•An ultrafast near-infrared fluorogenic probe DCI-BT was designed for the detection of ONOO-.
•Employing DCI-BT, Zn2+-induced endogenous ONOO- production in cells was successfully visualized for the first time.
•DCI-BT was successfully applied to monitor the changes in ONOO- levels in the Zn2+-induced ALI model.
•Sulforaphane was shown to be effective in protecting mice with Zn2+-induced ALI.
Abstract
Particulate matter derived from environmental pollution might contain zinc ions (Zn2+), and inhaling these particles exacerbates lung tissue's inflammatory response, impairing lung function and increasing the risk of acute lung injury (ALI). Zn2+ is known to contribute to oxidative stress, leading to elevated levels of reactive oxygen species such as peroxynitrite (ONOO-), which play a key role in the pathogenesis of ALI. Herein, a novel near-infrared fluorogenic probe, DCI-BT, was prepared for the specific detection of ONOO- based on the strategy of oxidative hydrolysis of imine to break into aldehyde. The response of DCI-BT to ONOO- was found to be extremely fast, and the addition of ONOO- would enhance its fluorescence intensity. Cell experiments showed that DCI-BT could efficiently indicate the changes in cellular ONOO- levels. Furthermore, employing DCI-BT, the Zn2+-induced endogenous ONOO- production in cells was successfully visualized, confirming that prolonged exposure to Zn2+ triggered cellular oxidative stress. Finally, the application of DCI-BT in the mice model of ALI was evaluated, and the results revealed that it had good biosafety and could effectively track the changes in ONOO- levels in the Zn2+-induced ALI model. Therefore, DCI-BT held promise as a valuable chemical tool for diagnosing and treating environmentally induced oxidative stress-related diseases.
Graphical Abstract
An innovative NIRF probe DCI-BT was capable of monitoring ONOO- in vitro and in vivo. In Zn2+-exposed ALI mice models, DCI-BT enabled real-time imaging of ONOO- levels and found that the degree of Zn2+-induced lung injury was positively correlated with the level of ONOO-.
1. Introduction
With rapid industrialization, environmental pollution has become a significant factor endangering human health. Zinc ions (Zn
2+), as a typical metal ion in the environment, originate from industrial emissions, mining, waste disposal, and other pathways. Particulate matter suspended in the atmosphere (e.g., PM2.5) may adsorb Zn
2+ on its surface and enter the lungs via the respiratory tract
[1],
[2]. It is well known that zinc is a widely distributed and essential trace element, critical for maintaining normal physiological functions in living organisms
[3]. The transport of Zn
2+ in cells mainly depends on the transmembrane zinc channel proteins solute-related carrier (SLC) 39 A and SLC30A
[4],
[5]. However, high concentrations of Zn
2+ may induce oxidative stress through multiple signaling mechanisms, resulting in potential toxicity. Imbalances in zinc homeostasis are correlated with the development of various diseases, including diabetes, alcoholic liver disease, lung injury, and traumatic brain injury
[6],
[7],
[8]. Previous studies have shown that environmental Zn
2+ concentrations are closely associated with acute lung injury (ALI), e.g., exposure to high-dose Zn
2+ up-regulates the expression levels of pro-inflammatory factors (e.g. IL-8 and COX-2) in human respiratory epithelial cells
[9],
[10],
[11].
Peroxynitrite (ONOO
-), as a common reactive oxygen species (ROS) in living organisms, is produced through the diffusion reaction of nitric oxide and superoxide anion
[12]. ONOO
- can undergo oxidative or nitrative reactions with a variety of biomolecules (proteins, DNA, and lipids), and participates in cellular signaling processes by modulating its structure and function, which ultimately leads to the occurrence of diseases, such as neurodegenerative, inflammatory, and cancerous diseases
[13],
[14],
[15]. It has been found that prolonged exposure to high-dose Zn
2+ triggers oxidative stress, apoptosis or necrosis, and damage to respiratory and lung tissues
[16],
[17]. Meanwhile, ONOO
- generated in the inflammatory response will further participate in the oxidative stress process and exacerbate oxidative cell damage
[18]. Therefore, clarifying the complex relationship between Zn
2+ exposure and ONOO
- helps to understand the mechanism of oxidative stress in Zn
2+-induced ALI, which is of great significance for environmental protection and timely intervention in ALI.
