Caspase-3和Caspase-8基因沉默保护大鼠肝脏缺血再灌注损伤

目的 观察Caspase-3和Caspase-8基因沉默对大鼠肝脏缺血再灌注损伤((IRI)的保护作用.方法 分别构建针对大鼠caspase-3和caspase-8基因的短发夹状RNA(shRNA)真核表达载体.肝脏IRI前48 h经门静脉注射磷酸盐缓冲液(PBS)、空载体或Caspase-3和Caspase-8shRNA,实验随机分为4组,假手术组、PBS组、空载体组和shRNA组.阻断大鼠70%入肝血流40min,再灌注6、12、24 h、3、5、7 d检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平,肝组织Caspase-3和Cmpase-8 mRNA的表达,Caspase-3和Caspase-8蛋白活性,细胞凋亡情况,丙二醛(MDA)以及超氧化物岐化酶(SOD)的含量.结果 与PBS组和空载体组比较,shRNA组再灌注6、12、24 h、3、5 d血清ALT和AST水平显著降低(P＜0.05),Caspase-3和Caspase-8 mRNA水平、Caspase-3和caspase-8蛋白活性(Cmpme-3活性:0.18±0.03比0.36±0.03和0.38±0.02,P＜0.05;caspase-8活性:0.16±0.03比0.32±0.02和0.34±0.03,P＜0.05)、肝细胞凋亡指数(22.46±3.25)%比(35.24±2.33)%和(37.36±2.51)%,P＜0.05]和肝组织中MDA含量(76.2±15.3)比(123.5±12.4)、(136.7±13.6)nmol/mg,P＜0.05)显著降低,SOD活性显著升高(24.1±3.2)比(12.2±3.1)、(11.4±2.9)U/mg,P＜0.05].结论 Caspase-3和Caspase-8基因沉默可以抑制细胞凋亡的发生,从而起到保护肝脏IRI的作用.

Caspase-3 and Caspase-8 gene silencing protected liver ischemia-reperfusion injury in rats

Objective To investigate the protective effect of Caspase-3 and Caspase-8 gene silencing on liver ischemia-reperfusion injury.MethodsThe eukaryotic expression vector of small hairpin RNA targeting rat Caspase-3 and Caspase-8 genes was constructed respectively.PBS,blank vector or Caspase-3 and Caspase-8 shRNA mixture were injected through portal vein 48 h before operation.The experiment was divided into 4 groups :sham group,PBS group,blank vector group and shRNA group.The left lateral and median lobes of the liver were subjected to ischemia for 40 min,resulting in a segmental (70%) hepatic ischemia.The serum ALT and AST levels were determined 6 h,12 h,24 h,3 days,5 days and 7 days after reperfusion respectively.The mRNA expression of Caspase-3 and Caspase-8,activity of Caspase-3 and Caspase-8 protein,apoptosis index,concentration of malondialdehyde (MDA) and superoxide dismutase (SOD) in the liver tissues were detected.Results Compared with PBS group and blank vector group,the ALT and AST levels were significantly decreased in shRNA group 6 h,12 h,24 h,3 days,5 days after reperfusion ( P＜0.05 ) ; Caspase-3 and Caspase-8 mRNA levels,protein activity ( Caspase-3 activity :0.18±0.03 vs 0.36±0.03 and 0.38±0.02,P＜0.05 ; Caspase-8 activity :0.16±0.03 vs 0.32±0.02 and O.34±0.03,P＜0.05 ),apoptosis index (22.46±3.25 ) % vs ( 35.24±2.33 ) % and ( 37.36±2.51 ) % ,P＜0.05 ] and concentration of MDA in the liver tissue (76.2±15.3 ) nmol/mg vs ( 123.5±12.4) nmol/mg and ( 136.7±13.6) nmol/mg,P＜0.05 ] were significantly decreased,whle the SOD activity was significantly increased (24.1±3.2) U/mg vs ( 12.2±3.1 ) U/mg and ( 11.4±2.9) U/mg,P＜0.05 ].Conclusion Caspase-3 and Caspase-8 gene silencing can protect the liver ischemia/reperfusion injury.