A novel fluorescent probe for detecting hydrogen sulfide in osteoblasts during lipopolysaccharide-mediated inflammation under periodontitis

Periodontitis, one of the most common chronic inflammatory diseases, affects the quality of life. Osteogenesis plays an important role in the disease. There is a connection between hydrogen sulfide (H2S) and periodontitis, but according to the study has been published, the precise role of H2S in inflammation remains in doubt. The main reason for the lack of research is that H2S is an endogenous gasotransmitter, difficult to discern through testing. So, we synthesized a novel fluorescence probe which can detect H2S in vitro. By using the novel H2S fluorescence probe, we found that H2S changes in osteoblasts mainly by cystathionine-γ-lyase, and H2S increases under LPS stimulation. H2S could be a potential marker for diagnosis of inflammatory diseases of bone, and might help deepen studies of the changes of H2S level and promote the progression on the researches about pathogenesis of periodontitis.

www.nature.com/scientificreports/ by regulating oxidative stress, mitochondrial function, and inflammation. It also has the ability to potentially prevent bone loss in periodontitis 10 . So, there is a connection between H 2 S and periodontitis, but until now, the precise role of H 2 S in inflammation remains unknown.
Most of the studies focus on the effect of the H 2 S, not many about H 2 S changes under stimulation. Researchers often use Western blot, immunohistochemical staining, and some other methods to detect the H 2 S changes indirectly. Recently, there are some direct techniques to detect H 2 S, such as chromatography, electrochemistry and colorimetry 11 . But a technique that can detect H 2 S directly in living cells is still needed. H 2 S-fluorescence probe, which is high-speed developing, is considered as one of the most helpful instrument areas in the field of H 2 S biology 12 . In recent years, many excellent fluorescent probes have been designed and synthesized throughin-depth-analysis of the structural features of biothiols 13,14 . We previously developed a H 2 S probe, which consists of a 1,8-naphthalimide as fluorophore and azido moiety as recognition site. The introduction of the electronwithdrawing azido group changes the push-pull system and quenches the fluorescence. It is noteworthy that the reaction is easy to carry out and the yield is high. When the probe reacts with hydrogen sulfide, the azido moiety is reduced to an amino group. Because the amino group acts as an electron-donating group, the effect of intramolecular charge transfer is enhanced, and the fluorescence is recovered (Fig. 1a). The probe is able to directly measure the real time H 2 S level in living cells. Overall, because of high resolution and sensitivity of the H 2 S probe make it a helpful tool. There are some studies showing that H 2 S fluorescence probe can detect endogenous H 2 S in real-time and in situ. However, most of them use tumor cells instead of somatic cells, if the probe could be used in somatic cells, it can broaden diagnose and treatment applications of H 2 S. By using a novel H 2 S fluorescence probe, we found that H 2 S changes in osteoblast mainly by CSE, and H 2 S increases under LPS stimulation.

Materials and methods
Regents. The hydrogen sulfide fluorescent probe was provided by Professor Baocun Zhu (School of Water Conservancy and Environment, University of Jinan, Jinan, China). 1 mg probe was dissolved in 100 μL dichloromethane, then was diluted with DMSO (Sigma-aldrich, USA) to a final concentration of 1 mM. α-MEM was used to dilute the mother liquor to get different concentrations. The test concentration was 10 μM and the experiment was carried out at room temperature (25 ℃).
Application of H 2 S probe to access exogenous H 2 S levels. The cells were pre-treated with NaHS (50, 100, 150, 500 μM) for 30 min, then, treated with the H 2 S probe (10 μM) for 30 min. Fluorescence and bright field images were collected after PBS washing for three times. Green fluorescence was observed under the confocal www.nature.com/scientificreports/ microscope at excitation wavelengths of 405 nm. In order to control exposure, Smart Gain was kept at the same voltage in every photographs.

