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

Abstract

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.

Figure 1

(a) The spectral changes of the probe before and after the reaction with H₂S. (b) Toxicity analysis.

Figure 2

Cell fluorescence imaging of different concentrations of exogenous H₂S (magnification 10). (a–e) Fluorescence imaging of cells incubated with different concentration of NaHS (0, 50, 100, 150, 500 μM) and probed by the H₂S probe (10 μM) for 30 min. (f) Fluorescence intensity analysis.

Figure 3

Laser confocal microscope images of H₂S probe in MC3T3-E1 cells (magnification 10). Cell fluorescence imaging of endogenous H₂S. (a) Fluorescence image of MC3T3-E1 cells incubated with Cys 100 μM for 30 min, then incubated with H₂S probe (10 μM) for 30 min. (b) Fluorescence image of MC3T3-E1 cells incubated with Cys 200 μM for 30 min, then incubated with H₂S probe (10 μM) for 30 min. (c) pre-treated MC3T3-E1 cells with 50 μM PAG for 30 min, then cells were treated with or without Cys for 30 min, then incubated with H₂S probe (10 μM) for 30 min. (d) Fluorescence intensity analysis.

Figure 4

Cell fluorescence imaging of LPS induced endogenous H₂S. (a) LPS 0 μM as control group. (b) Fluorescence image of MC3T3-E1 cells incubated with LPS (2 μM) for 30 min, then incubated with H₂S probe (10 μM) for 30 min. (c) Fluorescence intensity analysis.