measure the mRNA and protein level of collagen types I and III by cardiac fibroblasts incubated with AGEs(200ug/ml) and BSA (200ug/ml)respectively.(4)After inhibiting TRB3 expression by using TRB3 siRNA, the mRNA and protein level of collagen types I and III by cardiac fibroblasts incubated with AGEs(200ug/ml) and BSA (200ug/ml) were measured respectively. Results1. We first examined the expression of collagen types I and III stimulated by AGEs. After being incubated for 12h, the mRNA expression of collagen type I produced by cardiac fibroblasts increased 6.47 folds with AGEs compared with BSA(P<0.01), but AGEs decreased the mRNA expression of collagen type III by 16.5%(P<0.05). Incubation of cells in AGEs for 72h, the protein level of collagen type I increased 3.62 folds (P<0.01)and the protein level of collagen type III decreased by 53.1%(P<0.01).Which suggest that AGEs enhanced the expression of collagen type I but inhibit the expression of collagen type III.2.We next tested the activity of MAPKs induced by AGEs. Treatment with AGEs, but not BSA, resulted in the activation of ERK1/2 in a time-dependent manner, which was increased significantly at 30 minutes and declined to baseline levels at 60minutes.The same results were found in the activity of JNK. However, the activity of p38 was partly blocked by AGEs.3.We further examined the potential role of the MAPKs on the production of collagen types I and III stimulated by AGEs .(1)After being given ERK inhibitor (PD98059), mRNA expression of collagen types I and IIIby cardiac fibroblasts incubation with AGEs decreased by 62% and 13% (P<0.05) , the protein of collagen types I and III decreased by 50% (P<0.01) and 24.5% (P<0.05) respectively.(2)Treatment with p38 inhibitor(SB203580), the mRNA expression of collagen types I and III induced by AGEs reduced by 22% (P<0.05) and 33% (P<0.01) respectively, the protein of collagen types I and III decreased by 16.9%( P<0.05) and 53.7% (P<0.01) respectively.(3) Though JNK inhibitor SP600125 block the mRNA expression of collagen types I and III by 19% (P<0.05 ) and 19.4% (P<0.05 ) ,it enhanced the protein expression of collagen type I by 10.6%, and it have no effect on the protein expression of collagen type III.(4)We further investigated the role of TRB3 in collagens expression induced by AGEs. The resultes showed that incubation of cells in either AGEs or BSA stimulated the expression of TRB3 mRNA . The addition of AGEs increased TRB3 mRNA expression 4.53 folds of BSA (P<0.01) . After inhibiting the expression of TRB3 by siRNA, the mRNA and protein expression of collagen type I decreased by 74% (P<0.01) and 65.8% (P<0.01) ,the mRNA and protein expression of collagen type III increased by 25% (P<0.05) and 23.1% (P<0.05) respectively. We did not found the change of MAPKs activity after incubation with BSA.(5)We further explored whether TRB3 was involved in AGEs -induced collagens expression through MAPKs signaling pathway. Our data showed that after transfection with TRB3 siRNA, AGEs depressed the activity of ERK1/2 by 38% (P<0.05 ) and JNK by 46% (P<0.01) at 30min, the phosphorylation level raised by 53% (P<0.01) after incubation with AGEs for 60min. Conclusions(1)AGEs stimulate the expression of collagen type I but inhibit the expression of collagen type III.(2)ERKl/2, P38 and JNK are all involved in the expression of collagen type I induced by AGEs, and ERK1/2 play a more important role among them.(3)Both ERK1/2 and P38 involved in the expression of collagen type I can be inhibited by AGEs, and the role of P38 is more obviously than ERK1/2 and JNK. (4)TRB3 is involved in the collagens expression affected by AGEs by regulation of MAPKs specifically, TRB3 promote the activity of ERKl/2 and JNK while suppress the activity of p38.