Two-photon fluorescence imaging reveals a Golgi apparatus superoxide anion-mediated hepatic ischaemia-reperfusion signalling pathway

Hepatic ischaemia-reperfusion (IR) injury is mainly attributed to a burst of reactive oxygen species (ROS) that attack biological macromolecules and lead to cell death. The superoxide anion (O(2)˙(–)) is the first ROS to be generated and triggers the production of other ROS; thus, explorations of the role of O(2)˙(–) in the IR process are meaningful. Meanwhile, the Golgi apparatus generates O(2)˙(–)via Golgi-associated proteins, which might play an essential role in IR injury. However, the molecular mechanism by which O(2)˙(–) from the Golgi apparatus regulates hepatic IR injury is unclear. Therefore, to solve this problem, a two-photon (TP) excited fluorescence probe (CCA) was designed and prepared for the reversible detection of O(2)˙(–) in the Golgi apparatus. With the assistance of TP fluorescence microscopy, we observed a substantial increase in the levels of O(2)˙(–) in the Golgi apparatus of an IR mouse liver for the first time, as well as increased caspase-2 activity and apoptosis. Furthermore, we found that the tumour necrosis factor (TNF-α) functions as a positive mediator of O(2)˙(–) generation. Based on these data, we identified the potential signalling pathway in the Golgi that mediates O(2)˙(–) fluctuations in IR mice and revealed the related molecular mechanisms; we also provide a new target for treating IR injury.