Hepatic ischemia-reperfusion (IR) injury is dynamically regulated by intertwined superoxide anion (O2-)-peroxynitrite (ONOO-) cascaded molecules. Arginase 1 involves in O2-/ONOO- fluctuations and is strongly connected to IR injury. A few probes have been innovated to measure intracellular O2- or ONOO- by fluorescent imaging separately, but revealing the definite link of O2-, ONOO- and arginase 1 in situ remains unidentified in hepatic IR. Thus, a well-designed dual-color two-photon fluorescence probe (CyCA) was created for the in situ real-time detection of O2--ONOO-. Surprisingly, CyCA exhibited a suitable combination of high specificity, preeminent sensitivity, exclusive mitochondria-targeting and fast-response. On the basis of remarkable advantages, we successfully applied CyCA to visualize endogenous O2- and ONOO- in living cells and mice. The synergistic elevation of mitochondrial O2--ONOO- in IR mice was observed for the first time. Furthermore, three tyrosine nitration-sites in arginase 1 caused by ONOO- were identified in proteomic analysis, which was never reported previously. Attractively, nitro-modified arginase 1 could further promote ONOO- formation, ultimately exacerbating the intracellular redox imbalance and IR injury. These new findings decipher direct molecular links of O2--ONOO--arginase 1, and suggest effective strategies for the prevention and treatment of IR injury.
Keywords: Arginase 1; Hepatic ischemia-reperfusion injury; Peroxynitrite; Superoxide anion; Two-photon fluorescence imaging.
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