Effect of IRF-1 on murine liver ischemia-reperfusion injury by regulating mitophagy

Abstract

Objective To explore the effect of interferon regulatory factor-1 (IRF-1) on murine liver ischemia-reperfusion injury (IRI) by regulating mitophagy. Methods The living murine hepatic IRI model was established in the healthy male Male C57BL/6 mice. The male C57BL/6 mice were randomly allocated into sham group and IR group (n=6 each). Serum ALT and AST levels were determined in two groups by blood biochemical tests. The mitochondrial damage of liver cells was detected by Rh123 staining. The pathological changes of liver tissue were observed by HE staining. The apoptosis of liver cells was detected by TUNEL assay. The expression levels of IRF-1, Nix, LC3 and Caspase-3 proteins were detected by Western blotting. AML12 cells were cultured and divided into siRNA-NC group and siIRF-1 group. PI staining was used to detect the apoptosis of AML12 cells. Immunofluorescence was used to observe the formation of autophagy in AML12 cells during IRI. Western blotting was used to detect the expression of IRF-1, Nix, LC3 and Bax. Results IR group had significantly higher levels of serum ALT and AST than in sham group (P<0.001). Rhodamine 123 staining showed the decreased mitochondria membrane potential and mitochondrial damage. The hepatic histological changes were aggravated in IR group, consisted of hepatocytes swelling, fatty degeneration, sinusoids narrowing, neutrophils infiltration and large patchy necrosis. TUNEL staining showed IR group exhibited an increased hepatocellular apoptosis rate (P<0.001). Western blotting showed the expression of IRF-1 and caspase-3 proteins was significantly higher in IR group than that in sham group. The Nix and LC3 II expression levels were significantly lower in IR group than those in sham group. PI staining showed the apoptosis rate in siRNA-NC group was significantly higher than in siIRF-1 group. Immunofluorescence showed the number of autophagosomes was significantly less in siRNA-NC group than in siIRF-1 group. Western blotting showed the expression levels of IRF-1 and Bax in siRNA-NC group were significantly higher than those in siIRF-1 group, and those of Nix and LC3 II were significantly lower than those in siIRF-1 group. Conclusion IRF-1 promotes murine liver IRI, which may be related to the inhibition of mitophagy and induction of hepatocellular apoptosis. Inhibition of IRF-1 expression can improve mitophagy, and play a protective role in liver IRI. Key words: IRF-1; mitophagy; Ischemia-reperfusion injury