吡咯二硫氨基甲酸酯预处理对大鼠移植肺缺血再灌注损伤的保护作用

Influence of Pyrrolidine Dithiocarbamate Pretreatment on Function of the Transplanted Rat Lung

目的：吡咯二硫氨基甲酸酯（pyrrolidine dithiocarbamate,PDTC）通过阻断NF-κB下游炎性因子的释放,从分子水平、组织水平和移植肺功能评估等方面来考察其在大鼠移植肺缺血再灌注损伤中是否具有保护作用。方法：SD雄性大鼠30对,每只质量200~300g。分为两组（对照组和PDTC干预组）。每个组又按供肺的保存时间分为0h、2h、6h、8h和12h共5个亚组。每个亚组行6次成功的肺移植实验,收集实验标本。对照组供肺保存于LPD液中,实验组的保存液中加入逆转录聚合酶连反应PDTC（Sigma公司,40mmol.L-1）。移植术后30min,用Western blot方法检测两组供肺P50表达水平,凝胶迁移试验（EMSA）方法检测核转录因子κB（NF-κB）入核的变化。检测其下游因子肿瘤坏死因子-α（TNF-α）和一氧化氮合成酶（iN-OS）的表达水平。同时比较两组气道压力、血气分析氧合指数、湿干重比、HE染色、透射电镜等指标。结果：PDTC干预组和对照组相比,P50的表达无显著差异（P=0.276）。PDTC干预组和对照组相比,NF-κB入核明显的减少,其差异有统计学意义（P〈0.01）;,TNF-α和iNOS表达有显著差异（均P〈0.01）。PDTC干预组与对照组相比较,移植肺的NF-κB入核减少和下游炎症因子TNF-αi、NOS表达上的变化密切相关（TNF-α：r=0.636,P〈0.01;iNOS：r=0.633,P〈0.01）。PDTC干预组和对照组相比,肺移植后30min血气分析氧合指数（P=0.042）、湿干重比（P=0.001）、移植肺的肺损伤评分（P=0.017）,均有显著差异。结论：PDTC预处理能显著降低移植肺细胞因子水平,并进一步改善移植肺功能。其机制可能通过阻断NF-κB的入核来实现的,而非下调NF-κB的表达量。

Objective：Pyrrolidine dithiocarbamate（PDTC） which is the specific blocker of NF-κB were added into preservation solution to observe the possible protective effect of transplanted lung.Methods：Sixty male Sprague-Dawley rats,weigh ranging between 200-300g were randomized into two groups（PDTC group and control group）.Each group contained 5 sub-groups according to its preservation time（0,2,6,8,12 hours）.Six success lung transplantations were carried out in every subgroup.PDTC were added in the research group（Sigma,40mmol/L）.Thirty minutes after the rat lung transplantations,the expression of P50 were detected by western blot,and DNA binding activity of nuclear factor-kappa B（NF-кB）.Expression of inductible nitric oxide synthase（iNOS） and tumor necrosis factor alpha（TNF-α） were detected.In the aspect of lung function,airway pressure,oxygen indexes,wet/dry ratio,manifestation of lung injury on HE staining and transmission electron microscopy were compared between the two groups.Results：No significant difference in expression of P50 were yielded between the PDCT group and control group（P=0.276）.DNA binding activity of NF-кB of PDTC group reduced dramatically,compared with corresponding control groups（P0.01）as well as the expression of TNF-α（P0.01）and iNOS（P0.01）decreased dramatically,compared with corresponding control groups.In PDTC and control groups,Correlation analysis between decreasing DNA binding activity of NF-кB and down regulation of downstream inflammation signal factors TNF-α and iNOS showed significant difference（TNF-α： r=0.636,P0.01;iNOS： r=0.633,P0.01））.As to lung function after transplantation,in PDTC groups,the oxygen indexes increased（P=0.042）,wet/dry ratio deceased（P=0.001）,as well as lung injury assessment（P=0.017）,compared with corresponding control groups.Conclusions： PDTC pretreatment could down regulate the downstream inflammation factors dramatically and improve the function of transplanted rat lung.The declined expression of inflammation factors was close correlation with the improvement of transplant lung function.The process was approached by decreasing the DNA binding activity of NF-кB,not by down regulate NF-кB expression.