缺血后处理对肾缺血再灌注损伤及HO-1的影响

1、缺血后处理对肾缺血再灌注损伤及HO-1的影响刘先义摘 要目的：观察大鼠肾缺血再灌注后肾脏结构和功能的变化，以及肾组织中血红素氧合酶-1（heme oxygenase-1，HO-1）的表达情况；研究在体情况下，缺血后处理对肾缺血再灌注损伤及血红素氧合酶-1的影响，并探讨其可能机制。方法：健康SD大鼠32只,随机分为4组。组（n=8）为假手术组；组（n=8）为对照组，建立肾I/R模型；（n=8）、（n=8）组为实验组（即后处理组），先建立缺血模型，然后于缺血后再灌注前，行反复多次的、短暂灌注-停灌注。（1）组仅分离肾动、静脉，不阻断血供，45min后缝合切口；（2）组用无创动脉夹同时夹闭双侧肾动、

2、静脉45min，然后恢复灌注；（3）组用无创动脉夹同时夹闭双侧肾动、静脉45min，然后进行10s预灌注-10s停灌注，反复3次，最后恢复灌流；（4）组用无创动脉夹同时夹闭双侧肾动、静脉45min，然后进行2min预灌注-2min停灌注，反复3次，最后恢复灌流。自动生化仪检测血清尿素氮(BUN)、肌酐(Scr)，比色法检测血丙二醛(Malondialdehyde， MDA) 的含量、超氧化物岐化酶(superoxide dismutase ，SOD)活性，光镜下观察肾组织结构学，免疫组化检测肾组织HO-1表达水平，并进行图像分析。结果：1. 肾功能改变：缺血前各组大鼠肾功能均正常。再灌注24

3、h后，对照组(Scr=102.70±18.30µmol/L，BUN=15.80±5.88 mmol/L)、实验组Scr，BUN(: Scr=70.53±16.51µmol/L， BUN=8.77±3.82 mmol/L； : Scr=70.14±11.88µmol/L，BUN=9.55±3.57 mmol/L)值均显著升高，两组间差异有统计学意义；而实验组Scr、BUN值虽比正常组高，而比对照组明显降低，差异均有统计学意义(Scr：P<0.01；BUN：P<0.05)。2. MDA，SOD 水

4、平: 缺血再灌注后对照组MDA含量(10.60±3.30 nmol/ml)增高，SOD活性(71.78±22.32 U/ml)降低，与假手术组(MDA=3.82±1.77nmol/ml, SOD=152.17±27.12 U/ml)比较差异有统计学意义。与对照组相比，实验组MDA含量(:MDA=6.41±2.21 nmol/ml，: MDA=6.69±2.37nmol/ml)则明显降低，SOD活性(: SOD=115.87±18.63 U/ml, : SOD=117.11±20.81U/ml)增高，均有统计学差异(

5、MDA，P<0.05；SOD，P<0.01)。3. HO-1在肾小管的表达(免疫组化，×400)：HO-1在假手术组无明显表达(灰度值205.00±15.21）；对照组表达增强(156.90±36.26)；与对照组相比，实验组HO-1表达明显增强（:117.59±34.35, : 120.49±34.26,P < 0.05）。4. 肾组织形态学改变（HE染色）：假手术组肾小球、肾小管结构正常，肾组织未见明显的形态学改变；对照组肾小管上皮细胞多呈空泡变性和坏死，肾小管内见大量管型和坏死脱落细胞；实验组肾小管细胞水肿，仅少部分肾小

6、管上皮细胞坏死脱落，病理改变明显轻于对照组。结论：缺血后处理能增强HO-1表达，减少氧自由基（OFRs）的释放，刺激胞内抗氧化酶和自由基清除剂的释放,对大鼠肾缺血再灌注具有保护作用。关键词：肾，缺血再灌注损伤，自由基，血红素氧合酶-1，缺血后处理AbstractObjective To observe the structural and functional changes and expression of heme oxygenase-1 in kidney after ischemic reperfusion in rats. To investigate the effect of

7、postconditioning on renal ischemic reperfusion injury and expression of heme oxygenase-1 in kidney after ischemic postconditioning in rats .Study the characteristic and mechanism of renal injury after postconditioning in rats.Methods Methods Thirty-two healthy SD rats weighing 250±30g were divi

8、ded into four groups with eight animals in each group at random: sham-operation group without any condition but dissociating the renal blood vessel ,control group with 24 hours of reperfusing after 45 minutes of ischemia in two kidneys in which group the rat acute renal ischemia-reperfusion injury m

9、odel was established and two expremental groups with postconditioning.The experimental group 1 was induced by three cycles of 10 seconds reperfusion interspersed by 10 seconds of no-flow ischemia and the experimental group 2 was induced by three cycles of 2 minutes reperfusion interspersed by 2 minu

10、tes of no-flow ischemia after 45 minutes of ischemia in two kidneys.Blood was collected before operation and after 24 hours of reperfusion . The left kidney was collected after 24 hours of reperfusion. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD),blood urea nit

11、rogen (BUN) ,serum creatinine (Scr) in blood serum were examined. The structure of the kidney was observed under light microscopy. The expression of HO-1 was detected by immunohistochemistry technique and was evaluated by image pattern analysis system.Results Twenty-four hours after reperfusion , th

12、e blood serum levels of BUN (5.54±2.18mmol), Scr(36.64±11.57µmol/L) and content of MDA(3.28±1.77nmol/ml)in sham-operation group were significantly lower than those (BUN=15.80 ±5.88 mmol/ml, Scr=102.70±18.30µmol/L,MDA=10.60±3.30 nmol/ml) in control group.The ac

13、tivity of SOD in sham-operation group(152.17±27.12 U/ml) was more higher than that in control group (71.78±22.32 U/ml).The structure was changed badly in control group .The expressing of HO-1 in sham-operation group was much less than that in control group.Compared with control group , the

14、 blood serum levels of BUN (8.77±3.82 mmol/L), Scr(70.53±16.51µmol/L) and content of MDA(6.41±2.21nmol/ml) in exprimental group 1 and thoese(BUN=9.55±3.57 mmol/L,Scr=70.14±11.88µmol/L, MDA=6.69±2.37 nmol/ml) in exprimental group 2 were significantly lower. The

15、 activity of SOD in exprimental group 1(115.87±18.63 U/ml) and in exprimental group 2(117.11±20.81U/ml) were higher. The structure was changed less serious in exprimental group .The expressing levels of HO-1 protein was significantly higher in the exprimental groups than in the control group. Conclusio