| IntroductionWith the development of social economy and 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. Nowerdays, there are 92.4 milion adults of 20 years of age or older with diatebes in China, making China the No. 1 country with diabetics. Among the diabetic complications like cardiovascular and cerebrovascular diseases, retinopathy, diabetic nephropathy, and diabetic foot, etc., 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 (Trx) system is an important redox 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. Trx system is an important anti-free radical damage system, so we speculate that Trx might be involved in a variety of damages caused by DM. Our previous in vivo studies have shown that, in type 2 diatetic rat, there are cardiac injury and cardiomyocyte apoptosis, meanwhile, through the inhibition of TR (Thioredoxin reductase) activity and the increase of TXNIP (Thioredoxin interacting protein) protein expression to inhibit Trx ativity; even in the early stage in this model, we found the decrease expression of Trx and therefore the decrease of its activity, finally weakened its anti-apoptotic effects, causing the apoptosis of cardiomyocytes.The possible reasons for diabetes causing cardiac injury may be various, including high glucose and high fatty acid, inflammatory mediators like TNF-αproduced by neutrophils, coronary artery injury, etc. In order to know whether high glucose and high fatty acid metabolism in diatetes has direct damage on cardiomyocytes, whether Trx system in cardic cells changes, and whether the change mediates this injury, we choose cardiomyocyte culture to carry out the study. Although there is evidence in neonatal rat cardiomyocytes culture that high glucose and high fatty acid could cause injury, there are still differences in shape, structure, growth, and responses to the external stimuli between neonatal rat cardiomyocytes and adult rat cardiomyocytes. So we established the adult rat cardiomyocyte culture model, giving high glucose and high fatty acid to imitate the high glucose and high fatty acid metabolism in diabetes to investigate whether they could cause injury to the cardiomyocytes and explore the possible effects of Trx system and TXNIP in it.Objective1. To establish the model of cultured adult rat cardiomyocyte and observe whether high glucose and high fatty acid culture could cause apoptosis of cardiomyocytes and the extent of the damage and apoptosis.2. To observe the changes of the activity and expression of Trx system in cardiomyocyte injury caused by high glucose and high fatty acid culture; to analyze the relationship between the changes and the cultured-cardiomyocyte apoptosis.3. To observe whether TXNIP changes in high glucose and high fatty acid culture-induced cardiomyocyte apoptosis, and to analyse the relationship between the changes and the cultured-cardiomyocyte apoptosis.MethodsAfter given heparin and anaesthetic, the heart from male adult SD rat (about 250 g) was excised and the aorta was cannulated rapidly. The cannulated heart was mounted on a Langendorff perfusion apparatus with constant flow and then digested by a 0.05 % compound collagenase solution at 37 oC; the enzymatic digestion was terminated when the heart became softened. The heart was then cut off the cannula with the atria and aorta dissected away. The ventricular tissue was chopped in a stop buffer, and the single myocyte was dispersed gently by a wide tipped pipette. Then the extracellular calcium was restored stepwise to a final concentration of 1000μmol/L. Finally, after adjusting the cell suspension to an appropriate concentration, it was plated into 35mm culture dishes pre-coated with mouse laminin. The dishes were randomly divided into two groups: Control group: cultured by M199 with 5 mmol/L glucose and 20 mmol/L mannitol; High glucose and high fatty acid group: cultured by M199 with 25 mmol/L glucose and 600μmol/L palmitate. Cardiomyocytes were cultured in those two kinds of media for 48 hours and then the conditioned media and cardiomyocytes were collected respectively for different assays.The activity of lactate dehydrogenase (LDH) was measured by using an LDH Assay Kit; caspase-3 activity was measured to reflect the extent of cardiomyocyte apoptosis; caspase-8 activity and caspase-9 activity were measured to analyze the pathways of cardiomocyte apoptosis; the activities of Trx and TR in cardiac muscle cells were detected by insulin disulfide reduction assay and TR assay kit, respectively; the mRNA levels of Trx1, Trx2, TR1, TR2 and TXNIP were measured by Real-time PCR; the protein expressions of Trx1, Trx2, TR1, TR2 and TXNIP were measured by Western Blot.Results1. The morphology of cultured adult rat cardiomyocytes after 48hAfter being cultured