摘要
严重烧伤早期发生的“休克心”,是诱发或加重烧伤休克,导致其他组织器官缺血缺氧损害和功能障碍的重要原因之一,因此在烧伤早期实施有效的心肌保护措施对纠正休克、防治组织器官缺血缺氧损害具有重要意义。近年来的研究提出,游离的甘氨酸具有显著的细胞保护作用。甘氨酸可明显减轻缺血、再灌注所致的骨骼肌损伤;减轻细胞因子的释放及钙超载和氧自由基的产生;还能减轻内毒素血症,降低内毒素血症大鼠的死亡率。但甘氨酸是否对烧伤后心肌组织具有保护作用及其作用机制尚未见报道。
本研究通过建立体外培养的乳鼠心肌细胞缺血缺氧模型和大鼠30%烧伤模型,从心肌的能量、酶学及受体的证实等一系列指标,以明确甘氨酸对烧伤早期大鼠心肌损害的保护作用及心肌组织存在甘氨酸受体α1亚基。然后通过观察体外培养的心肌细胞,明确甘氨酸的最佳作用浓度,检测心肌细胞膜甘氨酸受体、钙离子、细胞膜电位、线粒体膜电位、mPTP开放、capase-3及其凋亡的变化,探讨甘氨酸保护作用机理。
材料与方法:
一、体外实验
1.模型与分组:离体培养的SD乳鼠心肌细胞,分为常氧组(N)、常氧加甘氨酸组(NG)、缺血缺氧组(1%氧浓度)(H)、甘氨酸处理加缺血缺氧组(GH组)、甘氨酸加甘氨酸受体激动剂Taurine加缺血缺氧组(GTH)、甘氨酸加anti-GlyRα1多克隆抗体加缺血缺氧组(iGH)。
2.观测指标和方法
(1)缺血缺氧1、3、6、12、24h后,刮取细胞用高效液相色谱检测ATP、ADP、AMP含量、EC及ATP/AMP比值。缺血缺氧1、3、6、12、24h后,刮取细胞,提取总RNA,采用RT-PCR方法检测TNF-αmRNA的表达;缺血缺氧6h后,取细胞培养上清液,紫外分光光度计测LDH含量,缺氧6h后心肌细胞存活率采用CCK-8检测及PI染色。
(2)离体培养的大鼠心肌细胞,采用特异荧光探针,在激光共聚焦下检测缺氧6h后心肌细胞游离钙、细胞膜电位、线粒体膜电位、mPTP开放及心肌细胞凋亡(Annex-V)的变化,生化方法检测capase-3的激活。
(3)心肌细胞甘氨酸受体各亚基检测,采用神经细胞做阳性对照,免疫组织化学荧光染色、RT-PCR及Western-Blot检测。
二、动物实验
1.模型与分组:采用SD大鼠致成30%TBSAⅢ°烫伤模型,试验分为正常组(N)、烧伤组(B)和甘氨酸处理组(G)。
2.观测指标和方法
(1)烧伤后1、3、6、12、24h后,心肌组织甘氨酸受体α1亚基表达变化规律,采用Western-Blot检测。
(2)烧伤后1、3、6、12、24h后,取心肌组织用高效液相色谱检测ATP、ADP、AMP、EC含量,常规方法检测血清中LDH含量。
(3) ELISA检测血清中TnI含量。
主要结果:
一、体外实验
1.缺血缺氧导致培养心肌细胞能量储备降低,包括减少ATP及EC含量,ATP/ADP比值减少,ADP、AMP含量增加,随缺氧时间的延长,心肌细胞能量储备损失逐渐加重,而加入甘氨酸可以明显改善缺血缺氧心肌细胞能量储备。
2.缺血缺氧使心肌细胞损伤明显,表现为LDH释放增加,PI染色阳性细胞明显增加,TNF-αmRNA表达明显增高,而加入甘氨酸可明显降低细胞培养液中LDH含量,PI染色阳性细胞明显减少,TNF-αmRNA表达较单纯缺血缺氧组显著减少。同时证实甘氨酸在5mmol/L时保护作用最大。
3. Annexin V检测心肌细胞早期凋亡,应用5mmol/L甘氨酸可以明显减轻心肌细胞的凋亡。
4.本实验首次证实在心肌细胞存在甘氨酸受体。以神经元细胞为阳性对照,RT-PCR检测心肌细胞表达GlyRα1、2及β亚基mRNA;Western-blot检测结果也证实在心肌细胞存在GlyRα1及β亚基;免疫组化证实在心肌细胞存在GlyR的α1亚基;缺血缺氧1、3、6、12、24h检测心肌细胞功能性亚基GlyRα1表达变化,可见在缺血缺氧6h后甘氨酸受体表达较正常组明显增强。表明在心肌细胞均存在甘氨酸受体。
5.心肌细胞缺血缺氧6h后,细胞膜电位荧光强度明显降低;钙离子荧光强度明显增强,线粒体膜电位荧光强度显著降低,mPTP开放增加,capase-3释放明显增加,而加入甘氨酸后,细胞膜电位荧光强度明显升高;钙离子荧光强度明显降低,线粒体膜电位荧光强度显著增强,mPTP开放减少,capase-3释放明显减少;加入甘氨酸受体激动剂Taurine后,各项指标变化与加入甘氨酸组变化一致;而加入anti-GlyRα1阻断甘氨酸受体α1亚基后,各项指标变化与加入甘氨酸组的结果相反。
二、动物实验
1.心肌组织有甘氨酸受体α1亚基蛋白表达,烧伤后1h开始其表达明显增强,烧伤后6h达高峰,12、24h逐渐降低。表明组织存在甘氨酸受体α1亚基。
2.烫伤后3h开始大鼠血清中LDH明显升高,伤后6h达高峰,后逐渐回落。而甘氨酸处理组与烧伤组比较,血清中LDH含量明显降低(P<0.01)
3.烧伤3h开始,烧伤后心肌组织ATP、EC明显降低,ADP、AMP含量明显升高,而甘氨酸干预后从伤后3h开始ATP、EC含量显著升高,ADP、AMP含量明显相应降低。
4.烫伤后1h,TnI即明显升高,伤后6h达高峰,后渐回落。而甘氨酸处理组血清TnI与烧伤组相比明显降低。
讨论与结论:
1.本研究证明,甘氨酸对体外培养的缺血缺氧心肌细胞具有显著的保护作用,表现为缺血缺氧心肌细胞ATP和EC含量、ATP/ADP比值均增加、ADP和AMP含量均减少,细胞培养上清液中LDH释放减少,细胞活力明显改善,PI染色阳性细胞明显减少,心肌细胞凋亡减少,TNF-αmRNA表达降低。甘氨酸在5mmol/L时,对缺血缺氧心肌细胞的保护作用最大。
2.证实了在心肌细胞存在甘氨酸受体。以神经元细胞为阳性对照,RT-PCR检测心肌细胞表达GlyRα1、2及β亚基mRNA;Western-blot检测结果也证实在心肌细胞存在GlyRα1及β亚基;免疫组化证实在心肌细胞存在GlyR的α1亚基;缺血缺氧1、3、6、12、24h检测心肌细胞功能性亚基GlyRα1表达变化,可见缺血缺氧6h后甘氨酸受体表达较正常组明显增强。
3.缺血缺氧后,心肌细胞膜发生去极化,细胞膜电压依赖性钙通道开放增加,钙离子内流增多,使线粒体膜电位降低及mPTP开放,激活凋亡激酶导致细胞凋亡。
