| Ischemia-reperfusion injury is inevitable in liver transplantation,and often causes liver failure after transplantation,delayed onset of liver dysfunction,bile duct injury and other complications,so how can we reduce the transplanted liver ischemia-reperfusion injury is regarded as one of hot issues in the field of liver surgery.Since 80s in the 20th century,the domestic and foreign scholars had done a lot of the pilot study and found several methods to reduce the hepatic ischernia-reperfusion injury in liver transplantation,in which the two methods that were ischemic preconditioning(IPC) and ischemic postconditioning(IPO)were the most important effective ones.Domestic and foreign scholars had found that given prior transient ischemic reperfusion,the liver could be induced to generate endogenous protective mechanisms that made it a significant tolerance in a longer period of time after ischemia. Thus they put forward to the concept of ischemic preconditioning.Currently the mechanisms of IPC on the liver transplantation were not yet entirely clear,but an increasing number of experiments had proved that they involves a lot of media,signal transduction pathways,cell activation and so on.Ischemic preconditioning was required to impose before ischemia,and it was not effectively controlled and clinically predicted,so its clinical application value was extremely limited.Followed by domestic scholars,they cultured adult rat cardiac myocytes in hypoxia - reoxygenation model and found that given repeated short-term reoxygenation—hypoxia before reoxygenation,anoxic myocardial cells could significantly increase the survival rates.So they put forward the concept of ischemic postconditioning.IPO has been confirmed the same protective effects in liver ischemia-reperfusion injury in animal models. However.the specific protection mechanisms of IPO were not entirely clear,the current trials confirmed that the period of protective effects of IPO occurred mainly in 2rain before ischemia-reperfusion,and the mechanisms may be associated with slowly controlled intermittent reoxygenation,decreasing a sudden of oxygen free radical outbreak when reoxygenation,stimulating the release of intracellular antioxidant enzymes and free radical curing agents,reducing cells membrane lipid peroxidation,inducing the expression of anti-inflammatory cytokines,reducing the calcium overload,increasing anti-apoptotic genes and so on.Recently,it was discovered that Heme oxygenase(HO) was regarded as a drastic enzyme for alleviating injury induced by oxygen radicals,and one of chief sensitive index of cells stress.HO had three isoenzymes of HO-1,HO-2和HO-3.HO-1 was induced.It up-regulated under many stress factors.HO-1 was a rate-limiting enzyme in degradating hemocrystallin.It degradated hemocrystallin into biliverdin,CO and Iron.So it had effects of resisting oxidation,maintaining microcirculation,regulating mitotic cycle,counteracting inflammatory reaction and so on.Some experiments of ischemia-reperfusion in liver transplantation confirmed that ischemic postconditioning induced HO-1 over-expression,so that transplanted liver had a good effectiveness of anti-ischemic / reperfusion injury.To investigate the role of heme oxygenase-1 on ischemic postconditioning of rat orthotopic liver transplantation can provide theory based on continuous study on protective mechanisms of ischemic postconditioning on hepatic graft and targets of anti-ischemia-reperfusion injury.Aim:1.To investigate the optimal time of ischemic postconditioning in 2min before reperfusion.2.To investigate the effects of ischemic postconditioning on the expression of heme oxyenase-1 in the liver graft with ischemia and reperfusion injury in rats.3.To investigate the protective effects and mechanisms of HO-1 on ischemic postconditioning of rat liver transplantation.Methods:1.To establish an improved model of rat liver transplantation based on Kamada's two-cuff technique.2.The best time of ischemic postconditioning in 2min.(1) Animal grouping:176 healthy male SD rats were randomly divided into two parts of 2h and 6h,meanwhile each part was randomly divided into six groups:①sham-operation group.②ischemia and reperfusion group(I/R),③10s/10s ischemic postconditioning group: animals were given six 10-second episodes of ischemia at 10-second intervals for reperfusion before persistent reperfusion of portal vein,④30s/30s ischemic postconditioning group:animals were given six 30-second episodes of ischemia at 30-second intervals for reperfusion before persistent reperfusion of portal vein,⑤60s/60s ischemic postconditioning group:animals were given six 60-second episodes of ischemia at 60-second intervals for reperfusion before persistent reperfusion of portal vein,⑥90s/90s ischemic postconditioning group:animals were given six 90-second episodes of ischemia at 90-second intervals for reperfusion before persistent reperfusion of portal vein.(2) Detection:At 2h,6h after portal vein reperfusion,blood samples were obtained from the abdominal aorta to determine the level of serum ALT,AST,simultaneously liver tissues were taken to determine the level of HO-1mRNA by RT-PCR.Calculated the brightness of HO-1/β-actin.The HO-1 positive cells were observed by immunohistochemistry. Calculated the rate of HO-1 positive cells.The changes of liver tissues were observed by HE staining and electronmicroscope.These methods were opted to investigate the optimal time of ischemic postconditioning in 2min before reperfusion.3.Effects of ischemic postconditioning on the expression of heme oxvenase-1 in the liver graft with ischemia and reperfusion injury in rats.(1) Animal grouping:56 healthy male SD rats were randomly divided into six groups:①sham-operation group,②ischemia and reperfusion group(I/R),③60s/60s ischemic postconditioning group,④CoPP+60s/60s ischemic postconditioning group(combining group):Rats were injected CoPP 5 mg/kg by intraperitoneal injection at 24 h before portal vein reperfusion to induce the expression of HO-1.