The Effects Of Glutamine, Glycine And Glycyl-glutamine Dipeptide On Cardiac Cytoprotection And Its Mechanism After Burn Injury

Background:Previous studies demonstrated that severe cardiac damage may occur in the early period of burn injury,resulting in the cardiac shock,and it is one of the frequent complications postburn.Cardiac damage not only cause myocardium dysfunction,but also induce or aggravate shock,lead to or exacerbate the damage to other organs due to ischemia and hypoxia,and even lead to multiple organ dysfunction syndromes(MODS).So,it is very important to effective protect myocardium in the early stage of burn injury on reversing shock,reducing the organ damage,and decreasing the internal organ complications.Nowadays,the fluid replacement and using cardiotonic were major therapeutic measure in burned patients for protecting myocardium.Although these measure may improve the cardiac contractile function at a certain degree,they can increase the load of the cardiac muscle.It is necessary to find a new drug to protect myocardium at early stage after burn injury.It was reported that glutamine(Gln) and glycine(Gly) were beneficial to protecting myocardium,minimizing cardiac damage and improving cardiac contractile function at the stage of ischemia and hypoxia damage.But glutamine could not be intravenous injected as an emergency medicine for cardiac damage at earlier stage of burn injury for its low solubility and weak heat stability.The glycyl-glutamine dipeptide(Gly-Gln),synthesis of glutamine and glycine,overcomes these shortcoming of glutamine and can minimize intestine injury and protect intestine mucosal barrier function generally.However,there is no report of the use of Gly-Gln to treat in heart damage after burns,and it is not clear whether the Gly-Gln is able to lessen myocardium damage,improve cardiac function up to now.Objective:To investigate the protective effects of glutamine,glycine and glycyl-glutamine dipeptide on myocardium damage and its signal mechanism after burn injury. Methods:In vitro experiment:One hundred and thirty six adult Wistar rats(weighing 200±20g,male or female) were obtained from the Laboratory Animal Center,Third Military Medical University.Rats were housed in individual cages in the animal care facility under controlled conditions of temperature and free access to water and standard pellet rat food for one week.All groups were fasted for 12 hours before burn injury.1.Animal groups and medicine supplementsRats were inflicted with 30%TBSA full-thickness burn and divided randomly into four groups:burned control(B) group,glutamine-treatment group(Gln),glycine-treatment group(Gly) and glycyl- glutamine dipeptide-treatment group(GG).Gln group supplemented with glutamine at 1.0g/kg.d,Gly group supplemented with glycine at 0.5g/kg.d,GG group supplemented with glycyl- glutamine dipeptide at 1.5g/kg.d.In order to maintain isonitrogenous and isocaloric intake,every group rats were supplemented with Tyrosine to control the intake of amino acids at 1.5g/kg.d.2.Experimental technique and index(1).Myocardial zymogram(CK,LDH,AST) was detected by automatic biochemistry analyzer.(2).The tissue section was stained with hematoxylin and eosin(3).Under anesthesia,the catheter connected with the press transducer of the four channel physiological recorder.Aortic systolic pressure(AOSP),aortic diastolic pressure (AODP) and left ventricular systolic peak pressure(LVSP) as well as±dp/dt_max were measured.(4).High-energy phosphate compound(AMP,ADP,ATP) was measured by high performance liquid chromatography.(5).Tissue glutathione and plasma lactic acid were detected by ultraviolet spectrophotometer.(6).Tissue heat shock protein 70 was detected by western-blotting.(7).Tissue Metallothionein was detected by immunofluorescence.In vivo experiment:Two hundred neonatal Wistar rats were obtained from the Laboratory Animal Center, Third Military Medical University.1.Primary cell culture and injury modelRats degermed by alcohol and the heart were quickly removed into cold PBS to wash out,the ventricles were cut into 1-3mm³ cubes and separated by trypsinization.Freed cells were collected into culture flask and cultured in DMEM culture medium with 10%fetal calf serum and 0.01M BrdU for 3 days.2.Cell groups and disposal(1).Cardiac cell were randomly divided into control group(C),burn group(B), administration group(Gln group supplied DMEM of final concentration 4,8,12,16 and 20mmol/L Gln;Gly group supplied DMEM of final concentration 0.4,0.8,1.2,1.6 and 2.0mmol/L Gly),and incubation with 10%burn serum for 30min to 6h.(2).The cells climbing on the carry sheet glass were randomly divided into control group(C),burn group(B),administration group(Gln group supplied DMEM of final concentration 12mmol/L Gln;Gly group supplied DMEM of final concentration 0.8mmol/L Gly) and blocking agent group(G+R).Blocking agent group added final concentration 25ng/ml rapamycin.Cells incubation with 10%burn serum for1,3,6h,formalin fixation.3.Experimental Technique and index(1).Cell survival was detected with trypan blue excluded experiment(2).The phosphorylation of mTOR,P70 S6k,4E-BP1 were revealed by western-blotting(3).HSP 70,MT and microtubule were detected through laser confocal microscope with immumofluorescence methodResults:1.The myocardial zymogram content was obviously increased after burn injury,CK, LDH and AST reaching a peak duing PBH12～72.Administration of Gln,Gly, Glycin-glutamine dipeptide could significantly decrease myocardial zymogram.2.Histological sections of burn-treatmed hearts showed widespread cardiac interstitial edema,muscle fibers discord at PBH12.Cardiac interstitial edema intensify,some muscle fibers break or waves arrange,focus dissolve at intracytoplasm.Cardiac muscle fibers necrosis and break,nucleus disappearance during PBH48～72.Administration of Gln,Gly, Glycin-glutamine dipeptide could lessen myocardial tissue pathological lesion obviously. 3.AOSP,AODP,LVSP and +dp/dtmax were decreased postburn,reached the lowest point at PBH72.Administration of Gln,Gly,Glycin-glutamine dipeptide could significantly improve the cardiac contractile function after burn injury.4.The ATP content and EC were obviously decreased and reached the lowest point at PBH72.The ADP and AMP content were increased and reached a peak at PBH12.The ATP, EC of treated groups were obviously higher than burned control group.5.The Lactic Acid content was increased and reached a peak at PBH24.The changes of administration groups were obviously less than the burned control group.6.The GSH content was decreased and reached the lowest point at PBH72.The changes of treated groups were obviously higher than burned control group.7.Administration of Gln,Gly and Glycin-glutamine dipeptide could significantly promote MT and HSP 70 protein synthesis.8.The degree of mTOR,P70 S6k and 4E-BP1 phosphorylation were significantly promoted in treated groups incubated with Gln and Gly.9.Gln and Gly could improve HSP 70 and MT protein synthesis,and the effects of Gln and Gly could be downregulated by using rapamycin.10.The microtubular structure of normal control group was integrity,network alinement,stain uniformitly.Microtubular was disruption,aniso-stain and structure blur after incubation with burn serum for 1h to 6h.Administration of Gln and Gly could obviously lessen microtubular damage.Conclusion:1.Gln and Gly can improve energy metabolism of myocardium,promote ATP synthesis and increase energy reserve.2.Gln and Gly can promote GSH and MT synthesis,decrease free radicle production and lessen lipid peroxidation damage.3.Gln and Gly can promote synthesize of HSP 70 and minimize microtuble damage.4.Gln and Gly can regulate translation of HSP 70 and MT through mTOR -P70S6k -4E-BP1 signal transduction pathway,and promote protein synthesis.