4.证明了甘氨酸抑制缺血缺氧心肌细胞凋亡的作用机制为:心肌细胞与组织存在甘氨酸受体,甘氨酸与其受体结合后,导致心肌细胞膜去极化明显减轻,从而使细胞膜电压依赖性钙通道开放减少,钙离子内流明显减少,升高缺血缺氧心肌细胞线粒体膜电位,减轻mPTP孔的开放,使Caspase-3活化减少,从而发挥抗凋亡作用。
5.证实在心肌组织存在甘氨酸受体α1亚基,大鼠烧伤30%TBSAⅢ°烫伤后,心肌组织甘氨酸受体α1亚基于烧伤后1h表达明显增强,烧伤后6h达高峰;甘氨酸对烧伤心肌组织具有保护作用,可使烧伤后3h开始ATP含量及EC明显升高,ADP和AMP含量显著降低;大鼠血清中LDH、TnI较烧伤组均明显下降。这些结果为烧伤早期“休克心”的防治,特别是烧伤后心肌的外源性保护提供了新的思路和方法。
“Shock heart”, occurring at the early stage following severe burns, is not only the pathophysiological basis of inducement and aggravation of burn shock, but also one of the main causes for 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 the mortality of rat treated with endotoxin, lessens injury of skeletal muscle from ischemic/reperfusion, decreases the release of mediators of inflammation, calcium overload and reactive oxygen species. But no reports concerning the protective effect of glycine on myocardium and its mechanism following severe burns have been found.
In the present study, the protective function of glycine on schemia/hypoxia cardiomyocytes and postburn myocardial damage and its mechanism is identified according to the changes of myocardial morphology, enzymology, energy, glycine receptor, free calcium ions, membrane potential, mitochondrial membrane potential, mPTP, capase-3 and apoptosis.
Materials and Methods
In vitro study
1. Cell model and groups
Neonatal murine cardiomyocytes were collected, the cells were cultured in a hypoxic mixed gas containing 1% oxygen and used as hypoxic model. Cells were divided into normal(N) group, glycine-treated group (GN), schemia/hypoxia group(H) , schemia/hypoxia combined with glycine grope(GH), schemia/hypoxia combined with glycine and anti- GlyRα1 polyclonal antibody grope(GHA), schemia/hypoxia treated with glycine and taurine group(GHT).
2. Observation of indexes and methods
①The cells were cultured in a ischemia/hypoxia .The content of ATP, ADP and AMP were tested by high performance liquid chromatogram(HPLC)and ATP/ADP and energy charge(EC) was calculated. The expression of TNF-αmRNA was detected by RT-PCR from cardiomyocytes after ischemia/hypoxia 1, 3, 6, 12 and 24h. LDH in the supernate fluid and the survival rate of cardiomyocytes after 6h ischemia/hypoxia were determined with routine methods. The cell survival rate was detected after 6h ischemia/hypoxia by CCK-8 and PI dyeing.
②The changes of free calcium ions, membrane potential, mitochondrial membrane potential, mPTP and apoptosis in the cardiomyocytes were determined using laser confocal microscopy following 6h ischemia/hypoxia. Capase-3 was determined with routine methods.
