| Objective To explore the feasibility of induction of tolerance to ischemia/ reperfusion in the rat liver by ethanol preconditioning and the related mechanisms.Materials, Methods and Results1. To establish and optimize the ethanol-preconditioning protocol, hepatic inflow occlusion under portal vein blood bypass mode and orthogonal design were adopted. Adult male Wistar rats were randomly divided into 4 groups:normal control group(N); ethanol group(E) ischemia-reperfusion group(IR); and ethanol preconditioning group(EPC) .The mortality, pathological changes of the liver, activities of serum ALT and AST, were determined. Results showed that (1) No rats died in the experiment; (2) Compared with N group, ALT in IR and EPC group were significantly increased after reperfusion, but ALT in EPC group was increased less higher at 6. 12. 24 h after reperfusion than in IR group. (3) Alterations of serum AST activity were similar to that of ALT. But compared with IR group, AST in EPC group was less elevated during reperfusion. (4) Except for many inflammatory cells and a few necrotic hepatocytes around centrolobular veins, no other abnormality was found in EPC group during the reperfusion , but severe liver damage was observed in ER group; (5) Less liver function and structure damage and higher liver ATP content were observed when rats were exposed to 40% ethanol at a dose of 5 g/kg body weight 24 h prior to ischemia.2. To investigate the protective effect of ethanol preconditioning against 90 min I/R liver injuries by optimized protocol. Adult male Wistar rats were randomly divided into five groups: the normal control group(N); ethanol group (E); ischemia/reperfusion?n-group(IR); 0.9%NS preconditioning combined with IR group (NPC) ;ethanol preconditioning combined with IR group (EPC) .The mortalities of the rats were observed and serum ALT, AST activity, pathological changes of the liver, apoptic hepatocytes and liver GSH content were measured. Results showed that (l)The rats in EPC group recovered completely 24 h after reperfusion, while those in NPC and IR group didn't recovery until 48 h; no death was found hi each group. (2) Activities of ALT were increased much less in EPC group than in IR and NPC group (p<0.05). (3) Changes of serum AST was similar to that of ALT in EPC group. (4) Content of liver GSH in EPC group was lower than that in N group at 3 > 6 h after reperfusion, but was significantly higher than that in N, NPC and IR group after 6 h ; (5) Compared with IR and NPC group, although extensively cell denaturalization were found, fewer necrotic and apoptic hepatocytes in EPC group, and no structural derogation was observed;3. To explore the mechanism of protective effect of ethanol preconditioning against I/R liver injuries, expression of the HSP70 protein and HSP72 mRNA in the reperfused or non-reperfused liver under optimized ethanol preconditioning condition were determined by means of immunochemistry staining, western blotting and RT-PCR. Results showed that (1) HSP70 was located mainly in cytoplasm and partially in nuclei at the early phase of reperfusion following 30 min ischemia, but mainly in cytoplasm 24 h after I/R with stronger positive staining around the centrolobular veins and expression of HSP70 was higher in EPC group than in NPC group.(2) Expression of HSP72 mRNA was elevated in EPC than in NPC group with peak values at 3 h and 1 h after reperfusion respectively. (3) Expression of HSP70 in the liver at the end of the 90 min ischemia was only slightly increased, while it was enhanced 1 h after reperfusion and reached its peak level after 6 h in EPC group. Much more HSP70 protein but fewer HSP72 mRNA expression were observed in EPC than in NPC group during the reperfusion (p<0.05).?in-Conclusions1. Ethanol preconditioning is a safe and effective protocol for pretreatment. It is showed that the optimized ethanol preconditioning protocol is A1B1C3 (40 %/5g/kg 724 h prior to ischemia) by orthogonal test design.2.
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