肺组织NF-κB和PUMA与SAP-ALI的关系及PDTC的干扰作用

Relationships of nuclear factor-κB and PUMA with lung injury in rats with severe acute pancreatitis and therapeutic effect of proline dithiocarbamate

目的探讨NF-κB(核因子-κB)和PUMA(p53正向凋亡调节因子)在大鼠重症急性胰腺炎致急性肺损伤(SAP-ALI)发病中的作用及脯氨酸二硫代氨基甲酸酯(PDTC)对此过程的影响。方法 SD大鼠随机分为假手术组(A组),SAP-ALI组(B组),SAP+PDTC干预组(C组),每组24只。各组再按6,12,24 h时点分为3个亚组,每亚组8只。A组开腹后翻动胰腺数次;B组采用胰胆管逆行注入5%牛磺胆酸钠(1 mL/kg)诱导SAP-ALI模型;C组在B组的基础上于术前1 h给予PDTC(15 mg/kg),各组按各时点处死大鼠。观察胰腺和肺脏病理变化。检测不同组肺脏组织中NF-κBp65,PUMA和Caspase-3的表达及Caspase-3活性;检测组织TNF-α,MIP-2和ICAM-1 mRNA的表达、髓过氧化物酶(MPO)的活性和肺泡上皮细胞凋亡指数。结果成功建立大鼠SAP-ALI模型。Western-blotting和RT-PCR结果显示,B组肺上皮细胞NF-κB p65,PUMA和Caspase-3蛋白表达在12 h后显著增加(P<0.05),NF-κB p65与PUMA呈高度正相关(r=0.987,P<0.01)。TNF-α,MIP-2,ICAM-1mRNA表达及MPO和Caspase-3活性明显增加(P<0.01)。C组肺损伤病理组织学评分在术后各时点较B组显著下降(P<0.05),C组12 h后的肺组织NF-κB p65,PUMA和Caspase-3蛋白表达及MPO和Caspase-3活性明显降低(P<0.05);TNF-α,MIP-2,ICAM-1 mRNA表达明显下降(P<0.05),C组细胞凋亡指数较B组明显降低(P<0.01)。结论大鼠SAP-ALI既与NF-κB活化引起的炎症因子释放有关又与NF-κB活化引起的PUMA上调促进细胞凋亡有关。PDTC通过抑制NF-κB活化,下调炎症因子释放及NF-κB活化引起的PUMA表达可抑制肺组织的细胞凋亡,从而减轻SAP-ALI的程度。

Objective To investigate the expression of NF-κB（nuclear factor-κB） and PUMA（p53 upregulated modulator of apoptosis）in severe pancreatitis-acute lung injury（SAP-ALI） and the effect of proline dithiocarbamate（PDTC） therapy.Methods SD rats weighing 200-250g were randomly divided into three groups： Sham operation group（A group,n=24）,SAP-ALI group（B group,n=24）,SAP＋PDTC treatment group（C group,n=24）.The model of SAP was established by injecting 1ml/kg of 5% sodium taurocholate into the pancreatic capsule of the rats in B group.On the basis of B group,C group was treated by PDTC at 1h after modeling.Eight rats of each group were killed in the following 6,12 and 24 h.The histopathologic changes in lung and pancreas were observed.The expressions of NF-κB,PUMA and caspase-3 in the lungs were detected by Western blotting,the expressions of TNF-α、MIP-2 and ICAM-1 mRNA in the lungs were detected by RT-PCR.The pulmonary injury was determined by myeloperoxidase（MPO） activity and TUNEL detection.Results Pathologic scores of lung in B group（6-24 h）were significantly higher than those in A and C group（P0.05）.The expressions of NF-κB p65,PUMA,Caspase-3 and TNF-α,MIP-2,ICAM-1 mRNA in B group in differant time point（12,24 h） were all higher than those in A and C group（P0.05）.NF-κB was correlated strongly with PUMA（r=0.987,P0.01）.Higher activity of MPO and caspase-3 active were seen in B group than that in A and B group.DNA ladder was easily seen in group B.Conclusions PDTC treatment can inhibit apoptosis of alveolar epithelial cells in SAP-ALI by inhibiting the activation of NF-κB,by which the PUMA was down-regulated,the bcl-2 was up-regulated and caspase-3 was down-regulated.On the other hand,PDTC can inhibit the activation of NF-κB,which in turn decreases the lung injury by inhibiion of the cytokine and inflammation medium