A near-infrared ratio fluorescent probe achieves mitochondrial hydrogen polysulfide imaging for monitoring ferroptosis in arthritis

Abstract

Reactive sulfur (RSS) is a type of sulfur-containing molecule widely present in biological systems. Hydrogen polysulfide (H₂S_n, n > 1), as a member of the active sulfur family, plays an indispensable role in many physiological and pathological processes. Ferroptosis is a special cell death mode driven by iron-dependent lipid peroxidation, which is involved in the occurrence and development of various human diseases. Ferroptosis is manifested by increased lipid peroxidation and elevated levels of reactive oxygen species (ROS), which further lead to an increase in H₂S_n content in cells. Emerging research suggests a close association between ferroptosis and arthritis related diseases. This work successfully constructed a mitochondrial-targeted ratiometric near-infrared fluorescent probe for the specific detection of H₂S_n. The experimental results show that the probe (Cy-S₄) has a large Stokes shift (∼218 nm), excellent optical properties, extremely fast response time (8 s), high sensitivity (DL = 0.23 μM), and strong specificity. This probe has been successfully applied to tracking the content of H₂S_n in ferroptosis process and fluorescence imaging of H₂S_n in inflammatory cell mitochondria. Pathological section data confirmed that the probe has good in vivo imaging ability, and more importantly, in vivo arthritis imaging experiments showed that the expression of H₂S_n plays an important role in ferroptosis. These experimental results will provide a reliable monitoring tool for the treatment and prevention of arthritis, enriching the theoretical research related to this disease.

1. Introduction

Active sulfur species (RSS), as a key class of sulfur-containing active molecules in living organisms, have gradually become a hot topic in recent years. Activated sulfur not only regulates cellular activity but also acts as an antioxidant in various cellular signaling pathways, which is crucial for maintaining normal physiological functions of organisms. Among these RSS, H₂S, as one of the most studied RSS, is considered the third key gas molecule after nitric oxide (NO) and carbon monoxide (CO) [[1], [2], [3], [4], [5], [6], [7], [8]], is closely related to various pathological processes such as cancer, Alzheimer's disease, inflammation, etc [[9], [10], [11], [12], [13], [14]]. In contrast, hydrogen sulfide (H₂S_n, n > 1) has received much less attention. However, recent current research has shown that the important endogenous gas signaling molecule is H₂S_n rather than H₂S, and H₂S_n has a higher protein thiolation efficiency [[15], [16], [17], [18], [19]].

The initial research on H₂S_n originated from the pioneering work of Ma et al.'s in 2014, which used 2-fluoro-5-nitrobenzoate as the recognition group [17]. With the continuous deepening of research on H₂S_n, it has been found that H₂S_n plays a crucial role in cell protection, signal transduction, anti-cancer and anti-inflammatory effects, redox reactions, and other aspects [15,[20], [21], [22], [23], [24]]. Therefore, the detection of H₂S_n has also become a major topic in the biomedical field. However, although many fluorescent probes have been reported for detecting H₂S_n, there are few near-infrared ratio type fluorescent probes related to H₂S_n [[25], [26], [27], [28], [29], [30], [31], [32], [33]]. Therefore, designing near-infrared ratio type fluorescent probes targeting H₂S_n, can avoid interference from its own background [23,[34], [35], [36]], further achieving dynamic monitoring and imaging of H₂S_n in living organisms.

The concept of ferroptosis was first proposed by Stockwell et al., in 2012. It is a cell death mode caused by iron dependent lipid peroxidation and the accumulation of reactive oxygen species [[37], [38], [39], [40], [41], [42]], and has received increasing attention in the past decade. Ferroptosis is closely related to various diseases, such as tumor suppression, cardiovascular disease, hepatitis, neuropathy, and urinary system diseases [[42], [43], [44], [45]]. Previous reports have shown that during the process of ferroptosis, the excessive accumulation of iron leads to the overproduction of lipid peroxidation, which typically results in an increase in intracellular ROS levels [41,43,[46], [47], [48]]. Excessive ROS oxidizes H₂S in cells to generate H₂S_n, leading to an increase in the content of H₂S_n in cells. By monitoring the content of H₂S_n in cells, the molecular physiological mechanism of ferroptosis can be better studied. It is worth mentioning that increasing evidence suggests a close relationship between iron death and various arthritis diseases [[49], [50], [51], [52]]. Excessive iron can accelerate chondrocyte apoptosis, affect the homeostasis of chondrocytes, and induce the occurrence of osteoarthritis [53,54]. Therefore, there is an urgent need to develop a fluorescent probe that can monitor the H₂S_n content in ferroptosis models in real time. In-depth study of its mechanism of action will open up more new avenues and targets for the treatment of arthritis.

