Unveiling the Crucial Roles of O2•- and ATP in Hepatic Ischemia-Reperfusion Injury Using Dual-Color/Reversible Fluorescence Imaging

Abstract

Hepatic ischemia-reperfusion injury (HIRI) is mainly responsible for morbidity or death due to graft rejection after liver transplantation. During HIRI, superoxide anion (O₂^•-) and adenosine-5'-triphosphate (ATP) have been identified as pivotal biomarkers associated with oxidative stress and energy metabolism, respectively. However, how the temporal and spatial fluctuations of O₂^•- and ATP coordinate changes in HIRI and particularly how they synergistically regulate each other in the pathological mechanism of HIRI remains unclear. Herein, we rationally designed and successfully synthesized a dual-color and dual-reversible molecular fluorescent probe (UDP) for dynamic and simultaneous visualization of O₂^•- and ATP in real-time, and uncovered their interrelationship and synergy in HIRI. UDP featured excellent sensitivity, selectivity, and reversibility in response to O₂^•- and ATP, which rendered UDP suitable for detecting O₂^•- and ATP and generating independent responses in the blue and red fluorescence channels without spectral crosstalk. Notably, in situ imaging with UDP revealed for the first time synchronous O₂^•- bursts and ATP depletion in hepatocytes and mouse livers during the process of HIRI. Surprisingly, a slight increase in ATP was observed during reperfusion. More importantly, intracellular O₂^•-─succinate dehydrogenase (SDH)─mitochondrial (Mito) reduced nicotinamide adenine dinucleotide (NADH)─Mito ATP─intracellular ATP cascade signaling pathway in the HIRI process was unveiled which illustrated the correlation between O₂^•- and ATP for the first time. This research confirms the potential of UDP for the dynamic monitoring of HIRI and provides a clear illustration of HIRI pathogenesis.

Figure 1

Structure and optical properties of UDP. (A) Luminescence reversible mechanisms of UDP in response to O₂^•– and ATP. (B) Fluorescence spectra of UDP (25 μM) after incubation with different concentrations of O₂^•– (0–65 μM) in PBS buffer solutions (10 mM, pH = 7.4) after 5 min. (C) Fluorescence intensity at 470 nm of UDP (25 μM) as a function of O₂^•– level. (D) Selectivity of UDP to common interfering substances (1–19: Blank, 1 mM Cys, 1 mM GSH, 1 mM Hcy, 10 mM K⁺, 10 mM Na⁺, 200 μM Ca²⁺, 200 μM Mg²⁺, 200 μM Cu²⁺, 200 μM Fe³⁺, 50 μM NO^•, 100 μΜ ^•OH, 10 mM H₂O₂, 100 μM ¹O₂, 100 μΜ TBHP, 100 μΜ ROO^•, 100 μM NaClO, 25 μM ONOO^–, and 65 μM O₂^•–) in the O₂^•– channel. (E) Fluorescence spectra of UDP (25 μM) after incubation with different concentrations of ATP (0–22 mM) in PBS buffer solutions (10 mM, pH = 7.4) after 25 min. (F) Fluorescence intensity at 588 nm of UDP (25 μM) as a function of ATP level. (G) Selectivity of UDP to common interfering substances (1–19: Blank, 1 mM Cys, 1 mM GSH, 10 mM K⁺, 10 mM Na⁺, 200 μM Ca²⁺, 200 μM Mg²⁺, 200 μM Zn²⁺, 10 mM PO₄^3–, 10 mM HPO₄^2–, 10 mM H₂PO₄^–, 10 mM SO₄^2–, 10 mM CO₃^2–, 10 mΜ UTP, 10 mΜ CTP, 10 mM GTP, 10 mM AMP, 10 mM ADP, and 10 mM ATP) in ATP channel. λ_ex = 380 nm for O₂^•–. λ_ex = 520 nm for ATP.

Figure 2

Reversibility, spectral crosstalk and biotoxicity evaluation of UDP. (A) Reversible response cycle for blue fluorescence of UDP (25 μM) with the addition of O₂^•– (65 μM) and GSH (200 μM). λ_ex/em = 380/470 nm. (B) In the presence of 22 mM ATP, fluorescence spectra of UDP (25 μM) after addition of different concentrations of O₂^•– (0–65 μM) in the O₂^•– channel. Inset: fluorescence spectra of UDP before and after adding 22 mM ATP in the O₂^•– channel. λ_ex = 380 nm. (C) Absorption spectra of UDP (25 μM) before and after reaction with 65 μM O₂^•– and 22 mM ATP. (D) Reversible response cycle for red fluorescence of UDP (25 μM) with the addition of ATP (22 mM) and apyrase (1 U/N). λ_ex/em = 520/588 nm. (E) In the presence of 65 μM O₂^•–, fluorescence spectra of UDP (25 μM) after addition of different concentrations of ATP (0–22 mM) in the ATP channel. Inset: fluorescence spectra of UDP before and after adding of 65 μM O₂^•– and the subsequent addition of 100 μM, 200 μM ATP in ATP channel. λ_ex = 520 nm. (F) Cell viability of HL-7702 cells after incubation with different concentrations of UDP (0–10^–3 M).

