联合预处理对大鼠缺血再灌注肾TNF-α、p38MAPK蛋白表达的影响

Effect of combination preconditioning on TNF-α,p38MAPK protein expression in renal ischemia-reperfusioninjury of rats

目的:观察抗TNF-α单抗联合p38MAPK反义寡核苷酸是否可以通过降低TNF-α、p38MAPK蛋白表达来减轻大鼠的肾脏缺血再灌注损伤。方法:将120只SD大鼠随机分成4组,分别为假手术组(Sham组)、缺血再灌注组(IR组)、缺血再灌注+抗TNF-α单抗组(抗TNF-α+IR组)、缺血再灌注+抗TNF-α单抗联合p38MAPK反义寡核苷酸组(联合预处理组),每组30只。Sham组于麻醉后分离肾动脉但不夹闭;IR组制作肾缺血再灌注模型时,于肾脏恢复血供前5 min通过尾静脉注射0.2 mL生理盐水,做阴性对照;联合预处理组于恢复血供前5 min注入抗TNF-α单抗(0.1 mg/kg,用生理盐水稀释至0.1 mL)和p38MAPK反义寡核苷酸(5 mg/kg,用生理盐水稀释至0.1 mL);抗TNF-α+IR组于肾脏再灌注前5 min,注射抗TNF-α单抗(0.1 mg/kg);采用HE染色,光镜观察肾组织病理学变化情况,采用免疫组化二步法检测TNF-α、p38MAPK蛋白在缺血再灌注肾组织中的表达情况。结果:联合预处理组再灌注后的肾组织损伤减轻。TNF-α蛋白在肾小管细胞浆中表达,在近曲小管上皮细胞表达最明显,缺血再灌注后表达即升高,至12 h时仍呈增高趋势。p38MAPK蛋白主要在远端肾小管上皮细胞浆中表达,于再灌注后6 h时表达最高,12 h时表达水平降低。联合预处理组再灌注后的肾组织TNF-α、p38MAPK蛋白水平均低于IR组(均P<0.05)。结论:TNF-α单抗和p38MAPK反义寡核苷酸联合应用能减轻大鼠缺血再灌注肾的病理损伤,其机制可能与降低肾组织TNF-α、p38MAPK蛋白的表达水平有关。

ObjectiveTo investigate the protective of AntiTNF-αcombined with p38MAPK antisense oligonucleotide on renal ischemia-reperfusion injuru in rats.Methods A total of 120 SD rats were randomly divided into 4 groups:Sham operation group(Sham group),ischemia reperfusion group(IR group),ischemia reperfusion+anti-TNF-αmab group(anti-TNF-A+IR group),and ischemia reperfusion+anti-TNF-αmab combined with p38MAPK antisense oligonucleotide group(combined pretreatment group),with 30 rats in each group.In Sham group,renal artery was separated but not clamped after anesthesia.When the renal ischemia reperfusion model was made in IR group,0.2 mL normal saline was injected through the tail vein 5 min before the renal blood supply was restored,and negative control was performed.The combined pretreatment group was injected with anti-TNF-αmonoclonal antibody(0.1 mg/kg,diluted with normal saline to 0.1 mL)and p38MAPK antisense oligonucleotide(5 mg/kg,diluted with normal saline to 0.1 mL)5 min before blood supply was restored.Anti-TNF-α+IR group was injected with anti-TNF-αmonoclonal antibody(O.1 mg/kg)5 min before renal reperfusion.Histopathological changes of kidney were observed by HE staining and light microscopy.Two-step inmunohistochemical methods were used to detect the changes of expression of TNF-α,p38MAPK.Results Pathological damage of kidney tissue was significantly alleviated in combination preconditioning group.TNF-αmainly expressed in renal proximal convoluted tubules,gradually upregulate with duration of ischemia-reperfusion to 12 h of reperfusion.p38MAPK mainly located at distal convoluted tubules,peaked at 6 h of reperfusion.But these effects were offset by administration in combination preconditioning group(P<0.05).Conclusions Renal ischemia-reperfusion injury can be alleviated by anti-TNF-αand p38MAPKantisense oligonucleotide treatment.