Due to its near-infrared absorption and emission, high molar absorptivity and fluorescence quantum yield, large Stokes shift, excellent photostability, low biological toxicity, and good biocompatibility, the semi cyanine structure is often used to develop fluorescent probes with excellent properties. Based on the above requirements, this article designs a near-infrared ratio fluorescent probe Cy-S₄ that can target mitochondria for imaging H₂S_n in and out of cells in vivo and in vitro during the ferroptosis process [[55], [56], [57], [58]]. As shown in Scheme 1, Cy-S₄ has a good proportional fluorescence effect. After reacting with H₂S_n, the probe undergoes a special addition reaction, causing the C N⁺ double bond to break and the fluorescence emission peak to shift from 728 nm blue to 530 nm, with a significant Stokes shift (∼218 nm). In addition, the probe has a very wide linear response range (0–400 μM), a low limit of detection (0.23 μM), and a fast response time (8 s). The results of cell imaging experiments showed that the addition of erastin (ferroptosis inducer) can promote the overexpression of H₂S_n in cells, and the addition of Fer-1 (ferroptosis inhibitor) can lead to a significant decrease in H₂S_n content in cells. We conducted an imaging study of H₂S_n in arthritis induced by ferroptosis by constructing an inflammatory mouse model. The use of Cy-S₄ to trace H₂S_n during ferroptosis provides new prospects for the diagnosis and treatment of arthritis.

Scheme 1.

A near-infrared ratio fluorescent probe achieves mitochondrial hydrogen polysulfide imaging for monitoring ferroptosis in arthritis.

2. Experimental

2.1. Reagents and methods

In the spectral experiment, fluorescence data were measured at an excitation wavelength of 510 nm, and the spectrum was scanned in the range of 520 nm–850 nm. The excitation and emission slit widths were both 3.00 nm. This work uses Na₂S₄ as a direct source for detecting polysulfides (H₂S₄), and dissolves Na₂S₄ in distilled water to prepare the test substance (concentration of 2 mM) and dissolves the probe in DMSO to prepare the mother liquor (concentration of 2 mM). All spectral testing experiments were conducted at room temperature using PBS buffer as the solvent system.

2.2. The synthesis route of Cy-S₄

The synthesis route of probe Cy-S₄ is shown in Scheme 2, and the detailed experimental steps are described in the supporting information. The structural characterization of the probe is shown in Figures S8-S10.

Scheme 2.

Synthesis step of the probe Cy-S₄ and its mechanism of interaction with H₂S_n.

Cy-S₄: ¹H NMR (600 MHz, DMSO-d₆) δ 8.56 (d, J = 14.9 Hz, 1H), 7.79–7.72 (m, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.52 (s, 2H), 7.48–7.40 (m, 2H), 6.94 (s, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.50 (d, J = 14.9 Hz, 1H), 3.85 (s, 3H), 2.70 (d, J = 29.7 Hz, 4H), 1.83 (s, 2H), 1.75 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆) δ 160.54, 158.81, 155.62, 143.19, 141.63, 135.93, 130.15, 129.92, 129.04, 126.01, 122.89, 115.22, 114.63, 112.27, 106.60, 102.83, 102.53, 102.27, 99.98, 49.74, 32.28, 30.12, 28.51, 27.97, 24.17, 20.72. MS: Calculated for C₂₆H₂₆NO₂⁺ [M]: m/z 384.20; found [M]: m/z 384.25. m.p. 193–196 °C.

