Zhang Jing, Yu Zhuo, Wang Mingjun, Kang Xiaoning, Wu Xiaoke, Yang Fengjiao, Yang Lu, Sun Shukai, Wu Li-An
State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi Clinical Research Center for Oral Diseases, Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi'an, 710032, China.
Cell Biosci. 2025 Feb 22;15(1):28. doi: 10.1186/s13578-025-01363-4.
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. The P2X7 receptor (P2X7R) on microglia can trigger the secretion of exosomes enriched with IL-1β, which is involved in inflammation. Thus, we hypothesized that the enhanced exosome secretion regulated by microglial P2X7R in the MDH contributes to pulpitis-induced pain.
An experimental pulpitis model was established in male SD rats to observe pain behaviors. Immunofluorescence staining, western blotting and quantitative real-time PCR were used to analyze the expression of IL-1β and Rab27a, a key protein secreted by exosomes during nociceptive processes. The effects of the exosome inhibitor GW4869 and the P2X7R antagonist Brilliant Blue G (BBG) on microglial P2X7R, exosome secretion and inflammation in the pulpitis model were analyzed. In vitro, microglial cells were cultured to collect exosomes, and stimulation with lipopolysaccharide (LPS), oxidized ATP (oxATP) and GW4869 altered the secretion of exosomes containing IL-1β.
In the experimental pulpitis model, the microglial exosome secretion and inflammatory factor release in the MDH were both correlated with the extent of pulpitis-induced pain, with the highest expression occurring on the 7th day. GW4869 and BBG inhibited Rab27a and IL-1β expression, reducing pulpitis-induced pain. In addition, exosomes were successfully extracted by ultracentrifugation in vitro, wherein LPS treatment promoted exosome secretion but GW4869 had the opposite effects on the secretion of exosomes and the IL-1β. Moreover, P2X7R inhibition by oxATP diminished exosome secretion, leading to a reduction in inflammatory responses.
This study highlights the regulatory role of microglial P2X7R in increased exosome secretion, indicating the potential utility of P2X7R as a promising target for pulpitis therapy. Our research highlights a new pulpitis mechanism in which exosomes enriched with IL-1β contribute to pulpitis-induced pain, suggesting the crucial roles of exosomes as pain biomarkers and harmful signaling molecules during pulpitis.
牙髓炎是一种以剧痛为特征的常见口腔疾病。据报道，延髓背角（MDH）中小胶质细胞的激活对于与牙髓炎相关的中枢敏化机制至关重要。小胶质细胞上的P2X7受体（P2X7R）可触发富含白细胞介素-1β（IL-1β）的外泌体分泌，IL-1β参与炎症反应。因此，我们推测MDH中小胶质细胞P2X7R调节的外泌体分泌增强促成了牙髓炎诱发的疼痛。
在雄性SD大鼠中建立实验性牙髓炎模型以观察疼痛行为。采用免疫荧光染色、蛋白质免疫印迹法和定量实时聚合酶链反应分析IL-1β和Rab27a（伤害感受过程中外泌体分泌的一种关键蛋白质）的表达。分析外泌体抑制剂GW4869和P2X7R拮抗剂亮蓝G（BBG）对牙髓炎模型中小胶质细胞P2X7R、外泌体分泌和炎症的影响。在体外，培养小胶质细胞以收集外泌体，并用脂多糖（LPS）、氧化三磷酸腺苷（oxATP）和GW4869刺激来改变含IL-1β的外泌体分泌。
在实验性牙髓炎模型中，MDH中小胶质细胞外泌体分泌和炎性因子释放均与牙髓炎诱发的疼痛程度相关，在第7天表达最高。GW4869和BBG抑制Rab27a和IL-1β表达，减轻了牙髓炎诱发的疼痛。此外，通过超速离心在体外成功提取了外泌体，其中LPS处理促进外泌体分泌，但GW4869对外泌体分泌和IL-1β有相反作用。此外，oxATP对P2X7R的抑制减少了外泌体分泌，导致炎症反应减轻。
本研究突出了小胶质细胞P2X7R在增加外泌体分泌中的调节作用，表明P2X7R作为牙髓炎治疗有前景的靶点的潜在效用。我们的研究突出了一种新的牙髓炎机制，即富含IL-1β的外泌体促成了牙髓炎诱发的疼痛，表明外泌体在牙髓炎期间作为疼痛生物标志物和有害信号分子的关键作用。
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