| Study one Effects of ischemic postconditioning on ischemic reperfusion injury and apoptosis of the graft after pancreas transplantation in ratObjective: To evaluate the effects of ischemic postconditioning on ischemic reperfusion injury and apoptosis of the graft after pancreas transplantation in the rat, and analyze the possible mechanism. Methods: Group Sham which consist 6 normol SD rats suffered with sham operation, 24 steptozozin-induced diabetic SD rats were randomly assigned to 2 groups: group I/R consisted of 6 diabetic rats which received pancreas transplantation normally, without additional intervention, Group IPO consisted of 18 diabetic rats received pancreas transplantation exposed IPO with 30-second reperfusion followed by 30-second reocclusion for once(IPO1, n=6), thrice (IPO2, n=6), six times(IPO3, n=6) at the onset of reperfusion after cold ischemia. The blood glucose, NO and TNF-αin serum, MDA, SOD, MPO, TUNEL cells, the expression of Bcl-2 and Bax protein (Western Blot) in graft were monitored at 2 hours after long-time reperfusion. Results: 1. The mean blood glucose level in Group IPO was lower than Group I/R (P<0.05, P<0.01, P<0.05), and that in Group IPO2 was lower than IPO1 and IPO3 2 hours after reperfusion (P<0.05, P<0.05). 2. The mean NO(P<0.01, P<0.01, P<0.01) and SOD(P<0.01, P<0.01, P<0.01) level in Group IPO was higher than Group I/R, that in Group IPO2 was higher than IPO1 and IPO3 2 hours after reperfusion(P<0.05, P<0.05). 3. The mean TNF-α(P<0.01, P<0.01, P<0.01), MDA(P<0.01, P<0.01, P<0.01) and MPO(P<0.01, P<0.01, P<0.01) level in Group IPO was lower than Group I/R, that in Group IPO2 was lower than IPO1 and IPO3 2 hours after reperfusion(P<0.05, P<0.05). 4. The apoptotic index in Group IPO was lower than Group I/R(P<0.01, P<0.01, P<0.01), and that in Group IPO2 was lower than IPO1 and IPO3 at 2 hours after reperfusion(P<0.05, P<0.05), the expression of the Bax protein in Group I/R was higher than Group IPO and that in Group IPO2 was lowest. 5. The expression of the Bcl-2 protein in Group IPO was higher than Group I/R, and that in Group IPO2 was highest, the expression of the Bax protein in Group IPO was lower than Group I/R, and that in Group IPO2 was lowest. Conclusions: 1. Ischemic postconditioning can protect graft from I/R injury during pancreas transplantation, which may achieve through increasing production of serum NO and tissular SOD, relieving conglutination and aggregation of PMNs in pancreas and restraining synthesis of TNF-α. 2. Ischemic postconditioning can reduce apoptosis of the graft, which may achieve through relieving conglutination and aggregation of PMNs, reducing oxygen radical and regulating expressions of Bcl-2 and Bax protein. 3. 30s ischemic and 30s reperfusion twice is the best way to induce ischemic preconditioning in rat pancreas transplantation. Study twoComparative study of the protective effects on ischemic reperfusion injury and apoptosis of the graft after pancreas transplantation among IPO, IPC and IPC-IPO in ratsObjectives: To study comparatively the protective mechanisms on ischemic reperfusion injury and apoptosis of the graft after pancreas transplant- tation between IPO and IPC and to evaluate whether the protective efects of IPO combined IPC (IPC-IPO) is additive of IPO and IPC in rat. Methods: Group control which consist 6 normol SD rats suffered with sham operation, 24 steptozozin- induced diabetic SD rats were randomly assigned to 4 groups: group I/R consisted of 6 diabetic rats which received pancreas transplantation normally, without additional intervention, Group IPC consisted of 6 diabetic rats received pancreas transplantation exposed IPC with 5min-occlusion followed by 5min- reperfusion for three times prior to cold ischemic reperfusion injury. (IPC, n=6), Group IPO consisted of 6 diabetic rats received pancreas transplantation exposed IPO with 30-second reperfusion followed by 30-second reocclusion for three times at the onset of reperfusion after cold ischemia. (IPO, n=6), Group IPC-IPO consisted of 6 diabetic rats received pancreas transplantation exposed IPC with 5min-occlusion followed by 5min-reperfusion for three times prior to cold ischemic reperfusion injury and 30-second reperfusion followed by 30-second reocclusion for three times at the onset of reperfusion after cold ischemia.The blood glucose, NO and TNF-ɑin serum, MDA, SOD, MPO, TUNEL cells, the expression of Bcl-2 and Bax protien (Western Blot) in graft were monitored at 2 hours after long-time reperfusion. Results: 1. The mean blood glucose level in Group IPO, IPC, IPC-IPO and I/R was lower than Sham group (P<0.01, P<0.01, P<0.01, P<0.01), that in Group IPO, IPC, IPC-IPO was lower than I/R group (P<0.01, P<0.01, P<0.01), and that in Group IPC, IPO and IPC-IPO were comparable at 2 hours after reperfusion (P>0.05). 