肝脏缺血再灌注损伤中线粒体膜氧化应激和电生理 功能障碍的分子机制研究

目的 探讨肝脏缺血再灌注损伤中线粒体膜氧化应激和电生理功能障碍的分子机制。方法 建立 SD 大 鼠肝移植 （冷缺血再灌注组， n=20） 和肝脏肝门部分阻断 （热缺血再灌注组， n=20） 的缺血再灌注损伤模型。以大鼠仅 作肝十二指肠韧带分离， 但不阻断肝脏肝门血液供应的 10 只大鼠为假手术组， 采集各组血标本测定丙氨酸转氨酶 （ALT）、 天冬氨酸转氨酶 （AST）、 肿瘤坏死因子-α （TNF-α） 及白细胞介素-1β （IL-1β） 水平； 采集胆汁标本用于测定胆 汁内葡萄糖的含量及 γ-谷氨酰转移酶（GGT）水平； 采集肝组织标本用于测定丙二醛（MDA）及超氧化物歧化酶 （SOD）， 流式细胞仪测定肝细胞线粒体膜电位， 采用酶联免疫吸附试验（ELISA）检测肝细胞内线粒体呼吸链复合体 活性。结果 冷、 热缺血再灌注组大鼠的 ALT、 AST、 胆汁葡萄糖、 GGT、 TNF-α、 IL-1β 及 MDA 水平均高于假手术组 （P＜0.01）， 且冷缺血再灌注组大鼠各项指标均高于热缺血再灌注组 （P＜0.05）。热缺血再灌注组和冷缺血再灌注组 大鼠肝脏 SOD 水平均明显低于假手术组， 并且热缺血再灌注组的 SOD 水平高于冷缺血再灌注组（P＜0.01）。与假 手术组相比， 热、 冷缺血再灌注损伤组再灌注 0 h（缺血 1 h）、 1 h、 12 h 线粒体膜电位细胞的比例均降低（P＜0.01）； 热、 冷缺血再灌注损伤组组内随时间延长其线粒体膜电位活性有逐渐恢复的趋势 （P＜0.01）。假手术组 0、 72 h 的线 粒体呼吸链复合体Ⅲ、 Ⅳ差异均无统计学意义； 热、 冷缺血再灌注组 72 h 较 0 h 线粒体呼吸链复合体活性均增高 （P＜0.01）； 0、 72 h 时， 假手术组， 热、 冷缺血再灌注损伤组线粒体呼吸链复合体Ⅲ呈依次降低， 而线粒体呼吸链复合 体Ⅳ呈依次增高 （P＜0.01）。结论 缺血再灌注损伤的强应激刺激可导致肝细胞线粒体膜氧化应激， 进而导致肝细 胞受到损害， 线粒体内相关酶系统活性受损， 最终导致线粒体内呼吸链酶复合体活性受损伤， 这可能是氧化应激导 致肝细胞线粒体膜氧化应激和电生理功能障碍， 继而肝细胞损伤和能量代谢障碍发生的分子机制。

The pathogenesis of oxidative stress damage to mitochondrial membrane and electrophysiological dysfunction of ischemia reperfusion injury of hepatocytes in rats

Objective To explore the pathogenesis of oxidative stress damage to mitochondrial membrane and electrophysiological dysfunction of ischemia reperfusion injury of hepatocytes in rats. Methods Seventy rats were randomly divided into three experimental groups: sham operation (SHAM) group, warm hepatic ischemia/reperfusion (wI/R) group and cold hepatic ischemia/reperfusion (cI/R) group. Blood samples were collected for the detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor- α (TNF-α) and interleukin (IL) -1 β levels. Bile samples were collected for the detection of glucose and γ-glutamyl transferase (GGT) levels. And liver samples were collected for the detection of malondialdehyde (MDA) and superoxide dismutase (SOD). Flow cytometry was used to detect mitochondrial membrane potential. The mitochondrial respiratory chain complex was examined using ELISA to assess ischemia reperfusion injury of hepatocytes in rats. Results The results indicated that ALT, AST, GLU, GGT, TNF-α, IL-1β and MDA levels were increased significantly in the wI/R group and cI/R group than those in SHAM group (P＜0.01), and those indexes were significantly higher in cI/R group than those of wI/R group (P＜0.05). The SOD level was significantly lower in wI/R group and cI/R group than that in SHAM group, which was significantly higher in wI/R group than that of cI/R group (P＜0.01). Compared with SHAM group, ratios of mitochondrial membrane potential were significantly decreased at 0, 1 and 12 h I/R in wI/R group and cI/R group (P＜0.01). The activity of mitochondrial membrane potential was gradually recovered with time in wI/R group and cI/R group (P＜0.01). There were no significant differences in mitochondrial respiratory chain complexes Ⅲ and Ⅳ between 0 h and 72 h in SHAM group. The activity of mitochondrial respiratory chain complexes was increased at 72 h than that of 0 h in wI/R group and cI/R group (P＜0.01). The activity of mitochondrial respiratory chain complexes Ⅲ was decreased ordinally at 0 h and 72 h, and the activity of mitochondrial respiratory chain complexes Ⅳ was increased ordinally in SHAM group, wI/R group and cI/R group (P＜0.01). Conclusion Mitochondrial membrane potential is significantly decreased after ischemia reperfusion injury, and the activity of mitochondrial respiratory chain complexes is significantly decreased as well, which might be the pathogenesis of oxidative stress damage to mitochondrial membrane and electrophysiological dysfunction of ischemia reperfusion injury of hepatocytes in rats.