缺血预处理对大鼠移植胰缺血再灌注损伤的保护作用及其与细胞凋亡的相关性

Effect of ischemic preconditioning on ischemia reperfusion injury of graft after pancreas transplantation and correlations with apoptosis in rat

目的:探讨缺血预处理对大鼠移植胰缺血再灌注损伤的早期保护作用及其与细胞凋亡的相关性.方法:正常大鼠6只为对照组,糖尿病SD大鼠24只随机分为缺血再灌注组(I/R组,n=6)和缺血预处理组(IPC组,n=18),IPC组又根据不同方法分为3个亚组:IPC1组(缺血5min再灌注5min1次,n=6)、IPC2组(缺血5min再灌注5min2次,n=6)和IPC3组(缺血5min再灌注5min3次,n=6),I/R组和IPC组均行单纯胰腺移植,24只SD大鼠为供体;检测各组再灌注前、后血糖;再灌注后2h血清中TNF-α和NO的含量、移植胰组织中SOD,MPO和MDA含量;用TUNEL法观察移植胰组织细胞凋亡情况,WesternBlot法检测移植胰组织Bax和Bcl-2蛋白表达情况.结果:再灌注后IPC1(14.3±1.1vs12.1±0.9mmol/L.P<0.05)组、IPC2(12.1±0.9vs16.5±1.4mmol/L,P<0.01)和IPC3组(14.7±1.3vs12.1±0.9mmol/L,P<0.05)相对于I/R组血糖低;IPC2组较IPC1组(12.1±0.9vs14.3±1.1mmol/L,P<0.05)和IPC3组(12.1±0.9vs14.7±1.3mmol/L.P<0.05)血糖低.再灌注后IPC1(1.41±0.17vs1.79±0.25kU/L,P<0.05)组、IPC2(1.05±0.16vs1.79±0.25kU/L,P<0.01)和IPC3组(1.43±0.20vs1.79±0.25kU/L,P<0.05)较I/R组血清中TNF-α含量低;IPC2组较IPC1组(1.05±0.16vs1.41±0.17kU/L,P<0.05)和IPC3组(1.05±0.16vs1.43±0.20kU/L,P<0.05)TNF-α含量低.再灌注后IPC1(13.13±2.87vs8.91±1.23μg/L,P<0.05)组、IPC2(18.79±2.39vs8.91±1.23μg/L,p<0.01)和IPC3组(14.36±1.78vs8.91±1.23μg/L,P<0.05)较I/R组血清中NO含量高;IPC2组较IPC1组(18.79±2.39vs13.13±1.87μg/L,P<0.05)和IPC3组(18.79±2.39vs14.36±1.78μg/L,P<0.05)NO含量高.再灌注后IPC1(179.82±19.54vs153.47±17.67mU/g,P<0.05)组、IPC2(213.64±22.97vc153.47±17.67mU/g,P<0.01)和IPC3组(181.68±20.32vs153.47±17.67mU/g,P<0.05)较I/R.组移植胰组织中SOD活性高;IPC2组较IPC1组(213.64±22.97vs179.82±19.54mU/g,P<0.05)和IPC3组(213.64±22.97vs181.68±20,32mU/g.P<0.05)SOD活性高.再灌注后IPC1(0.70±0.26vs0.87±0.31mmol/g,P<0.05)组、IPC2(0.46±0.18vs0.87±0.31mmol/g,P<0.01)和IPC3组(0.67±0.15vs0.87±0.31mmol/g,P<0.05)较I/R组移植胰组织中MDA含量低;IPC2纽较IPC1组(0.46±0.18vs0.70±0.26mmol/g,P<0.05)和IPC3组(0.46±0.18vs10.67±0.15mmol/g,P<0.05)MDA含量低.再灌注后IPC1(0.81±0.23vs0.96±0.34A/g,P<0.05)组、IPC2(0.51±0.16vs0.96±0.34A/g,P<0.01)和IPC3组(0.78±0.22vs0.96±0.34A/g,P<0.05)较I/R.组移植胰组织中MPO活性低;IPC2组较IPC1组(0.51±0.16vs0.81±0.23A/g,P<0.05)和IPC3组(0.51+0.16vs0.78+0.22A/g,P<0.05)MPO活性低.再灌注后IPC1(25.21±3.47vs35.65±4.78%,P<0.01)组、IPC2(15.47±2.09vs35.65±4.78%,P<0.01)和IPC3组(24.89±3.56vs35.65±3.78%,P<0.01)较I/K组移植胰组织中AI值低;IPC2组较IPC1组(15.47±2.09vs25.21±3.47%,P<0.05)和IPC3组(15.47±2.09vs24.89±3.56%,P<0.05)AI值低.再灌注后I/R组胰组织Bax蛋白高表达,Bcl-2蛋白低表达,IPC各组再灌注后移植胰组织Bax蛋白低表达,Bcl-2蛋白高表达,而IPC2组Bcl-2蛋白表达最高Bax蛋白表达最低.结论:缺血预处理对大鼠移植胰的缺血再灌注损伤具有早期保护作用,可能于提高SOD的活性、增加内源性NO的合成、下调TNF-α和减轻PMNs黏附与聚集有关;缺血预处理可以减少移植胰缺血再灌注后的细胞凋亡,可能于减轻PMNs黏附与聚集、减少氧自由基、上调Bcl-2蛋白和下调Bax蛋白有关;缺血5min再灌注5min2次是最佳的大鼠移植胰缺血预处理诱导办法.

