依赖天冬氨酸特异性半胱氨酸蛋白酶-3的肝细胞rn凋亡在肝硬化大鼠肝缺血再灌注损伤中的作用

摘要

AIM: To investigate whether hepatocyte apoptosis is contributed to liver ischemia-reperfusion (I/R) injury and the relationship between liver caspase-3 activity and hepatocyte apoptosis in cirrhotic rats. METHODS: Liver ischemia-reperfusion is induced by Pringle maneuver. The cirrhotic rats were randomized into two groups: Group A: simple hepatic blood inflow occlusion (HBIO); Group B: HBIO + inhibitor, before HBIO, ZVAD-fmk 15 mg/kg was injected via dorsal penis vein; Group C: healthy rat, simple HBIO. The ischemia time was 30 min in these groups. Serum aspartate aminotransferase(AST), liver caspase-3 activity, and apoptotic hepatocytes were examined in the three groups. RESULTS: After 6 h of reperfusion, the liver caspase-3 activity was markedly elevated and reached its peak, which was statistically higher than that of before I/R ［(18.1±1.8 ) μmol*h-1*g-1 (tissue) vs (6.6±2.0) μmol*h-1*g-1 (tissue), P＜0.01］. The same change occurred in hepatocyte apoptosis between 6 h of reperfusion and before I/R (20.9%±4.9% vs 0.5%±0.3%, P＜0.01). As the reperfusion prolonged, the caspase-3 activity and apoptotic hepatocyte decreased gradually. The 7th-day survival rate was 62.5% in group A. The serum AST, liver caspase-3 activity and apoptotic hepatocytes were significantly higher in group A than those in group B and C, representing the most severe liver injury among the three groups. CONCLUSION: Hepatocyte apoptosis is the major form of cell death in liver ischemia-reperfusion injury in cirrhotic rats. Hepatoctye apoptosis induced by I/R is caspase-3 dependent, and inhibiting caspase-3 can alleviate liver injury. The caspase-3 dependent hepatocyte apoptosis is highly contributed to the pathological phenomenon that the ischemic sensitivity of cirrhotic liver is higher than normal liver.%目的：探讨肝硬化大鼠肝缺血/再灌注（I/R）损伤是否与肝细胞凋亡相关及天冬氨酸特异性半胱氨酸蛋白酶-3（caspase-3）活性变化与肝细胞凋亡的关系。方法：Pringle法复制肝I/R模型，将肝硬化大鼠随机分为2组：A组：单纯肝门阻断；B组：血流阻断+抑制剂:N-苯甲基氧化碳酰-缬氨酸-丙氨酸-天冬氨酸-氟化丙酮(ZVAD-fmk) 15 mg/kg；取无肝硬化大鼠，作单纯肝门阻断为C组。各组肝门阻断时间均为30 min，再灌注72 h。比较3组的血清天冬氨酸转氨酶(AST)、肝组织的caspase-3活性和肝细胞凋亡数。结果：A组大鼠肝组织caspase-3活性、肝细胞凋亡数在再灌注后6 h达高峰，分别为(18.1±1.8) μmol AMC*h-1*g-1(tissue)和20.9%±4.9%，与I/R前的(6.6±2.0) μmol AMC*h-1*g-1 (tissue)和0.5%±0.3%相比，P＜0.01。肝细胞凋亡数、caspase-3的活性随灌注时间的延长而减低，两者随时间的变化一致。3组中A组肝损伤最严重，表现为再灌注后6 h血清AST最高，与B、C组比较有显著差异，大鼠7 d生存率只为62.5%。进一步研究表明，再灌注后6 h，A组的肝组织caspase-3活性、肝细胞凋亡数亦明显比B、C组高。结论：肝细胞凋亡是肝硬化大鼠肝I/R损伤的主要病理改变。肝细胞凋亡的发生可能主要依赖于肝组织caspase-3活性的改变，抑制caspase-3能明显减轻肝I/R损伤。肝硬化肝脏比无硬化肝脏对缺血损伤敏感性高的病理机制与依赖caspase-3的肝细胞凋亡密切相关。