| Objective: Liver ischemia reperfusion(IR)injury is a common organ and tissue damage in clinical work.Liver IR is critical to the development of liver disease,liver surgery and survival prognosis.How to protect the ischemic liver cells,reduce the incidence of liver IR,are hot spots at home and abroad.The aim of this study was to investigate the changes of IRF-1 expression and autophagy activity in IR of mouse liver,and to explore the effect of IRF-1 on JNK on IR autophagy and injury induced by IR treatment to reveal hepatic ischemia-reperfusion.The potential mechanism of the injury process provides a new idea for the prevention and treatment of liver IR.Methods: Male adult C57BL/6 mice were randomly divided into sham group,ischemia reperfusion 0h,2h,6h,12 h,24h group.The serum levels of ALT and AST were measured in 70% hepatic ischemia-reperfusion model of the left lobe and the middle hepatic portal.HE staining was used to observe the histopathological changes of liver and observe the apoptosis of hepatocytes by TUNEL method.Immunohistochemistry was used to detect the expressions of PCNA,Bcl-2 and p-JNK in liver tissue.The changes of autophagy of hepatocytes were observed by transmission electron microscopy.The expression of IRF-1,JNK,p-JNK,Beclin-1,p62,LC3,Bcl-2,Caspase-3 and Cleave Caspase-3.(H / R)cell model was established by using hepatocyte line AML12 cells to simulate hepatic ischemia-reperfusion,and further confirmed that IRF-1 regulated JNK-induced autophagic death in hypoxia / reoxygenation.Western blot was used to detect the expression of IRF-1,JNK,p-JNK,Beclin-1,p62,LC3,Bcl-2,Caspase-3 and Cleave Caspase in AML12 cells by Ad IRF-1 and JNK si RNA pretreatment.The expression of p-JNK in AML12 cells was detected by immunofluorescence.Confocal microscopy and transmission electron microscopy were used to investigate the autophagic changes of AML12 cells.(H / R)cell model was established by using the autophagy activator rapamycin and autophagy inhibitor 3-MA.The survival rate of AML12 cells was detected by MTT assay.Western blot was used to detect the survival rate of AML12 cells.The expression of Beclin-1,p62,LC3,Bcl-2,Caspase-3 and Cleave Caspase-3 in AML12 cells were detected by immunohistochemistry.The effect of autophagy on the apoptosis of AML12 cells induced by H / R was detected.Results: The levels of serum ALT and AST in IR group were significantly higher than those in Sham group(P <0.05),and increased with the increase of reperfusion time.IR group increased with the increase of reperfusion time,and liver Cell edema,balloon-like changes,hepatic sinus stenosis,central venous congestion and liver cell necrosis and other pathological changes.TUNEL-labeled apoptotic cells in the liver tissue of the Sham group were less,while the apoptotic cells in the IR group increased significantly with the increase of reperfusion time.Compared with Sham group,the number of PCNA positive cells in liver tissue of IR group was reached and peaked at 6 h.The number of Bcl-2 positive cells in liver tissue of IR group decreased with the increase of reperfusion time,and reached the lowest at 12 h.Compared with Sham group,the number of p-JNK positive cells in liver tissue of IR group reached the peak at 2h after reperfusion,and then decreased with the increase of reperfusion time.The number of autophagosomes in the liver of the IR 6h group was more than that in the Sham group.The levels of IRF-1,JNK and p-JNK protein in IR group increased significantly with the increase of reperfusion time,and peaked at 2h.The level of Cleave Caspase-3 protein in liver tissue of IR group increased with the increase of ischemia-reperfusion time point,and reached the peak at 6h,Caspase-3 protein level has no significant change at each point in time,while the level of anti-apoptotic protein Bcl-2 decreased.The levels of autophagy-related proteins Beclin-1,p62 and LC3 were increased,and reached the peak at 6h.Western blot analysis showed that the levels of IRF-1,JNK and p-JNK protein in Ad IRF-1 group were higher than those in Ad GFP group,and the levels of Beclin-1,p62 and LC3 were increased in Caspase-3 And the level of Bcl-2 protein was decreased,and the expression of p-JNK in Ad ML group was higher than that in Ad GFP group.JNK and p-JNK expression in JNK si RNA group decreased,and the levels of Beclin-1,p62,LC3,Caspase-3 and Cleave Caspase-3 were decreased and the level of Bcl-2 protein was increased.The results of confocal and transmission electron microscopy showed that the autophagy of Ad Fl M group 1 was increased,while that of JNK si RNA group was decreased.The survival rate of AML12 cells in Rap group was significantly lower than that in IR group(P <0.001),but the survival rate of AML12 cells in 3-MA group was significantly higher than that in IR group(P <0.01).The expression levels of Beclin-1,p62,LC3 and Caspase-3 and Cleave Caspase-3 in the Rap group were significantly higher than those in the IR group,while the 3-MA group was significantly lower than the IR group.Conclusion: The expressions of IRF-1 and JNK signaling pathway induced by hepatic ischemia-reperfusion in mice increased,the number of autophagy increased and the expression of autophagy-related protein Beclin-1,p62 and LC3 were upregulated.At the same time,the expressions of Cleave Caspase-3 protein was increased.In the process of hepatic ischemia-reperfusion,IRF-1 increased hepatocyte autophagy by regulating JNK signaling pathway,promoted hepatocyte apoptosis and aggravated ischemia-reperfusion injury. |
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