| Abstract:Objective:In this in-vitro experiment, to explore Deferoxamine (DFO) on the regulation of hepatocytes in the process of cold ischemia and it’s possible mechanisms, we simulated the cold ischemia and reperfusion process of the hepatocytes, for further improving liver preservation conditions, and providing theoretical support for improvement of organ preservation solution. Method:Low temperature, glucose-oxygen deprivation, subsequently, rewarming and reoxygenation were used to induce ischemia-reperfusion injury model in cultured normal hepatocyte cell line (HL-7702) which were divided into4groups:control group and DFO-treated group (25μmol/L,50μmol/L and100μmol/L group). After cold ischemia8h and reperfusion8h, cell morphology change were observed by inverted microscope, apoptosis rate were determined by flow cytometry (FCM) respectively, the content of lactate dehydrogenase (LDH) and malondialdehyde (MDA) were tested. Atomic absorption spectrometry was used to test the intracellular concentration of Fe3+. Experimental data expressed as (x±s). Experimental statistical data applied SPSS16.0for windows statistical package for the statistical analysis. P<0.05showed statistical significance. Result:(1) After cold ischemia8h, MDA content of hepatocytes was (1.83±0.63) nmol/ml,(2.60±0.26) nmol/ml,(2.74±0.32) nmol/ml in DFO-treated groups of lower, medium and high concentration, respectively, which were significantly lower than (3.57±0.45) nmol/ml in control group(P<0.05); then, after reperfusion8h, MDA content of hepatocytes was (2.48±0.36) nmol/ml,(3.07±0.35) nmol/ml,(3.56±0.25) nmol/ml in DFO treated groups of lower, medium and high concentration, respectively, which were significantly lower than (4.16±0.24) nmol/ml in control group (P<0.05).Both after cold ischemia8h and reperfusion8h, the MDA content of high concentrations DFO-treated group is lower than the medium and lower DFO-treared group(P<0.05), medium concentration group and the low concentration group was no significant difference (P<0.05).(2) After cold ischemia8h, LDH content in the supernatant was (187.50±15.97) U/L,(230.99±14.04) U/L,(247.01±25.49) U/L in DFO treated groups of lower, medium and high concentration, respectively, which were significantly lower than (287.16±28.82) U/L in control group(P<0.05); then, after reperfusion8h, LDH was (216.67±22.22) U/L,(263.68±7.16) U/L,(297.57±21.06) U/L, in DFO treated groups of lower, medium and high concentration, respectively, which were significantly lower than (354.51±31.02) U/L in control group (P＜0.01). Both after cold ischemia8h and reperfusion8h, the LDH content in the supernatant of high concentrations DFO-treated group is lower than the medium and lower DFO-treared group (P＜0.05), DFO-treared groups of medium concentration and the low concentration was no significant difference (P>0.05).(3) Apoptosis rate in DFO-treated groups of high, medium and low concentration was (6.37±1.28)%,(14.07±1.61)%and (16.07±1.71)%, respectively, which were significantly lower than control group (20.53±1.58)%(P＜0.01). Normal hepatocytes rate in DFO-treated groups of high, medium and low concentration was (92.62±0.56)%,(89.470.63)%and (88.52±0.96)%respectively, which were significantly higher than control group (75.32±1.67)%(P＜0.01). The Apoptosis rate of high concentrations DFO-treated group is lower than medium and lower DFO-treared group (P＜0.01), DFO-treared groups of medium concentration and the low concentration was no significant difference (P>0.05). The normal hepatocytes rate of high concentrations DFO-treated group is higher than medium and lower DFO-treared group (P<0.01), DFO-treared groups of medium concentration and the low concentration was no significant difference (P>0.05).(4) After cold ischemia8h, The intracellular Fe3+concentration were (2.09±0.28) mmol/L,(1.92±0.24) mmol/L, and (1.36±0.14) mmol/L in DFO-treated groups of lower, medium and high concentration, respectively, which were significantly lower than (2.58±0.27) mmol/L in control group (P<0.05). The intracellular Fe3+concentration of high concentrations DFO-treated group is lower than medium and lower DFO-treared group (P＜0.01), DFO-treared groups of medium concentration and the low concentration was no significant difference (P>0.05). Conclusion:(1) It can generate a lot of chelation iron in the process of cold ischemia of cultured hepatocytes.(2) DFO has reduced the MDA and LDH levels, it indicates that DFO can reduce oxidative stress injury of cultured hepatocytes from cold ischemia and reperfusion.(3) DFO has inhibited apoptosis induced by cold ischemia injury, and its mechanism related that DFO reduce the chelation state iron content during the process of cold ischemia.(4) The protective effect of high concentration DFO treated group was most significant than other concentrations group and control groups.(5) In this study, the protective effect of DFO was significantly, but further research is needed to confirm the effect of the in vivo experiments. |
PDF Full Text Request