Cy-S₄+H₂S₄: ¹H NMR (600 MHz, DMSO-d₆) δ 8.29 (s, 1H), 8.08 (d, J = 18.8 Hz, 2H), 7.76 (s, 1H), 7.68 (s, 1H), 7.54 (s, 1H), 7.41 (s, 2H), 6.80 (s, 1H), 6.75 (s, 1H), 2.65 (s, 2H), 2.62 (s, 2H), 1.94 (s, 6H), 1.79 (s, 2H), 1.18 (s, 3H), 0.80 (s, 1H).

3. Results and discussion

3.1. Spectral sensing properties of H₂S₄ probe

In order to evaluate the optical properties of the probe Cy-S₄, UV visible absorption and fluorescence spectra of different concentrations of H₂S₄ before and after reaction with Cy-S₄ were studied in PBS buffer. Fig. 1a shows that the maximum absorption peak of Cy-S₄ is at 685 nm. With the addition of H₂S₄ at different concentrations (0–200 μM), the absorption peak of the solution undergoes a blue - shift to around 365 nm. At the same time, as the concentration of H₂S₄ increases, the absorption at this peak gradually increases, while the absorbance of the probe around 685 nm decreases. The data in Fig. S1 show that under excitation light of 510 nm, the fluorescence intensity of the probe does not change with time within 1800 s, indicating good fluorescence stability of the probe. The fluorescence spectrum in Fig. 1b shows that Cy-S₄ emits bright near-infrared fluorescence in PBS. When H₂S₄ is added, the fluorescence of Cy-S₄ gradually decreases at around 728 nm, and the fluorescence emission at 530 nm is significantly enhanced. This is attributed to the breaking of the C N⁺ double bond in the probe molecule structure, which results in a smaller conjugated structure, a significant blue - shift in the fluorescence peak, a significant ratio signal, and a large Stokes shift (∼218 nm). The linear relationship between the fluorescence intensity of the probe at I_{728 nm}/I_{530 nm} and the concentration of H₂S₄ was analyzed. From Fig. 1c, it can be seen that when the concentration of H₂S₄ is between 0 and 400 μM, there is a good linear relationship between the fluorescence intensity and H₂S₄ concentration (correlation coefficient R² = 0.98), and the calculated detection limit is 0.23 μM. Compared with the H₂S₄ fluorescent probe reported in Table S1, the above experimental results indicate that the probe has excellent characteristics such as a wide linear range and low detection limit.

Fig. 1.

(a) UV visible absorption spectra before and after the reaction between Cy-S₄ and H₂S₄; And fluorescence spectrum of (b); (c) The fluorescence linear correction curve; (d) Time dependence curve of the interaction between Cy-S₄ and H₂S₄; (e) Selective curves of Cy-S₄ for various biomolecules; (f) The anti-interference experiment of Cy-S₄ with 31 analytes; (0)Blank, 1)Na⁺, 2)K⁺, 3)Ca²⁺, 4)Zn²⁺, 5)Mn²⁺, 6)Mg²⁺, 7)Cu²⁺, 8)Ba²⁺, 9)Fe²⁺, 10)Cd²⁺, 11)Fe³⁺, 12)Al³⁺, 13) Cl⁻, 14) I⁻, 15)S^2-, 16)NO₃⁻17)CO₃^2-, 18)SO₃^2-, 19)SO₄^2-, 20)H₂S, 21)GSH, 22)Cys, 23)Hcy, 24)H₂O₂, 25)·OH, 26)¹O₂, 27)O₂⁻, 28)NO₂⁻, 29)NO, 30)ONOO⁻, 31)H₂S₄). Cy-S₄ = 10 μM, λ_ex = 510 nm.