Application of H 2 S probe to access endogenous H 2 S levels.
In the periodontium of mammalian host, H 2 S is produced using Cys mainly by CSE and CBS. The cells were pre-treated with Cys (100 μM, 200 μM) for 30 min, then, treated with the H 2 S probe (10 μM) for 30 min. Fluorescence and bright field images were collected after PBS washing for three times. Green fluorescence was observed under the confocal microscope at excitation wavelengths of 405 nm. In order to control exposure, Smart Gain was kept at the same voltage in every photographs. PAG is an irreversible inhibitor of CSE. It can block the produce of endogenous H 2 S in MC3T3-E1. Therefore, we pre-treated cells with 50 μM PAG, 30 min, then cells were treated with or without Cys for 30 min. Last, fluorescence was examined as before, Smart Gain was kept at the same voltage in every photographs.
Addition of lipopolysaccharide (LPS) for inducing inflammation and assessment with H 2 S probe: The cells were incubated with 1, 2 μg/mL LPS for one day. Subsequently, the culture dish was washed with PBS for three times and incubated with 10 μM probe for 30 min. Then, the cells were washed with PBS, then the fluorescence imaging was examined by confocal microscope, Smart Gain was kept at the same voltage in every photographs.

Results
Probe spectra and toxicity analysis: As shown in Fig. 1a, the probe itself had almost no fluorescence, but it showed a significant fluorescence enhancement after the addition of H 2 S (100 μM). The cell's viable and healthy during the detection is a key concern. Figure 1b showed that cell viability was almost not affected by the probe at 10 μM. Toxicity is mainly introduced by solvent, DMSO and dichloromethane. The result verify that the H 2 S probe is harmless to the cell. Thus, the H 2 S probe can be used in living cells for fluorescence imaging analysis.
Cell fluorescence imaging of different concentrations exogenous H 2 S: As shown by Fig. 2, with the different concentrations (0, 50, 100, 150, 500 μM) of NaHS, a gradual increase of intensive green fluorescence was observed using 405 nm as an excitation wavelength. Consistent with previous studies, the amount of H 2 S is one third of exogenous of NaHS. Thus, the probe was estimated detection of accuracy to 10 μM. Fluorescent intensity is stable during the progress of taking pictures under the confocal laser scanning microscopy. That indicated that www.nature.com/scientificreports/ our probe is sensitive to H 2 S, and it also prove that H 2 S probe was cell membrane permeable and can be used in the normal cells for detecting intracellular H 2 S. Cell fluorescence imaging of endogenous H 2 S: According to the previous research, for osteoblasts, CSE-H 2 S might be the major path for the H 2 S produced 13 . As shown by Fig. 3, the incubation of cells with 100 μM Cys produced intensive green fluorescence, but the fluorescence decreased when cells were incubated with 200 μM Cys. That means that low dose of Cys could increase H 2 S production, but high dose of Cys inhibited H 2 S production. In order to verify whether the CSE-H 2 S pathway is the main pathway to produce the H 2 S, we used PAG as the irreversible inhibitor to CSE. Figure 3 showed that the intensity of fluorescence was decreased, which means the H 2 S was decreased, because of the pretreatment of the inhibitor, and the intensity of PAG group was as weak as the control group, indicating that the production of endogenous H 2 S was significantly inhibited with CSE inhibitor.
Cell fluorescence imaging of LPS induced endogenous H 2 S: when cells were treated with LPS (2 μg/mL) to produce inflammation, as shown by Fig. 4, intensive green fluorescence were produced compared to the control group. This indicates that when inflammation occurs, a lot of H 2 S was produced. In other words, the increase of H 2 S level can serve as an indicator for cells that are under the inflammation state. The production of endogenous H 2 S induced by lipopolysaccharide-mediated inflammation was successfully monitored with this H 2 S probe.

