Effects Of Glucagon-like Peptide 1 On Cardiomyocyte Apoptosis In Scalded Rats During The Early Stage Post Injury

Mode of cell death typically includes necrosis and apoptosis. Compared with necrosis, apoptosis has its unique morphological and biochemical changes. It is generally believed that apoptosis is a kind of cellular suicide process caused by the cell death programs, which are stored in the cell and are used by the external factors. Apoptosis is a fundamental cellular event, and is a widespread phenomenon on life. Appropriate apoptosis is normal physiological events during the development of tissues and organs, but excessive apoptosis can lead to the organic disease and dysfunction of tissue and organ. As the terminal differentiation cells, cardiomyocytes basically lose the proliferative potential after the birth. Excessive apoptosis of cardiomyocytes will result in the decrease of myocardial effective contractility units and the change of the function. Therefore, excessive apoptosis of cardiomyocytes have an important impact in cardiac structures and functions. The cardiomyocyte apoptosis is affected by a lot of factors, including acute myocardial ischemia, mechanical pressure, oxygen radical, intracellular calcium overload and some neurological endocrine hormones and some cytokines. It suggests that in the injury of cardiac myocytes caused by ischemia and hypoxia, cell necrosis is subsequent to apoptosis, proving that cardiomyocyte apoptosis is one of the main form of cell death in the early stage post injury. It is also identified that apoptosis is an important reason on rats'myocardial injury and myocardial dysfunction in the early stage post scald. Therefore, in this case, how to control the cardiomyocyte apoptosis is an important measure to protecting the myocardials injury and myocardial dysfunction after burn.Glucagon-like peptide 1 (GLP-1) is a polypeptide-ducdenin that is mainly secreted by Langerhans cells in the distal ileum, the colon or the rectum. Its effect is mainly upon the specific receptor-glucagon-like peptide 1 (GLP-1R). Its major functions are:stimulating glucose-dependent insulin secretion; inhibiting glucagon secretion; inhibiting gastrointestinal motility and gastric secretion, delaying gastric emptying; promoting P-cells of islet's regeneration and inhibiting their apoptosis, etc. In addition to express in the pancreatic tissue, GLP-1 R also exists in brain, lung, heart, kidney and other organs. The wide distribution determines the diversity of effect. In the research of normal rat's heart and cardiac ischemia/reperfusion model, Zhao and his colleagues have found that besides the direct effect on the heart, GLP-1 decreased the left ventricular developed pressure and LV dP/dt in normal hearts, it also significantly increased the uptake of myocardial glucose and translocation of glucose transporter-1 through increased synthesis of nitric oxide. GLP-1 also accelerated cardiac function recovery after 30 min'ischemia, especially the left ventricular end-diastolic pressure was significantly improved. Recent studies have confirmed that GLP-1 can be acted on cardiomyocytes In vitro directly, having some protective effects on Hypoxia/reoxygenation-induced myocardium injury, the mechanism may be major generated from the inhibition of cardiomyocyte apoptosis.There are so many studies have shown that GLP-1 can inhibits cardiomyocyte apoptosis, but the GLP-1 can easily be decomposed by dipeptidase IV (DPPIV) in vivo and its Half-life is less than 2 minutes. It's hard to detect the dynamic condition from the animal's experiment, which limits its application in vivo experiments. Afterwards, some researchers isolated Exendin-4 from American lizard saliva, and it is a long-acting analogue of GLP-1. Preliminary studies indicate that Exendin-4 and GLP-1 have similar biological effect, such as blood glucose regulation, insulin secretion, improving the function of islet, inhibitingβ-cell apoptosis and so on. GLP-1 has only got 31 amino acid residue, but Exendin-4 has eight more amino acid residue at the end of C. Comparing with GLP-1, the second part of amino acid is amino acid residue which will not be decomposed by Dipeptidyl peptidaseⅣ, and the plasma half-life time may even longer. Does the impact of Exendin-4 participate in the process of cardiomyocyte apoptosis after scald? There are not relevant reports yet at home or abroad so far. In this dissertation, it discusses the effect and mechanism of Exendin-4 and GLP-1 on cardiomyocyte apoptosis in the early stage post burn, through detecting the expression of GLP-1 R in myocardium, cardiomyocyte apoptosis index and Caspase-3 activity in myocardium in the early stage post injury in terms of the administration of Exendin-4 intervent in 30% TBSAⅢ°scalded rats.Objectives1. To explore the changes of myocardial GLP-1 R expression and its distribution feature in rats in the early stage post scald as well as those after administration of Exendin-4.2. To observe the change of cardiomyocyte apoptotic index and myocardial Caspase-3 activity of rats in the early stage post scald.3. To observe the change of cardiomyocyte apoptotic index and myocardial Caspase-3 activity of rats after administration of Exendin-4, and attempt to clarify the effect of Exendin-4 