3.2. Study on the selectivity and response time of Cy-S₄ towards H₂S₄

Fig. 1d shows that in the absence of H₂S₄, the fluorescence intensity of the probe remained almost unchanged within 1 min, indicating that the probe itself has good stability and is not easily affected by environmental factors that cause spontaneous fluorescence changes. As the concentration of H₂S₄ increases, the ratio of fluorescence intensity also significantly increases. This proves that there is a specific reaction between the probe Cy-S₄ and H₂S₄, resulting in a change in the fluorescence signal that reaches its maximum value around 8 s, indicating that Cy-S₄ has an extremely fast response speed. Compared with the previously reported probe (Table S1), this response time is shorter, indicating that the probe Cy-S₄ has significant advantages in detecting H₂S₄. In summary, the fluorescent probe exhibits ultra-fast detection capability for H₂S₄, demonstrating good stability and high sensitivity. It has potential application value in biomedical fields such as real-time imaging of H₂S₄ metabolism processes in organisms, environmental monitoring, etc.

To test whether Cy-S₄ can specifically and sensitively respond to H₂S₄, the selectivity of Cy-S₄ towards H₂S₄ in PBS buffer system was evaluated. As shown in Fig. 1e, Cy-S₄ was reacted with various types of analytes and their fluorescence changes were observed. The measurement results show that there is no significant change in the fluorescence of the system, indicating that these substances, especially sulfur-containing substances, have no significant effect on the fluorescence properties of Cy-S₄. Furthermore, through anti-interference experiments (Fig. 1f), it can be seen more intuitively that the addition of other analytes other than H₂S₄ almost does not cause a change in fluorescence ratio. The results show that only the H₂S₄ solution changes from blue to colorless, and the fluorescence color of the solution also changes significantly compared to other analytes under UV light. This color change not only provides a visual indication for the detection of H₂S₄, but also makes Cy-S₄ a “naked eye” colorimetric sensing probe.

3.3. Study on the reaction mechanism of Cy-S₄ and H₂S₄

Since H₂S₄ is a stronger nucleophile than other biological thiols, it is more likely to react with electron deficient moieties (such as C N⁺). The C N⁺ portion in the Cy-S₄ structure is an active electrophilic center that is susceptible to attack by nucleophilic reagents. Therefore, it is speculated that H₂S₄ has undergone a special nucleophilic addition reaction at the C N⁺ position of the probe's semi cyanine unit. To verify this hypothesis, nuclear magnetic titration experiments were conducted (Fig. S11), and significant changes were observed through the analysis of the hydrogen spectra of Cy-S₄ before and after the addition of H₂S₄. A new proton signal (δ 0.85 ppm) appeared in the spectrum upon the addition of H₂S₄, which was attributed to the hydrogen added to the probe structure by H₂S₄. As shown in the HPLC data of Fig. S6, the retention time of the probe is 2.79 min, and the retention time after reacting with H₂S₄ is 4.75 min, indicating that a new substance was indeed generated after the reaction. And due to the double bond breakage in the probe structure, its polarity decreases and hydrophobicity increases, resulting in an extended retention time of the substance under the same testing conditions. In addition, the mass spectrometry data in Fig. S12 further confirm the mechanism of interaction between the probe Cy-S₄ and H₂S₄. This reaction site not only deepens our understanding of the interaction between the probe and H₂S₄, but also provides new ideas for developing more sensitive and rapid probes.

3.4. Experimental exploration of Cy-S₄ targeting cell mitochondria

Mitochondria are important organelles within cells that play a crucial role in cellular metabolism. By constructing fluorescent probes targeting mitochondria, these probes can be used to study mitochondrial related functions such as energy metabolism and redox reactions. For example, in the process of ferroptosis, the function of mitochondria is severely affected. Mitochondria become smaller and their membrane density increases, which affects their normal energy production function. At the same time, an increase in mitochondrial reactive oxygen species also promotes ferroptosis. By constructing fluorescent probes that can target mitochondria, we can better explore the relationship between mitochondria and ferroptosis. To verify whether the probe Cy-S₄ has the function of localizing to mitochondria, a co-localization experiment was conducted on Cy-S₄ using the commercial mitochondrial localization dye MitoTracker Green. As shown in Fig. 2, cells labeled with Cy-S₄ exhibit red fluorescence, while cells labeled with MitoTracker green exhibit green fluorescence. From the normalized contour lines, it can be seen that the spectral peak positions in the overlapping areas of cells are the same. After analysis using Image J software, the co-localization Pearson coefficient of the probe in mitochondria reached 0.98. This indicates that Cy-S₄ has significant mitochondrial targeting ability.