Discussion
The main aim of the experiment is to solve the problem of detection of the inflammation of osteoblast, furthermore, we found that H 2 S produced by osteoblast is mainly via CSE-H 2 S pathway. In our study, we proved that our probe can be used in the normal cell to detect the H 2 S changes, which is rarely studied. There are already a lot of fluorescent probes that have been devised to detect intracellular H 2 S levels, however, to our knowledge, most of these probes were successfully applied to show alteration of H 2 S levels of tumor cells or living animals [15][16][17] .   18 . This is consistent with our results. For the inflammation of bone, there are two proved sources of H 2 S: bacteria and macrophage. When inflammation occurs, some bacteria produced and released H 2 S, including various common gram-negative pathogens in osteomyelitis such as Escherichia coli, Enterococcus faecalis, Enterobacter cloacae, and Klebsiella Pneumoniar. For macrophage, research shows that the level of H 2 S was improved and the expression of CSE mRNA increased because of the stimulate of LPS 19 . Our study shows that osteoblasts is the third source of H 2 S. Different sources of H 2 S might have interaction effect, for example, H 2 S production by osteoblast might modulate macrophage polarization and contribute to bone reparation. Keeping physiological level of endogenous H 2 S in PDLSCs/ periodontal tissue is beneficial to maintain the homeostasis of periodontal tissue 20 . An appropriated level of H 2 S may play a vital role in maintaining the homeostasis of the bone marrow system. A previous study has clarified that BMSCs can produce H 2 S, regulate osteogenic differentiation and cell self-renewal, and that the lack of H 2 S could lead to defects in their differentiation 21 . Exogenous H 2 S could protect cell injury by regulating oxidative stress, mitochondrial function, and inflammation. While when inflammation occurs, H 2 S from bacteria disturbs the endogenous H 2 S of osteoblast cells, leads to a negative effect. In periodontitis studies, drugs that can release H 2 S have been used for the treatment, such as ATB-352, a kind of ketoprofen that can releasing H 2 S. The main aim is to minimize the presence of side-effect at the gastrointestinal tract. Meanwhile they found that the reduction of the inflammation even had a beneficial effect on bone resorption or tissue damage. ATB-346, releasing H 2 S like ATB-352, is beneficial for improving bone quality too 10 . Since H 2 S also can promote the development of periodontitis, there are still many questions about the biological mechanisms of H 2 S. It is well-know that there are many kinds of cell playing important roles in periodontitis, such as periodontal ligament stem cells, osteoclasts, and immune cells. Independent detection of H 2 S changes in living cell might facilitate the study of the role of H 2 S in diseases.
It was found previously that CBS and CSE were both increased in human gingival tissue during periodontitis through the technology of PCR and Western blot. However, H 2 S level or H 2 S synthesis in gingivitis and periodontitis was detected not increase after tissue homogenate 22 . This can be problematic for many reasons, such as the synthesis capacity decreased or consume increased of H 2 S in inflammation. But as a gasotransmitter, half of H 2 S can escape from medium in five minutes in tissue culture wells, which makes it hard to detect 23 . Under physiological conditions, H 2 S presents in three chemical ionization forms, about 18.5% H 2 S, 81.5% HS − and minute quantities of S 2−24 . Different detection methods might lead to different results. H 2 S is more permeable in plasma membranes, the solubility of H 2 S in lipophilic solvents is quintuple greater than in water 25 , thus, fluorescence probe in theory could detect H 2 S more precisely. Our H 2 S probe might help deepen studies of the changes of H 2 S level and promote the progression on the researches about pathogenesis of periodontitis.
Fluorescence techniques is gaining widespread attention as sensors offering excellent sensitivity, good selectivity, and rapid response to changes. First of all, our probe has been shown to be sensitive for endogenous H 2 S detection and real-time monitoring of the changes in H 2 S in living cells, and it reacts quickly under physiological conditions. There are some things that can be improved, for example, a more precise target of probes to certain subcellular organelles, certain cells, tissues, or organs, which may be achieved by using near-infrared emit to get a greater tissue penetration and minimize the interference from background auto-fluorescence 26 . The probe might be improved, like detect Hcy/Cys/GSH/H 2 S at the same time 27 . For clinical use, H 2 S has a potential to www.nature.com/scientificreports/ be used as an appropriate biomarker for the related investigations of inflammation response. However, it still requires further development.

Conclusion
In conclusion, it is the first experiment using H 2 S probe to detect H 2 S changes under stimulation in osteoblast in real time. We used a new hypotoxic H 2 S probe for exogenous and endogenous H 2 S detection in living osteoblast cells. Moreover, the results indicate that in osteoblast cells, H 2 S is produced mainly by CSE-H 2 S pathway directly, it also shows that under inflammation stimulation, endogenous H 2 S production will increase. The results suggest that H 2 S could be a potential marker for diagnosis of inflammatory diseases of bone, and it might help further studies for understanding the synthesis and change of H 2 S level in pathogenesis of periodontal disease.

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.