③Using nerve cell as positive control, glycine receptor on membrane of cardiomyocytes were detected by immunohistochemistry, RT-PCR and Western-blot.
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 expression of GlyRα1 in myocardium were detected by western-blot after burn 1, 3, 6, 12 and 24h.
②The content of ATP, ADP, AMP and EC in myocardium tissue were tested by high performance liquid chromatogram(HPLC). The contents of LDH 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. The changes of energy in cardiomyocytes: In the ischemia/hypoxia group, ATP, EC and ATP/ADP levels were decreased gradually obviously after1, 3, 6, 12 and 24h ischemia/hypoxia. At the same time,ADP and AMP levels were increased gradually and obviously. However, the changes of energy in the glycine treated group were markedly improved when1, 3, 5 and 10mmol/L of glycine were added.
2. The cardiomyocytes were obviously damaged by ischemia/hypoxia, including the levels of LDH increased in supernate fluid; the quantity of positive cells of PI dyeing increased and the expression of TNF-αmRNA increased obviously. However, the levels of LDH decreased in supernate fluid, the quantity of positive cells of PI dyeing decreased and the expression of TNF-αmRNA decreased obviously in the glycine treated group. Meanwhile, the glycine protection was the best in 5mmol/L.
3. Apoptosis of cardiomyocytes was detected by Annexin-V. The 5mmol/L glycine can obviously lighten apoptosis after ischemia/hypoxia 6h.
4. Glycine receptors exist in cardiomyocytes. Nerve cells as positive control, the expression of GlyRα1, GlyRα2 and GlyRβmRNA were founded by RT-PCR in cardiomyocytes. The expression of GlyRα1 and GlyRβwere founded by western-blot in cardiomyocytes. GlyRα1 subunits on membrane of cardiomyocytes were stained by immunohistochemistry. The changes of expression of GlyRα1 subunits on membrane of cardiomyocytes were checked after 1, 3, 6, 12 and 24h ischemia/hypoxia .
5. Fluorescence intensity of membrane potential of cardiomyocytes after 6h schemia/hypoxia became lower than that in normal cardiomyocytes; Fluorescence intensity of calcium of cardiomyocytes after 6h schemia/hypoxia became stronger than that in normal cardiomyocytes; Fluorescence intensity of mitochondrial membrane potential of cardiomyocytes after 6h schemia/hypoxia became lower than that in normal cardiomyocytes; The opening of mPTP of cardiomyocytes after 6h schemia/hypoxia became stronger than that in normal cardiomyocytes; The release of capase-3 of cardiomyocytes after 6h schemia/hypoxia became lower than that in normal cardiomyocytes. However, the changes of membrane potential, calcium, mitochondrial membrane potential, mPTP and capase-3 when glycine was added were opposites to the groups of schemia/hypoxia 6h. The changes of membrane potential, calcium, mitochondrial membrane potential, mPTP and capase-3 when Taurine(0.5mmol/L) was added were concoed with the groups of glycine treated. The changes of membrane potential, calcium, mitochondrial membrane potential, mPTP and capase-3 when GlyRα1(1:100) was added were opposites to the groups of glycine treated.
In vivo study:
1. The GlyRα1 exist in myocardium, the expression of GlyRα1 became stronger after burn 1h, reached the maximum at 6h then depressed gradually.
2. In the burn untreated rats, levels of LDH in serum increased obviously, reached the maximum at 6h then recovered gradually. However, the levels of LDH in serum decreased obviously in the glycine-treated group.
3. The content of ATP and EC were decreased and TDP and TMP were increased obviously after 3h burns. However, the content of ATP were increased and TDP and TMP were decreased obviously after 3h burns when glycine were added.
4. 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.
Discussion and conclusion
1. The results of cardiomyocytes culture in vito on schemia/hypoxia studies indicate that glycine has an obvious protective effect, including ATP, EC and ATP/ADP levels increased gradually and obviously after ischemia/hypoxia, at the same time,ADP and AMP levels decreased gradually and obviously, the levels of LDH decreased in supernate fluid, the quantity of positive cells of PI dyeing decreased, apoptosis of cardiomyocytes decreased and the expression of TNF-αmRNA decreased obviously. Meanwhile, the glycine protection was the best in 5mmol/L.
2. Glycine receptors exist in cardiomyocytes. Nerve cells as positive control, the expression of GlyRα1, GlyRα2 and GlyRβmRNA were founded by RT-PCR in cardiomyocytes, the expression of GlyRα1 and GlyRβwere founded by western-blot in cardiomyocytes, GlyRα1 subunits on membrane of cardiomyocytes were stained by immunohistochemistry. The expression of GlyRα1 subunits on membrane of cardiomyocytes were founded after 1, 3, 6, 12 and 24 h ischemia/hypoxia.
3. Cardiomyocytes membrane depolarized after 6h ischemia/hypoxia. Opened L-type voltage-dependent calcium channels and inflow of calcium increased. Mitochondrial membrane potential of cardiomyocytes decreased. The opening of mPTP increased. Kinase of apoptosis activated. In the end, led to apoptosis in cardiomyocytes.
