莱菔硫烷对大鼠心脏移植冷缺血再灌注损伤的保护作用

The protection of Sulforaphane on myocardial cold ischemia reperfusion injury in rat heart transplantation

目的 建立大鼠同种异体异位心脏移植模型,莱菔硫烷（Sulforaphane,SFN）预处理受体大鼠,观察并探讨其对大鼠心肌冷缺血再灌注损伤的保护作用.方法 健康雄性Lewis大鼠140只,其中100只采用数字表法随机分为对照组、SFN0.5组、SFN0.75组、SFN1.0组和SFN25组5组,每组20只（供、受体各10只）.各组受体大鼠分别于受体移植前24h经尾静脉注射等量生理盐水、SFN0.5mg· kg-1 bw-1、SFN 0.75mg·kg-·bw-、SFN 1.0mg·kg-1 ·bw-和SFN 2.5 mg·kg-1·bw-.将冷藏于4C HTK液18h的供心,移植到受体大鼠腹腔内,建立大鼠同种异体异位心脏移植模型.移植后6、24 h分别从受体鼠眼内眦静脉或腔静脉采血,检测血清中乳酸脱氢酶（LDH）、肌酸激酶同工酶（CK-MB）、肌钙蛋白（TnT）水平;移植后24 h,取供心心肌组织,检测心肌组织中脂质过氧化物（lipid hydroperoxide,LPO）含量及超氧化物歧化酶（superoxide dismutase,SOD）的活性,应用免疫组织化学检测移植心肌组织诱导型一氧化氮合酶（inducible Nitric Oxide,iNOS）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）、低氧诱导因子-1oα（Hypoxia in-ducible factor,HIF-1α）的表达,电镜观察心肌超微结构变化.另外40只大鼠应用Kaplan-meier分析其生存率.结果 与对照组大鼠比较,其他4组经SFN预处理大鼠的中位生存期明显高;血清中LDH、TnT、CK-MB水平均有不同程度下降;心肌LPO含量均降低（P＜0.05）;SOD/LPO比值高（P＜0.01）;心肌超微结构损伤程度轻.另外,与对照组比较,SFN0.75组、SFN10组（P＜0.05）、SFN2.5组（P＜0.05）大鼠的心脏功能评分较高;移植后6、24 h,SFN0.5组心肌组织SOD活性升高（P＜0.05）;SFN0.75组、SFN1.0组、SFN2.5组心肌组织iNOS表达降低（P＜0.01）;SFN1.0组、SFN2.5组Caspase-3表达低（P＜0.05）;SFN10组、SFN25组HIF-1α表达低（P＜0.05）.结论 SFN可通过抗氧化、抗细胞凋亡机制,减轻心脏移植中冷缺血再灌注损伤引起的氧化应激和心肌细胞凋亡,从而减轻移植心脏的缺血再灌注损伤.SFN剂量增加,保护作用增强.

Objective To explore the protection of Sulforaphane on myocardial cold ischemia reperfusion injury in the isogeneicheterotopic heart transplantation model of rats.Methods 100 healthy male Lewis rats were randomly divided into 5groups,20 rats in each group（donor and recipient 10 rats respectively）.Control group：recipient rats were injected the equal amountsaline via tail vein 24 h before heart transplantation.SFN0.5 group：recipient rats were injected sulforaphane 0.5 mg· kg-1 · bw-1 via tail vein 24 h before heart transplantation.SFN0.75 group：recipient rats were injected sulforaphane 0.75 mg · kg-1 · bw-1 via tail vein 24 h before heart transplantation.SFN1.0 group：recipient rats were injected sulforaphane 1.0 mg · kg-1 · bw-1 via tail vein 24 h before heart transplantation.SFN2.5 group：recipient rats were injected sulforaphane 2.5 mg · kg-1 · bw-1 via tail vein 24 h before heart transplantation.Donnor rats＇ hearts were stored in 4℃ HTK solution for 18 h in control and experimental groups.Heart transplantation was performed.Lactate dehydrogenase （LDH）,Creatine kinaseMB （CK-MB）,Troponin T（TnT） were determined at 6 h and 24 h after heart transplanatation.Levels of LPO and activity SOD were tested at 24 h after heart transplanatation.The protein expression of iNOS,Caspase-3,HIF-1α was detected by immunohistochemistry.The changes of myocardial ultrastructural were detected by transmission electron microscope.The survival of two groups was estimated by kaplan-meier analysis using 40 healthy male Lewis rats.Results The median survival time of SFN preconditioning group was obviously higher than that of control group （7d vs.5d）,graft function in SFN0.75,SFN1.0,SFN2.5 groups was significantly improved comnpared with control group.The levels of LDH,CK-MB,TnT at 24 h were significantly reduced in SFN preconditioning groups compared with control group.The level of LPO was significantly decreased in SFN preconditioning groups compared with control group,SOD/LPO was significantly increased in SFN preconditioning groups,but the activity of SOD was significant increased in SFN0.5 group.The protein expression of iNOS,Caspase-3,HIF-1 α was significantly reduced in SFN1.0and SFN2.5 groups compared with control group.Myocardial ultrastructure also proved that SFN preconditioning groups was better than control group.Conclusion SFN reduced oxidative stress and myocardial cells apoptosis caused by cold ischemia-reperfusion injury in heart transplantation through its anti-oxidation and anti-apoptotic,which reduced ischemia-reperfusion injury in heart transplantation,and its protective effect increased with dose increased.