Abstract
Background
Pulpitis is a prevalent oral disease characterized by severe pain. The activation of microglia in the medullary dorsal horn (MDH) is reportedly essential for the central sensitization mechanism associated with pulpitis. And the P2X7 receptor (P2X7R) on microglia can trigger secretion of exosome enriched in IL-1β, which is involved in the inflammation. Thus, we hypothesized that enhanced exosome secretion regulated by microglial P2X7R in the MDH contributes to pulpitis-induced pain.
Methods
The male SD rats were chosen as experimental animals and the experimental pulpitis model was established to observe the rat’s pain behavior. Immunofluorescence staining, western blot and quantitative real-time PCR, were used to analyze the expression of Rab27a and IL-1β. The exosome inhibitor GW4869 and P2X7R antagonist Brilliant Blue G (BBG) were performed to analyze the correlation between microglial P2X7R, exosome secretion and inflammation in the pulpitis model. In vitro, microglia cell lines were cultured to collect exosomes, and stimulation of lipopolysaccharide (LPS), oxidized ATP (oxATP) and GW4869 detected changes in exosome secretion and inflammatory factors.
Results
In the experimental pulpitis model, the degree of microglial exosome secretion and inflammatory factor release in the MDH was correlated with the degree of pulpitis-induced pain, with the highest expression on the 7th day. GW4869, as well as BBG, could inhibit Rab27a and IL-1β expression, reducing pulpitis-induced pain. In addition, exosomes were successfully extracted by ultracentrifugation in vitro, LPS treatment could promote the exosome secretion, while GW4869 had an opposite role on the secretion of exosomes and inflammatory factor IL-1β. Moreover, P2X7R inhibition by oxATP also diminished exosome secretion, leading to a reduction in inflammatory responses.
Conclusion
This study indicates the regulatory role of microglial P2X7R in increased exosome secretion, implicating the potential utility of P2X7R as a promising target for pulpitis therapy. And our research provides a new pulpitis mechanism that exosomes enriched in IL-1β contributed to pulpitis-induced pain, suggesting the crucial role of exosomes as pain biomarkers and harmful signal bearers in pulpitis.