氯胺酮复合乌司他丁对内毒素两次打击诱导大鼠急性肺损伤的影响

Effect of Ketamine combined with Ulinastatin on “double-hit” rat model of acute lung injury induced by Lipopolysaccharide

目的探讨氯胺酮复合乌司他丁对内毒素"两次打击"诱导大鼠急性肺损伤中NF-κB、IκB的影响及其与TNF-α、IL-6的关系。方法 25只SPF级雄性成年SD大鼠随机分为5组,分别是对照组(C组)、内毒素组(L组)、氯胺酮组(K组)、乌司他丁组(U组)、氯胺酮复合乌司他丁组(K+U组),每组5只。动物腹腔注射0.03%内毒素(lipopolysaccharide,LPS)1mg/kg,16h后,经尾静脉注射0.l%LPS 1.5mg/kg,建立"两次打击"ALI模型。U组、K+U组分别在第二次LPS注射后经尾静脉给予乌司他丁50000U/kg,K组、K+U组经尾静脉给予Ket 10mg/(kg·h),C组、L组、U组给予等量的生理盐水。4h后,抽腹主动脉血0.5ml,测Pa O2;抽下腔静脉血2ml,检测血清肿瘤坏死因子(tumor necrosis factor,TNF)α和白介素(interleukin,IL)6浓度;取肺组织标本,采用电泳迁移率实验(Electrophoretic Mobility Shift Assay,EMSA)检测NF-κB活性;采用(streptavidin biotin-peroxidase complex,SABC)免疫组化方法检测肺组织IκBα蛋白的表达;并对结果进行相关分析。结果与C组比较,各组动脉血Pa O2、肺组织IκB含量下降低(P<0.05),血清TNF-α、IL-6与肺组织NF-κB含量均升高(P<0.05);与L组比较,K组、U组、K+U组Pa O2及肺组织IκB含量升高(P<0.05),血清TNF-α、IL-6与肺组织NF-κB含量均下降(P<0.05);K组、U组及K+U组两两比较,K+U组中Pa O2及肺组织中IκB含量升高(P<0.05),血清TNF-α、IL-6与肺组织NF-κB含量均下降(P<0.05)。NF-κB与TNF-α(r=0.945,P<0.01)、IL-6(r=0.952,P<0.01)呈正相关,与IκB(r=-0.949,P<0.01)呈负相关。结论复合应用氯胺酮与乌司他丁可使LPS"两次打击"诱导大鼠急性肺损伤中NF-κB、TNF-α、IL-6含量下降,使Pa O2、IκB含量上升,减轻肺损伤,较两者单独应用效果更佳。

Objective To investigate the effect of ketamine combined with ulinastatin on nuclear factor κB（NF-κB）,inhibitor of nuclear factor-κB（IκB）,tumor necrosis factor-α（TNF-α） and interleukin 6（IL-6） in ＂double-hit＂ rat model of acute lung injury（ALI） induced by lipopolysaccharide（LPS）. Methods Twenty-five SPF levels male SD rats were randomly divided into 5 groups equally： control group（Group C）,lipopolysaccharide group（Group L）,ketamine group（Group K）,ulinastatin group（Group U）,ketamine combined with ulinastatin group（Group K＋U）. The rats received two doses LPS to establish ＂double-hit＂ model of ALI,which was intraperitoneal LPS 1mg/kg and intravenous LPS 1.5mg/kg at 16 h interval. The rats received ulinastatin 50000 U iv after second dose of LPS in group U and group K ＋U. Ketamine was infused iv at 10mg·kg-1·h-1in group K and group K ＋U,normal saline was given in group C,group L and group U. After 4h,Pa O2 was measured at 0.5ml of blood in abdominal aorta,the concentration of TNF-α and IL-6 was measured at 2ml of blood in inferior vena cava. NF-κB was detected by electrophoretic mobility shift assay（EMSA） and IκB was detected by streptavidin biotin-peroxidase complex（SABC） in lung tissue specimen of rats. The correlation analysis of NF-κB with IκB,TNF-α and IL-6 were observed. Results Compared with group C,Pa O2 and the protein expression of IκB in lung tissue in other groups decreased significantly（P〈0.05）,the concentration of TNF-α and IL-6 and the activity of NF-κB in lung tissue in other groups increased significantly（P〈0.05）. Pa O2 and IκB in group K,group U and group K＋U increased significantly（P〈0.05）,TNF-α,IL-6 and NF-κB in group K,group U and group K＋U decreased significantly（P〈0.05）compared to group L. The multiple comparison among group K,group U and group K ＋U,Pa O2 and IκB in group K ＋U was the highest（P〈0.05）,TNF-α,IL-6 and NF-κB in group K＋U was the lowest（P〈0.05）. The relationship between NF-κB and TNF-α（r=0.945,P〈0.01） or IL-6（r=0.952,P〈0.01） was positive,NF-κB and IκB（r=-0.949,P〈0.01） showed negative correlation. Conclusion Ketamine combined with ulinastatin have protective effects against ＂double-hit＂ rat model of ALI induced by LPS through inhibiting the activity of NF-κB,attenuating the concentration of TNF-α and IL-6,increasing Pa O2 and the protein expression of IκB. The effect of combined application is better than separate application.