The Effect Of Octreotide On Hepatic Ischemia-reperfusion Injury In The Rabbit And Its Possible Mechanism

Part one The Effect of Octreotide on Hemodynamics andBlood Biochemiology and Inflammatory Cytokine fromIschemia-reperfusion in the Rabbit LiverObjective: To observe the protective effect of octreotide onhemodynamics, blood biochemiology and inflammatory cytokine fromischemia-reperfusion injury in the rabbit liver and its possible mechanism.Methods: Pringle's maneuver rabbit hepatic ischemia-reperfusion modelswere established. 24 adult New Zealand rabbits were randomly divided into equal 3groups: sham operative group (group A), isehemia-reperfusion group (group B) andoctreotide preconditioning group (group C). recorded the changes of MAP, HR inevery rabbit at the time before ischemia(T₁), 15min(T₂), 30min(T₃) afterischemia, 15min(T₄), 30min(T₅), 60min(T₆), 120min(T₇), 240min(T₈) after reperfusion;detected the Alanine aminotransferase (ALT), Aspartate aminotransferase(AST), Lactate dehydrogenase (LDH), Endotoxin (ETX) in the serum and Tumornecrosis factor-alpha (TNF-α), Interleukin-1beta (IL-1β) in the plasma in every rabbitat T₁, T₃,T₅, T₆, T₇, T₈.Results:1. From 15min after ischemia to 60min after reperfusion the MAP, HR ofgroup B were lower than that of group A (P＜0.01), from 15min after ischemia to 15min after reperfusion the MAP, HR of group C were lower than that of group A(P＜0.01), From 15min after ischemia to 60min after reperfusion the MAP, HR ofgroup C were higher than that of group B (P＜0.05 or P＜0.01).2. From 30min after ischemia to 240min after reperfusion the ALT, AST, LDH ofgroup B, C were higher than that of group A(P＜0.05 or p＜0.01), the highest time pointwas 120min after reperfusion, and the ALT, AST, LDH of group C were lower thanthat of group B in this period(P＜0.05 or p＜0.01); from 30min after ischemia to240min after reperfusion plasma ETX of group B, C were higher than that of group A(P＜0.05 or p＜0.01),the ETX of group C were lower than that of group B in thisperiod (P＜0.01).3. From 30min after ischemia to 240min after reperfusion the TNF—αofgroup B were higher than that of group A (P＜0.05 or p＜0.01), the highest timepoint was 60min after reperfusion, but the TNF—αof group C started toincrease until 30min after reperfusion, from 30min after ischemia the TNF—αof group C were lower than that of group B (P＜0.05 or p＜0.01); from 30min afterischemia the IL-1βof group B were higher than that of group A(p＜0.01), from 30minafter ischemia to 120min after reperfusion the IL-1βof group C were higher than thatof group A (P＜0.05 or p＜0.01), the IL-1βof group C at any time of reperfusionwere lower than that of group B(P＜0.05 or p＜0.01).Conclusion:1. Oetreotide can protect rabbit liver injury from ischemia-reperfusion.2. The protective of Octreotide on rabbit hepatic ischemia-reperfusioninjury is related to the two aspects: one is direct hepatocellular conservation; thesecond is down-regulative Inflammation medium-TNFα, IL-1β) and decreasing theETX in the plasma.Part two The Effect of Octreotide on Pathomorphology andHepatocellular Ultrastructure and Apoptosis from Ischemia-rep-erfusion Injury in Rabbit LiverObjective: To observe the protective effect of octreotide onpathomorphology, hepatocellular ultrastructure, apoptosis and Bcl-2/ Bax fromischemia-reperfusion injury and its possible mechanism in rabbit liver.Methods: Pringle's maneuver rabbit liver ischemia-reperfusion models wereestablished. 