冷保存再灌注期间离体肝组织内氧自由基及[Ca^(2+)]_i 对 p38MAPK激活的影响

Effect of OFR and [Ca^(2+)]_i on the activation of p38 MAPK in isolated rabbit liver during cold preservation and reperfusion period

目的:了解离体肝脏缺血再灌注期间氧自由基及钙离子超载是否是激活 p38MAPK 的因素之一,为进一步揭示肝脏缺血再灌注损伤的信号转导机制打下基础.方法:通过自行建立的兔离体肝脏缺血再灌注模型,根据冷保存液中别嘌呤醇浓度的不同分为 A、B、C、D4组;根据冷保仔液中维拉帕米浓度的不同又分为 E、F、G、H4组;分别于离体前、冷保存末及再灌注5min、10min、60min、120min 获取离体肝组织,分别应用免疫映迹杂交(westem-blot)和免疫沉淀法测定磷酸化 p38MAPK 的表达及活性水平;并进行肝组织内氧自由基(oxygen free radicals,OFR) 含量的测定(A、B、C、D组);用 Fura-2/AM 负载法进行肝细胞内钙离子浓度测定(A、E、F、G 组).结果:于再灌注5min 各组离体肝组织的氧自由基水平升高至峰值,但各组两两之间存在显著性差异(A、D、C、D组:2.32±0.22,1.82±0.15,1.63±0.11,1.29±0.10,P<0.05,t=2.57);于再灌注10min 供肝组织 p38MAPK磷酸化水平及活性均升高至峰值,但各组两两之间存在显著性差异(A、B、C、D 组 p38MAPK 磷酸化水平:76.2±7.0,61.4±5.9,47.3±2.5,37.7±3.0,P<0.05,t=2.61;A、B、C、D 组 p38MAPK 活性水平:82.7±6.8,69.7±5.2, 54.5±5.5,41.2±3.1,P<0.05,t=2.61;A、E、F、G 组 p38MAPK 磷酸化水平:80.3±8.7,63.3±42,50.4±5.6,39.2±5.7,P<0.05,t=2.61;A、E、F、G 组p38MPK 活性水平:80.8±8.9,66.7±4.2,53.7±4.1,39.4±5.5,P<0.05,t=2.61);再灌注5min 时氧自由基及[Ca^(2+)]_i越高的离体肝,则再灌注10min 时离体肝组织p38活性峰值越高,二者之间呈显著性相关关系.(P<0.05,R_(OFR)=0.976,R_(Ca)=0.970)结论:别嘌呤醇能显著性抑制离体肝缺血再灌注期间肝组织内 OFR 水平,而维拉帕米能显著性抑制离体肝缺血再灌注期间肝细胞内钙离子浓度超载;而且 OFR 水平及钙离子与离体肝组织 p38MAPK 的激活密切相关.

AIM:To study the effect of OFR(oxygen-derieved free radicals)and calcium on the activation of p38 MAPK in isolated rabbit liver during cold preservation and reperfusion period. METHODS:Based on the cold preservation-reperfusion model of isolated rabbit liver,isolated livers were divided into 4 groups according to the concentration of allopurinol or verapamil in the preservation solution.Liver tissue samples were harvested at the time points of before cold preservation,at the end of cold preservation,and during reperfusion period(5 min,10 min,60 min,120 min).The phosphorylation level of p38MAPK and its activity in liver tissue were detected by Western-Blotting and immuno- precipitation respectively.The content of OFR and con- centration of intracellular calciumion([Ca^(2+)]i)were also measured by the corresponding method. RESULTS:The content of OFR was increased to its peak value at 5 min during reperfusion period,the difference among the four groups was significant(groups A,B,C,D: 2.32±0.22,1.82±0.15,1.63±0.11,1.29±0.10,P<0.05, t=2.57).At 10 min during reperfusion period,the phos- phorylation level and its activity of p38MAPK reached its peak value,and there was significant difference among the four groups(Groups A,B,C,D:p38MAPK phospho- rylation level(76.2±7.0,61.4±5.9,47.3±2.5,37.7±3.0,P<0.05, t=2.61.Groups A,B,C,D:p38MAPK activity)82.7±6.8, 69.7±5.2,54.5±5.5,41.2±3.1,P<0.05,t=2.61.Groups A, E,F,G:p38MAPK phosphorylation level(80.3±8.7,63.3±4.2, 50.4±5.6,39.2±5.7,P<0.05,t=2.61.Groups A,E,F,G: p38MAPK activity(80.8±8.9,66.7±4.2,53.7±4.1,39.4±5.5, P<0.05,t=2.61).The peak value of OFR content and [Ca^(2+)]i was significantly and positively correlated with the peak value of p38MAPK phosphorylation level and activity (P<0.05,R_(OFR)=0.976,R_(Ca)=0.970). CONCLUSION:Allopurinol can significantly inhibit the OFR content of isolated liver tissues while verapamil can signifi- cantly inhibit the overload of Ca^(2+)concentration of isolated liver cells.The OFR content and overload of intracellular calciumion concentration are closely related with p38MAPK activation during cold preservation and reperfusion period. So activation of p38MAPK signal pathway may be an im- portant mechanism of OFR and Ca(2+),which play a critical role in the ischemia-reperfusion injury of donor liver.