褪黑素预处理受体对移植肾的保护作用及其机制探讨

Protection and mechanism of kidney graft after recipient pretreated with melatonin in rats kidney transplantation

目的 探讨褪黑素预处理受体对移植肾的保护作用及其可能的机制. 方法 40只Lewis大鼠应用随机数字表随机分为对照组和实验组,每组20只（供、受体各10只）.实验组：50 mg/kg褪黑素＋5 ml牛奶在移植前2h经胃管注入受体大鼠;对照组：5 ml牛奶在移植前2h经胃管注入受体大鼠.移植后0、6和24 h分别采血检测血尿素氮（BUN）和肌酐（Cr）及天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）、乳酸脱氢酶（lactate dehydrogenase,LDH）;移植后24 h取移植肾检测肾组织中超氧化物歧化酶（superoxide dismutase,SOD）及脂质过氧化物（lipid hydroperoxide,LPO）;过碘酸-雪夫染色（PAS染色）观察组织学变化,免疫组化法检测肾移植物中核转录因子-κB （nuclear factor kappa B,NF-κB）和半胱氨酸蛋白酶-3（caspase-3）阳性肾小管数. 结果 实验组与对照组移植后6 h BUN分别为（36.26± 19.05）、（53.64± 16.57） mmol/L,移植后24 h血Cr分别为（144.09±76.91）、（259.01±132.60） μmol/L,组间比较差异均有统计学意义（P＜0.05）.移植后24 h,实验组血清AST、ALT、LDH均低于对照组,组间比较差异均有统计学意义（P＜0.05）.移植后24 h实验组和对照组移植肾组织中SOD分别为（8.91±4.38）、（2.21 ±0.45） U/mg,LPO分别为（9.5±5.8）、（60.8±23.7） pmol/mg,组间比较差异均有统计学意义（P＜0.05）.两组肾小管损伤指数分别为1.50±0.22、3.00±0.15,NF-κB阳性小管评分为2.00±0.21、3.60±0.16,caspase-3阳性小管评分为2.77±0.48、3.38±0.52,组间比较差异均有统计学意义（P＜0.05）. 结论 褪黑素预处理受体可能通过降低体内的氧化应激反应,提高抗氧化酶活性,增强清除自由基的能力,抑制脂质过氧化反应和凋亡的发生来减轻移植肾的缺血再灌注损伤,提高移植肾功能.

Objective To investigate the protection and mechanism of melatonin against the adverse effects of ischemia reperfusion injury （IRI） in kidney graft tissue.Methods Forty Lewis rats were randomly divided into 2 groups （donor and recipient individually 10 animals）.Melatonin （50 mg/kg.bw） and 5 ml milk were given by gavage for recipient 2 h before kidney transplantation in experiment group.Controls were given the same volume of milk only.Subsequently,left kidney grafts stored in 4 ℃ Histidine-Tryptophan-Ketoglutarate solution for 24 h were transplanted into bilaterally nephrectomized syngeneic recipients.Blood was detected 0,6,and 24 h after reperfusion from recipient＇s eye vein or vena cava for analysis of serum blood urea nitrogen （BUN）,creatinine,transaminases,and lactate dehydrogenase （LDH）.Biopsies were taken 24 h after reperfusion to evaluate tubular damage,the enzymatic activity of superoxide dismutase （SOD）,lipid hydroperoxide （LPO）,nuclear factor kappa B （NF-κB）,caspase-3 as indices of oxidative stress,necrosis and apoptosis,respectively.Results Serum BUN decreased 6 h after reperfusion and serum creatiuine decreased 24 h after transplantation in melatonin group compared with those in controls （36.26±19.05 mmol/L versus 53.64±16.57 mmol/L,144.09t76.91 μmol/L versus 259.01 ± 132.60 μ.mol/L,P＜0.05）.Serum aspartate aminotransferase,alanine aminotransferase,and LDH decreased significantly 24 h after reperfusion in melatonin group compared with those in controls （P＜0.05）.At the same time,kidney graft tubular damage index,NF-κB and caspase-3 immunohistochemistry expression also decreased than those in controls （1.50±0.22 versus 3.00t0.15,2.00±0.21 versus 3.60±0.16,2.77±0.48 versus 3.38±0.52,P＜0.05）.Kidney graft tissue SOD activation increased,while LPO concentration decreased in melatonin group （8.91±4.38 U/mg versus 2.21±0.45 U/mg and 9.5±5.8 pmol/mg versus 60.8±23.7 pmol/mg,P＜0.05）.℃onclusions Recipient preconditioning with melatonin might reduces significantly adverse effects of IRI and improves graft function in experimental kidney transplantation,likely through reduce of anti-oxidation,increase of anti-apoptosis,improvement of clean up reactive oxygen species,and inhibition of lipid peroxidation.