Fig. 2.

Co-localization imaging of Cy-S₄ in HeLa cell mitochondria. (a) Bright channel; (b) Green channel (Mito-tracker, λ_ex = 488 nm, λ_em = 500–540 nm); (c) Red channel (Cy-S₄, λ_ex = 605 nm, λ_em = 650–750 nm); (d) Overlay channel; (e) Pearson's coefficient; (f) Line profile. Scale bar = 20 μm.

3.5. Confocal Imaging of Cy-S₄ in cells

3.5.1. Confocal imaging of H₂S_n in cell mitochondria by Cy-S₄

Due to the excellent spectral characteristics exhibited in in vitro testing by the probe Cy-S₄, we investigated its ability to detect endogenous and exogenous H₂S_n at the cellular level. Firstly, we determined the cytotoxicity of Cy-S₄ through cell viability experiments. Fig. S2 shows that even when Cy-S₄ at a high concentration of 100 μM, the cell survival rate still exceeds 80 %, indicating that Cy-S₄ has low cytotoxicity. This probe demonstrates good practicality and safety in subsequent cell or in vivo imaging experiments. Fig. S3 shows that the fluorescence signal of the probe in the cell is basically stable. Fig. S4 shows the stability of Cy-S₄'s ability to image H₂S_n. It was found that the ratio of fluorescence signals in the red and green channels remained stable over a period of 30 min, indicating that Cy-S₄ has excellent ability to image H₂S_n. Subsequently, different concentrations of H₂S_n were added to HeLa cells for incubation, and it was observed that as the concentration of H₂S_n increased, the fluorescence of the red channel of the probe gradually decreased, while the fluorescence of the green channel gradually increased (Fig. 3a). This change can also be seen more intuitively in Fig. 3b and c. The above experimental results indicate that the probe can effectively conduct semi quantitative imaging experiments on H₂S_n inside cells using the ratio signal of dual-emission channels, improving the accuracy of fluorescence imaging.

Fig. 3.

(a) Cy-S₄ imaging of exogenous H₂S₄ in HeLa cells; (b) Fluorescence intensity in the yellow and red channels; (c) Fluorescence intensity ratio (I_Green/I_Red); The collected green channel is 550–650 nm, and the red channel is 650–750 nm. The fluorescence intensity data are expressed as mean ± standard deviation. λ_ex = 488 nm, Scale bar: 50 μm.

3.5.2. Confocal imaging of H₂S₄ in cells induced by LPS using Cy-S₄

As is well known, lipopolysaccharide (LPS) induces overexpression of cystathionine lyase (CSE) mRNA to promote the expression of H₂S_n. As shown in Fig. 4a and b, cells treated only with Cy-S₄ exhibit bright red fluorescence, while the cell group pre-incubated with LPS shows a significant decrease in fluorescence in the red channel and a significant increase in fluorescence in the green channel. The fluorescence ratio values of the two channels reached ∼1.7 (Fig. 4c), indicating that LPS indeed induces the production of endogenous H₂S_n in cells, leading to a significant increase in its content in the cells. When cells pretreated with LPS were treated again with the reactive oxygen species inhibitor NAC, the fluorescence of the red channel was restored, while the fluorescence of the green channel was significantly reduced. This phenomenon indicates that NAC effectively inhibits the decrease in reactive oxygen species levels in cells induced by LPS, thereby reducing the content of H₂S_n. When the thiol inhibitor NEM is pre-added to the cells, the red channel shows obvious red fluorescence, while the green channel has weak fluorescence. Next, based on the previous cell experiment, LPS is further added, and cell imaging revealed that both the red and green channels have certain fluorescence signals. The ability of Cy-S₄ to monitor endogenous H₂S_n production in real-time at the cellular level is verified through the above cell confocal imaging experiments, and the effects of LPS and thiol inhibitor NEM on H₂S_n production were explored. This provides a powerful tool for understanding the mechanism of LPS induced endogenous H₂S_n production and developing diagnostic methods for related diseases.