AIM: To investigate the effect of ischemic preconditioning on ischemia reperfusion injury of the pancreas graft in rat, and to analyze the possible mechanism. METHODS: Six normal SD rats in the control group received sham operation. Twenty-four SD rats with steptozozin-in-duced diabetes were randomly assigned to 2 groups: Group I/R consisted of 6 diabetic rats which received pancreas transplantation; Group IPC consisted of 18 diabetic rats which received pancreas transplantation and were exposed to 5 min ischemia and 5 min reperfusion once (IPC1, n = 6), twice (IPC2, n = 6) or thrice (IPC3, n = 6) before ablating donors. Blood glucose, serum NO and TNF-α, MDA, SOD, MPO, TUNEL cells, and the expression of Bcl-2 and Bax pretien (Western Blot) in graft were monitored. RESULTS: The mean blood glucose levels in Group IPC1 (14.3±1.1 vs 12.1±0.9 mmol/L,P<0.05), Group IPC2(12.1±0.9 vs 16.5±1.4 mmol/L, P<0.01) and Group IPC3(14.7±1.3 vs 12.1±0.9 mmol/L, P<0.05) were lower than that in Group I/R, and the glucose level in Group IPC2 was lower than those in IPC1(12.1±0.9 vs 14.3±1.1 mmol/L, P<0.05)and IPC3 (12.1±0.9 vs 14.7±1.3 mmol/L, P<0.05) 2 hours after reperfusion. The mean NO (13.13±2.87 vs 8.91±1.23 μg/L, P<0.05,18.79±2.39 vs 8.91±1.23 μg/L, P<0.01,14.36±1.78 vs 8.91 ±1.23 μg/L, P<0.05) and SOD (179.82±19.54 vs 153.47±17.67 mU/g, P<0.05,213.64±22.97 vs 153.47±17.67 mU/g, P<0.01,181.68±20.32 vs 153.47±17.67 mU/g,P<0.05) levels in Group IPC1, IPC2and IPC3were higher than that in Group I/R, and the levels in Group IPC2 were higher than those in Group IPC1 and Group IPC3 2 hours after reperfusion (18.79±2.39 vs 13.13±2.87 μg/L, 18.79±2.39 vs 14.36±1.78 μg/L, 213.64±22.97 vs 179.82±19.54 mU/g, 213.64±22.97 vs 181.68±20.32 mU/g, P<0.05). The mean levels of TNF-α(1.41±0.17 vs 1.79±0.25 kU/L, P<0.05, 1.05±0.16 vs 1.79±0.25 kU/L,P<0.01, 1.43±0.20 vs 1.79±0.25 kU/L,P<0.05, MDA (0.70±0.26 vs 0.87±0.31 mmol/g, P<0.050.46±0.18 vs0.87±0.31 mmol/g,P<0.01; 0.67±0.15 vs 0.87±0.31 mmol/g,P<0.05) and MPO (0.81± 0.23 vs 0.96±0.34 A/g, P<0.05, 0.51±0.16 vs0.96±0.34 A/g,P<0.01,0.78±0.22 vs 0.96±0.34 A/g,P<0.05) in Group IPC1, IPC2 and IPC3 were lower than those in Group I/R, and those in Group IPC2 were lower than in IPC1 and IPC3 2 hours after reperfusion (1.05±0.16 vs 1.41±0.17 kU/L, 1.05±0.16 vs 1.43±0.20 kU/L, 0.46±0.18 vs 0.70±0.26 mmol/g, 0.46±0.18 vs 10.67±0.15 mmol/g, 0.51±0.16 vs 0.81±0.23 A/g, 0.51±0.16 vs 0.78±0.22 A/g,P<0.05). The apoptotic indexes in IPC1(25.21±3.47 vs 35.65±4.78 %, P<0.01), IPC2 (15.47±2.09 vs 35.65±4.78 %,P<0.01) and IPC3 (24.89±3.56 vs 35.65±4.78 %,P<0.01) were lower than Group I/R, and that in Group IPC2 was lower than IPC1 (15.47±2.09 vs 25.21±3.47%,P<0.01) and IPC3 (15.47±2.09 vs 24.89±3.56%,P<0.01) 2 hours after reperfusion. The expression of the Bax protein in Group I/R was higher than that in Group IPC, while that in Group IPC2 was the lowest. The expression of Bcl-2 protein in Group IPC was higher than in Group I/R, and that in Group IPC2 was the highest. The expression of the Bax protein in Group IPC was lower than Group I/R, and that in Group IPC2 was the lowest. CONCLUSION: Ischemic preconditioning can protect pan- creas graft from I/R injury. The possible mechanism may be related to the increased production of serum NO and tissue SOD, reduced conglutination and aggregation of PMNs in pancreas and diminished synthesis TNF-α. Is-chemic preconditioning can reduce apopotosis of the graft, which may be resulted from alleviated conglutination and aggregation of PMNs, increased oxygen radical, increased Bcl-2 protein and reduced P53 protein expression. Five min ischemia and five min reperfusion twice is the best way to induce ischemic preconditioning in rat pancreas transplantation.