异丙肾上腺素预处理对大鼠胰腺移植后缺血再灌注损伤的抑制作用

Preconditioning of pancreatic graft with isoproterenol reduces posttransplant ischemia/reperfusion injury in rats

目的:探讨减轻大鼠胰腺移植后缺血再灌注损伤的保护作用机制.方法:IPC后动态检测大鼠胰腺组织热休克蛋白表达.建立大鼠胰腺移植缺血再灌注模型,选择表达热休克蛋白高峰时段供体大鼠胰腺移植作为实验组,未预处理供体大鼠胰腺移植作为对照组.移植后6h,采集静脉血及移植胰腺.热休克蛋白70(HSP70)分别用Westernblot法及免疫组织化学法检测.免疫组织化学法测定肿瘤坏死因子-α(TNF-α)表达.流式细胞仪检测胰腺细胞凋亡率.碘淀粉比色法检测血淀粉酶水平.结果:IPC后供体大鼠胰腺中HSP70的表达在24h达到高峰,与其他各时段比较具有显著差异(0.92±0.25vs0.24±0.04,0.34±0.06,0.58±0.07,0.62±0.11,0.25±0.09,均P<0.05),IPC后6h,12h,24h,36h大鼠胰腺中HSP70的表达与未预处理组相应时段比较差异也显著(0.34±0.06vs0.28±0.07,0.58±0.07vs0.25±0.04,0.92±0.25vs0.27±0.05,0.62±0.11vs0.25±0.06,均P<0.05),48h恢复到原来水平.而未预处理组各时段间比较差异无统计学意义(P>0.05).HSP70主要表达于胰腺腺泡细胞及血管壁.对照组胰腺组织中TNF-α、细胞凋亡率、中性粒细胞、血淀粉酶的水平明显升高,与假手术组相比差异显著(均P<0.01).实验组降低了胰腺组织中TNF-α、细胞凋亡率、白细胞数、血淀粉酶的水平,与对照组比较差异具有统计学意义(11929±1220vs46111±3127,26.7%±4.5%vs37.4%±4.7%,3308±531vs6668±1506,1057IU/L±148IU/Lvs1408IU/L±195IU/L,均P<0.05).结论:IPC减轻了大鼠胰腺移植后缺血再灌注损伤,IPC保护作用与HSP70的诱导生成有关.

AIM： To determine the protective effects of isoproterenol preconditioning （IPC） against ischemia/reperfusion injury in rats after pancreas transplantation and to explore mechanisms involved. METHODS： The expression of heat shock protein 70 （HSP70） in the pancreas of rats undergoing IPC was detected at different time points after IPC. A rat model of posttransplant pancreatic ischemia/reperfusion injury was established.The donor rats that showed high expression of HSP70 in the pancreas were used as experiment group, while donor rats that did not undergo IPC were used as control group. The blood and pancreatic samples were taken 6 h after pancreas transplantation. The expression of HSP70 in the pancreas was detected by Western blot and immunohistochemistry. The expression of TNF-α in the pancreas was detected by immunohistochemistry. Serum amylase was determined by iodine colorimetry. The apoptosis rate of pancreatic cells was determined by flow cytometry. RESULTS： The expression level of HSP70 in the pancreas of donor rats reached the peak at 24 h after IPC, which was significantly higher than those at other time points （0.92 ± 0.25 vs 0.24 ± 0.04, 0.34 ± 0.06, 0.58 ± 0.07, 0.62 ± 0.11 and 0.25 ± 0.09, respectively; all P 0.05）. The expression levels of HSP70 in the experimental group at 6, 12, 24 and 36 h after IPC were significantly higher than those in the control group at corresponding time points （0.34 ± 0.06 vs 0.28 ± 0.07, 0.58 ± 0.07 vs 0.25 ± 0.04, 0.92 ± 0.25 vs 0.27 ± 0.05 and 0.62 ± 0.11 vs 0.25 ± 0.06, respectively; all P 0.05） but returned to normal level at 48 h. No significant differences were noted in the expression levels of HSP70 among each time point in the control group. HSP70 was mainly expressed in pancreatic acinar cells and the vessel wall. The expression level of TNF-α, apoptosis rate, neutrophil count and serum amylase significantly increased in the control group when compared with those in sham-operated group （all P 0.01）. However, the levels of these parameters significantly decreased in the experiment group when compared with those in the control group （11 929 ± 1 220 vs 46 111 ± 3 127, 26.7% ± 4.5% vs 37.4% ± 4.7%, 3 308 ± 531 vs 6 668 ± 1 506 and 1 057 IU/L ± 148 IU/L vs 1 408 IU/L± 195 IU/L, respectively; all P 0.05）. CONCLUSION： Isoproterenol preconditioning reduces ischemia/reperfusion injury in rats after pancreas transplantation perhaps by inducing the production of HSP70.