4. The possible mechanism that glycine inhibited apoptosis of cardiomyocytes on schemia/hypoxia may be that glycine receptors existing in cardiomyocytes and myocardial tissues. Membrane of cardiomyocytes was depolarized after schemia/hypoxia. As a consequence, agonist-induced opening of L-type voltage-dependent calcium channels and inflow of calcium increased. Mitochondrial membrane potential of cardiomyocytes decreased. The opening of mPTP increased. The releasing of caspase-3 inreased. In the end, these responses led to apoptosis in cardiomyocytes. However, the membrane potential, calcium, mitochondrial membrane potential, mPTP and capase-3 were changed markdely when glycine were added. Then, these results decreased apoptosis in cardiomyocytes after schemia/hypoxia.
5. The GlyRα1 exist in myocardium. In a model of 30% TBSA of full-thickness burns in SD rats, the expression of GlyRα1 became stronger after burn 1h and reached the maximum at 6h then depressed gradually. Glycine has an obvious protective effect on myocardial tissues in burn rats, including increasing the content of ATP and EC, decreasing the content of TDP and TMP obviously after 3h burns, decreasing the content of LDH and TnI in serum dramatically after burns. In a word, the new strategy and methods were provided from the results for preventing and treatment“shock heart”after burn early, especially for providing exogenous protection to myocardium of postburn.
引文
1.闫柏刚,黄跃生,杨宗城等。烧伤后立即切痂对30%Ⅲ°烧伤大鼠心肌力学的影响。中华整形烧伤外科杂志,1998,14(5):334-337
2.黄跃生.严重烧伤后早期心肌损害的细胞分子机制与防治策略研究进展。中华烧伤杂志,2006,22(3):161-163.
3.黄跃生,李志清,吴庆云,等.缺血缺血缺氧在烧伤后休克心中的作用及其机理探讨.中华创伤杂志,2002,18:205-209.
4.黄跃生,杨宗城,迟路湘,等.烧伤后“休克心”的研究.中华烧伤杂志,2000,16:275-278.
5. Wang G., Zhao M, Wang E H. Effects of glycine and methylprednisolone on hemorrhagic shock in rats. Chin Med J (Engl). 2004 Sep; 117(9): 1334-41.
6. Mauriz J L, Matilla B, Culebras J M. Dietary glycine inhibits activation of nuclear factor kappa B and prevents liver injury in hemorrhagic shock in the rat. Free Radic Biol Med. 2001 Nov 15; 31(10): 1236-44.
7. Bruck R, Wardi J, Aeed H et al. Glycine modulates cytokine secretion, inhibits hepatic damage and improves survival in a model of endotoxemia in mice. Liver Int. 2003 Aug; 23(4): 276-82.
8. Zhong Z, Wheeler M D. L-Glycine: a novel antiinflammol/ Latory, immol/ Lunomodulatory, and cytoprotective agent. Curr Opin Clin Nutr Metab Care. 2003 Mar; 6(2): 229-40.
9. Kallakuri S, Ascher E, Pagala M et al. Protective effect of glycine in mesenteric ischemia and reperfusion injury in a rat model. J Vasc Surg. 2003 Nov; 38(5): 1113-20.
10. Jacob T, Ascher E, Hingorani A et al. Glycine prevents the induction of apoptosis attributed to mesenteric ischemia/reperfusion injury in a rat model. Surgery. 2003 Sep; 134(3): 457-66.
11. Lee MA, McCauley R D, Kong S E et al. Influence of glycine on intestinal ischemia-reperfusion injury. JPEN J Parenter Enteral Nutr. 2002 Mar-Apr; 26(2): 130-5.
12. Ascher E, Hanson JN, Cheng,W et al. Glycine preserves function and decreases necrosis in skeletal muscle undergoing ischemia and reperfusion injury. Surgery. 2001 Feb; 129(2): 231-5.
13. Li X, Bradford BU, Wheeler MD, et al. Dietary glycine prevents peptidoglycan polysaccharide-induced reactive arthritis in the rat: role for glycine-gated chloride channel. Infect Immun 2001; 69:5883-5891.
14. Michael D. Wheeler and Ronald G. Thurman, Production of superoxide and TNF-αfrom alveolar macrophages is blunted by glycine. Am-J-Physiol. 1999 Nov; 277(5 Pt 1): L952-9.
15. Yin M, Ikejima K, Arteel GE, et al. Glycine accelerates recovery from alcohol-induced liver injury [J]. J Pharmacol Exp Ther, 1998,286(2):1014-1019.
16. Iimuro Y, Bradford BU, Forman DT. Glycine prevents alcohol–induced liver injury by decreasing alcohol in the rat stomach[J]. Gastroenterology, 1996, 110(5):1536-1542.
17.周琼陆大祥付咏梅王华东甘氨酸对小鼠心肌缺血性损伤的防治作用研究,中国病理生理杂志,2002,18(4),360-362.
18. Young AB, Mac-Donald RL (1983) Glycine as a spinal cord neurotransmitter in Handbook of the Spinal Cord, (Davidoff RA, ed) pp 1-43
19. Froh M, Thurman RG, Wheeler MD. Molecular evidence for a glycine-gated chloride channel in macrophages and leukocytes. Am J Physiol Gastrointest Liver Physiol 2002; 283:G856-G863.
