| 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. The latest epidemiological survey has shown that there are92.4million adults of20years of age or older with diabetes in China, making China the No.1country with diabetics. The major harm of DM lies in its complications in various organs, especially of the heart, which becomes a main cause of death for diabetics. Researchers have done a lot in studying the mechanisms of diabetic cardiac complications, but mainly focused on the impact of coronary vascular injury and inflammatory response, and direct injury of high glucose and high fatty acid to myocardial cells received very little attention.Thioredoxin (Trx) system is an important redox protein system in the body, widely expressed in various cells. It has such functions as anti-free radical damage, anti-apoptosis, and pro-growth, and has played an important role in the occurrence and development of diseases involving free radical damage like reperfusion injury. During2009and2010, we had observed significant myocardial apoptosis accompanied by decline of Trx activity both in in vitro model of type2diabetic rats established by high-sugar and high-fat diet and streptozotocin (STZ) injection and in vivo model of cultured cardiomyocytes treated with high glucose and high fatty acid. In analyzing the reasons for the decline of Trx activity, it is noted that the expression of endogenous Trx-related protein-Trx interacting protein (TXNIP) was increased greatly. TXNIP can inhibit Trx function through combining with it. TXNIP is an endogenous inhibiting protein of Trx, and its expression can be increased significantly during diabetic cardiac injury, which indicates that TXNIP plays an important role in diabetic myocardial injury.Objective1. To induce cardiomyocyte injury by giving high glucose and high fatty acid, giving exogenous Trx to increase Trx activity, and to observe whether this injury can be reduced, in order to determine the function of Trx in high glucose and high fatty acid induced cell injury. 2. To induce cardiomyocyte injury by giving high glucose and high fatty acid, using RNA interference to inhibit the expression of endogenous TXNIP, and to observe whether high glucose and high fatty acid culture-induced cardiomyocyte apoptosis could be reduced.Methods1. Giving exogenous Trx for an interferenceH9c2cells in the logarithmic phase were randomly divided into three groups:Normal control group:cultured by DMEM with5mmol/L glucose and20mmol/L mannitol; High glucose and high fatty acid group:cultured by DMEM with25mmol/L glucose and600μmol/L palmitate; and Trx interference group:cultured by DMEM with25mmol/L glucose,600μmol/L palmitate and3μmol/L Trx. After cultured for48hours, the conditioned media and cardiomyocytes were collected to determine the activity of lactate dehydrogenase (LDH), caspase-3, and Trx; the expression of Trx was tested by western blot.2. Verifying the inhibitory effect of the recombined plasmidH9c2cells in the logarithmic phase were randomly divided into five groups:Normal control group:non-transfected normal cells; Negative control group (RNAi-HK group):empty plasmid HK transfected cells; RNAi-TXNIP1group:plasmid TXNIP1transfected cells; RNAi-TXNIP2group:plasmid TXNIP2transfected cells; and RNAi-TXNIP3group:plasmid TXNIP3transfected cells. Notes:we first transfected cells with RNAi-HK, and observed under fluorescent microscope at24h,48h, and72h to look for the highest transfection efficiency time point. Cells of five groups were transfected and cultured according to these time points, and collected when they were at the highest transfection efficiency time point to determine the expression of TXNIP by RT-PCR and western blot, in order to analyze the effect of RNA interference.3. Analyzing the role of TXNIP in high glucose and high fatty acid culture-induced myocardial apoptosisFrom the first part of the experiment, cells from RNAi-TXNIP3group were obtained for they were proved of the highest transfection efficiency for studying in this part. They were divided into four groups:(1) cells transfected with RNAi-HK were randomly divided into two groups:Normal+RNAi-HK group (cultured by DMEM with5mmol/L glucose and20mmol/L mannitol); High glucose and high fatty acid+RNAi-HK group (cultured by DMEM with25mmol/L glucose and600μmol/L palmitate).