摘要：
Objective To investigate the effect of Anti TNF-α combined with p38 MAPK antisense oligonucleotide on renal funtion and TNF-α,p38MAPK protein expression at different ischemia-reperfusion points.Methods One hundred and twenty normal male Sprague-Dawley rats were divided into 4 groups by simple randomization:sham-operated (sham) group,ischemia-reperfusion (IR) group,Anti TNF-α + ischemia-reperfusion (Anti TNF-α + IR) group,Anti TNF-α and p38 MAPK antisense oligonucleotide + ischemia-reperfusion (combination preconditioning + IR) group.Each group comprised 30 rats.Anti TNF-α + IR group was subjected to ischemia-reperfusion injury with intravenous administration of Anti TNF-α (0.1 mg/kg) 5 min before reperfusion.Combination preconditioning + IR group was subjected to ischemia-reperfusion injury with intravenous administration of Anti TNF-α(0.1 mg/kg)and p38MAPK antisense oligonucleotide (5 mg/kg)5 min before reperfusion.IR group with the same injury was followed by saline administration in the same manner.Sham group was subjected to only anesthetization but not to ischemia.Detecting the plasma creatinine and plasma urea nitrogen and two-step inmunohistochemical methods were used to detect the changes of expression of TNF-α,p38 MAPK.The measurement data were compared with the t test and the count data were compared with Chi-square test.The date were expressed by ((x) ± s).Intergroup comparison translated by variance analysis.Results After reperfusion,the plasma urea nitrogen in the IR group was (15.86 ± 2.41),(21.13 ± 2.21),(25.47 ± 2.29),(30.51 ± 2.03),(35.56±2.47) μmol/L at 0,1,3,6 and 12 h,respectively,and was (25.61 ±5.40),(32.48 ±2.30) (68.20±1.20),(84.42 ±2.43),(96.15 ±2.23)at 0,1,3,6 and 12 h respectively,and still showed an increasing trend at 12 h.TNF-α mainly expressed in renal proximal convoluted tubules,gradually upregulate with duration of ischemia-reperfusion to 12h of reperfusion.p38MAPK mainly located at distal convoluted tubules,peaked at 6h of reperfusion.But these effects were offset by administration in combination preconditioning group (P ＜ 0.05).Conclusion Renal ischemia-reperfusion injury can be alleviated by Anti TNF-α and p38 MAPK antisense oligonucleotide treatment.%目的 探讨抗肿瘤坏死因子-α(TNF-α)单抗联合p38丝裂原活化蛋白激酶(p38 MAPK)反义寡核苷酸预处理对缺血再灌注肾不同时间肾功能的变化和TNF-α、p38MAPK蛋白表达的影响.方法 健康雄性SD大鼠120只,按简单随机化分成4组,假手术(Sham)组、缺血再灌注(IR)组、抗TNF-α单抗+缺血再灌注(抗TNF-α单抗+IR)组、抗TNF-α单抗联合p38 MAPK反义寡核苷酸+缺血再灌注(联合预处理+IR)组.每组30只.假手术组于麻醉后分离肾动脉但不夹闭.抗TNF-α单抗+IR组于恢复血供前5 min通过尾静脉注入抗TNF-α单抗(0.1 mg/kg,用0.9％氯化钠溶液稀释至0.1 ml).联合预处理组于恢复血供前5 min通过尾静脉注入抗TNF-α单抗(0.1 mg/kg)和p38 MAPK反义寡核苷酸(5 mg/kg两者用0.9％氯化钠溶液稀释至0.1 ml).IR组于恢复血供前5 min通过尾静脉注入0.1ml 0.9％氯化钠溶液.全自动生化分析仪检测血浆尿素氮和肌酐水平,采用免疫组化二步法检测肾组织TNF-α、p38 MAPK蛋白表达.计量资料采用t检验,计数资料采用x2检验,数据以均数±标准差((x)±s)表示,组间比较采用方差分析.结果 IR组于再灌注后血浆尿素氮在0、1、3、6、12 h分别为(15.86±2.41)、(21.13±2.21)、(25.47±2.29)、(30.51±2.03)、(35.56±2.47) μmol/L、肌酐在0、1、3、6、12 h分别为(25.61±5.40)、(32.48 ±2.30)、(68.20±1.20)、(84.42±2.43)、(96.15±2.23) μmol/L,至12h仍呈升高趋势.联合预处理组再灌注后血浆尿素氮、肌酐上升幅度低于缺血再灌注组相应时间点(P＜0.05).TNF-α蛋白在近端小管上皮细胞表达为著,随着再灌注时间延长的明显增加,再灌注12h时仍呈增高趋势.p38MAPK主要在远端小管上皮细胞表达,再灌注后6h后达高峰.联合预处理组TNF-α、p38MAPK蛋白水平显著低于IR组(P＜0.05).结论 抗TNF-α单抗及p38MAPK反义寡核苷酸联合应用,通过降低TNF-α、p38MAPK蛋白表达水平,减轻肾缺血再灌注损伤.