20. Zhong Z, Connor HD, Yin M, et al. Dietary glycine and renal denervation prevents cyclosporin A-induced hydroxyl radical production in rat kidney. Mol Pharmacol 1999;
21. Li X, Bradford BU, Wheeler MD, et al. Dietary glycine prevents peptidoglycan polysaccharide-induced reactive arthritis in the rat: role for glycine-gated chloride channel. Infect Immun 2001; 69:5883-5891.
22. Wheeler MD, Rose ML, Yamashima S, et al. Dietary glycine blunts lung inflammatory cell influx following acute endotoxin. Am J Physiol Lung Cell Mol Physiol 2000; 279:L390-L398.
23.林函王祥瑞心肌缺血再灌注损伤中线粒体的作用。《国外医学》麻醉学与复苏分册2002年第23卷第5期.
24. Chlopcikova S, Psotova J, Miketova P. Neonatal rat cardiomyocytes- a model for the study of morphological, biochemical and electrophysiological characteristics of the heart[J]. Biomed Pap Fac Univ Palacky Olomouc Czach Repub, 2001, 145(2), 49–55.
25. Jun-ichi Sadoshima, Lothar et al. Molecular Characterization of the Stretch-induced Adaptation of Cultured Cardiac Cells. The Journal of Biological Chemistry, 1992, 267(15): 10551-10560.
26. Wheeler MD, Stachlewitz RF, Yamashina S, et al. Glycine-gated chloride channels in neutrophils attenuate calcium influx and superoxide production. FASEB J 2000; 14:476-484.
27. Qu W, Ikejima K, Zhong Z, et al. Glycine blocks the increase in intracellular free Ca2+ due to vasoactive mediators in hepatic parenchymal cells. Am J Physiol Gastrointest Liver Physiol 2002; 283:G1249-G1256.
28. Yamashina S, Konno A, Wheeler MD, et al. Endothelial cells contain a glycine-gated chloride channel. Nutr Cancer 2001; 40:197-204.
29. Miller GW, Schnellmann RG. A putative cytoprotective receptor in the kidney: relation to the neuronal strychnine-sensitive glycine receptor. Life Sci 1994; 55:27-34.
30. Seppet E, Eimre M, Peet N et al. Compartmentation of energy metabolism in atrial myocardium of patients undergoing cardiac surgery. Mol Cell Biochem, 2005 Feb; 270(1-2): 49-61.
31. Telkova I L, Tepliakov AT. Relationships between changes of coronary blood flow, energy metabolism of the myocardium, and hyperinsulinemia in patients with ischemic heart disease. Kardiologiia, 2005; 45(8): 61-8.
32. Hajime F, Toru K, Yoji M, et al. Involvement of inducible nitric oxide synthase in cardiac dysfunction with tumor necrosis factor-α. Am J Physiol Heart Cire Physiol.2002, 282:H2159-2166
33. Pfeiffer F, Betz H. Solubilisation of the glycine receptor from the rat spinal cord. Brain Res. 1981; 226:273-279.
34. Yamashina S, Konno A, Wheeler MD, et al. Endothelial cells contain a glycine-gated chloride channel. Nutr Cancer 2001; 40:197-204.
35. Zhang Y, Ikejima K, Honda H, et al. Glycine prevents apoptosis of rat sinusoidal endothelial cells caused by deprivation of vascular endothelial growth factor. Hepatology 2002; 32:542-546.
36. Stachlewitz RF, Seabra V, Bradford BU, et al. Glycine and uridine prevent D-galactosamine hepatotoxicity in the rat: role of Kupffer cells. Hepatology 1999; 29:737-745.
37. Zhong Z, Connor HD, Yin M, et al. Dietary glycine and renal denervation prevents cyclosporin A-induced hydroxyl radical production in rat kidney. Mol Pharmacol 1999; 56:455-463.
38. Ortells, M.O., Lunt, G.G., 1995. Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci. 18,121-127.
39. Pfeiffer F, Betz H. Solubilisation of the glycine receptor from the rat spinal cord. Brain Res. 1981; 226:273-279.
40. Prior P, Schroitc B, grenoinglob G, et al,Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tublin linker protein.Neuron,1992,8(6):1161-l170
41. Meyer G, Kirsch J, Betz H, et al. Identification of a gephyrin binding motif on the glycine receptorβsubunit. Neuron,1995,15(3):563-572
42. Ren-Qi Huang and Glenn H. Dillon.Direct inhibition of glycine receptors by genistein, a tyrosine kinase inhibitor. Neuropharmacology .Volume 39, Issue 11 , October 2000, Pages 2195-2204
43. Mascia, M.P., Wick, M.J., Martinez, L.D., Harris, R.A., 1998. Enhancement of glycine receptor function by ethanol: role of phosphorylation. Br. J. Pharmacol. 125, 263-270.
44.李萍徐样敏杨雄里甘氨酸受体的研究进展。生物化学与生物物理进展。2001,28(5),609-614.
45. Molon A, Di-Giovanni S, Hathout Y. Functional recovery of glycine receptors in spastic murine model of startle disease. Neurobiol Dis. 2006 Feb; 21(2): 291-304.
46. Lynch JW, Callister RJ. Glycine receptors: a new therapeutic target in pain pathways. Curr Opin Investig Drugs. 2006 Jan; 7(1): 48-53.
47. Christopher P. Baines and Jeffery D. Molkentin. STRESS signaling pathways that modulate cardiac myocyte apoptosis. Journal of Molecular and Cellular Cardiology,38(2005)47-62.
