Protective effect of ischemic preconditioning on the grafted pancreas and its correlation with apoptosis in rats / 中国组织工程研究

ABSTRACT

BACKGROUND: During pancreas transplantation, ischemia/reperfusion (I/R) injury can lead to many complications, which directly threaten the survival of the donor pancreas and the receptor itself, and the serious ones may result in the failure of transplantation. Ischemic preconditioning can protect the target organs in the following ischemia, which has become one of the hot spots in investigating organ transplantation.OBJECTIVE: To observe the early protective effect of ischemic preconditioning on the I/R injury of the grafted pancreas in the rat, and analyze its correlation with apoptosis.DESIGN: A randomized control animal experiment.SETTINGS Department of Gastrointestinal Surgery, Department of Anesthesiology, Xijing Hospital of the Fourth Military Medical University of Chinese PLA.MATERIALS Seventy male SD rats of 3-6 months, weighing 250-320 g, were used.METHODS: The experiments were conducted in the laboratory of Department of Gastrointestinal Surgery between September 2001 and April 2004.Six normal rats were taken as the control group, and 24 successful diabetic models were divided into I/R group and 1, 2 and 3-time ischemic preconditioning groups (n=18) according to the method of random number table,with 6 rats in each. The rats in the latter three groups were treated with 5-minute ischemia and 5-minute reperfusion for once, twice and three times respectively, all the rats underwent the pancreas transplantation. Twentyfour SD rats served as donors.MAIN OUTCOME MEASURES: ① Blood glucose before and after reperfusion in each group; Serum contents of tumor necrosis factor alpha (TNF-α) and nitric oxide; Activities of superoxide dismutase (SOD) and myeloperoxidase (MPD) and content of malondialdehyde (MDA) in the grafted pancreatic tissue; ② Apoptosis in the grafted pancreatic tissue observed by means of in situ end-labeling; Expressions of Bax and Bcl-2 proteins in the grafted pancreatic tissue with the method of Western blotting.RESULTS: ① Changes of blood glucose before and after reperfusion The levels of blood glucose were decreased as compared with those before reperfusion in the I/R group and ischemic preconditioning groups. It was significantly lower in the 2-time ischemic preconditioning group than in the I/R group, 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ②Serum content of TNF-α at 2 hours after reperfusion It was lower in the ischemic preconditioning groups than in the I/R group; It was lower in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ③ Serum content of nitric oxide after reperfusion It was higher in the ischemic preconditioning groups than in the I/R group; It was higher in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ④SOD activity in the grafted pancreatic tissue after perfusion It was higher in the ischemic preconditioning groups than in the I/R group; It was higher in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ⑤ MAD content and MPD activity in the grafted pancreatic tissue after perfusion Those were lower in the ischemic preconditioning groups than in the I/R group, also lower in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups. ⑥ Apoptosis in the grafted pancreatic tissue The apoptosis index after perfusion was lower in the ischemic preconditioning groups than in the I/R group; It was significantly lower in the 2-time ischemic preconditioning group than in the 1 and 3-time ischemic preconditioning groups (P ＜ 0.05). ⑦ Expressions of Bax and Bcl-2proteins in the grafted pancreatic tissue There was high expression of Bax protein and low expression of Bcl-2 protein in the grafted pancreatic tissue after perfusion in the I/R group; Low expression of Bax protein and high expression of Bcl-2 protein in the grafted pancreatic tissue after perfusion were observed in the ischemic preconditioning groups; In the 2-time ischemic preconditioning group, the expression of Bcl-2 protein was the highest but that of Bax protein was the lowest.CONCLUSION: Ischemic preconditioning can protect the grafted pancreas from I/R injury at early pancreas transplantation, which maybe correlated with the elevation of SOD activity, increase of the synthesis of endogenous nitric oxide, down-regulation of TNF-α and the alleviations of the adhesion and aggregation of polymorphonuclear leukocytes. Ischemic preconditioning can reduce the apoptosis of the grafted pancreas, and the the possible mechanism may be correlated with the alleviations of the adhesion and aggregation of polymorphonuclear leukocytes, reduce of oxygen-derived free radicals, up-regulation of Bcl-2 protein and the down-regulation of Bax protein. 5-mintue ischemia and 5-minute reperfusion for twice is the best way to induce ischemic preconditioning in rat pancreas transplantation.