(2) Cells transfected with RNAi-TXNIP were randomly divided into two groups:Normal+RNAi-TXNIP3group (cultured by the medium of Normal+RNAi-HK group); High glucose and high fatty acid+RNAi-TXNIP3group (cultured by the medium of High glucose and high fatty acid+RNAi-HK group). Four groups were cultured for48h, and then collected the supernatant and cells for determining the activity of LDH, caspase-3, Trx, and p38kinase.Results1. Results of giving exogenous Trx for interference1.1LDH activity assayCompared with Normal control group, LDH activity of High glucose and high fatty acid group was increased significantly (260.50±11.35vs.69.20±8.04,P<0.01), which indicated high glucose and high fatty acid could induce myocardial cell apoptosis; but LDH activity was decreased greatly in Trx interference group (90.63±6.03vs.260.50±11.35,P<0.01), which indicated that giving exogenous Trx could decrease myocardial cell injury induced by high glucose and high fatty acid (figure1).1.2Cardiomyocyte apoptosis detectionThe apoptosis of cardiomyocytes was detected by using caspase-3activity assay. Caspase-3is the final common pathway of caspse-dependent apoptosis, through specific cleavage of downstream poly ADP-ribose polymerase PARP, and activating Ca2+/Mg2+-dependent nucleic acid enzymes, and finally leads to cell apoptosis, therefore, its activity can be used for detecting the extent of cell apoptosis.Compared with Normal control group, high glucose and high fatty acid culture could increase caspase-3activity of H9c2cardiomyoytes markedly (1.68±0.04vs.0.67±0.09,P<0.01), while Trx interference decreased myocardial cell apoptosis induced by high glucose and high fatty acid (caspase-3activity:0.87±0.05vs.1.68±0.04,P<0.01)(figure2).1.3Trx activity of H9c2cells assayTrx activity was assayed by using insulin reduction method. Compared with Normal control group, Trx activity of High glucose and high fatty acid group was decreased significantly (0.28±0.048vs.1.008±0.10, P<0.01); but compared with High glucose and high fatty acid group, Trx activity of Trx interference group was increased significantly (0.87±0.127vs.0.28±0.04, P<0.01)(figure3).1.4Measurement of expression of Trx protein by Western blotWestern blot belts analysis showed that, as compared with Normal control group, there was no significant difference in Trx expression of High glucose and high fatty acid group (1.07±0.07vs.1.06±0.07,P>0.05)(figure4), which indicated that high glucose and high fatty acid culture had not caused the level of Trx protein changed. Since great amount of exogenous Trx was added to Trx interference group, its useless to analyze Trx expression in this group.2. Results of verifying the inhibitory effects of recombinant plasmids2.1observation of transfection efficiency by fluorescence microscopeAfter H9c2cells were transfected with RNAi-HK, the transfection efficiency was observed under fluorescence microscope at24h,48h, and72h, and all were found green fluorescence of GFP in the plasma of cells, and this indicated the success of transfection. From calculating the proportion of green fluorescence, it was found that the transfection efficiencies of24h,48h and72h were10.94±1.02%,81.86±5.43%, and48.30±5.67%, respectively, among which, that of48h was the highest one (as compared with24h:P<0.01; with72h:P<0.01)(figure8), so we chose this time point for analysis of gene expression inhibitory rate after transient transfection of target gene.2.2Detection of interference effect on TXNIP after recombinant plasmid transfection2.2.1Effects of RNAi on the level of TXNIP mRNACompared with Normal control group, the level of TXNIP mRNA in RNAi-HK group was not changed significantly (0.92±0.05vs.1.025±0.025), but in RNAi-TXNIPl group, RNAi-TXNIP2group and RNAi-TXNIP3group, they were decreased significantly, respectively as0.34±0.02vs.1.02±0.02(P<0.01),0.62±0.082vs.1.02±0.02(P<0.01), and0.24±0.05vs.1.02±0.02(P<0.01)(figure11), which indicated that the three recombinant plasmids could reduce the level of TXNIP mRNA, with the inhibitory effects of66%,38%and76%, among which the highest was in RNAi-TXNIP3group.2.2.2Effects of RNAi on the expression of TXNIP proteinCompared with Normal control group, the expression of TXNIP protein in RNAi-HK group was not changed significantly (0.84±0.04vs.0.92±0.02), but in RNAi-TXNIPl group, RNAi-TXNIP2group and RNAi-TXNIP3group, they were decreased significantly, respectively as0.44±0.03vs.0.92±0.02(P<0.01),0.68±0.06vs.0.92±0.02(P<0.01) and0.26±0.01vs.0.92±0.02(P<0.01)(figure12). These results