抑制Caspase-8基因表达对减轻大鼠肝脏缺血再灌注损伤的作用

目的 观察抑制半胱氨酸天冬氨酸特异性蛋白酶8(Caspase-8)基因的表达对减轻大鼠肝脏缺血再灌注损伤的作用.方法 构建针对大鼠Caspase-8基因的短发夹状RNA(shRNA)真核表达载体.将Lewis大鼠随机分为3组,每组8只.(1)假手术组:麻醉后,取腹部正中切口,缝合关腹;(2)磷酸盐缓冲液(PBS液)组:阻断肝门血流前48 h经门静脉注射PBS液1 ml,然后行肝脏缺血再灌注;(3)shRNA组:阻断肝门血流前48 h经门静脉注射Caspase-8 shRNA 50 μg(总体积为1 ml),然后行肝脏缺血再灌注.肝脏缺血再灌注的方法为阻断大鼠70%入肝血流40min.于再灌注6 h、12 h、24 h、3 d、5 d、7 d时检测血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平;检测肝组织中Caspase-8 mRNA的表达、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液组比较,shRNA组再灌注6、12、24 h,血清中ALT和AST水平显著降低(P<0.05);肝组织中Caspase-8 mRNA水平、肝细胞凋亡指数(shRNA组和PBS液组分别为22.33%±4.28%和35.24%±2.33%)以及肝组织中MDA含量shRNA组和PBS液组分别为(94.5±11.2)nmol/mg和(133.5±12.4)nmol/mg]均显著降低(P<0.05),而肝组织中SOD活性显著升高shRNA组和PBS液组分别为(21.6±3.7)U/mg和(12.2±3.1)U/mg,P<0.05].结论 通过RNA干扰Caspase-8基因的表达可以抑制肝细胞凋亡的发生,减轻肝脏缺血再灌注损伤.

Liver ischemia/reperfusion injury in rats was relieved by inhibiting the Caspase-8 expression

Objective To investigate the alleviation of liver ischemia/reperfusion injury (IRI) by inhibiting the Caspase-8 expression. Methods The eukaryotic expression vector of small hairpin RNA targeting rat Caspase-8 gene was constructed. The Lewis rats were randomly divided into 3 groups (n = 8 each):(1) sham group: the rats with laparotomy did not undergo the liver IRI; (2) PBS group:PBS was injected through portal vein 48 h before operation; (3) shRNA group: Caspase-8 shRNA was injected through portal vein 48 h before operation. The left lateral and median lobes of the liver were rendered ischemia for 40 min resulting in a segmental (70%) hepatic ischemia. The serum ALT and AST levels were detected 6 h, 12 h, 24 h, 3 days, 5 days and 7 days after reperfusion respectively.The mRNA expression level of Caspase-8, apoptosis index, concentrations of the malondialdehyde (MDA) and superoxide dismutase (SOD) in the liver tissue were detected. Results Compared with PBS group, the ALT and AST levels were significantly decreased in shRNA group 6 h, 12 h and 24 h after reperfusion (P<0.05), Caspase-8 mRNA expression level, apoptosis index (22.33%±4.28% vs 35.24%±2.33%, P<0.05) and concentrations of MDA in the liver tissue (94.5±11.2 vs 133.5±12.4 nmol/mg, P<0.05) were significantly decreased, and the SOD activity was significantly increased (21.6±3.7 vs 12.2±3.1 U/nag, P < 0.05). Conclusion Inhibiti0on of the Caspase-8 expression by RNA interference can protect the liver IRI.