肝缺血再灌注时葡萄糖调节蛋白78、葡萄糖调节蛋白94、磷酸化蛋白激酶B1及半胱氨酰天冬氨酸特异性蛋白酶-12的表达及意义

目的动态观察大鼠肝缺血再灌注（HIRI）不同时限葡萄糖调节蛋白78（GRP78）、葡萄糖调节蛋白94（GRP94）、磷酸化蛋白激酶B1（p-Akt1）及半胱氨酰天冬氨酸特异性蛋白酶12（Caspase-12）的表达变化，并探讨其意义。方法将55只Wistar雄性大鼠随机分为正常组（n=5）、假手术组（n=25）、缺血再灌注组（n=25），无损伤动脉钳阻断肝血流45min后使其复流，用免疫组织化学法检测GRP78、GRP94及Caspase-12在再灌注0、3、12、24、72h时的蛋白表达，用Western blot法检测同期p-Akt1蛋白表达。结果GRP78在缺血再灌注组的表达显著高于正常组与假手术组（P〈0．01），在缺血再灌注0h时表达开始上升（393．04±79．06），12h时达峰值（2369．38±182．53），24h（1023．02±185．61）及72h（505．19±102．89）表达开始下降，但仍高于前述各组表达水平。GRP94在缺血再灌注组的表达显著高于正常组与假手术组（P〈0．01），在缺血再灌注0h时表达开始上升（168．47±21．08），12h时达峰值（678．80±52．62），24h（425．78±38．63）及72h（173．99±36．82）表达降低。Caspase-12在缺血再灌注组的表达显著高于正常组与假手术组（P〈0．01），在缺血再灌注0h时表达开始上升（647．00±73．68），12h达峰值（6867．67±601．11），24h（2356．12±210．65）及72h（675．89±109．3）表达开始降低。p-Akt1在缺血再灌注组各观察时限点的表达趋势与GRP78一致，显著高于正常组及假手术组。结论大鼠肝缺血再灌注损伤可触发经内质网途经的Caspase级联反应活化；GRP78与GRP94可能通过降低内质网负荷和促进糖异生而发挥细胞保护作用；p-Akt1可能是此过程中机体通过磷酸肌醇3激酶（P13K）／Akt信号通路上调GRP78表达的重要信号分子。

Objective To dynamically observe the changes of glucose regulated protein 78 (GRP78),glucose regulated protein 94 (GRP94),p-protein kinase B1 (p-Akt1) and Caspase-12 expression during the periods of hepatic ischemia/reperfusion (I/R) in rats.Methods Fifty-five Wistar rats were randomly divided into 3 groups:normal control group (n =5),sham operated group (n =25) and I/R group (n =25).The rat model of hepatic I/R was established by clamping hepatic pedicle for 45 min by using no damage artery clamp and then reperfusion.The expression of GRP78,GRP94 and Caspase-12 was detected by using immunohistochemistry SABC at 0,3,12,24-and 72 h after I/R.The expression of p-Akt1 was detected by using Western blotting at the same time points.Results The expression levels of GRP78,GRP94 and Caspase-12 in I/R group were significantly higher than in normal control group and sham operated group (P ＜0.01).In I/R group,the expression of GRP78 was increased at 0 h (393.04±79.06) after reperfusion,peaked at 12 h (2369.38 ± 182.53),and began to decline at 24 h (1023.02 ± 185.61) and 72 h (505.19 ± 102.89),but still significantly higher than the other two groups.In I/R group,the expression of GRP94 was increased at 0 h (168.47 ±21.08) after reperfusion,peaked at 12 h (678.8 ±52.62),and hegan to decline at 24 h (425.78 ±38.63) and 72 h (173.99 ±36.82).In I/R group,the expression of Caspase-12 was increased at 0 h (647±73.68) after reperfusion,peaked at 12 h (6867.67 ± 601.11),and began to decline at 24 h (2356.12 ±210.65) and 72 h (675.89 ± 109.3).The expression trend of p-Aktl resembled GRP78 in I/R group at different time points,and significantly higher than the other two groups.Conclusion Hepatic I/R injury can activate Caspase cascade reaction via the endoplasmic reticulum.GRP78 and GRP94 may play a protective effect during the periods of hepatic I/R in rats by reducing the burden of endoplasmic reticulum and promoting gluconeogenic pathway.p-Akt1 probably is an important signal molecule in the phosphatidylinositol 3 kinase (PI3K)/Akt signal path by which the expression of GRP78 is up-regulated.