缺血预处理抑制大鼠胰腺移植缺血再灌注胰腺细胞凋亡:活性氧与线粒体DNA修复酶的作用

Ischemic precondition inhibits pancreatic acinar cells apoptosis in rats with ischemia/reperfusion injury following pancreas transplantation:Role of reactive oxygen and mitochondrial DNA repair enzyme

背景:缺血预处理可诱发机体内源性保护机制,可全面有效地防治器官移植缺血再灌注损伤。在胰腺移植过程中冷、热缺血均可导致移植胰腺缺血再灌注损伤,线粒体结构及功能与胰腺病变密切相关,近些年研究发现,线粒体DNA存在修复体系,其与线粒体DNA损伤之间的平衡决定了疾病的发生和转归。目的:观察缺血预处理对大鼠胰腺移植缺血再灌注损伤时的细胞凋亡的影响,分析线粒体DNA修复酶8-氧鸟嘌呤DNA糖基化酶和氧化应激在其中的变化规律及可能途径。方法:纳入健康雄性SD大鼠50只,其中20只为供体,10只为假手术组,另20只糖尿病造模后分为缺血再灌注组和缺血预处理组,每组10只。假手术组只行开、关腹手术,缺血再灌注组和缺血预处理组行异位全胰十二指肠移植。缺血再灌注组对应供体大鼠于获取供胰前以4℃UW液灌洗20min;缺血预处理组对应供体大鼠于获取供胰前阻断腹主动脉5min,再灌注5min,共2次。供胰均控制热缺血时间为15min,冷缺血时间为180min。再灌注后12h检测血浆淀粉酶活性、血糖浓度及Caspase-3,9活化水平,流式细胞法检测腺泡细胞凋亡率,罗丹明123法检测线粒体膜电位,二氯荧光素法检测线粒体过氧化氢产生速率,高效液相色谱法检测线粒体DNA中8-氧鸟嘌呤质量浓度,荧光定量聚合酶链反应法检测8-氧鸟嘌呤DNA糖基化酶mRNA的表达,Western-blotting法检测细胞色素C释放、磷酸化Akt及线粒体8-氧鸟嘌呤DNA糖基化酶蛋白表达水平。结果与结论:缺血预处理可降低线粒体氧化应激,提高Akt磷酸化水平,从而上调8-氧鸟嘌呤DNA糖基化酶表达,减少线粒体DNA氧化损伤,抑制腺泡细胞凋亡,减轻移植胰缺血再灌注损伤。

BACKGROUND：Ischemic preconditioning（IPC）can induce endogenous protection mechanism,which effectively prevent ischemia/reperfusion injury following organ transplantation.Cold and warm ischemia may induce ischemia/reperfusion injury of pancreas transplantation,and apoptosis of pancreatic acinar cells is one of the important reasons of pancreas graft functional defect after transplantation.Mitochondrial DNA has repair system,and its balance with mitochondrial DNA injury influences disease occurrence and outcome.OBJECTIVE：To observe the effect of IPC on apoptosis of transplanted pancreatic acinar cells,and the possible role of reactive oxygen（ROS）and mitochondrial DNA repair enzyme.METHODS：A total of 50 health,male,Sprague-Dawley rats were randomly divided into three groups：sham operated（n = 10）,donors（n = 20）and recipients（n = 20）.The recipients were randomly divided into ischemia/reperfusion group（IR,n = 10）and IPC group（n = 10）.The sham operated group was subjected to abdominal open and close operation.IR group and IPC group received establishment of diabetic model by streptozotocin injection.IR rats received whole pancreatic-duodenal transplantation alone.IPC rats received whole pancreatic-duodenal transplantation exposed ischemic preconditioning with 5 minutes ischemia and 5 minutes reperfusion twice.All grafts were keep with warm ischemia time 15 minutes and cold ischemia（in 4 ℃ UW preservation solution）time 180 minutes.Twelve hours after reperfusion,serum amylase,blood glucose,Caspase-3,-9 activity were detected.Pancreatic acinar cell apoptosis was measured by flow cytometry.Mitochondrial cross-membrane potential（△Ψ）was measured by monitoring the fluorescence spectrum of rhodamine 123.Mitochondrial H2O2 generation was determined using dichlorofluorescein as a probe.8-oxodG in mitochondrial DNA（mtDNA）was measured with HPLC system.Release of cytochrome C,phosphorylation of Akt and mitochondrial OGG1 protein expression were determined by Western-blotting.RESULTS AND CONCLUSION：The ischemia preconditioning can relieve the pancreatic acinar cell apoptosis in pancreas graft and relieve IR injury by decreasing mitochondrial oxidative stress,mtDNA injury,and increasing phosphorylation of Akt and mitochondrial OGG1 expression.