Dental Stem Cell Biology Research Unit and Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
Center of Excellence for Regenerative Dentistry, Chulalongkorn University, Bangkok, Thailand.
J Periodontol. 2024 Mar;95(3):281-295. doi: 10.1002/JPER.23-0389. Epub 2023 Nov 6.
Various stimuli, that is, mechanical stresses or inflammation, induce the release of adenosine triphosphate (ATP) by human periodontal ligament cells (HPDLCs). Extracellular adenosine triphosphate (eATP) affects HPDLCs' functions such as immunosuppressive action and inflammatory responses. Lipopolysaccharide (LPS) is the key factor involved in periodontal inflammation. However, the possible correlation and detailed mechanism of inflammation-mediated eATP by LPS and inflammatory cascade formation in HPDLCs is unclarified. This study aims to examine the role of eATP on the HPDLCs' responses concerning inflammatory actions after LPS treatment.
HPDLCs were stimulated with Porphyromonas gingivalis LPS and polyinosinic:polycytidylic acid (poly I:C). The amount of ATP release was measured at different time points using a bioluminescence assay. HPDLCs were treated with eATP. The expression of pro-inflammatory and anti-inflammatory genes was determined. Specific PX purinoreceptor 7 (PX) inhibitors (brilliant blue G [BBG] and KN62), a specific PY purinoreceptor 1 (PY) inhibitors (MRS2179), calcium chelator (EGTA), protein kinase C (PKC) inhibitors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF𝜅B) activation inhibitors, and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) inhibitors (H89 dihydrochloride) and activators (forskolin) were used to dissect the mechanism of eATP-induced HPDLCs' inflammatory responses.
LPS and poly I:C induced ATP release. A low concentration of eATP (50 µM) increased pro-inflammatory genes (COX2, IL1B, IL6, IL8, IL12, and TNFA), while a high concentration (500 µM) enhanced anti-inflammatory genes (IL4 and IL10). BBG, KN62, and NF𝜅B activation inhibitors impeded eATP-induced pro-inflammatory genes. MRS2179 and H89 markedly suppressed eATP-induced anti-inflammatory genes. Forskolin induced IL4 and IL10.
HPDLCs respond to LPS by releasing ATP. eATP has dose-dependent dual functions on HPDLCs' inflammatory responses via different pathways. As regulation of inflammation is important in regeneration, eATP may help to limit inflammation and trigger periodontal regeneration.
各种刺激物，即机械应力或炎症，会诱导人牙周韧带细胞（HPDLCs）释放三磷酸腺苷（ATP）。细胞外三磷酸腺苷（eATP）会影响 HPDLCs 的功能，如免疫抑制作用和炎症反应。脂多糖（LPS）是牙周炎症涉及的关键因素。然而，LPS 介导的炎症引起的 eATP 与 HPDLCs 中炎症级联形成的可能相关性和详细机制尚不清楚。本研究旨在探讨 eATP 在 LPS 处理后 HPDLCs 炎症反应中的作用。
用牙龈卟啉单胞菌 LPS 和聚肌苷酸：聚胞苷酸（poly I:C）刺激 HPDLCs。使用生物发光法在不同时间点测量 ATP 释放量。用 eATP 处理 HPDLCs。测定促炎和抗炎基因的表达。使用特定的 PX 嘌呤能受体 7（PX）抑制剂（亮蓝 G [BBG]和 KN62）、特定的 PY 嘌呤能受体 1（PY）抑制剂（MRS2179）、钙螯合剂（EGTA）、蛋白激酶 C（PKC）抑制剂、核因子 kappa-轻链增强子的激活 B 细胞（NF-κB）激活抑制剂、环腺苷酸依赖性蛋白激酶 A（PKA）抑制剂（H89 二盐酸盐）和激活剂（forskolin）来剖析 eATP 诱导的 HPDLCs 炎症反应的机制。
LPS 和 poly I:C 诱导 ATP 释放。低浓度的 eATP（50 µM）增加促炎基因（COX2、IL1B、IL6、IL8、IL12 和 TNFA），而高浓度（500 µM）增强抗炎基因（IL4 和 IL10）。BBG、KN62 和 NF-κB 激活抑制剂阻止了 eATP 诱导的促炎基因。MRS2179 和 H89 显著抑制了 eATP 诱导的抗炎基因。forskolin 诱导了 IL4 和 IL10 的表达。
HPDLCs 通过释放 ATP 对 LPS 作出反应。eATP 通过不同途径对 HPDLCs 的炎症反应具有剂量依赖性的双重作用。由于炎症的调节在再生中很重要，因此 eATP 可能有助于限制炎症并触发牙周再生。
