乌司他丁对全脑缺血再灌注大鼠海马神经元程序性坏死的影响

Effect of ulinastatin on programmed necrosis in hippocampal neurons in a rat model of global cerebral ischemia-reperfusion

目的评价乌司他丁对全脑缺血再灌注大鼠海马神经元程序性坏死的影响。方法清洁级健康成年雄性SD大鼠48只,8周龄,体重280~320g,采用随机数字表法分为3组(n=16):假手术组(Sham组)、全脑缺血再灌注组(I/R组)和乌司他丁组(UT组)。I/R组和UT组均采用四血管阻断法制备大鼠全脑缺血再灌注模型。UT组于缺血即刻经尾静脉注射10万U/kg乌司他丁,Sham组和I/R组注射等容量生理盐水。于再灌注6、12和24 h时行神经功能缺损评分(NDS)。于再灌注24h时处死大鼠取海马组织,光镜下观察病理学结果,采用分光光度法测定MDA含量和SOD活性,采用Western blot法检测受体相互作用蛋白激酶1(RIPK1)、受体相互作用蛋白激酶3(RIPK3)和混合系列蛋白酶样结构域(MLKL)的蛋白表达。结果与Sham组比较,I/R组和UT组各时点NDS升高,海马MDA含量升高,SOD活性降低,RIPK1、RIPK3和MLKL表达上调(P<0.05);与I/R组比较,UT组NDS降低,海马MDA含量降低,SOD活性升高,RIPK1、RIPK3和MLKL表达下调(P<0.05)。UT组海马病理学损伤较I/R组减轻。结论乌司他丁减轻大鼠全脑缺血再灌注损伤的机制与抑制海马神经元程序性坏死有关。

Objective To evaluate the effect of ulinastatin on programmed necrosis in hippocampal neurons in a rat model of global cerebral ischemia-reperfusion(I/R).Methods Forty-eight clean-grade healthy adult male Sprague-Dawley rats,aged 8 weeks,weighing 280-320 g,were divided into 3 groups(n=16 each)using a random number table method:sham operation group(Sham group),global cerebral I/R group(I/R group)and ulinastatin group(UT group).Global cerebral I/R was produced by 4-vessel occlusion method in chloral hydrate-anesthetized rats in I/R and UT groups.Ulinastatin 100 000 U/kg was injected via the tail vein at the onset of ischemia in group UT,and the equal volume of normal saline was given instead in Sham and I/R groups.Neurological deficit score(NDS)was estimated at 6,12 and 24 h of reperfusion.Animals were sacrificed at 24 h of reperfusion,brains were removed and the hippocampi were obtained for examination of pathological changes(with a light microscope)and for determination of the malondialdehyde(MDA)content and superoxide dismutase(SOD)activity(by spectrophotometry),and expression of receptor-interacting protein kinase 1(RIPK1),RIPK3,and mixed lineage kinase domain-like protein(MLKL)in hippocampal tissues(by Western blot).Results Compared with Sham group,the NDS was significantly increased at each time point,the MDA content was increased,the SOD activity was decreased,and the expression of RIPK1,RIPK3 and MLKL was up-regulated in I/R and UT groups(P<0.05).Compared with I/R group,the NDS was significantly decreased at each time point,the MDA content was decreased,the SOD activity was increased,and the expression of RIPK1,RIPK3 and MLKL was down-regulated in UT group(P<0.05).The pathological changes of hippocampi were significantly attenuated in UT group when compared with I/R group.Conclusion The mechanism by which ulinastatin ameliorates global cerebral I/R injury is related to inhibiting programmed necrosis in hippocampal neurons of rats.