indicated that the three interference fragments could exert an interfering effect on the expression of TXNIP protein, with the inhibitory rates of56%,32%and74%. Taking results from RT-PCR and western blot together into consideration, it was concluded that the most effective inhibition to TXNIP gene of H9c2cells was in RNAi-TXNIP3group. Therefore, RNAi-TXNIP3was selected for later experiments as interference plasmid.3. Analysis of effects of TXNIP on high glucose and high fatty acid-induced cardiomyocyte apoptosis by use of TXNIP interference plasmid3.1Measurement of expression of TXNIP3.1.1Measurement of expression of TXNIP mRNACompared with Normal+RNAi-HK group, the level of TXNIP mRNA in High glucose and high fatty acid+RNAi-HK group was increased significantly (1.40±0.03vs.1.0±0.06,P<0.01); but in Normal+RNAi-TXNIP3group was decreased greatly (0.24±0.15vs.1.0±0.06, P<0.01); compared with High glucose and high fatty acid+RNAi-HK group, the level of TXNIP mRNA in High glucose and high fatty acid+RNAi-TXNIP3group was decreased greatly (0.47±0.04vs.1.40±0.03,P<0.01)(figure14).3.1.2Measurement of expression of TXNIP protein by Western blotCompared with Normal+RNAi-HK group, the level of TXNIP protein in High glucose and high fatty acid+RNAi-HK group was increased significantly (1.80±0.05vs.0.92±0.05, P<0.01); but in Normal+RNAi-TXNIP3group was decreased greatly(0.26±0.04vs.0.92±0.05, P<0.01); compared with High glucose and high fatty acid+RNAi-HK group, the level of TXNIP mRNA in High glucose and high fatty acid+RNAi-TXNIP3group was decreased greatly (0.30±0.04vs.0.92±0.05,P<0.01)(figure15).The results indicated that High glucose and high fatty acid could induce myocardial cell TXNIP expression; but RNAi could reduce myocardial cell TXNIP expression.3.2LDH activity assayCompared with Normal+RNAi-HK group, LDH activity of High glucose and high fatty acid+RNAi-HK group was increased significantly (260.50±11.35vs.69.20±8.04, P<0.01); compared with High glucose and high fatty acid+RNAi-HK group, LDH activity in High glucose and high fatty acid+RNAi-TXNIP3group was decreased greatly (106.90±13.16vs.260.50±11.35,P<0.01)(figure16). The results indicated that TXNIP expression decrease could reduce myocardial cell injury induced by high glucose and high fatty acid.3.3Caspase-3activity assayCompared with Normal+RNAi-HK group, caspase-3activities of High glucose and high fatty acid+RNAi-HK group was increased significantly (1.68±0.04vs.0.67±0.09(P<0.01)). Compared with High glucose and high fatty acid+RNAi-HK group, caspase-3activities of High glucose and high fatty acid+RNAi-TXNIP3group was even decreased (1.03±0.08vs.1.68±0.04(P<0.01)(Figure17). This indicated that TXNIP expression decrease could reduce myocardial cell injury induced by high glucose and high fatty acid.3.4Analysis of Trx activityCompared with Normal+RNAi-HK group, Trx activity of High glucose and high fatty acid+RNAi-HK group was decreased significantly (0.28±0.04vs.1.00±0.09, P<0.01); compared with High glucose and high fatty acid+RNAi-HK group, Trx activity in High glucose and high fatty acid+RNAi-TXNIP3group was increased greatly (0.44±0.12vs.0.28±0.04(P<0.05))(figure18). The results indicated that under high glucose and high fatty acid condition, TXNIP induced myocardial injury and apoptosis through inhibiting Trx activity.3.5Analysis of p38activityCompared with Normal+RNAi-HK group, p38activity of High glucose and high fatty acid+RNAi-HK group was increased significantly (1.86±0.09vs.1.05±0.09,P<0.01); compared with High glucose and high fatty acid+RNAi-HK group, p38activity in High glucose and high fatty acid+RNAi-TXNIP3group was decreased significantly (1.24±0.16vs.1.86±0.09,(P<0.01))(figure19).Conclusions1. High glucose and high fatty acid culture could cause injury and cell apoptosis of H9c2cardiomyocytes, and this may result from the decrease of Trx activity induced by high glucose and high fatty acid; supplying exogenous Trx could increase Trx activity significantly, therefore reduce myocardial injury and apoptosis.2. Inhibitory effect of high glucose and high fatty acid on Trx activity is related to its inducing effect on TXNIP expression. Using RNA interference plasmid to inhibit high glucose and high fatty acid-induced TXNIP expression could increase Trx activity and reduce activation of p38kinase, therefore reduce myocardial injury and apoptosis. |
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