With high spatial and temporal resolution, fluorescence imaging enables tracking the dynamic changes of target molecules in organisms in real-time, providing more accurate results for disease diagnosis
[19]. Compared with UV-vis fluorescent probes, near-infrared fluorogenic (NIRF) probes have higher tissue penetration ability, lower photodamage, and background fluorescence, making them a promising tool for studying the physiological distribution and concentration changes of reactive species
[20],
[21],
[22],
[23],
[24],
[25]. Harnessing various chemical reaction strategies, including
N-dearylation
[26],
[27],
[28],
[29], oxidative cleavage of hydrazine
[30], boronic acids/boronic esters oxidation
[31],
[32],
[33], and oxidative breaking of unsaturated bonds
[34],
[35], several fluorescent probes for the detection of ONOO
- have been constructed
[36],
[37],
[38],
[39],
[40]. However, despite the existing ONOO
- fluorogenic probes being used in different disease models, no reports of NIRF probes have been applied to investigate Zn
2+ exposure-induced changes in ONOO
- levels (
Table S1). Moreover, current studies have paid less attention to environmentally induced ALI, which differs significantly from modeling pathogen-induced ALI. Therefore, exploring the impact of Zn
2+ exposure on ONOO
- levels is of great significance for understanding environmentally induced ALI. In this work, a novel NIRF probe, namely DCI-BT, for ONOO
-, was designed and synthesized. The first positive correlation between the elevated ONOO
- and the degree of lung injury was demonstrated in Zn
2+-induced cell and ALI mice models (
Scheme 1).
Scheme 1. Schematic illustration of DCI-BT for specific detection of ONOO- in Zn2+ exposure-induced ALI (by Figdraw).
Fig. 1. (a) Emission spectra of DCI-BT (10 μM) upon the addition of different concentrations of ONOO- (0–14 μM). (b) Fluorescence responses of DCI-BT (10 μM) at 640 nm after adding varied concentrations of ONOO- (0–28 μM). (c) The linear relationship between fluorescence intensities of DCI-BT at 640 nm and ONOO- concentrations ranging from 0 to 14 μM. (d) Fluorescence intensities change of DCI-BT (10 μM) at 640 nm in the absence or presence of ONOO- (14 μM) as a function of time. (e) pH effect on fluorescence intensities of DCI-BT at 640 nm before and after the addition of ONOO- (14 μM). (f) Selectivity studies of DCI-BT (10 μM) with various biological interfering analytes (100 μM): 1. blank, 2. ONOO-, 3. K+, 4. Ca2+, 5. Mg2+, 6. Zn2+, 7. Cu2+, 8. CO32-, 9. NO3-, 10. SO42-, 11. HSO3-, 12. HS-, 13. Cys, 14. GSH, 15. 1O2, 16. O2.-, 17. .OH, 18. H2O2, 19. -OCl. Data are expressed as the mean ± SD (n = 3).
Fig. 2. Visualization of exogenous ONOO- with DCI-BT (λex = 488 nm). (a) First row: HPMEC cells preincubated with SIN-1 (0, 50, 100 μM), SIN-1 (100 μM) + AG (1 mM) for 30 min were stained with DCI-BT (10 μM) for 30 min. Second row: RAW264.7 cells preincubated with SIN-1 (0, 50, 100 μM), SIN-1 (100 μM) + AG (1 mM) for 30 min were stained with DCI-BT (10 μM) for 30 min. Scale bar: 50 μm. (b) The pixel intensity of DCI-BT labeled HPMEC cells. (c) The pixel intensity of DCI-BT labeled RAW264.7 cells. Data are expressed as the mean ± SD (n = 3).
Fig. 3. Time course fluorescence images of RAW264.7 cells under ZnSO4 exposure. Blue channel: the cells were stained with Hoechst 33258 (5 μg/mL) for 30 min (λex = 405 nm). From the first to the fifth column, the cells stimulated with ZnSO4 (100 μM) for 0, 15, 30, 60, and 90 min were incubated with DCI-BT (10 μM) for 30 min. Sixth column: the cells pretreated with ZnSO4 (100 μM) and AG (1 mM) for 90 min were incubated with DCI-BT (10 μM) for 30 min. Merge channel: the images overlap with blue and red channels. Scale bar: 50 μm.
