| "Shock heart"(cardiac shock), occurring at the early stage of severe burns, is not only the pathophysical basis of inducement and aggravation of burn shock, but also the main cause of ischemia/hypoxia and dysfunctions of other organs such as kidney and gut. Therefore, it is of importance to protect myocardium from damage in the early stage postburn. Evidence has been found to indicate the notable protective effect of free glycine. Glycine obviously relieves endotoxemia and lowers rat mortality, lessens injury of skeletal muscle from ischemic/reperfusion, decreases the release of mediators of inflammation, calcium overload and reactive oxygen species. However, whether or not there is a protective effect of glycine on myocardium and its mechanism following severe burns remains unknown. In the present study, protective function of glycine on hypoxic cardiomyocytes and postburn myocardial damage and its mechanism is identified according to the changes of myocardial morphology, enzymology, free calcium ions, membrane potential and glycine receptor. Materials and Methods In vitro study 1.Cell model and groups Neonatal murine cardiomyocytes were harvested and divided into normal(N) group, glycine-treated group (GN), hypoxic group(H) , hypoxia combined with glycine grope(GH), hypoxia combined with glycine and anti-GlyRα1 polyclonal antibody grope(GHA), hypoxia treated with glycine and taurine group(GHT). 2. Observation of indexes and methods ①The cells were cultured in a hypoxic mixed gas containing 1% oxygen and used as hypoxic model. LDH,CK in the supernate fluid and the survival rate of cardiomyocytes after 1,3,6,12,24 h hypoxia were determined with routine methods. ②The changes of free calcium ions, membrane potential and immunofluoresecence histochemistry in the cardiomyocytes were determined using laser confocal microscopy following 1,3,6,12,24 h hypoxia. ③GlyRα1 subunits on membrane of cardiomyocytes were stained by immunohistochemistry and observed under Olympus fluorescence inverted microscope. In vivo animal study 1.Establishment of burn model and groups The study was carried out using a model of 30% TBSA of full-thickness burns in SD rats. The burn rats were grouped as normal (N),glycine-treated(G) and untreated(B) groups. 2.Observation indexes and methods ①The changes of pathological observations in myocardial tissues were obtained by HE staining. ②The contents of LDH,CK in serum were checked by normal methods. ③The content of TnI in serum was determined by enzyme-linked immunospecific assay. Results: In vitro study: 1. Levels of LDH in supernate fluid: In the hypoxic group, LDH levels were increased gradually and obviously after1,3,6,12,24 h hypoxia. However, LDH release in the glycine treated group were markedly decreased (P<0.01) when 0.1,0.5,1.0mmol/L of glycine were added. The level of LDH in the glycine treated group was negatively correlated to the concentration of glycine. 2. Levels of CK: Increased release of CK in cardiomyocytes after 1,3,6,12,24 h hypoxia was found in the hypoxic untreated group. However, The level of CK in the glycine-treated group were markedly decreased (P<0.01). 3. The survival rate of cardiomyocytes: The survival rate of cells was obviously decreased after 6h hypoxia, while the survival rate of cardiomyocytes markedly improved in the glycine treated group(P<0.01). 4. GlyRα1 subunits : GlyRα1 subunits were found on membrane of cardiomyocytes. 5. Concentration of calcium ions in cardiomyocyte: The calcium overload incardiomyocyte was detected after 6h hypoxia but considerably reduced when treated with glycine. The anti-GlyRα1 subunits was able to block the glycine receptor and inhibit the protective effect of glycine, intensifying the calcium overload. The calcium overload was blocked obviously when Taurine (glycine receptor agonist ) was given. 6. Membrane potential of cardiomyocyte: Fluorescence intensity of membrane potential of cardiomyocytes after 6h-hypoxia became lower than that in normal cardiomyocyte. The fluorescence intensity of membrane potential became stronger when glycine was added. This demonstrated that there is an obvious depolarization of membrane potential in cardiomyocytes after hypoxia, and the depolarization can be considerably blocked by glycine. In vivo study: 1. In the burn untreated rats, levels of LDH,CK in serum increased obviously, reached the maximum at 6 and 12 h respectively, then recovered gradually. However, the levels of LDH,CK in serum decreased obviously in the glycine-treated group. 2. The content of TnI in serum was elevated dramatically at 1 h after burns and came to the summit in 6 h, slightly recovered at 12 and 24 h, which was consistent with the pathological changes of myocardial tissues.The content of TnI in serum was decreased obviously in burn rats treated with glycine. 3. Changes of pathological observations in myocardial tissues were mild in the glycine-treated group than those found in the untreated. Conclusion: The results of both in vito and vivo studies indicate that glycine has an obvious protective effect on hypoxic cardiomyocytes and myocardial tissues in burn rats. The possible mechanism may be that glycine receptors existing in cardiomyocyte can be conjugated by glycine to attenuate membrane potential depolarization, which depletes voltage-dependent calcium channel on the cellular membrane, and blocks calcium overload after hypoxia。... |
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