Müller Christian, Dünschede Friedrich, Koch Elke, Vollmar Angelika M, Kiemer Alexandra K
Dept. of Pharmacy, Center of Drug Research, Univ. of Munich, Germany.
Am J Physiol Gastrointest Liver Physiol. 2003 Oct;285(4):G769-78. doi: 10.1152/ajpgi.00009.2003. Epub 2003 Jun 19.
In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether alpha-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (+/-wortmannin), followed by ischemia (1 h, 37 degrees C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-kappaB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 micromol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-kappaB and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.
在肝切除和肝移植中，缺血再灌注损伤（IRI）是器官功能障碍或无功能的主要原因之一。本研究的目的是确定α-硫辛酸（LA）是否能够减轻IRI。用含或不含LA（±渥曼青霉素）的克氏-亨氏缓冲液灌注大鼠肝脏，随后进行缺血（1小时，37℃）和再灌注（90分钟）。通过酶法测定乳酸脱氢酶（LDH）和嘌呤核苷磷酸化酶（PNP）的流出以及肝脏ATP含量。通过电泳迁移率变动分析（EMSA）检测核因子κB（NF-κB）和活化蛋白1（AP-1）的激活情况，通过蛋白质印迹法检测蛋白质磷酸化情况。半胱天冬酶-3样活性作为凋亡过程的指标。静脉注射500微摩尔LA的动物经历90分钟的部分无血流缺血，随后再灌注长达7天。LA预处理显著降低了离体灌注大鼠肝脏再灌注期间的LDH和PNP流出。LA处理的肝脏中ATP含量显著增加。LA预处理的器官中缺血后NF-κB和AP-1的激活显著降低。LA预处理显著增强了Akt磷酸化。它对内皮型一氧化氮合酶和Bad磷酸化均无影响。重要的是，同时给予渥曼青霉素（一种磷脂酰肌醇（PI）3激酶/Akt途径的抑制剂）可阻断LA对IRI的保护作用，证明Akt激活与LA的肝保护作用之间存在因果关系。有趣的是，尽管Akt被激活，但LA并未减少缺血后凋亡细胞死亡。LA治疗在体内的疗效表现为用LA预处理的动物血浆谷胱甘肽S-转移酶（GST）水平降低以及肝脏组织学改善。本研究首次表明PI3激酶/Akt途径在大鼠肝脏IRI中起核心保护作用，并且给予LA可通过该途径减轻IRI。