Figure 3

Confocal fluorescence imaging of O₂^•– and ATP fluctuations in hepatocytes stimulated by 2-ME or oligomycin A. (A) Confocal fluorescence images of O₂^•– (blue channel, λ_ex = 405 nm, λ_em = 420–490 nm) and ATP (red channel, λ_ex = 514 nm, λ_em = 525–668 nm) in hepatocytes by UDP staining (40 μM, 20 min) after incubation of 2-ME (3 μg/mL, 1 h) and Tiron (10 μM, 1 h). (B) Confocal fluorescence images of O₂^•– (blue channel, λ_ex = 405 nm, λ_em = 420–490 nm) and ATP (red channel, λ_ex = 514 nm, λ_em = 525–668 nm) in hepatocytes by UDP staining (40 μM, 20 min) after incubation of oligomycin A (50 μM, 1 h) and ATP (10 mM, 1 h). (C, D) Relative blue and red fluorescence intensity output of (A) and (B), respectively. The blue fluorescence intensity of control group was defined as 1. The data are expressed as the mean ± SD. ***P < 0.001. Concordant results were obtained from five independent experiments.

Figure 4

Dynamic visualization of O₂^•– and ATP fluctuations in hepatocytes during the whole process of HIRI and effect of intervention by HIRI drug NAC. (A) Fluorescence imaging of O₂^•– (blue channel, λ_ex = 405 nm, λ_em = 420–490 nm) and ATP (red channel, λ_ex = 514 nm, λ_em = 525–668 nm) by UDP (40 μM) in hepatocytes undergoing 0, 20, or 40 min of ischemia and 20 or 40 min of reperfusion after 40 min of ischemia, and pretreatment with 0.5 or 1 mM NAC. (B, C) Relative blue and red fluorescence intensity output of (A). The blue fluorescence intensity of control group was defined as 1. (D) Relative ROS levels in different phases of HIRI by ROS content assay kit. (E) ATP levels in different phases of HIRI by the ATP content assay kit. The data are expressed as the mean ± SD. ***P < 0.001. Concordant results were obtained from five independent experiments.

Figure 5

Real-time visualization of O₂^•– and ATP dynamics in mouse livers during HIRI. (A) Procedure diagram. (B) Fluorescence imaging of O₂^•– (blue channel, λ_ex = 405 nm, λ_em = 420–490 nm) and ATP (red channel, λ_ex = 514 nm, λ_em = 525–668 nm) by UDP (100 μM) in mouse livers undergoing 0, 20, or 40 min of ischemia and 20 or 40 min of reperfusion after 40 min of ischemia. (C, D) Relative blue and red fluorescence intensity output of (B). The blue fluorescence intensity of the control group was defined as 1. (E) H&E staining of heart, spleen, kidney, lung, and liver in control and HIRI mice. The data are expressed as the mean ± SD. ***P < 0.001. Concordant results were obtained from five independent experiments.

Figure 6

Proteomic analysis of the reaction of SDH with O₂^•– through LC–MS/MS. (A) Oxidation of C68, M71. (B) Oxidation of W47. (C) Oxidation of H57. (D) Oxidation of W218. Residues represented by * and red color are modification sites by O₂^•–.

Figure 7

Potential signaling pathways involving O₂^•– and ATP in HIRI. (A) SDH activity assays in hepatocytes under different treatments. (B, C) Analyses of mitochondrial NADH contents in hepatocytes under various treatments. (D) Mitochondrial ATP contents analyses of hepatocytes in different treatment groups. (E) Fluorescence imaging of ATP (red channel, λ_ex = 514 nm, λ_em = 525–668 nm) in hepatocytes by UDP under the incubation of 3-NPA. (F) Relative red fluorescence intensity output of (E). (G) Intracellular ATP contents analyses of hepatocytes in different treatment groups. (H) Schematic of intracellular O₂^•–—SDH—Mito NADH—Mito ATP—intracellular ATP cascade signaling pathway during HIRI.