in two different media for 48h, the cardiomyocytes were observed under a light microscope and we found that there were 90 % of rod-shaped cardiomyocytes in Control group being quiescent and had visible cross striations and sharp edges; while there were less cardiomyocytes in High glucose and high fatty acid group, and the cross striations became indistinct, with more shortened and round shape cells in the dishes, the latter of which reperesent the dying ones (Fig. 1). This suggested that the cultured adult rat cardiomyocytes model was successfully established.2. LDH activity assayThe LDH activity of Control group is (715.2±128.2) U/L, while that of High glucose and high fatty acid group is much higher: (1054.6±122.9) U/L, and there is statistical difference between them (P<0.01). This result suggests that membranes of the cultured adult rat cardiomyocytes were injured, causing LDH released greatly outside the cells, which represents the necrotic cardiac cell injury (Table 1).3. Increased cardiomyocytes apoptosis in High glucose and high fatty acid group3.1 Caspase-3 activity assayAfter being cultured in two different media for 48h, caspase-3 activity was assayed, with the results of (0.0829±0.0115) nmol?h-1?mg-1pro. in High glucose and high fatty acid group and (0.0543±0.0118) nmol?h-1?mg-1pro. in Control group, showing significant increase in the former, and there is a statistical difference between them (P<0.01) (Fig.2).3.2 Caspase-8 activity and Caspase-9 activity assay After 48h culture, caspase-8 activity and caspase-9 activity in High glucose and high fatty acid group were significantly increased compared with those of Control group, [caspase-8 activity : (0.1991±0.0334) nmol?h-1?mg-1pro. vs. (0.1446±0.0279) nmol?h-1?mg-1pro., P<0.01; caspase-9 activity : (0.1036±0.0146) nmol?h-1?mg-1pro. vs. (0.0653±0.0170) nmol?h-1?mg-1pro., P<0.01)] (Fig.2)4. The activities of Trx system in High glucose and high fatty acid group were decreased4.1 The activity of Trx in High glucose and high fatty acid group was decreased Compared with that of Control group, the relative Trx activity of High glucose and high fatty acid group was decreased after being cultured for 48h (0.24±0.17 vs. 1.00±0.37, P<0.01) (Fig.3).4.2 The activity of TR in High glucose and high fatty acid group was decreased Compared with that of Control group, the relative TR activity of High glucose and high fatty acid group was significantly decreased after being cultured for 48h (0.60±0.22 vs. 1.00±0.28, P<0.01) (Fig. 4).5. The changes of mRNA level and protein expressions of Trx system in 48h-cultured adult rat cardiomyocytes5.1 The changes of mRNA levels of Trx system Compared with those of Control group, there was no significant difference in mRNA levels of Trx1 and Trx2 in High glucose and high fatty acid group after 48h culture; but for TR1 and TR2, their mRNA levels were decreased, and the differences are of statistical significance (Trx1: 1.052±0.189 vs. 1.000±0.000, P>0.05; Trx2: 0.999±0.258 vs.1.000±0.000, P>0.05; TR1: 0.862±0.126 vs. 1.000±0.000, P<0.05; TR2: 0.803±0.174 vs. 1.000±0.000, P<0.05) (Fig. 7, 10). Compared with those of Control group, there was no significant difference in the relative protein expressions of Trx1 and Trx2 in High glucose and high fatty acid group; but for TR1 and TR2, the expressions were significantly decreased (Trx1:0.74±0.26 vs. 1.00±0.44, P>0.05; Trx2:0.68±0.21 vs. 1.00±0.37, P>0.05; TR1: 0.55±0.19 vs. 1.00±0.40, P<0.05; TR2: 0.49±0.20 vs. 1.00±0.55, P<0.05) (Fig.11, 12, 13, 14).6. The changes of mRNA level and protein expression of TXNIP in 48h-cultured adult rat cardiomyocytes6.1 The change of mRNA level of TXNIP Compared with that of Control group, the mRNA level of TXNIP in High glucose and high fatty acid group was significantly increased after 48h culture (1.418±0.302 vs. 1.000±0.000, P<0.01) (Fig.17).6.2 The change of protein expression of TXNIP Compared with that of Control group, the relative protein expression of TXNIP in High glucose and high fatty acid group was significantly increased after being cultured for 48h (1.63±0.57 vs. 1.00±0.13, P<0.05) (Fig.18).Conclusions1.High glucose and high fatty acid culture could cause injury and cell apoptosis in adult rat cardiomyocytes, and caspase-8- and caspase-9-dependent apoptotic pathways were both involved in this apoptosis.2. High glucose and high fatty acid culture could decrease Trx activity, therefore attenuated the anti-apoptotic function of Trx and induced cardiomyocytes apoptosis.3. The inhibition of high glucose and high fatty acid culture to Trx was related to its decreasing TR activity and increasing TXNIP expression effects. 5.2 The changes of protein expressions of Trx system... |
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