N-乙酰半胱氨酸对大鼠心脏移植缺血再灌注损伤细胞凋亡的影响

EFFECTS OF N-ACETYLCYSTEINE ON APOPTOSIS INDUCED BY MYOCARDIAL ISCHEMIA REPERFUSION INJURY IN RATS' HEART TRANSPLANTATION

目的观察N-乙酰半胱氨酸（N-acetylcysteine，NAC）对大鼠心脏移植后心肌缺血再灌注损伤导致的细胞凋亡的影响，并探讨其可能机制。 方法健康雄性Lewis大鼠60只，体重200～220 g，随机分为3组，每组20只，供、受体各10只。对照组：摘取供心前30 min经供体大鼠下腔静脉注射生理盐水1 mL；供体预处理组：摘取供心前30 min经供体大鼠下腔静脉注射NAC（300 mg/kg），受体不作处理；受体预处理组：于移植前30 min经受体大鼠下腔静脉注射NAC（300 mg/kg），供体不作处理。各组均建立同种异体异位心脏移植模型。术后观察受体大鼠存活情况；于移植后6、24 h取静脉血检测天冬氨酸转氨酶（aspartate aminotransferase，AST）、丙氨酸转氨酶（alanine aminotransferase，ALT）、乳酸脱氢酶（lactate dehydrogenase，LDH）含量；移植后24 h，取心肌组织检测超氧化物歧化酶（superoxide dismutase，SOD）、脂质过氧化物（lipid hydroperoxide，LPO）活性，观察心肌组织学及超微结构，免疫组织化学染色检测活性半胱氨酸天冬氨酸蛋白酶3（active Caspase-3）蛋白表达，TUNEL法检测心肌细胞凋亡指数（apoptosis index，AI）。 结果各组心脏移植手术均成功，大鼠均存活至实验完成。移植后6 h，供、受体预处理组AST、ALT、LDH均显著低于对照组（P 〈 0.05），24 h时供体预处理组AST、ALT及受体预处理组AST、LDH低于对照组（P 〈 0.05）；供、受体预处理组间各指标比较差异均无统计学意义（P 〉 0.05）。移植后6、24 h组内比较，除受体预处理组ALT差异无统计学意义外（P 〉 0.05）外，移植后24 h两组各指标均显著低于6 h（P 〈 0.05）。供、受体预处理组心肌组织中SOD含量及SOD/LPO均高于对照组（P 〈 0.05），供、受体预处理组比较差异无统计学意义（P 〉 0.05）；各组LPO比较差异均无统计学意义（P 〉 0.05）。组织学及透射电镜观察显示，受体预处理组心肌损伤程度均明显轻于对照组和供体预处理组；受体预处理组active Caspase-3蛋白表达量显著高于对照组和供体预处理组（P 〈 0.05）；供、受体预处理组AI显著低于对照组（P 〈 0.05），供、受体预处理组间差异无统计学意义（P 〉 0.05）。 结论NAC可以通过提高心肌SOD含量、降低active Caspase-3活性抑制心肌细胞凋亡来减轻大鼠心脏移植缺血再灌注损伤，进而保护供体心脏。

ObjectiveTo investigate the effect of N-acetylcysteine （NAC） on the apoptosis during myocardial ischemia reperfusion injury in rats’ heart transplantation, and to explore the possible role of NAC in myocardial apoptosis. MethodsSixty healthy male Lewis rats （weighing, 200-220 g） were randomly divided into 3 groups, 20 rats each group （10 donors and 10 recipients）. In control group, 1 mL normal saline was infused via inferior vena cava at 30 minutes before donor harvesting; in donor preconditioning group, NAC （300 mg/kg） was infused via inferior vena cava at 30 minutes before donor harvesting, but no treatment in recipients; and in recipient preconditioning group, NAC （300 mg/kg） was infused via inferior vena cava at 30 minutes before recipient transplantation, but no treatment in donors. Heart transplantation was established in each group. Blood was drawn at 6 and 24 hours after reperfusion for analysis of aspartate aminotransferase （AST）, alanine aminotransferase （ALT）, lactate dehydrogenase （LDH） as markers of graft injury; myocardial tissue was harvested to determine the superoxide dismutase （SOD） and lipid hydroperoxide （LPO） activity at 24 hours after reperfusion and to observe the histology and ultrastructural changes. Graft active Caspase-3 protein expression was measured by immunohistochemistry staining, and apoptosis index （AI） was calculated by TUNEL. Results The heart transplantation operation was successfully completed in all groups, and the rats survived to the end of the experiment. The serum levels of AST, ALT, and LDH in donor and recipient preconditioning groups were significantly lower than those in control group at 6 hours after reperfusion （P 〈 0.05）; the levels of AST and ALT in donor preconditioning group and the levels of AST and LDH in recipient preconditioning group were significantly lower than those in control group at 24 hours （P 〈 0.05）; and no significant difference was found between donor and recipient perconditioning groups （P 〉 0.05）. The levels of AST, ALT, and LDH at 24 hours were significantly lower than those at 6 hours in each group （P 〈 0.05） except the level of ALT in recipient preconditioning group （P 〉 0.05）. SOD activity and SOD/LPO in donor and recipient preconditioning groups were significantly higher than those in control group （P 〈 0.05）, but no significant difference between donor and recipient preconditioning groups （P 〉 0.05）; there was no significant difference in LPO activity among 3 groups （P 〉 0.05）. Histological staining and transmission electron microscope showed that myocardial injury in recipient preconditioning group was obviously lighter than that in donor preconditioning group and control group. Active Caspase-3 in recipient pretreatment group was significantly higher than that in donor preconditioning group and control group （P 〈 0.05）. AI of donor and recipient preconditioning groups was significantly lower than that of control group （P 〈 0.05）, but no significant difference was found between donor and recipient preconditioning groups （P 〉 0.05）. ConclusionNAC can relieve ischemia reperfusion injury in rats’ heart transplantation by improving myocardial SOD content, and reducing active Caspase-3 activity and AI, which has a protective effect on myocardial cell of donor heart.