| Introduction:With the improvement of people's living standards, Diabetes Mellitus (DM) has become a threat to the health of mankind. Among different types of DM, Type 2 diabetes (T2DM) is more common, and it accounts for more than 90% of the total diabetes in China. Although people of the middle-aged and the elderly are more likely to develop T2DM, it alarms us that there are more and more young people getting it. Among the diabetic complications, cardiac events are of the highest risk, and it has become a main cause of death of diabetics. Researchers have done a lot in studying the mechanisms of diabetic cardiac complications, but mainly in the coronary vascular injury, and direct injury to myocardial cells received very little attention.Thioredoxin (Thioredoxin, Trx) system is an important protein system in the body, widely expressed in various cells, and has played an important role in the occurrence and development of diseases involving free radical damage and apoptosis such as inflammation, tumor, and reperfusion injury. Patients with diabetes show significant glucose and lipid metabolic abnormalities, with the generation of a large number of free radicals, including peroxynitrite (ONOO-); at the same time, others and our preliminary researches have showed that there is also apoptosis of myocardial cells. Considering that Trx system is an important anti-free radical damage system and can inhibit cell apoptosis in vivo, we speculate that Trx might be involved in the occurrence of a variety of damages caused by DM. However, studies on the role of Trx in diabetes have just been started and many basic issues need to be resolved. We still lack systematic studies of depth in the expression of Trx system, its activity, and its changes in anti-free radical and anti-apoptosis effects in DM.Objective:1. To establish animal models of type 2 diabetic rats and observe whether diabetes could cause apoptosis of myocardial cells; to find out the extent of the damage and apoptosis in development of the disease and choose the best observing time point. 2. To observe the changes of the activity and expression of Trx system in myocardial injury in type 2 diabetic rats and analyze the relationship between the changes and myocardial apoptosis.Methods:Male adult SD rats (about 180 g) were randomly divided into two groups:sham group (NC), diabetes group (DM). Rats of DM group were subjected to high-sugar and high-fat diet and streptozotocin (STZ) injection in order to successfully establish type 2 diabetic model of rats. Rats of NC group were only given normal diet and equal amount of citric acid buffer injection. One week after injection, we measured fasting blood glucose (FBG) from tail vein blood, FBG≥16.7 mmol/L, and observed no significant blood sugar decrease, indicating successful establishment of diabetic model of rats. Then the rats in DM group and NC group were randomly divided into five groups of different time (1st,2nd,4th,12th, and 24th w),10 in each group.4 rats died during the experiment, leaving 46 in DM group and 50 in NC group. The rats were sacrificed in batches after we measured the indicators for cardiac function, including LVDP, +dp/dtmax and-dp/dtmax at different time points (1st,2nd,4th,12th, and 24th w) after STZ injection.The levels of insulin, CK-MB and cTnl in serum were measured by ELISA method; caspase-3 activity was measured to reflect the extent