Introduction: Hepatic Ischemia-Reperfusion Injury(HIRI), an inevitable complication of liver surgery including hepatectomy or liver transplantation(LT), is responsible for liver dysfunction or allograft rejection. Glycogen synthase kinase 3 beta(Gsk3β) is a negative regulator involving in glucose homeostasis, energy metabolism and mitochondrial dysfunction, our preliminary data showed that myeloid Gsk3β knockout(Gsk3β-mKO) mice represented ameliorated HIRI, comparing with the WT counter-parts. However, the underlying mechanisms remained to be studied. Kupffer cells(KCs) are critical immune regulators under either steady state or inflammatory strike. During acute phase of HIRI, KCs undergo necroptotic loss, resulting in impaired self-maintenance of liver homeostasis. In addition, our previous works demonstrated that depletion of KCs in advance resulted in severe murine HIRI or even death, suggesting that conservation of KCs was of significance in liver protection. In this current study, we aimed to investigate whether Gsk3β regulated HIRI via KCs necroptosis.
Methods: Male WT(C57/BL6J) and Gsk3β-mKO(Lyz-Cre-Flox) mice were employed to performed murine liver partial warm ischemia-reperfusion(IR) model. Clodronate liposome(200μl/mouse) were injected i.p. 48h prior to the onset of liver ischemia. Mice were sacrificed at 0/6h after 90min of ischemia. Kupffer cells were stained by F4/80 and CD11b in FACS, or Clec4f in immunofluorescence. To eliminate the interference of infiltrating macrophages, we performed KC reconstitution. In brief, KCs were isolated from WT/ Gsk3β-mKO liver by 0.27% collagenase IV and purified by percoll resolution. Cells were injected into WT mice pre-treated with CL, then the reconstituted mice were performed IR surgery. Expression of necroptosis related protein RIPK3 were stained by Western blotting.
Results: Numbers and proportion of KCs in Gsk3β-mKO were higher than WT groups, and RIPK3 expression level was lower in KO liver. In addition, reconstitution of Gsk3β deficiency KCs represented significant protection effect from HIRI than WT.
Conclusion: Gsk3β deficiency protected KCs from necroptosis, resulting in ameliorated HIRI. This study not only advanced our understanding of the pathophysiology of liver IRI, but also provided a novel dimension in developing therapeutic strategy in liver inflammatory diseases.