In situ and real-time imaging of superoxide anion and peroxynitrite elucidating arginase 1 nitration aggravating hepatic ischemia-reperfusion injury.

Abstract

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.