超氧化物歧化酶对大鼠肝脏缺血再灌注损伤保护作用的体外实验

The protective effect of superoxide dismutase on non heart beating donor livers during ischemia and reperfusion in vitro

目的探讨氧自由基清除剂对大鼠肝脏缺血再灌注损伤的保护作用及其机理。方法将Wistar大鼠分成三组,各组通过门静脉插管,对肝脏进行原位灌洗,心脏搏动组以4℃HTK液灌洗;心脏停跳组在心脏搏动停止60 min后,以同样方法灌洗肝脏;SOD实验组灌洗方法同心脏停跳组,但灌洗液中含有超氧化物歧化酶(SOD)7500 IU。灌注结束后,快速切取肝脏,于4℃HTK液中保存24 h,然后在体外以Krebs-Henseleit缓冲液再灌注45 min。测定再灌注过程中的门静脉压力,收集再灌注液,进行血气分析,测定其中丙氨酸转氨酶(ALT)、谷氨酸乳酸脱氢酶(GLDH)及脂质过氧化物(LPO)的含量;切取肝组织,检测其能量底物总和(TAN)及细胞凋亡情况。结果再灌注期间,SOD实验组的门静脉压力为(6.4±0.9)cm H_2O,明显低于心脏停跳组的(12.1±0.7)cm H_2O(P<0.01)。随着再灌注时间的延长,灌注液中ALT和GLDH的浓度不断升高,但SOD实验组明显低于心脏停跳组(P<0.05)。心脏停跳组肝脏氧消耗量明显低于心脏搏动组(P<0.01),而SOD实验组氧消耗量明显增加(P<0.01)。SOD实验组的TAN为(7.6±0.4)μmol/g,明显高于心脏停跳组的(5.3±0.7)μmol/g(P<0.05)。SOD实验组灌注液中LPO的含量为(0.42±0.10)nmol/g,明显低于心脏停跳组的(0.98±0.18)nmol/g(P<0.01)。心脏搏动组只有极少量的肝窦内皮细胞和肝细胞凋亡,SOD实验组中凋亡细胞的数量也非常有限,而在心脏停跳组,则有大量的肝窦内皮细胞发生凋亡。结论在体外,SOD能显著减轻大鼠肝脏的缺血再灌注损伤,这可能与SOD抑制了氧自由基所致的细胞凋亡有关。

Objective To investigate the protective effect and mechanism of oxygen free radical scavenger in non heart beating donor （NHBD） liver grafts during ischemia and reperfusion injury. Methods Wistar rats were divided into 3 groups （n = 6 in each group）, and livers were perfused via the portal vein with 60 ml of cold （4 ℃） HTK solution from heart beating donors （HB） and from NHBD after 60 min cardiac arrest. A third group consisted of NHBD livers which were perfused with HTK containing 7500 IU of superoxide dismutase （SOD）. Livers were harvested and reperfused with Krebs-Henseleit buffer in a recirculating system at 37 ℃ for 45 rain after 24 h cold storage. The portal venous pressure and alanine aminotransferase （ALT）, glutamate-lactate dehydrogenase （GLDH）, lipoperoxides （LPO） in perfusate were examined. At the end of reperfusion, perfusate gas-blood analysis and total adenine nucleotides （TAN）, apoptosis in liver tissue were tested. Results During reperfusion, the portal venous pressure in SOD group was （6. 4 ± 0. 9） cm H2 O, significantly lower than that in NHBD group （12. 1 ± 0. 7 cm H2 O） （P〈0. 01 ）. The ALT and GLDH in perfusate were increased with the prolongation of reperfusion time, and those in SOD group were lower than in NHBD group （P〈0. 05）. Hepatic oxygen utilization in NHBD was less than that in HB （P〈0. 01）, but increased significantly in SOD group （P〈0. 01 ）. TAN in SOD group （7. 6 ± 0. 4/μmol/g） was higher than that in NHBD group （5. 3±0. 7/μmol/g） significantly （P〈0. 05）. LPO in SOD group （0. 42 ± 0. 10 nmol/g） was lower than that in NHBD （0. 98 ± 0. 18 nmol/g） （P〈0. 01 ）. In the HB and SOD groups apoptotic cells were rarely seen, while in NHBD group, there were great number of apoptotic cells present mainly in sinusoidal lining cells and hepatocytes. Conclusion In vitro, SOD can prevent the rats from liver ischemia-reperfusion injury, which may be contributed to the suppression of apoptosis induced by oxygen free radicals.