Fig. 4. Confocal images of RAW264.7 cells under ZnSO4 stress. (a) Blue channel: the cells were stained with Hoechst 33258 (5 μg/mL) for 30 min; red channel: the cells were stimulated with various concentrations of ZnSO4 (0, 50, 100, 200 μM), ZnSO4 (200 μM) + ebselen (200 μM) for 90 min, then stained with DCI-BT (10 μM) for 30 min; merge channel: the images were the overlap of blue and red channels. Scale bar: 50 μm. (b) The pixel intensity of DCI-BT labeled RAW264.7 cells in the red channel in (a). (c) The protein levels of iNOS, IL-6, IN-1β, and TNF-α were detected by western blot analysis. (d) The relative protein expression level of iNOS, IL-6, IN-1β, and TNF-α shown in (c). Data are expressed as the mean ± SD (n = 3).
Fig. 5. (a) NIRF fluorescence imaging of control and various Zn2+ concentrations exposure mice after intratracheal drip injection of DCI-BT (200 μM, 40 μL) for 30 min. λex/em = 535/650 nm. (b) Photographs and fluorescence imaging of dissected major organs (heart, liver, spleen, lung, kidney) from mice in plane (a). (c) H&E staining of lung tissue in Zn2+ exposure-induced ALI model. Sections of lung tissue were immunostained with an antibody that detected 3-NT. (d) Quantification of average FL intensities in plane (a). (e) Lung wet/dry ratio in control and Zn2+ exposure mice. (f) Proportion of 3-NT positive area in control and Zn2+ exposure mice. Bars represented mean ± S.D. (n = 3). Statistical analysis was performed using a one-way ANOVA and multiple comparison test of significant differences (*P<0.05, **P <0.01, ***P <0.001).
Fig. 6. (a) NIRF fluorescence imaging of control and high-dose Zn2+ exposure mice at different times after intratracheal drip injection of DCI-BT (200 μM, 40 μL) for 30 min. λex/em = 535/650 nm. (b) Photographs and fluorescence imaging of dissected major organs (heart, liver, spleen, lung, kidney) from mice in plane (a). (c) Quantification of average FL intensities in plane (a). (d-f) Serum levels of inflammatory factors IL-6, IL-1β, and TNF-α in control and Zn2+ exposure mice. (g) Different concentrations of Zn2+ exposure induced the protein levels of COX-2, IL-6, and IL-1β in mice lung tissues. Statistical analysis was performed using a one-way ANOVA and multiple comparison test of significant differences (*P<0.05, **P <0.01, ***P <0.001, ****P <0.0001).
Conclusion
In conclusion, a novel NIRF probe DCI-BT for ONOO- was successfully designed and synthesized through the condensation and dehydration reaction of DCI-CHO with 2-hydrazinobenzothiazole. The fluorescence of DCI-BT was dramatically enhanced upon the oxidative cleavage of imine moiety by ONOO-, which mainly originated from the release of fluorophore DCI-COOH. The highly sensitive nature of DCI-BT, with its near-infrared fluorescence capabilities, allowed for real-time, non-invasive imaging of ONOO-. One of the most promising clinical applications of DCI-BT was in the early diagnosis of ALI, particularly in patients exposed to environmental pollutants like Zn2+. ONOO- in lung tissues could serve as a biomarker for oxidative stress-induced lung injury. In the Zn2+-induced cell model, the level of ONOO- was significantly up-regulated, and DCI-BT allowed dynamic visual monitoring of this process. We found that Zn2+-exposed mice induced ALI. Leveraging DCI-BT, we confirmed that i) the degree of lung injury in mice treated with high-dose Zn2+ was significantly higher than that in mice treated with low-dose Zn2+, ii) the degree of Zn2+-induced lung injury was positively correlated with the level of ONOO-, and iii) SFN had a protective effect against ALI induced by Zn2+ exposure. These findings illustrated the importance of DCI-BT as a promising chemical tool in the study of the occurrence and development of Zn2+-induced ALI.