2. The mean NO and SOD level in Group IPO, IPC and IPC-IPO was higher than Group I/R (P<0.01, P<0.01, P<0.01) at 2 hours after reperfusion (P>0.05). that in Group IPC, IPO and IPC-IPO were comparable 3. The mean TNF-α( P<0.01, P<0.01, P<0.01), MDA(P<0.01, P<0.01, P<0.01) and MPO(P<0.01, P<0.01, P<0.01) level in Group IPO, IPC and IPC-IPO was lower than Group I/R at 2 hours after reperfusion, that in Group IPC, IPO and IPC-IPO were comparable (P>0.05). 4. The apoptotice index in Group IPO, IPC and IPC-IPO was lower than Group I/R at 2 hours after reperfusion, that in Group IPC, IPO and IPC-IPO were comparable (P>0.05). The expression of the Bax protein in Group I/R was higher than Group IPO, IPC and IPC-IPO. The expression of the Bcl-2 protein in Group IPO, IPC and IPC-IPO was higher than Group I/R. Conclusions: 1. IPO and IPC are associated with comparable effective to protect graft from I/R injury during pancreas transplantation. 2. increasing production of serum NO and tissular SOD, relieving conglutination and aggregation of PMNs in pancreas and restraining synthesis TNF-α, increase Bcl-2 protein and reduce Bax protein expression are the common path of the protection of IPO and IPC. 3. The combined protective effects of IPC and IPO do not appear to be additive, which is equal to IPC or IPO alone.Study three The protective effects of non-wounded leg ischemic postconditioning on ischemic reperfusion injury of donor'pancreas graft in ratsObjectives: To investigate the effects of non-wounded leg ischemic postconditioning on ischemic reperfusion injury of donor'pancreas graft in rats, and analyze the possible mechanism. Methods: Group control which consist 6 normol SD rats suffered with sham operation, 12 steptozozin-induced diabetic SD rats were randomly assigned to two groups: group I/R (n=6) received pancreas transplantation normally, without additional intervention, Group RIPO (n=6) received pancreas transplantation exposed IPO with 5 minutes ischemic and 5 minutes reperfusion thrice before ablating donors in the posterior limbs. The blood glucose, TNF-αin serum, MDA and MPO in graft tissue were monitored, and apoptotic cells were stained by TUNEL technique at 2 hours after long-time reperfusion. Results: 1. The blood glucose, TNF-ɑ, MDA and MPO of group I/R were higher than Group RIPO after reperfusion (P<0.01).The levels of SOD and NO of group I/R were lower than Group RIPO after reperfusion (P<0.01).2. The apoptotic index of group I/R were higher than Group RIP (P<0.01). Conclusions: 1. non-wounded leg ischemic postconditioning can protect rat pancreas graft from ischemic reperfusion injury during pancreas transplantation. 2. The possible mechanisms relate to releasing NO, oxygen radical and TNF-αafter reperfusion.Study fourThe effects of the ischemic postconditioning on intestinal mucosal barrier after the rats'pancreas transplantation Objectives: To investigate the protective effects of the ischemic postconditioning on intestinal mucosal barrier after the rats'pancreas transplantation and analyze the possible mechanism. Methods: Group control included 12 normal rats received sham operation, 24 steptozozin-induced diabetic SD rats were randomly assigned to two groups: Group I/R consisted of 12 diabetic rats which received pancreas transplantation normally, without additional intervention; Group IPO consisted of 12 diabetic rats received pancreas transplantation exposed IPO with 30-second reperfusion followed by 30-second reocclusion thrice at the onset of reperfusion after cold ischemia. The intestinal permeability, absorptional function and morphology, intestine tissue water (wet weight /dry weight), the levels of Endotoxin, TNF-α, NO and SOD in serum, MDA, SOD and MPO in intestinal tissue were monitored at 5 days after reperfusion. Simultaneity, the bacteria translocational ratio of mesentery lymph node, liver and spleen were observed. Results: 1. The levels of TNF-αand Endotoxin in serum (P<0.01), the intestinal permeability (P<0.01), the bacteria transclocational ratio (P<0.01), the degree of intestinal mucous injury, the apoptosis index (P<0.01), W/D and MPO activity (P<0.01) in intestinal mucous of Group IPO after reperfusion were lower than those in Group I/R. 2. The NO level and SOD activity in serum, the intestinal absorptional function of Group IPO after reperfusion were higher than those in Group I/R (P<0.01). Conclusions: Ischemic postconditioning in donor pancreas graft can protect recipient intestinal mucous barrier, low the bacteria translocation. The possible mechanisms related to restraining synthesis of TNF-α, relieving conglutination and aggregation of PMNs, increasing production of endogenous NO and SOD.
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