(2) Detection:At 6h after portal vein reperfusion.blood samples were obtained from the abdominal aorta to determine the level of serum ALT.AST,simultaneously liver tissues were taken to determine the level of HO-1 mRNA by RT-PCR.Calculated the brightness of HO-1/β-actin.The HO-1 positive cells were observed by immunohistochemistry. Calculated the rate of HO-1 positive cells.The changes of liver tissues were observed by HE staining.These methods were opted to investigate the effects of ischemic postconditioning on the expression of heme oxyenase-1 in the liver graft with ischemia and reperfusion injury in rats.4.The protective effects and possible mechanisms of Heine oxygenase-1 in rats liver transplantation with the treatment of ischemic precondition.(1) Animal grouping:56 healthy male SD rats were randomly divided into six groups:①sham-operation group,②ischemia and reperfusion group(I/R),③60s/60s ischemic postconditioning group,④ZnPP+60s/60s ischemic postconditioning group(combining group):Rats were injected zinc protoporphyrin IX(ZnPP) 20 mg/kg by intraperitoneal injection at 24 h before portal vein reperfusion to induce the expression of HO-1.(2) Detection:At 6h after portal vein reperfusion,blood samples were obtained from the abdominal aorta to determine the level of serum ALT,AST,simultaneously liver tissues were taken to determine the level of HO-1mRNA by RT-PCR.Calculated the brightness of HO-1/β-actin.The HO-1 positive,cells were observed by immunohistochemistry. Calculated the rate of HO-1 positive cells.Liver tissues and blood samples were taken to determine the level of MDA,SOD,TGF-β1,TNF-α,IL-1.The changes of liver tissues were observed by HE staining and electronmicroscope.These methods were opted to investigate the protective effects of HO-1 on ischemia and reperfusion graft,and the relationship between HO-1 and TGF-β1,TNF-α,IL-1.Results:1.The modified Kamada's "double-cuff technique"shortened the period of operation and anhepatic phase,but the survival rate of rat after transplantation was not significantly different than orthodox methods.2.The best time of ischemic postconditioning in 2min:In 2h and 6h after transplatation. Compared with Sham group,the levels of ALT and AST in I/R group and 10s/10s. 30s/30s,60s/60s,90s/90s IPO groups were significantly higher(P＜0.05);The levels of HO-1 were significantly higher(P＜0.05);The injury in liver tissues aggravated. Compared with 2h,the levels of ALT and AST in 6h was significantly higher(P＜0.05); The levels of HO-1 were significantly higher(P＜0.05);The injury in liver tissues aggravated.Compared with I/R group,10s/10s group was significantly higher(P＜0.05); The levels of HO-1 were significantly higher(P＜0.05);The injury in liver tissues aggravated;30s/30s,60s/60s groups were significantly lower(P＜0.05),The levels of HO-1 were significantly higher(P＜0.05);The injury in liver tissues lessened,meanwhile the 60s/60s group was lowest.There was no difference between 90s/90s IPO group and group I/R(P＞0.05).3.Effects of ischemic postconditioning on the expression of heme oxyenase-1 in the liver graft with ischemia and reperfusion injury in rats:After CoPP interference,compared with Sham group,the levels of ALT and AST in I/R group,60s/60s IPO group and CoPP+60s/60s IPO group(combining group)were significantly higher(P＜0.05);The levels of HO-1 were significantly higher(P＜0.05);The injury in liver tissues aggravated. Compared with I/R group,the levels of ALT and AST in 60s/60s IPO group and CoPP+ 60s/60s IPO group(combining group)were significantly lower(P＜0.05);The levels of HO-1 were significantly higher(P＜0.05);The injury in liver tissues lessened.There was no difference between 60s/60s IPO group and combining group(P＞0.05).4.The protective effects and possible mechanisms of Heme oxygenase-1 in rats liver transplantation with the treatment of ischemic precondition:After ZnPP interference, compared with Sham group,the levels of ALT and AST in I/R group,60s/60s IPO group and ZnPP+60s/60s IPO group(combining group)were significantly higher(P＜0.05);The levels of HO-1 were significantly higher(P＜0.05);The levels of MDA,IL-1β,TGF-β1 and TNF-βwere significantly higher(P＜0.05);The levels of SOD was significantly lower(P＜0.05);The injury in liver tissues aggravated.Compared with I/R group,the levels of ALT and AST in 60s/60s IPO group was significantly lower(P＜0.05);The levels of HO-1 were significantly higher(P＜0.05);The levels of MDA,IL-1βand TNF-αwere significantly lower(P＜0.05);The levels of SOD and TGF-β1 were significantly higher(P＜0.05);The injury in liver tissues lessened.There was no difference between combining group and I/R group(P＞0.05);Compared with 60s/60s IPO group, the levels of ALT and AST in combining group was significantly higher(P＜0.05);The levels of HO-1 were significantly lower(P＜0.05);The levels of MDA,IL-1βand TNF-αwere significantly higher(P＜0.05);The levels of SOD and TGF-β1 were significantly lower(P＜0.05);The injury in liver tissues aggravated.Conclusions:1.The modified Kamada's "double-cuff"mold was a stable,reliable,convenient and practicable mold.2.The optimal time of ischemic postconditioning in 2min before reperfusion was 60s/60s.It was the most effective way to reduce the liver ischemia-reperfusion injury after transplantation.3.CoPP+60s/60s IPO and 60s/60s IPO increased the expression of HO-1,but the two methods had the same effection on inducing the expression of HO-1.4.CoPP interference induced the HO-1 overexpression,and lessened the ischemia-reperfusion injury in rats;ZnPP interference inhibited the HO-1 overexpression,and added the ischemia - reperfusion injury.5.The relationship between HO-1 and TGF-β1,TNF-α,IL-1 was close.When HO-1 overexpress,the level of TGF-β1 increased and TNF-α,IL-1 depressed;When HO-1 underexpress,the level of TGF-β1 depressed and TNF-α,IL-1 increased.The mechanisms of HO-1 in anti-ischemia-reperfusion injury were related to the generation of the inflammatory cytokines.
|
PDF Full Text Request