Fig. 4.

(a) Confocal imaging of endogenous H₂S₄ in cells induced by LPS using Cy-S₄; (b) Bar chart of cell fluorescence intensity in two channels; (c) Bar chart of fluorescence intensity ratio of five groups of cells (I_Green/I_Red). The collected green channels are 550–650 nm, and the red channels are 650–750 nm. All fluorescence data in the figure are represented as mean ± standard deviation. λ_ex = 488 nm, Scale bar: 50 μm.

3.5.3. Cell confocal imaging of Cy-S₄ during ferroptosis process

As mentioned in the introduction, there are numerous reports in the literature that ferroptosis is a form of cell death closely related to abnormal iron metabolism. Excessive Fe²⁺ generates a large amount of reactive oxygen species (ROS) through the Fenton reaction. Excessive ROS and H₂S present in cells can react through oxidative processes to generate H₂S_n, while endogenous H₂S_n production mainly occurs in mitochondria, accounting for more than 50 % of the total H₂S_n production in the cell. Therefore, based on the above research background, it is helpful for a deeper understanding of the imaging ability of Cy-S₄ on H₂S_n in cell mitochondria during ferroptosis. Erastin is a traditional inducer of iron toxicity that can inhibit the uptake of cysteine, leading to depletion of cellular GSH and ultimately inducing ferroptosis. As shown in Fig. 5a–c, when cells are treated with erastin, the fluorescence intensity in the red channel decreases significantly, while the fluorescence signal in the green channel increases significantly. Fer-1 is a commonly used potent antioxidant that can effectively eliminate ROS in living cells. After treatment with Fer-1, the fluorescence of the red channel increases and the fluorescence of the green channel decreases significantly, indicating that the inhibitor effectively reduces the expression level of H₂S_n. In summary, Cy-S₄ can effectively monitor the expression of H₂S_n during ferroptosis in real-time, providing a powerful practical tool for studying the physiological mechanisms of H₂S_n during ferroptosis.

Fig. 5.

(a) Cy-S₄ confocal imaging of endogenous H₂S₄ in cells during ferroptosis; (b) Fluorescence intensity bar chart in yellow and red channels; (c) Bar chart of fluorescence intensity ratio in yellow and red channels (I_Green/I_Red). The collected fluorescence range is 550–650 nm for the green channel and 650–750 nm for the red channel. The fluorescence data in the figure are represented as mean ± standard deviation. λ_ex = 488 nm, Scale bar: 50 μm.

3.6. Pathological evaluation of Cy-S₄ in living organs

Fig. S5 shows that during the 7-day experimental period, mice injected with Cy-S₄ showed a smaller change in body weight compared to the PBS control group, indicating that Cy-S₄ had no significant effect on the physiological status of mice. Through H&E staining analysis of the heart, liver, spleen, lungs, and kidneys, it was found that there was no significant difference in tissue morphology between the Cy-S₄ group and the blank group, and no obvious signs of tissue damage or inflammation were observed. Based on weight changes and histopathological results, Cy-S₄ showed good biocompatibility in mice and did not cause significant toxic reactions. Therefore, Cy-S₄ is suitable for long-term in vivo imaging studies. These findings provide reliable safety support for the further application of Cy-S₄, indicating its potential value in in vivo imaging research (see Fig. 6).

Fig. 6.

Pathological evaluation of Cy-S₄ tissue sections in mice, scale bar: 200 μm.