48. Saeko Tada-Oikawa, Yusuke Hiraku, et al. Mechanism for generation of hydrogen peroxide and change of mitochondrial membrane potential during rotenone-induced apoptosis. Life Sciences.73(2003)3277-3288.
49. Zhonghai Li and Jingze Wang . A forskolin derivative, FSK88, induces apoptosis in human gastric cancer BGC823 cells through caspase activation involving regulation of Bcl-2 family gene expression, dissipation of mitochondrial membrane potential and cytochrome c release. Cell Biology International, 30(2006)940-946.
50. Dauffenbach L M, Khan S M, Yeh J. Corpus luteum regression in the rat--in vivo and in vitro studies of apoptotic mechanisms. J Med. 2003; 34(1-6): 87-100.
51. Kraft C A, Efimova T, Eckert RL. Activation of PKCdelta and p38delta MAPK during okadaic acid dependent keratinocyte apoptosis. Arch Dermatol Res, 2007 May; 299(2): 71-83.
52. Javier Farinas, Andrea W. Chow, and H. Garrett Wada . A Microfluidic Device for Measuring Cellular Membrane Potential. Analytical Biochemistry 295, 138–142 (2001).
53. Kristi L. Whiteaker, Rachel Davis-Taber et al. potassium channel activation in cultured neonatal rat ventricular myocytes Journal of Pharmacological and Toxicological Methods 46 (2001) 45– 50.
54. Mai Prochnow A, Webb J S, Ferrari B C et al. Ecological advantages of autolysis during the development and dispersal of Pseudoalteromonas tunicata biofilms. Appl Environ Microbiol. 2006 Aug; 72(8): 5414-20.
55. Mork H K, Sjaastad I, Sande J B et al. Increased cardiomyocyte function and Ca2+ transients in mice during early congestive heart failure. J Mol Cell Cardiol. 2007 Aug; 43(2): 177-86.
56. Kapur N, Banach K. Inositol-1,4,5-trisphosphate-mediated spontaneous activity in mouse embryonic stem cell-derived cardiomyocytes. J Physiol. 2007 Jun 15; 581(Pt 3): 1113-27
57. Ruiz Meana M, Garcia Dorado D, Miro Casas E, et al. Mitochondrial Ca2+ uptake during simulated ischemia does not affect permeability transition pore opening upon simulated reperfusion. Cardiovasc Res. 2006 Sep 1; 71(4): 715-24.
58. Sundararajan Jayaraman. Flow cytometric determination of mitochondrial membrane potential changes during apoptosis of T lymphocytic and pancreatic beta cell lines: Comparison of tetramethylrhodamineethylester (TMRE), chloromethyl-X-rosamine (H2-CMX-Ros) and MitoTracker Red 580 (MTR580). ournal of Immunological Methods,306(2005)68-79.
59. Mulhern M L, Madson C J, Kador P F, et al. Cellular osmolytes reduce lens epithelial cell death and alleviate cataract formation in galactosemic rats. Mol-Vis. 2007; 13: 1397-405.
60. Tanioka H, Hieda O, Kawasaki S et al. Assessment of epithelial integrity and cell viability in epithelial flaps prepared with the epi-LASIK procedure. J Cataract Refract Surg. 2007 Jul; 33(7): 1195-200.
61. Soraya S.Smaili,et al.Mitochondria in Ca2+ singnaling and apoptasis[J].Journal of Bioenergetic and Biomembranes,2000,32(1):35-46.
62. Yang S, Thor A D, Edgerton S et al, Caspase-3 mediated feedback activation of apical caspases in doxorubicin and TNF-alpha induced apoptosis. Apoptosis. 2006 Nov, 11(11): 1987-97.
63.张家平,黄跃生,周新。严重烧伤大鼠心肌细胞凋亡与心功能损害的关系。中华烧伤杂志,2002,18(5):272-274.
64. Anders A. Jensena, Uffe Kristiansen. Functional characteris- ation of the humana1 glycine receptor in a fluorescence-based membrane potential assay. Biochemical Pharmacology 67 (2004) 1789–1799.
65.房冬梅,冯美云。运动对心肌组织能量代谢的影响。体育科研,2002 Dec ,23(4):16-18。
66.梁晚益,林一民。严重烧伤早期心肌线粒体Ca2+转运变化及外源性ATP的影响。重庆医学,2002,12(31):1188-1190.
67. Huang Yuesheng, Yang Zongcheng, Li Ao. R.Crowther. Pathogenesis of early cardiac myocyte damage after severe burns [J]. J Trauma,1999,46:428-432
68.闫柏刚,黄跃生,杨宗城等。烧伤后立即切痂对30%Ⅲ°烧伤大鼠心肌力学的影响。中华整形烧伤外科杂志,1998,14(5):334-337。
69. Oksala N, Alhava E, Paimela H. Heat shock preconditioning and eicosanoid pathways modulate caspase 3-like activity in superficially injured isolated guinea pig gastric mucosa. Eur Surg Res. 2004 Mar-Apr; 36(2): 67-73.
70. Fallavollita J A, Jacob S, Young R F et al. Regional alterations in SR Ca(2+)-ATPase, phospholamban, and HSP-70 expression in chronic hibernating myocardium. Am J Physiol. 1999 Oct; 277(4 Pt 2): H1418-28.
