An Experimental Research For The Protective Effect Of Deferoxamine On Cold Ischemia Reperfusion Injury In Rats Liver

Objective: By the acyclic isolated reperfused rat liver model(AIPRL)experiments,we simulated the cold ischemia and reperfusion of theliver in liver transplantation,to explore DFO on the regulation of liver in theprocess of cold ischemia-reperfusion and it’s protection mechanisms,for furtherimproving liver preservation conditions, and providing theoretical support forimprovement of organ preservation solution.Method:120adult male SD rats weighting220g to280g were used inthis study. Rats were divided randomly into four groups: control group andDeferoxamine(DFO)-treated groups (25μmol/L,50μmol/Land100μmol/L group),then simulated the cold ischemia and reperfusion of the liver in livertransplantation.After cold ischemia12h and reperfusion2h,liver pathologicalchange were observed by microscope;Determine cell apoptosis of the hepatictissue by site DNA terminal deoxynucleotydy transferase assay(TUNEL method)after cold ischemia12h; Determination the content of lactate dehydrogenase(LDH)、alanine transaminase (ALT)、aspartate aminotransferase (AST) andmalondialdehyde (MDA) in perfusion outflow fluid of each group after coldischemia12h and reperfusion2h;Atomic absorption spectrometry was used totest the hepatic tissue concentration of Fe3+after cold ischemia12h;Determination the bile volume secreted by the liver of each group duringreperfusion2h in vitro after cold ischemia12h; Inflammatory cytokine expression including Tumor Necrosis Factor-alpha(TNF-α）and Interleukin-6(IL-6) were assayed by Enzyme-Linked Immunosorbent Assay(ELISA) aftercold ischemia12h and reperfusion2h;After cold ischemia12h,the0.4percenttrypan blue solution was poured into the liver by portal vein catheterization andrecode the time when the left lateral of the liver achieve uniform blue dye.Fromthe different of the time above,we can determine the damage of the livermicrocirculation during cold ischemia. Experimental data expressed as (x±s).Experimental statistical data applied IBM SPSS Statistics19for the statisticalanalysis. P<0.05showed statistically significant.Result:1. Pathology examination revealed:after cold ischemia12h,the pathologyscores of the DH group、DM group、DL group and control group were0.90±0.66、1.10±0.57、1.75±0.59、2.05±0.55.The scores of DH and DM groupwere lower than its in DL and control group (P<0.05);After reperfusion2h,thepathology scores of the DH、DM、DL and control group were2.05±0.55、2.50±0.67、3.40±0.46、3.70±0.59,The scores of DH and DM group were obviouslower than its in DL and control group(P<0.01).2. TUNEL results show,after cold ischemia12h the apoptosis density inDH、DM、DL and control group were (23.8±4.9)cell number／mm2、(25.7±5.1)cell number／mm2、(30.2±4.9）cell number／mm2and (33.9±3.9）cell number／mm2.The apoptosis density in DH group and DM group weresignificantly lower than the control group and the DL group(P<0.05). 3. After cold ischemia12h, the content of LDH in DH、DM、DL andcontrol group were(229.53±41.03)U/L、(256.01±42.52)U/L、(310.94±41.58)U/L、(341.42±56.18)U/L,the ALT were (80.46±12.61)U/L、（91.28±12.99)U/L、(118.84±13.56)U/L、(130.16±14.40)U/L,the AST were （111.89±15.60）U/L、（124.72±14.94）U/L、（161.31±17.98)U/L、(174.39±13.62)U/L.Accordingto the data above,the content of LDH、ALT、AST in the DH and the DM groupsignificant lower than its in the DL group and the control group(P<0.01). Then,after reperfusion2h,the content of LDH in DH、DM、DL and control groupwere (356.26±54.20)U/L、(399.40±43.87)U/L、(463.68±68.91)U/L、(528.13±37.03) U/L，the ALT were(143.20±22.72)U/L、(159.22±15.87)U/L、(211.04±25.23)U/L、(223.90±26.12)U/L，the AST were(203.90±31.13)U/L、(216.97±16.53)U/L、(309.95±29.68)U/L、(327.70±28.91)U/L, groups of DH and DM,respectively, which were also significantly lower than control group and DLgroup(P<0.01).4. After cold ischemia12h, the MDA content of hepatic tissue in DH groupand DM group were (1.35±0.25)nmol/mgprot,(1.49±0.15)nmol/mgprot,respectively, lower than in control group(2.55±0.25)nmol/mgprot and in the DLgroup (2.40±0.27)nmol/mgprot(P<0.05); then, after reperfusion2h,the MDAcontent of hepatic tissue in DH group、DM group、DL group and the controlgroup were(3.46±0.40)nmol/mgprot)、(3.85±0.42)nmol/mgprot、(5.37±0.58)nmol/mgprot、(5.78±0.39) nmol/mgprot. The MDA content substantiallyincreased in each group compared to the content above,and in DH group and DM group, respectively, which were also significantly lower than in controlgroup and the DL group(P<0.05).5. The bile secreted by the rats liver of each group during reperfusion2h invitro,the DM group and the DH group were (42.8±9.3)μl／g�'liver�'2h and(46.3±7.2)μl／g�'liver�'2h,respectively, which were significantly more than(32.5±5.5)μl／g�'liver�'2h of the control group and(34.8±6.2)μl／g�'liver.2h of the DL group (P<0.01).6. After cold ischemia12h, The concentration of Fe3+in hepatic tissuewere (22.13±1.24)mmol/L,(23.84±2.23) mmol/L in the DH group and theDM group, respectively, which were significantly lower than(30.97±2.19)mmol/L in control group and （29.19±2.08）mmol/L in the DL group(P<0.01).7. Elisa results show,after reperfusion2h,the content of the IL-6in hepatic tissue of the DH group、the DM group were (89.18±14.60)pg/ml、(99.71±23.13)pg/ml, significantly lower than the control group （144.91±23.76）pg/ml and the DL group（134.05±16.84）pg/ml（P<0.01);The content of the TNF-α in hepatic tissue of the DH group、the DM groupwere (83.18±10.31)pg/ml、(85.07±7.34)pg/ml lower than the control group （145.52±29.36）pg/ml and the low group（142.61±27.86）pg/ml（P<0.05）.8. After cold ischemia12h,poured the0.4percent trypan blue solution intothe liver by portal vein catheterization and recode the time when the left lateral of the liver achieve uniform blue dye,then,the time of the DH group、DMgroup、DL group and the control group were(64.5±10.6）s、（69.1±7.9）s、（75.3±9.2）s and（79.9±6.6）s.According to the time above, the DH group andthe DM group were quite short than the control goup and the DL group（P<0.05).Conclusion:1.DFO intervention can reduce the generation of chelation iron in the coldpreservation process, reducing the level of ROS and MDA, inhibition ofapoptosis induced by cold ischemic injury,finally, reduce the cold ischemiareperfusion injury of the rat liver.2.DFO intervention can mitigate the acute injury of the rat liver duringcold preservation phase and had a protective effect to rat liver function.3.DFO had a protective effect on the hepatic microcirculation caused bythe cold preservation.4.The protective function of DFO to rat liver ischemia reperfusion wasconcentration-dependent.