ZBP1-mediated apoptosis and inflammation exacerbate steatotic liver ischemia/reperfusion injury

Abstract Background & Aims: In liver transplantation, steatotic donor livers are rapidly increasing but are more susceptible to ischemia-reperfusion (I/R) injury. This study aimed to investigate the underlying mechanisms and identify therapeutic targets. Methods: Cell death markers were determined in donor livers and animal models. Casp8f/f;Alb-Cre, Ripk1D138N/D138N, Tnf-/-, Ripk3-/-, Trif-/-, Zbp1-/- mice were fed high fat diet or choline-deficient high fat diet to induce steatotic livers and were then subjected to I/R injury to investigate the mechanisms of cell death and inflammation. Primary mouse or human hepatocytes were isolated and subjected to hypoxia-reoxygenation challenge to further elucidate the regulation mechanisms. Results: Apoptosis- and inflammation-induced injury was exacerbated in I/R process of steatotic livers, with inflammation-induced injury playing a more predominant role. In both normal and steatotic livers, caspase-8 ablation mitigated I/R injury by reducing apoptosis but not inflammation while RIPK1 kinase inhibition more effectively protected against I/R injury by alleviating both apoptosis and inflammation. Z-DNA binding protein 1 (ZBP1) but not TNF-a deficiency inhibited RIPK1 activation and protected against I/R injury in steatotic livers but not normal livers. In steatotic livers, overmuch palmitic acid triggered ZBP1 transcription through activating JNK pathway. During liver transplantation, excessive reactive oxygen species were generated during I/R injury and triggered ZBP1 oligomerization, which led to the kinase activation of RIPK1 and the subsequent aggravation of apoptosis- and inflammation-induced injury. Conclusions: ZBP1-mediated apoptosis and inflammation exacerbate steatotic liver I/R injury, which could be leveraged to protect steatotic donor livers in transplantation. Overall design: The RNA-seq data includes the following two sections. 1) Zbp1+/+ and Zbp1-/- mice were fed high fat diet for 4 months and subjected to 1 h ischemia/6 h reperfusion operation. The livers were harvested and RNA were extracted for RNA-seq. 2) Wildtype mice were injected AAV8 to overexpress Zbp1 in the livers (Vector was the nagative control). 1 month later, 10 mg/kg Nec-1s was injected peritoneally to the mice. After 1 h, the mice were subjected to 1 h ischemia/6 h reperfusion operation. The livers were harvested and RNA was extracted for RNA-seq.