The Role Of TGR5 In High-glucose Induced- Apoptosis Of Myocardial Cell In Mice And Its Mechanism

Background: The latest statistics of The International Diabetes Federation(IDF) in 2011 November shown that the patients in China, who have diabetes, are more than 90 millions and ranked first in the world. Till 2030,the number will be increased to 13 billions if without any measures[1]. As diabetes mellitus complications, diabetic cardiomy-opathy(diabetic cardiomyopathy, DCM) is not only widely recognized by more and more medical professionals, but also has been gain highly focus by clinicians and epidemiologists. At present, the major reasons why DCM caused the occurrence of apoptosis are related to the following mechanisms:(1) the main signal for myocardial energy metabolic abnormalities, is glycolipids myocardial abnormalities, which will let the heart increases using fat and lessens the use of sugar.(2) The intracellular calcium abnormally adjusts.(3) The microvascular lesion and microcirculation.(4) The reason why high blood glucose increases oxidative stress, may be strengthened by the signal transduction pathways so as to activate the programmed cell death[2]. Although the pathogenesis of DCM is complicated, the popular studies are tending to the oxidative stress in diabetic cardiomyopathy and the position and role of the occurrence and development[3]. High blood sugar induced by oxidative stress can start and runs through the whole stage of diabetic cardiomyopathy, which is also important to damage myocardial cell apoptosis[4].As one of the bile acid receptor, G protein coupled bile acid receptor 1(G protein-Coupled bile acid receptor 1, TGR5) plays an important role in bile acid signaling network. Recently, the activation of TGR5 can be involved in the body energy metabolism, and alleviated oxidative stress and inflammatory reaction[5]. Therefore, TGR5 may become a new target for the treatment of diabetic cardiomyopathy. TGR5 is widely expressed in many tissues, such as brown adipose tissue, liver, muscle and the central nervous system, but the effects are significantly different in various tissues. The highest effect is shown on gallbladder that there is evidence confirmed by the study on myocardial cells [6].The research takes the myocardial cells of neonatal mice as sample and use modern molecular biology technique to discuss under a high glucose circumstance, after agitating TGR5 receptor on myocardial cell, what’s the membrane effect on myocardial cell apoptosis and any possible mechanism. Objective: This experiment if studied at myocardial cells of neonatal mice, to investigate whether the agitated membrane TGR5 receptor on myocardial cell membrane can alleviate the damage of high glucose on the myocardial cells and its possible mechanism, so as to provide treatment of ideas and a new theoretical foundation for the diabetic cardiomyopathy.Methods: 1.Culturing of myocardial cells of neonatal mice Obtained heart under sterile conditions, cleaned it & inserted 4°C then let it stay overnight, stopped digesting the next day, got the single myocardial cell by times with the prepared collagenase digestion liquid, collected the cells then inoculated inthe dish, with differential adhesion method to remove cardiac fibroblasts, relatively high purity of myocardial cell was obtained for this experiment.2. The establishment of myocardial apoptosis model induced by high glucose Myocardial cells in the control group, the blood glucose concentration(5.5mmol/L)Cultured 2-3 days medium, selection of adherent cell density above 90%, beating rhythm of myocardial cells in 120-140 /min for experiment. With high glucose(40 mmol/L) DMEM affect myocardial cells after 72 h, using the difference in detection of myocardial cell apoptosis and the normal group cytometry flow.3. Expression of TGR5 m RNA and HO-1 m RNA real-time PCR detection of myocardial cell membrane According to experimental design, divided myocardial cells in different groups after intervention. RNA was extracted from each group, after the identification of RNA purity and concentration, reverses transcribed c DNA gene sequence data, checked the serial number from Gene Bank, designed corresponding primers by computer software Prime Premier 5.0, and then started fluorescent quantitative detection of target gene. 4. By using western-blot to inspect how TGR5 protein shows on myocardial cells membrane. According to experimental design, divided myocardial cells into different intervention group. Then extracted each group myocardial cell membrane protein after cultivated by different methods. Next, gained the protein concentration determined by BCA method. With Tubul in as the refe-rence, these protein samples were separated, transferred film, clos ed, followed by incubation 1 anti-body and 2 anti-body, and color imaged Final ly, the colored image by image processing software Quantity One was detectedand analyzed the band gray value to detecting the expression level of target gen e and protein determination.5. Sh RNA silencing TGR5 gene on myocardial cell membrane The cell concentration of 2×l07/ hole, inoculated in 6-well plate, normal cultured for 3 days, when the adherent cells reached 90%, took the good status cell for experiment. Polybrene transfection reagent mixed with high glucose + agonists medium according to 5 μg/m L concentration, incubated for 15 mins in room temperature, took sh RNA diluents 20 μL solution then added into the culture hole and mixed them, finally added the transfection medium. Extra necessary RNA from harvested transfected cells detected the expression of TGR5 and flow cytometry measurement of apoptosis by real-time PCR.6. Myocardial cell apoptosis rate was examined by flow cytometry. After 72 hours when processed each myocardium cell in different group, by using 0.25% trypsin digested a unicell and do the collection. Then using ITC Annexin V/Propidium Iodide stained separately, performed at room temperature, avoids light, reacted 15 min, then added Binding Buffer, mixing to detect apoptosis rate with flow cytometry.Results:1. Myocardial cells of mice of is successfully cultured.2. High glucose induced cardiomyocyte apoptosis in mice model established successfully.3. Real-time PCR results:3.1 Expression of TGR5 m RNA in different groups: control group, high glucose and high glucose addition of agonist group expression gradually increased, the inter group comparison of P < 0.01; compared with the control group, low sugar hypertonic group expressed lower, P > 0.05; compared with the control group, the expression increased significantly in liver tissue of group, P < 0.01; compared with high glucose +agonists, high glucose + agonists + virus groups of expression decreased significantly, P < 0.01.3.2 HO-1 m RNA expression in different groups: control group, high glucose group, high glucose +Nomilin group, the expression increased in turn, each group P < 0.01. 4. Expression of TGR5 protein with Western blot method, analysis and measuring of objective optical density with value. Compared with the control group, high glucose group is more highly expressed, P< 0.05; compared with high glucose group, high glucose + agonist group expression increased significantly, P < 0.01. 5. Sh RNA infected myocardial cells successfully. 6. Cardiomyocyte apoptosis was detected with flow cytometry. The rate of myocardial cells in each group(divided to early and terminal stage) is tested by flow cytometry to compare the difference between the groups. The high glucose group got more apoptotic cells than the control group, P < 0.01; high glucose +Nomilin group got less apoptotic cells obviously than the high glucose group, P< 0.01; high glucose +Nomilin+ virus group significantly increased apoptotic cells than high glucose +Nomilin group, P< 0.01; high glucose +Nomilin+Zn PP group significantly increased apoptotic cells than high glucose +Nomilin group, P < 0.01.Conclusions: 1. High glucose can increase the expression of TGR5 m RNA and protein of mice myocardial cells.2. Activation of TGR5 can reduce the apoptosis of myocardial cells of mice in high glucose and its mechanism may be related to increasing intracellular level of HO-1 m RNA so as to protect the myocardial cells.