71. Mehrabi M R, Serbecic N, Tamaddon F et al. Clinical benefit of prostaglandin E1-treatment of patients with ischemic heart disease: stimulation of therapeutic angiogenesis in vital and infarcted myocardium. Biomed Pharmacother. 2003 May-Jun; 57(3-4): 173-8.
72.黄跃生烧伤早期脏器损害防治的研究进展。中华烧伤杂志。2003,10(5),257-260。
73. Alain L, Guy H. Serum cardiac troponin I and T in early posttraumatic phaodomyolysis[J].Clin Chem,1998,449(3)-667-668.
74. Arteaga G M, Warren C M, Milutinovic S, et al. Specific enhancement of sarcomeric response to Ca2+ protects murine myocardium against ischemia-reperfusion dysfunction. Am J Physiol Heart Circ Physiol. 2005 Nov; 289(5): H2183-92.
75. Murpy J T,Horton J W,Purdue G F.et al.Evaluation of Troponin-I as an indicator of cardiac dysfunction after thermal injury.J trau.1998;45(4):700-704.
1. Weinhrrg JM, Davis JA, Abarzua M, et al. J C1in Invest, 1987, 80:1446-54
2. Aprison MH and Werman R. The distribution of glycine in cat spinal cord and roots. Life Csi, 1965, 4: 2075-83
3. Rajendra S,Lynch JW, Schofield PR. The glycine receptor. Pharmacol Ther,1997;73(2):121-146
4. Ikejima K, Iimuro Y, Forman DT, Thurman RG. A diet containing glycine improves survival in endotoxin shock in the rat. Am J Physiol Gastrointest Liver Physiol 1996; 271:G97-G103。
5. Ascher,-E; Hanson,-J-N; Cheng,-W; Hingorani,-A; Scheinman,-M. Glycine preserves function and decreases necrosis in skeletal muscle undergoing ischemia and reperfusion injury. Surgery. 2001 Feb; 129(2): 231-5
6. Li X, Bradford BU, Wheeler MD, et al. Dietary glycine prevents peptidoglycan polysaccharide-induced reactive arthritis in the rat: role for glycine-gated chloride channel. Infect Immun 2001; 69:5883-5891.
7. Michael D. Wheeler and Ronald G. Thurman, Production of superoxide and TNF-αfrom alveolar macrophages is blunted by glycine. Am-J-Physiol. 1999 Nov; 277(5 Pt 1): L952-9。
8. Yin M, Ikejima K, Arteel GE, et al. Glycine accelerates recovery from alcohol-induced liver injury [J]. J Pharmacol Exp Ther, 1998,286(2):1014-1019
9. Iimuro Y, Bradford BU, Forman DT. Glycine prevents alcohol–induced liver injury by decreasing alcohol in the rat stomach[J]. Gastroenterology, 1996,110(5):1536-1542
10. Stachlewitz RF, Seabra V, Bradford B, et al. Glycine and uridine prevent D-galactosamine hepatotoxicity in the rat: role of Kupffer cells. Hepatology, 1999;29(3):737-745
11. Koo DJ, Chaudry IH, Wang P. Kupffer cells are responsible for producing inflammatory cytokines and hepatocellular dysfunction during early sepsis. J Sutg Res, 1999:83(2) :151-157
12. Yang S,Koo DJ, Chaudry IH, et al. Glycine attenuates hepatocellular depression during early sepsis and reduces sepsis-induced mortality. Crit Care Med, 2001;29 (6);1201-1206
13. Su GL. Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation.Am J Physiol Gastrointest Liver Physiol, 2002;283(2): inG256-65
14. Ikejima K, Iimuro Y, Forman DT, Thurman RG. A diet containing glycine improves survival in endotoxin shock in the rat. Am J Physiol Gastrointest Liver Physiol 1996; 271:G97-G103。
15. Mauriz fL, Matilla B, Dietary glycine inhibits activation of nuclear factor kappa B and prevents liver injury in hemorrhagic shock in the rat. Free Radic Biol Med, 2001:31(10);1236-1244.
16. Young AB, Mac-Donald RL (1983) Glycine as a spinal cord neurotransmitter in Handbook of the Spinal Cord, (Davidoff RA, ed) pp 1-43
17. Pfeiffer F, Betz H. Solubilisation of the glycine receptor from the rat spinal cord. Brain Res. 1981; 226:273-279.
18.李萍徐样敏杨雄里甘氨酸受体的研究进展。生物化学与生物物理进展。2001,28(5),609-614。
19. Wheeler MD, Rose ML, Yamashima S, et al. Dietary glycine blunts lung inflammatory cell influx following acute endotoxin. Am J Physiol Lung Cell Mol Physiol 2000; 279:L390-L398.
20. Stachlewitz RF, Li X, Smith S, et al. Glycine inhibits growth of T lymphocytes by an IL-2-independent mechanism. J Immunol 2000; 164:176-182.2.
21. Yamashina S, Konno A, Wheeler MD, et al. Endothelial cells contain a glycine-gated chloride channel. Nutr Cancer 2001; 40:197-204.
22. Qu W, Ikejima K, Zhong Z, et al. Glycine blocks the increase in intracellular free Ca2+ due to vasoactive mediators in hepatic parenchymal cells. Am J Physiol Gastrointest Liver Physiol 2002; 283:G1249-G1256.