3.7. Imaging of Cy-S₄ in arthritis mice associated with ferroptosis process

As mentioned in the introduction, H₂S₄ is closely associated with arthritis caused by ferroptosis. Therefore, the study further systematically evaluated the imaging performance of Cy-S₄ in arthritis models associated with ferroptosis. The experiment was divided into five groups: blank group (PBS), control group (Cy-S₄), experimental group (LPS + Cy-S₄), inflammation inhibitor group (NEM + Cy-S₄), and ferroptosis inhibitor group (LPS + Fer-1 + Cy-S₄). Experimental data analysis shows that the blank group has no specific fluorescence interference. The fluorescence intensity of the control group was stronger than that of the blank group and reached its peak at 20 min, indicating that Cy-S₄ has good enrichment ability in this tissue. In the LPS induced arthritis model, the fluorescence signal is significantly enhanced and reaches its maximum value at around 20 min. This indicates that an increase in reactive oxygen species (ROS) or lipid peroxidation products during ferroptosis leads to upregulation of H₂S₄ expression, thereby significantly activating the fluorescence response of Cy-S₄. The fluorescence intensity of the inflammation inhibitor group decreased by half compared to the experimental group, indicating that NEM effectively suppressed the expression level of H₂S₄ in mice. In the final group of ferroptosis inhibitors, the fluorescence intensity was significantly reduced compared to the experimental group, confirming that Fer-1 alleviated LPS induced upregulation of H₂S₄ expression by inhibiting ferroptosis related inflammatory pathways, effectively blocking probe activation. These imaging data can also be visually observed from the fluorescence intensity histogram in Fig. 7b. In order to more comprehensively and accurately verify the reliability of the in vivo fluorescence experiment results, we further tested ferroptosis-related biomarkers (GSH, MPO) and detected three inflammatory-factor-related pathological and biochemical indicators (TNF-α, IL-6, and 1L-1β) in LPS-induced arthritis using ELISA (Fig. S7). These experimental parameters confirm the successful construction of an inflammatory model related to ferroptosis. The above results indicated that Cy-S₄ efficiently and accurately monitored the expression level of H₂S₄ in arthritis models. The intervention effect of Fer-1 further clarifies the regulatory mechanism of ferroptosis in this disease, providing an effective monitoring and imaging tool for the diagnosis of arthritis related to ferroptosis.

Fig. 7.

Imaging of Cy-S₄ in mice with arthritis. (a) Blank group: Subcutaneous injection of PBS in mice for imaging; Control group: Mice were subcutaneously injected with Cy-S₄ for imaging; Inflammation group: Mice were pretreated with LPS subcutaneously for 14 h, followed by imaging with Mito-S4 injection; Ferroptosis inhibitor group: Mice were subcutaneously injected with LPS for 14 h, followed by further subcutaneous injection of Fer-1 for 3 h, and then injection of Cy-S₄ for imaging. (b) Histograms of fluorescence intensity of four groups of mice in (a) at different times. All fluorescence data in the figure are represented as mean ± standard deviation.

4. Conclusion

In summary, based on the nucleophilic addition mechanism, we have designed an accurate and sensitive near-infrared ratio fluorescent probe Cy-S₄ for mitochondrial targeting in response to H₂S₄. The probe has a large Stokes shift of ∼218 nm, and after interacting with H₂S₄, it emits strong red fluorescence in the near-infrared region at 728 nm, which gives it the advantages of strong tissue penetration and low background interference. Cy-S₄ can respond sensitively (DL = 0.23 μM) and specifically to H₂S₄ within 8 s. Cy-S₄ not only efficiently monitors and images the expression levels of H₂S₄ during ferroptosis, but also successfully verifies its ability to track H₂S₄ in real-time in LPS induced inflammatory cells. Even better, Cy-S₄ demonstrated excellent imaging performance in arthritis tissues associated with ferroptosis due to H₂S₄ overexpression. This provides a visualization tool for further research on ferroptosis in the pathogenesis of arthritis and lays an experimental foundation for optimizing treatment strategies.

CRediT authorship contribution statement

Ting Cao: Writing – review & editing, Writing – original draft, Visualization, Supervision, Methodology, Funding acquisition, Data curation, Conceptualization. Ziwen Xiao: Methodology, Formal analysis, Data curation, Conceptualization. Wenhua Dong: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Hong Ma: Writing – original draft, Software, Methodology, Funding acquisition, Data curation. Deyan Gong: Supervision, Funding acquisition, Data curation, Conceptualization. Zhefeng Fan: Supervision, Resources, Funding acquisition.

Ethics approval

This work has received approval for research ethics from Hefei University of Technology and a proof/certificate of approval is available upon request.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: