Background: Axin2 is a multiple function protein involved in many biological pathways, and its overexpression might regulate Β-catenin activity. The Notch signaling is known to modulate cell proliferation, differentiation and survival. However, the functional roles of Axin2/Β-catenin and Notch1 axis in liver injury due to ischemia and reperfusion (IRI) remain unknown. This study was designed to explore the underlying mechanisms of Axin2/Β-catenin/Notch1 signaling cascade in regulating innate immunity in vitro and in a mouse model of liver IRI. Methods: Using a partial lobar liver warm ischemia model, C57BL/6 wide-type (WT) mice (n=5/gr) were injected with Axin2 siRNA/nonspecific control siRNA (2 mg/kg, i.v.). Sham control WT mice underwent the same procedure but without vascular occlusion. Mice were sacrificed after 6 h of reperfusion. For in vitro study, bone marrow-derived macrophages (BMM) were transfected with Axin2 siRNA/Notch1 siRNA, or nonspecific siRNA (100 nM) and incubated with LPS (100 ng/ml). Results: Mice treated with Axin2 siRNA were resistant to liver IRI, in parallel with increased hepatic expression of Β-catenin, Notch1, p-Akt but diminished PTEN/TLR4 expression. In contrast, untreated or nonspecific siRNA-treated WT controls revealed enhanced PTEN/TLR4 activity, increased sGPT levels and exacerbated hepatic damage, as evidenced by increased hepatocellular edema, sinusoidal congestion, cytoplasmic vacuolization, and severe hepatocellular necrosis (30-50%). Unlike in controls, Axin2 siRNA treatment significantly decreased local neutrophil accumulation, macrophage activation, and hepatocellular apoptosis. Furthermore, Axin2 siRNA treatment in LPS-stimulated BMM upregulated the expression of Β-catein and Notch1 and its target gene Hes1, whereas Notch1 knockdown in BMM resulted in increased PTEN/TLR4 expression, with enhanced NF-kB activity via MAPKs pathway. Conclusion: This study demonstrates that Axin2 can provide a negative feedback to modulate Β-catenin activity in the mechanism of hepatic IRI. Axin2 blockade activates Β-catenin/Notch1 signaling and prevents hepatic IRI through regulation of Notch1-mediated PTEN/TLR4 functions. Our novel findings: 1/ underscore the critical role of Axin2/Β-catenin/Notch1 cascade in regulating innate immune responses in hepatic IRI; and 2/ provide new and refined therapeutic options for the management of organ IRI in transplant recipients.

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