ZBP1-sensed hypoxic stress triggers intrinsic necroptosis in hepatocytes, aggravating hepatic ischemia-reperfusion injury: an experimental study

Background: Hepatic ischemia-reperfusion injury, an inevitable complication in hepatic surgery, significantly compromises patient outcomes. Hypoxia-induced hepatocyte death is a critical early event that triggers inflammatory cascades and leads to organ damage during ischemia-reperfusion injury. However, the underlying mechanisms remain poorly defined. Necroptosis is widely recognized as a form of regulated cell death. Here, we show that hypoxia activates Z-DNA binding protein 1 (ZBP1), an intracellular Z-DNA sensor protein, leading to intrinsic necroptosis in hepatocytes. Methods: We established a hepatic ischemia-reperfusion mouse model and a primary hepatocyte hypoxia-reoxygenation system. Moreover, we applied gene interference and conditional knockout to investigate the molecular mechanism. Results: We found that hepatic ischemia-reperfusion specifically induced receptor-interacting protein kinase-3 (RIP3)-dependent necroptosis in hepatocytes, rather than non-parenchymal cells. The specific knockout of RIP3 in hepatocytes or the use of RIP3 inhibitor significantly alleviated hepatocyte death and reduced inflammatory response. Moreover, hypoxic conditions alone were sufficient to induce RIP3-dependent necroptosis in hepatocytes, with reoxygenation being dispensable. Mechanistically, our results showed that hypoxia resulted in mitochondrial damage and Z-DNA accumulation, thereby activating ZBP1/RIP3 signaling. Mitochondrial protectant, mitochondrial DNA remover, or the knockdown of ZBP1 rescued hepatocytes from necroptosis. Furthermore, AAV8-ZBP1-shRNA infection completely blocked hepatocyte necroptosis and mitigated hepatic ischemia-reperfusion injury. Taken together, we found that mitochondrial DNA damage leads to hepatocyte ZBP1/RIP3-dependent necroptosis, aggravating ischemia-reperfusion injury. Conclusions: Here, we reveal that hypoxia-induced mitochondrial Z-DNA/ZBP1/RIP3-dependent necroptosis acts as a key driver in hepatic ischemia-reperfusion injury, expanding the knowledge on the mechanism of hypoxia-induced hepatocyte death. Crucially, therapeutic targeting Z-DNA/ZBP1/RIP3 signaling significantly attenuated hepatic ischemia-reperfusion injury, providing preclinical evidence for clinical translation.