of myocardial apoptosis; caspase-8 activity and caspase-9 activity were measured to analyze the pathways of myocardial apoptosis; the activities of Trx and TR in cardiac muscles were detected by insulin disulfide reduction assay and TR assay kit, respectively; the mRNA expressions of Trxl,Trx2, thioredoxin reductase 1 (TR1), thioredoxin reductase 2 (TR2) and thioredoxin interacting protein (TXNIP) were measured by Real-time PCR method; the protein expressions of Trxl,Trx2,TR1,TR2 and TXNIP were measured by Western Blot method.Results:1 The successful establishment of DM rat modelAfter one week STZ treatment, the level of blood glucose in rats of DM group became significantly higher than that of NC group (1 w:16.9±1.80 mmol/L vs.5.40±0.20 mmol/L, P<0.01; 2 w:17.1±1.90 mmol/L vs.5.30±0.30 mmol/L, P<0.01; 4 w:17.2±1.70 mmol/L vs. 5.30±0.40 mmol/L, P<0.01; 12 w:17.8±1.90 mmol/L vs.5.50±0.30 mmol/L, P<0.01; 24 w: 22.7±1.80 mmol/L vs.5.30±0.20, P<0.01); meanwhile, the serum insulin level of DM group was not significantly different from that of NC group, even higher at the 24th week (24 w:0.68±0.05 ng/ml vs.0.45±0.02, P<0.01)(table 1). It suggested that the type 2 diabetic rats model was successfully established.2 The cardiac function in DM group was aggravatedIn the 12th and 24th week, compared with those of NC group, the levels of LVSP,±dP/dtmax DM group were significantly decreased (12 w:LVSP:13.74±1.69 Kpa vs.16.10±1.28 Kpa, P<0.01;+dp/dtmax:456.49±168.95 Kpa/s vs.596.91±143.68 Kpa/s, P<0.05;-dp/dtmax: 475.74±172.54 Kpa/s vs.607.74±165.41 Kpa/s, P<0.05; 24 w:LVSP:12.56±2.12 Kpa vs. 16.79±1.86 Kpa, P<0.01;+dp/dtmax:432.85±186.43 Kpa/s vs.607.36±149.52 Kpa/s, P<0.05;-dp/dtmax:453.28±194.58 Kpa/s vs.575.39±176.32 Kpa/s, P<0.05)(table 2).3 The changes of biochemical indicators for myocardial injury in DM rats3.1 CK-MB content in serum of DM rats was increasedCompared with that of NC group, CK-MB content in DM group increased significantly in the 2nd,4th and 12th week (2 w:23.67±3.02 ng/ml vs.11.62±0.99 ng/ml, P<0.01; 4 w:24.53±1.79 ng/ml vs.11.78±1.29 ng/ml, P<0.01; 12 w:25.07±4.07 ng/ml vs.5.50±0.30 ng/ml, P<0.01), and became worse as the disease progressing (table 3).3.2 cTnI content in serum of DM rats was increasedCompared with that of NC group, cTnl content in DM group was increased significantly at the 12th and 24th week (12 w:1.50±0.25 ng/ml vs.0.77±0.10 ng/ml, P<0.05; 24 w:0.46±0.13 ng/ml vs.0.24±0.02 ng/ml, P<0.05), and became worse with the course of the disease prolonged (table 3).4 Increased myocardial apoptosis in DM rats4.1 Caspase-3 activity assayCompared with that of NC group, caspase-3 activity in DM group was significantly increased at the 4th and 12th week (4 w:0.55±0.05 nmol/h/mg vs.0.12±0.01 nmol/h/mg, P<0.05; 12 w:0.92±0.05 nmol/h/mg vs.0.11±0.02 nmol/h/mg, P<0.01)(Fig.1).4.2 Caspase-8 and Caspase-9 activity assay Compared with those of NC group, caspase-8 activities in DM group were significantly increased at the 4th and 12th week (4 w:0.67±0.05 nmol/h/mg vs.0.30±0.02 nmol/h/mg, P<0.01; 12 w:0.92±0.10 nmol/h/mg vs.0.25±0.04 nmol/h/mg, P<0.01)(Fig.2); while caspase-9 activities in DM group were also significantly increased at the 12th and 24th week (12 w:0.13±0.04 nmol/h/mg vs.0.06±0.01 nmol/h/mg, P<0.05; 24 w:0.19±0.03 nmol/h/mg vs.0.06±0.01 nmol/h/mg, P<0.01)(Fig.3).5 The activity of Trx system in DM rats was decreased5.1 The activity of