on cardiomyocyte apoptosis of rats in the early stage post scald as well as the mechanism.Materials and Methods1. Experimental animals and grouping:54 healthy adult SD rats of either sex (supplied by the Experimental Animal Center of Southern Medical University), weighting (200±20) g, According to random number table, the cases were randomized into three groups:normal control group (N group, n=6), simple burn group (S group, n=24), administered with Exendin-4 group (C group, n=24).2. Animal model production:30 g/L sodium amytal was injected into the abdominal cavity for anesthesia with the dosage of (40 mg/kg) after weight. Back hair on rats was depilated by 100 g/L sodium sulfide. Calculate the TBS A of the rats according to the formula of S=K·W2/3(K=9.0, S Units:cm2, W Units:g), Scald area equals to overall area multiply 30%. Then 30% TBSA deep third degree scald was caused by immersing rats in 100℃hot water for 15 s. After injury, according to Parkland formula, that is, per-kilogram of body weight for every 1% of the burn area 4 ml intraperitoneal injection of compound sodium lactate Ringer's solution would be half of the total is divided into two equal portions, immediately after injury and injected 4 h after injury, the other half is divided into four copies after injury 8 h,12 h,16 h, and 24 h into the injured after second 24-hour, halve the liquid volume and divided it into 3 sub-uniform rehydration. E group Administration Methods:Exendin-4 was dissolved in 0.2 mL water for injection, with Exendin-4 5 ug/kg,2 times a day, injected subcutaneously, and giving the first injection immediately after injury.3. Indication and Methods:Respectively, Kill the rats in 6,12,24 and 48 h with the depth of anesthesia at total of four observation time points. Take left ventricular myocardial tissue for standby. Use immunohistochemical staining method to detect the expression of myocardial GLP-1R distribution characteristics; Use the TUNEL way to detect cardiomyocyte apoptosis and calculate the apoptotic index; fluorescence assay Caspase-3 activity (pNA content indicated the results).4. Statistics processing:All the data presented by X士S, and analyzed by statistical software of SPSS 13.0. This experiment is 3×4* 6 factorial design, The lines completely randomized two factor factorial analysis (two-way analysis of variance), to understand the main effects and interaction of the various variables; do one-way analysis of variance among time groups and treatment groups, Level data use Kruskal-Wallis H way to test. Associativity among variables was analyzed by correlation method of pearson. Statistical significance was assumed at P<0.05.Results1. There were numerous myocardial GLP-1R express in the normal, simple scalded and scalded rats with administration of exendin-4 respectively. GLP-1R mainly distributed in the cells membrane, cytoplasm and nucleus of cardiomyocytes. There was no significant changes about the expression of myocardial GLP-1R in the three groups.2. There were only a few myocardial apoptotic cells in N group (0.23±0.32)%. Compared with that in N group, the cardiomyocyte apoptotic index in S group increased at 6 post scald hour (PSH), reached the peak at 12 PSH (14.80±5.46%), then decreased gradually, but it was still significantly higher at 24,48 PSH (10.27±4.48%,7.07±2.90%). After administration of exendin-4, myocardial apoptotic index was significantly decreased at 6 PSH [(4.03±1.18)%],12 PSH [(6.50±1.41)%],24 PSH [(3.70±1.05)%] and 48 PSH [(2.90±1.11)%] with comparison of that in S group (P＜0.05).3. Myocardial caspase-3 activity in S group increased significantly at 6,12,24 PSH. Compared with N group (47.86±16.09 mmol/L), myocardial Caspase-3 activity in S group was significantly enhanced at 6 PSH (104.33±26.92 mmol/L), reached the maximum value at 12 PSH (162.87±43.53 mmol/L), then gradually reduced, but continued at a high level at 24 PSH (111.65±20.43 mmol/L). Myocardial Caspase-3 activity in E group was significantly decreased at 6 PSH (65.86±7.80 mmol/L),12 PSH (83.01±16.85 mmol/L) and 24 PSH (66.82±13.61 mmol/L) than that in S group respectively (P＜0.05).4. The apoptotic index of cardiomyocyte was significantly positively correlated with myocardial caspase-3 activity in group S and group E, coefficient correlation is 0.674 and 0.511 (P<0.05).Conclusions1. There are myocardial GLP-1R expression in both simply scalded rats and scalded rats with administration of exendin-4, GLP-1R is mainly distributed in the cell membrane, cytoplasm and nucleus of cardiac myocytes.2. There is no significant changes about the expression of myocardial GLP-1R in the three groups, which indicate the anti-apoptotic effect of exendin-4 on cardiomyocyte may not be by regulating the number of receptor, but should be due to activating downstream molecular signals induced by combination with the receptor.3. Apoptotic index of cardiomyocyte and myocardial caspase-3 activity were increased respectively in scalded rats during the early stage post injury. Administration of exdendin-4 can significantly reduce the apoptotic index and myocardial caspase-3 activity.4. Exdendin-4 may can combinate with the GLP-1R, then activate the cell signaling pathways, and decrease caspase-3 activity, ultimately inhibit cardiomyocyte apoptosis.