内质网应激与大鼠肝脏冷缺血再灌注时细胞凋亡的关系

Relationship between endoplasmic reticulum stress and cell apoptosis during liver cold ischemia- reperfusion in rats

目的评价内质网应激与大鼠肝脏冷缺血再灌注时细胞凋亡的关系。方法SPF级健康成年雄性SD大鼠32只，体重200～250g，采用随机数字表法，将其分为2组（n=16）：假手术组（s组）和肝脏冷缺血再灌注组（I／R组）。I／R组肝缺血后于肝下下腔静脉血管夹上方灌注4℃乳酸钠林格液6～8ml／min，灌注时间为30min，灌注结束后恢复肝脏血流。I／R组于再灌注6h（s组于关腹后6h）时，取下腔静脉血标本，测定血清ALT和AST浓度；采集血标本后，取肝组织，进行肝组织病理学评分；分别采用硫代巴比妥酸法测定MDA含量，采用黄嘌呤氧化酶法测定SOD活性；采用TUNEL法确定细胞凋亡率；采用免疫组化法检测肝组织内质网分子伴侣蛋白46（ERP46）、免疫球蛋白重链结合蛋白（BiP）和caspase-12的表达；采用实时定量PCR法检测肝组织ERP46、BiP和caspase-12的mRNA表达。结果与s组比较，I／R组血清ALT和AST浓度、肝组织病理学评分、MDA含量和细胞凋亡率升高，SOD活性降低，ERP46、BiP和caspase-12及其mRNA表达上调（P〈0．05）。结论大鼠肝脏冷缺血再灌注时细胞凋亡的机制可能与内质网应激过度激活有关。

Objective To evaluate the relationship between endoplasmic reticulum stress and cell apoptosis during liver cold ischemia-reperfusion （I/R） in rats. Methods Thirty-two SPF healthy adult male Sprague-Dawley rats, weighing 200-250 g, were divided into 2 groups （n= 16 each） using a random number table： sham operation group （ group S） and liver cold I/R group （ group I/R）. In group I/R, the liver was perfused through the portal vein with 4℃ lactated Ringer＇s solution 6-8 ml/min for 30 rain after liver ischemia, and the liver blood flow was restored after the end of perfusion. At 6 h of reperfusion in group I/R or at 6 h after peritoneum closure in group S, blood samples from the inferior vena cava were collected for determination of serum alanine transaminase and aspartate transaminase concentrations. After blood sampling, liver tissues were obtained for examination of pathological changes and for determination of malondialdehyde content （by thiobarbituric acid method） , superoxide dismutase activity （using xanthine oxidase method ）, cell apoptosis （using TUNEL ）, expression of endoplasmic reticulum protein 46 （ERP46） , immunoglobulin heavy chain binding protein （BiP） and caspase-12 （by immunohistochemistry）, and expression of ERP46, BiP and caspase-12 mRNA （using quantitative real-time polymerase chain reaction）. The pathological changes were scored. Apoptosis rate was calculated. Results Compared with group S, the serum alanine transaminase and aspartate transaminase concentrations, pathological scores, malondialdehyde content and apoptosis rate were significantly increased, the activity of superoxide dismutase was decreased, and the expression of ERP46, BiP and caspase-12 protein and mRNA was up- regulated in group I/R （P〈0.05）. Conclusion The mechanism of cell apoptosis during liver cold I/R may be related to excessive activation of endoplasmic reticulum stress in rats.