Trx in DM rats was decreasedCompared with those of NC group, Trx activities of DM rats were decreased at the 2nd,4th, 12th, and 24th week (2 w:0.84±0.14 vs.1.00±0.13, P<0.01; 4 w:0.74±0.13 vs.1.00±0.13, P<0.01; 12 w:0.63±0.14 vs.1.00±0.13, P<0.01; 24 w:0.50±0.19 vs.1.00±0.13, P<0.01), and continuously decreased with the development of the disease (Fig.4).5.2 The activity of TR in DM rats was decreasedCompared with those of NC group, TR activities of DM rats were significantly decreased at the 2nd,4th,12th, and 24th week (2 w:0.79±0.20 vs.1.00±0.17, P<0.01; 4 w:0.71±0.22 vs. 1.00±0.17, P<0.01; 12 w:0.60±0.23 vs.1.00±0.17, P<0.01; 24 w:0.47±0.17 vs.1.00±0.17, P<0.01), and continuously decreased with the development of the disease (Fig.5).6 The changes of mRNA and protein expressions of Trx system in myocardial tissue of DM rats6.1 The change of mRNA expression of Trx system in myocardial tissue of DM ratsCompared with those of NC group, the mRNA expressions of Trx1, Trx2, TR1 and TR2 in DM group were not significantly different at the 1st and 2nd week, while decreased at the 4th week, increased at the 12th week, and kept at a high level in the 24th week, and the differences were of statistical significance except those of TR1 and TR2 at the 24th week (Trx1:4 w:0.63±0.14 vs. 1.00±0.14, P<0.01; 12 w:1.71±0.29 vs.1.00±0.21, P<0.01; 24 w:1.29±0.27 vs.1.00±0.17, P<0.05; Trx2:4 w:0.70±0.13 vs.1.00±0.19, P<0.01; 12 w:1.59±0.21 vs.1.00±0.12, P<0.01; 24 w:1.24±0.19 vs.1.00±0.21, P<0.05; TR1:4 w:0.62±0.20 vs.1.00±0.16, P<0.01; 12 w:1.41±0.19 vs.1.00±0.14, P<0.01; TR2:4 w:0.42±0.06 vs.1.00±0.14, P<0.01; 12 w:1.56±0.12 vs. 1.00±0.19, P<0.01)(Fig.8,9,12,13). 6.2 The change of protein expression of Trx system in myocardial tissue of DM rats at the 12th weekAt the protein level, we only used Western Blot method to detect the expression of each protein in the cardiac muscles at the 12th week. Compared with those of NC group, the protein expressions of Trx1, Trx2, TR1 and TR2 in DM group were significantly increased (Trxl: 1.62±0.15 vs.1.00±0.17, P<0.01; Trx2:1.53±0.11 vs.1.00±0.16, P<0.01; TR1:1.34±0.13 vs. 1.00±0.11,P<0.01;TR2:1.34±0.15 vs.1.00±0.14,P<0.01)(Fig.14,15,16,17).7 The change of mRNA and protein expressions of TXNIP in myocardial tissue of DM rats7.1 The change of mRNA expression of TXNIP in myocardial tissue of DM ratsCompared with those of NC group, the mRNA expression of TXNIP in DM group was significantly increased at the 4th,12th, and 24th week (4 w:1.43±0.18 vs.1.00±0.16, P<0.01; 12 w: 1.94±0.23 vs.1.00±0.17,P<0.01;24w:1.23±0.28 vs.1.00±0.21,P<0.05)(Fig.20).7.2 The change of protein expression of TXNIP in myocardial tissue of DM rats at the 12th weekCompared with that of NC group, the protein expression of TXNIP in DM group was significantly increased at the 12th week (2.08±0.15 vs.1.00±0.15, P<0.01)(Fig.21).Conclusion1. Diabetes may cause myocardial injury and cell apoptosis, accompanying with cardiac function decrease, and the extent of which was aggravated with the development of the disease. And this apoptosis correlates with caspase-8-and caspase-9-dependent apoptotic pathways.2. Diabetes may attenuate the anti-apoptotic function of Trx by inhibiting TR activity, up-regulating TXNIP expression, and down-regulating Trx expression in the early stage, then induce myocardial cell apoptosis and heart injury.
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