96 adult New Zealand rabbits were randomly divided into sham operativegroup (group A), ischemia-reperfusion group(group B)and octreotide preconditioninggroup (group C), according the different reperfusion time (30min,60min, 120min,240min) the group A,B,C were divided into equal sub-groupA_â… , A_â…¡, A_â…¢, A_â…£, B_â… ,B_â…¡, B_â…¢,B_â…£and C_â… , C_â…¡, C_â…¢, C_â…£,observed the pathomorphology ofevery sub-group under light microscope, the hepatocellular ultrastructure in groupA_â…¢,B_â…¢,C_â…¢by electromicroscope, the apoptosis of hepatocellular in groupA_â…¢,B_â…¢,C_â…¢in TUNEL and flow cytometer and the Bcl-2/Bax of hepatocellular ingroup A_â…¢,B_â…¢,C_â…¢in immunohistochemical method.Results:1.Under light microscope, the injury of pathomorphology of every sub-group ofgroup C was slighter than that of group B,and the heaviest time of group Bwas at 120min and 240min after reperfusion.2. By electromicroscope could see the injury of hepatocellular ultrastructureof group C_â…¢was slighter than that of group B_â…¢.3.The TUNEL positive cell counts and percentage of apoptosis in flow cytometerof group C_â…¢were lower than that of group B_â…¢.4.The expression of Bcl-2 in group C_â…¢was higher than that of group B_â…¢, theBax in group C_â…¢was lower than that of group B.Conclusion:1.Octreotide can alleviate the changes of pathomorphology and hepatocellularultrastructure from hepatic ischemia-reperfusion injury, limit the hepatocellularapoptosis,up-regulative the expression of apoptosis inhibitor Bcl-2, down-regulativeapoptosis promote protein Bax, indicate that Octreotide can protect the liver fromischemia-reperfusion injury.2.The protective of Octreotide on hepatic ischemia-reperfusion injury isrelated to mitochondrion-mediated Bcl-2/Bax apoptosis pathway.Part three The Effect of Octreotide on the Expression Proteinsfrom Ischemia-reperfusion Injury in RabbitLiverObjective: Constructed the two dimensional electrophoresis (2-DE) pictures of rabbit hepatic tissues from sham operative group (group A),ischemia-reperfusiongroup(group B)and octreotide preconditioning group(group C),analyzed and verifiedthe differential expression proteins in order to illuminate the mechanisms ofoctreotide on hepatic ischemia-reperfusion injury.Methods: Pringle's maneuver rabbit liver ischemia-reperfusion models wereestablished. 24 adult New Zealand rabbits were randomly divided into equal 3 groups:sham operative group (group A),ischemia-reperfusion group (group B)and octreotidepreconditioning group (group C),resected hepatic tissue of every rabbit after 30minischemia then 120min reperfusion; the total proteins of hepatic tissue of every rabbitfrom different group were extracted and separated by 2-DE, by colloidal Coomassiebrilliant blue staining and Image Scanner scanning 2-DE profiles wereconstructed,the differential expression proteins of three groups were analyzed usingPDQuest image analysis, the peptide mass fingerprints were established and analyzedby Surface-enhanced laser desorption/ionization time Of flight massspectrometry(MALDI-TOF-MS),verified the differentially expressed proteins byWestern blot.Results:1.Two-DE profiles with clear background and well reproducibility were obtained,analyzed and compared using PDQuest image analysis then found 31 differentiallyexpressed protein spots, there were 20 up-regulated proteins in group B,17up-regulated proteins in group C,down-regulated proteins in group B and 11down-regulated proteins in group C.2.The 31 proteins were identified by MALDI-TOF-MS and a total of 18differential expression proteins were successfully identified,these proteins weremetabolism enzymes involving mitochondrial, molecular chaperone,anti-apoptoticproteins and other proteins or initiation factors involving cellular structures,Signaltransduction and transcript translation.3.verified the differentially expressed proteins HSP70 and HSP27 by Westernblot,the HSP70 and HSP27 up-regulated in group B and group C,especially in groupC.the results were similar to proteomics. Conclusion:The protective mechanisms of octreotide on hepatic ischemiareperfusion injury are complex, relatived to changes of many proteins.Octreotide can regulate metabolism enzymes involving mitochondrial such asNADH-ubiquinone oxidoreductase and triosephosphate isomerase, increasethe substance of against injury such as HSP70 and HSP27, up-regulatephosphatidylethanolamine-binding protein to decrease hepatocellular secretory andregulate the hepatocellular apoptosis and so on. Some other differentially expressedproteins need be futher researched and verified.