Regulatory Effects Of IRF-1 On Autophagy In Murine Hepatic Ischemia Reperfusion Injury

Objective: Ischemia/reperfusion(IR)injury(IRI),an organ damage occurs in the settings of liver transplantation and partial liver resection,which directly affects patients' prognosis and survival.Researchers have been working to explore better ways to protect livers from IRI.This study is intended to reveal the relationships between autophagic activation and IRF-1 expression during the course of hepatic IRI,by which to demonstrate the potential mechanism of IRF-1 in regulating IR-induced activation of autophagic signaling and liver damage.Methods: A model of segmental(70%)murine hepatic ischemia and reperfusion was administered as described previously.Liver function(assessed by serum ALT level)and histology(assessed by H&E staining)were observed after reperfusion of 2 h,6 h,12 h and 24 h respectively.Hepatocellular apoptosis was determined by TUNEL assay,IRF-1 m RNA was detected using RT-PCR.Expression of IRF-1 and autophagic marker(LC3II and P62)was detected using western blot analysis.Autophagosomes were observed by transmission electron microscopy(TEM).In order to evaluate the role of IRF-1 in regulating IR-induced liver damage,IRF-1 was up-regulated by propagating Ad IRF-1 into mice via tail vein injection,and down-regulated by knocking out IRF-1 gene.After hepatic ischemia and reperfusion injury model was build,blood samples were tested for serum ALT level,H&E staining and TUNEL assay were used to detect hepatocellular damage and apoptosis.Expression of p-P38,LC3 II and P62 proteins was detected using western blot analysis.Autophagosomes were observed by TEM.To elucidate the mechanism of IRF-1 in regulating IR-induced autophagy,hypoxia and reoxygenation(H/R)model was simulated after Ad IRF-1 or IRF-1 si RNA was transfected into AML12 cells.TEM was used to observe the formation of autophagosomes.Expression of p-P38,LC3 II and P62 proteins was detected by western blot analysis.To further evaluate the effect of IRF-1 on H/R induced-autophagic activation by regulating P38 signaling pathway,AML12 cells were transfected with Ad IRF-1 and SB203580 to inhibit the phosphorylation of P38 MAPK at the same time before H/R model was simulated.Autophagosomes formation was observed by TEM and expression of LC3 II and P62 was detected by western blot analysis.Results: Both serum ALT level and hepatic IRF-1 m RNA and protein expression in IR-treated mice were significantly higher than that in Sham,peaked at 12 h and maintained high until 24 h after reperfusion.Livers exposed to IR revealed hepatocyte edema,sinusoidal stenosis and congestion,necrosis and increased hepatocellular apoptotic rates as compared with Sham(P<0.001).The expression of autophagic marker(LC3II and P62)was increased after reperfusion,and peaked at 6 h and 12 h after reperfusion respectively.The amount of autophagosomes in hepatocytes was significantly higher in livers exposed to IR than that in Sham(P<0.001).Compared with Ad GFP,Ad IRF-1 group exhibited a significant higher serum ALT level(P<0.001),increased hepatocellular apoptotic rate(P<0.001),larger extent of typical sinusoidal stenosis,congestion and necrosis.The expression of p-P38,LC3 II,P62 and the amount of autophagosomes were also significantly increased in Ad IRF-1 group.Compared with the wildtype(WT),IRF-1 gene knockout(KO)mice exhibited a significantly decreased serum ALT level,hepatocellular pathology and apoptosis after exposed to IR.And the expression of p-P38,LC3 II,P62 and autophagosomes were both decreased in KO mice after exposed to IR.Moreover,the expression of p-P38,LC3 II,P62 proteins and autophagosomes formation in AML12 cells were increased in GFP-IRF-1 as compared with GFP-NC group while decreased in si RNA-IRF-1 as compared with si RNA-NC group.In comparison with GFP-IRF-1,the expression of LC3 II and P62 and the amount of autophagosomes were decreased in GFP-IRF-1+SB203580 when the phosphorylation of P38 was inhibited by SB203580.Conclusion: IR induces the expression of IRF-1 and activation of autophagy.IRF-1 promotes the IR-induced autophagic activation and liver damage,which may be related to the activation of P38 MAPK and induction of hepatocellular autophagic and apoptotic activation,which effect can be mitigated by IRF-1 inhibition.