| Liver ischemia reperfusion injury(IRI)is a very complex pathophysiology process in development of many liver diseases.A lot of factors can influence the process.Calorie restriction or starvation(fasting)has some beneficial effects in terms of prolonging life and increasing resistance to stress.It has also been shown that calorie restriction has a protective role during ischemia-reperfusion injury(IRI)in several organs,but the underlying mechanism has not been elucidated.Our studies have demonstrated that short-term starvation(STS)preconditioning can reduce release of TNF-??IL-6 and increase release of IL-10 compared with that in the IR group.Our previous experiments have showed that short-term starvation preconditioning can reduce the expression of inflammatory cytokines gene and the release of inflammatory cytokines.Also,during the reperfusion stage,compared with the IR group,the expression of Sirtl was increased in STS group.Thus,we will investigate that short-term starvation preconditioning regulates Sirtl-autophagy signaling pathways during liver ischemia reperfusion injury.The success of the project will be a great significance in ischemia diseases,provide a rationale for a novel therapeutic strategy for managing liver IRI.The subject includes the following three parts:The First Part:Low-dose LPS preconditioning reduces Liver Ischemia Reperfusion Injury in MiceBiochemical detection,Pathological histology detection,TUNEL,caspase-3 detection and qRT-PCR detection were analyzed in ischemic liver tissue using a murine liver partial warm ischemia mode.Compared with sham group,the levels of ALT and AST are significantly higher in IR group(P<0.01),on the contrary,the levels of ALT and AST in STS+IR group is lower than that in IR group(P<0.05),especially in 2d-ST and 3d-ST groups(P<0.01).Pathological histology detection shows IR group have severe hepatic lobule distortion,sinusoidal congestion,apparent edema,vacuolization and massive necrosis;all of which were significantly improved in the 1d-ST,2d-ST,and 3d-ST groups.These results indicate that starvation for 1,2,and 3 days effectively attenuates liver IRI,especially in 2d-ST and 3d-ST mice.TUNEL staining shows that the frequency of TUNEL-positive cells in total hepatocytes was 43.17±6.63%,17.50±3.63%,18.53±3.34%,and 67.17±7.80%in the ld-ST,2d-ST,3d-ST,and control groups,respectively.the numbers of positive cells in STS groups are less than that in IR group(P<0.01).It shows that short-term starvation preconditioning can inhibit apoptosis obviously.the expression of BCL-2,BCL-xl and P-Akt was significantly higher in the starvation-treated groups than in the control group.In addition,2d-ST and 3d-ST mice had higher expression of BCL-2,BCL-x1 and P-Akt than 1d-ST mice,but there was no significant difference between 2d-ST and 3d-ST mice.In contrast to anti-apoptotic proteins,expression of cleaved capase-3 a pro-apoptotic protein,was effectively inhibited in the starvation-treated groups.These findings indicate that STS significantly alleviate liver IR injury and inhibits apoptosis in ischemic liver,especially in 2d-ST and 3d-ST mice.The second part:Short-term starvation reduces Liver Ischemia Reperfusion Injury by enhancing autophagy in MiceSeveral studies have reported that starvation,fasting,or DR may induce expression of autophagy in different organs,including liver,brain,heart,and kidney.We investigated whether STS preconditioning induced autophagy in liver tissues.LC3B and P62 markers of autophagy in liver were measured by western blotting.As expected,starvation for 2 days significantly increased LC3B expression(2.38±0.23 vs.1.00±0.13;P<0.01)and decreased P62 expression(0.50±0.07 vs.1.000±0.15;P<0.05)compared to controls.To confirm the western blotting results,autophagosomes were measured via LC3B staining,which revealed that starvation effectively increased LC3B staining compared to controls.These data strongly support the notion that STS effectively induces autophagy in liver.To determine whether STS-induced autophagy regulated STS-mediated protection against liver IRI,we injected 3-methyladenine(3-MA)to block autophagy expression in 2d-ST mice.We found that starvation for 2 days markedly attenuated IR-enhanced sALT(8824.2±2470.6 vs.876.4±129.2;P<0.01),but the beneficial effect was effectively abolished by 3-MA treatment(4821.3±946.2 vs.876.4±129.2;P<0.01).These data were consistent with HE staining,which showed that starvation for 2 days effectively reduced IR-induced edema,sinusoidal congestion,structural damage,and hepatocellular necrosis,but 3-MA treatment abolished these effects in ischemic liver.Suzuki scores for the IR,ST+IR,and 3-MA+ST+IR groups were 3.6±0.3,1.5±0.2,and 3.2±0.3,respectively.We further analyzed hepatocellular apoptosis in ischemic livers via TUNEL staining,which revealed that 3-MA almost reversed the starvation-decreased frequency of TUNEL-positive cells in ischemic liver.The frequency of TUNEL-positive cells was 60.17±7.36%,24.00±3.51%,and 54.33±5.89%in the IR,ST+IR,and 3-MA+ST+IR groups,respectively.Taken together,the above findings demonstrate that autophagy is critical for STS-mediated protection against liver IRI.The third part:Short-term starvation reduces Liver Ischemia Reperfusion Injury by SIRT1-autophagy Signaling in MiceIt has been reported that starvation can induce expression of Sirtl.Sirtl could be an important factor in the process of autophagy induced by starvation.However,whether short-term starvation induce autophagy by Sirt-1 signaling remains unclear.We detect Sirtl mRNA expression in liver tissue after IR injury,and found that Sirtl mRNA expression was increased 1.6-fold after 1-day,2.5-fold after 2-day,and 2.0-fold after 3-day starvation.Western blotting confirmed these data;Sirtl protein expression was markedly enhanced after 2-and 3-day starvation.We injected mice with sirtinol(a Sirtl inhibitor)before and during starvation.Western blotting showed that sirtinol effectively inhibited STS-induced LC3B expression and stabilized P62 expression in liver.Immunohistologic staining further confirmed that STS-induced autophagy was mediated by Sirtl.Biochemical detecting showed that sirtinol effectively restored IR-enhanced sALT(2949.0±920.9 vs.8006.0±1291.0;P<0.01)and sAST(4060±965.3 vs.8854.0±2279;P<0.01)from starvation treatment.Consistent with biochemical markers,HE staining showed that the sirtinol treatment group had similar histologic damage as the IR group.These results demonstrate that Sirtl plays a vital role during STS-mediated protection against liver IRI. |
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