23. Ascher,-E; Hanson,-J-N; Cheng,-W; Hingorani,-A; Scheinman,-M. Glycine preserves function and decreases necrosis in skeletal muscle undergoing ischemia and reperfusion injury. Surgery. 2001 Feb; 129(2): 231-5
24. Schemmer P, Schoonhoven R, Swenbero JA, et al. Transplatation, 1998, 65(8): 1015-20
25. Henderson JM. Hepato Gastroenterol, 1999, 46 ( suppl 2 ) : 1462-9
26. Ferdinand Serracino-Inglott et al. The American Journal of Surgery,2001, 181: 160-8
27.蒋莉,吴翠贞,徐柯。国外医学生理病理科学与临床分册,1998, 18 (4):349-52I1
28. Schemmer P, Enomoto N, Bradford BU, et al. Am J Physiol Gastroinsest Liver Yhsiol 2001, 280(6): 61075- 825
29. Wheeler MD, Ikejema K, Enomoto N, et al. Cell Mol Life Sci, 1999, 56: 843-56
30. Michaelw,Robert F, Shunhei Y, et al. FASEB J, 2000, 14: 476-84
31. Zhang Z, Ikejima K, Honda H, et al. Hepatology, 2000, 32 ( 3 ) : 542-6
32. Goldman R, Ferber E, Zor U. Involvement of reactive oxygen species in phospholipase A2 activation: inhibition of protein tyrosine phosphatases and activation of protein kinases. Adv Exp Med Biol 1997; 400A:25-300.
33. Hensley K, Robinson KA, Gabbita SP, et al. Reactive oxygen species, cell signaling, and cell injury. Free Radic Biol Med 2000; 28:1456-1462.
34. Zhong Z, Connor HD, Yin M, et al. Dietary glycine and renal denervation prevents cyclosporin A-induced hydroxyl radical production in rat kidney. Mol Pharmacol 1999; 56:455-463.
35. Zhong Z, Enomoto N, Connor HD, et al. Glycine improves survival after hemorrhagic shock in the rat. Shock 1999; 12:54-62.
36. Mauriz JL, Matilla B, Culebras JM, et al. Dietary glycine inhibits activation of nuclear factor kappa B and prevents liver injury in hemorrhagic shock in the rat. Free Radic Biol Med 2001; 31:1236-1244.
37. Baldwin AS. The NF-[kappa]B and I[kappa]B proteins: new discoveries and insights. Annu Rev Immunol 1996; 14:649-681.
38. Bunzendahl H, Yin M, Stachlewitz RF, et al. Dietary glycine prolongs graft survival in transplant models. Shock 2000; 13:163-164.
39. Schilling M, den Butter G, Marsh DC, et al. Glycine inhibits phospholipolysis of hypoxic membranes. Tex Med 1994; 6:140-143.
40. Yin M, Zhong Z, Connor HD, et al. Protective effect of glycine on renal injury induced by ischemia-reperfusion in vivo. Am J Physiol Renal Physiol 2002; 282:F417-F423.
41. Schemmer P, Bradford BU, Rose ML, et al. Intravenous glycine improves survival in rat liver transplantation. Am J Physiol Gastrointest Liver Physiol 1999; 276:G932.
42. Mangino JE, Kotadia B, Mangino MJ. Characterization of hypothermic intestinal ischemia-reperfusion injury in dogs: Effects of glycine. Transplantation 1996; 62:173-178. Ovid Full Text Bibliographic Links
43. Zhong Z, Li X, Yamashina S, et al. Cyclosporin A causes a hypermetabolic state and hypoxia in the liver: prevention by dietary glycine. J Pharmacol Exp Ther 2001; 299:858-865.
44. Nichols JC, Bronk SF, Mellgren RL, Gores GJ. Inhibition of nonlysosomal calcium- dependent proteolysis by glycine during anoxic injury of rat hepatocytes. Gastroenterology 1994; 106:168-176.
45. Venkatachalam MA, Weinberg JM. Mechanisms of cell injury in ATP-depleted proximal tubules. Role of glycine, calcium, and polyphosphoinositides. Nephrology Dialysis Transplantation 1994; 9:15-21.
46. Nishimura Y, Romer LH, Lemasters JJ. Mitochondrial dysfunction and cytoskeletal disruption during chemical hypoxia to cultured rat hepatic sinusoidal endothelial cells: the pH paradox and cytoprotection by glucose, acidotic pH, and glycine. Hepatology 1998; 27:1039-1049.
47. Weinberg JM, Roeser NF, Davis JA, Venkatachalam MA. Glycine-protected, hypoxic, proximal tubules develop severely compromised energetic function. Kidney Int 1997; 52:140-151.
48. Qian T, Nieminen AL, Herman B, Lemasters JJ. Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am J Physiol Cell Physiol 1997; 273:C1783-C1792.
49. Deters M, Strubelt O, Younes M. Protection by glycine against hypoxia-reoxygenation induced hepatic injury. Res Commun Mol Pathol Pharmacol 1997; 97:199-213.
50. Nishimura Y, Lemasters JJ. Glycine blocks opening of a death channel in cultured hepatic sinusoidal endothelial cells during chemical hypoxia. Cell Death Differ 2001; 8:850-858.