| Objective: Through setting up the model of rats’ hepatic ischemic/reperfusion injury, to investigate the effect of early limb remote ischemic preconditioning combined with delayed limb remote ischemic preconditioning on hepatic ischemic/reperfusion injury in rats.Methods: Adult male Sprague-Dawley rats were randomly divided into 5 groups.The S group(n=8):24h before the surgery the rats received the first anesthesia without other processing and then were performed with the second anesthesia in order to separate hepatic pedicle with no more process.The I/R group(n=8):Before the surgery what the rats were peformed with was similar to S group. When the operation was beginning, using nondestructive microvascular clip to block the blood flow into the left and middle liver for 30 min and then loosing the microvascular clip for the sake of reperfusion with 3h. The ELRIPC group(n=8):The rats were performed the same process before the second anesthesia and after the second anesthesia binding hind limb of the rat by tourniquet so that the colour of hind limb’s skin became purple and femoral artery missed arteriopslmus,subsequently the tourniquet was loosen for 10 min when the blood flow was blocked 10 min for 3 cycles.Then performing the same method about I/R group to establish the station of hepatic ischemic reperfusion.The DLRIPC group(n=8) :To perform limb remote ischemic preconditioning after the first anesthesia and establish the station ofhepatic ischemic/reperfusion by the method like I/R group after the second anesthesia.The EDLRIPC group(n=8):To perform limb remote ischemic preconditioning after the first anesthesia and after the second anesthesia take the same action like what methods the ELRIPC group used after the second anesthesia. Collecting 2ml from postcava at post-reperfusion 3h and cutting part left and middle of liver for testing in each group: Testing the activity of ALT and AST in serum by automatic biochemical analyzer; To perform HE staining on liver section and observe the liver microstructural changes in light glass; Testing the positive rate of the expression of Bcl-2 and Bax protein(BI) by immunhistochemistry; Testing the hepatocyte apoptosis index(AI) by TUNEL.Results: 1.The activity of ALT and AST in serum:Compared with S group, the activity of AST and ALT in I/R group,ELRIPC group,DLRIPC group,EDLRIPC group were significantly increased(P<0.05).Compared with I/R group, the activity of AST and ALT in I/R group,ELRIPC group,DLRIPC group,EDLRIPC group were significantly decreased(P<0.05). However, there were no significant difference in ELRIPC group,DLRIPC group,EDLRIPC group(P>0.05). 2.The liver microstructural changes in light glass:In S group, the hepatic lobule structure had a clear outline, complete liver cell line and there was no obvious blood stasis between liver blood sinus.In I/R group,the liver cell line was inordinate and the hepatocytes were swelling and the cytoplasm was loosen. What the worst was hepatocytes were ballooming degeneration partly.Compared with I/R group, the station of liver microstructure was better in ELRIPC group,DLRIPC group and EDLRIPC group. The liver cell line was clear and the cellular swelling was gently. 3.The positive rate of the expression of Bcl-2 and Bax protein(BI):Compared with I/R group, the BI of Bcl-2 protein was significantly increased in ELRIPC group,DLRIPC group and EDLRIPC group(P<0.05).There was no significant difference in ELRIPC group,DLRIPC group and EDLRIPC group(P>0.05).Compared with I/R group, the BI of Bax protein was significantly decreased in ELRIPC group,DLRIPC group and EDLRIPCgroup(P<0.05).There was no significant difference in ELRIPC group,DLRIPC group and EDLRIPC group(P>0.05). 4.The hepatocyte apoptosis index(AI):Compared with S group, the AI was increased in I/R group, ELRIPC group,DLRIPC group and DELRIPC group(P<0.05).Compared with I/R group, the AI was decreased in ELRIPC group,DLRIPC group and EDLRIPC group(P<0.05).There was no signigicant difference in ELRIPC group,DLRIPC group and EDLRIPC group(P>0.05).Conclusion: The protection was effective in terms of liver ischemic/reperfusion in rats through early limb remote ischemic preconditioning combined with delayed limb remote ischemic preconditioning, early limb remote ischemic preconditioning and delayed limb remote ischemic preconditioning.The extent of protection was similar between early limb remote ischemic preconditioning and delayed limb remote ischemic preconditioning.However,the protection wasn’t enhanced through early limb remote ischemic preconditioning combined with delayed limb remote ischemic preconditioning. The upregulation of Bcl-2 protein and the downregulation of Bax protein were involved in the protection of limb remote ischemic preconditioning. |
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