Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan; School of Dentistry, College of Medicine, National Taiwan University, Taipei, Taiwan.
School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Dentistry, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
J Formos Med Assoc. 2023 Jan;122(1):47-57. doi: 10.1016/j.jfma.2022.08.005. Epub 2022 Aug 26.
BACKGROUND/PURPOSE: The signaling mechanisms for Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammation in human dental pulp cells are not fully clarified. This in vitro study aimed to evaluate the involvement of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in PgLPS-induced pulpal inflammation.
Human dental pulp cells (HDPCs) were challenged with PgLPS with or without pretreatment and coincubation with a PI3K/Akt inhibitor (LY294002). The gene or protein levels of PI3K, Akt, interleukin (IL)-6, IL-8, alkaline phosphatase (ALP), osteocalcin and osteonectin were analyzed by reverse transcription polymerase chain reaction (PCR), real-time PCR, western blotting, and immunofluorescent staining. In addition, an enzyme-linked immunosorbent assay was used to analyze IL-6 and IL-8 levels in culture medium.
In response to 5 μg/ml PgLPS, IL-6, IL-8, and PI3K, but not Akt mRNA expression of HDPCs, was upregulated. IL-6, IL-8, PI3K, and p-Akt protein levels were stimulated by 10-50 μg/ml of PgLPS in HDPCs. PgLPS also induced IL-6 and IL-8 secretion at concentrations higher than 5 μg/ml. Pretreatment and co-incubation by LY294002 attenuated PgLPS-induced IL-6 and IL-8 mRNA expression in HDPCs. The mRNA expression of ALP, but not osteocalcin and osteonectin, was inhibited by higher concentrations of PgLPS in HDPCs.
P. gingivalis contributes to pulpal inflammation in HDPCs by dysregulating PI3K/Akt signaling pathway to stimulate IL-6 and IL-8 mRNA/protein expression and secretion. These results are useful for understanding the pulpal inflammation and possible biomarkers of inflamed pulp diagnosis and treatment.
背景/目的：牙龈卟啉单胞菌脂多糖（PgLPS）诱导人牙髓细胞炎症的信号机制尚未完全阐明。本体外研究旨在评估磷脂酰肌醇 3-激酶（PI3K）/蛋白激酶 B（Akt）通路在 PgLPS 诱导的牙髓炎症中的作用。
用人牙髓细胞（HDPCs）用 PgLPS 刺激，或用 PI3K/Akt 抑制剂（LY294002）预处理和共孵育。通过逆转录聚合酶链反应（PCR）、实时 PCR、western blot 和免疫荧光染色分析 PI3K、Akt、白细胞介素（IL）-6、IL-8、碱性磷酸酶（ALP）、骨钙素和骨粘连蛋白的基因或蛋白水平。此外，酶联免疫吸附试验用于分析培养基中 IL-6 和 IL-8 的水平。
在 5μg/ml PgLPS 刺激下，HDPCs 的 IL-6、IL-8 和 PI3K mRNA 表达上调，但 Akt mRNA 表达未上调。10-50μg/ml 的 PgLPS 刺激 HDPCs 中 IL-6、IL-8、PI3K 和 p-Akt 蛋白水平。PgLPS 也诱导高于 5μg/ml 的 IL-6 和 IL-8 分泌。LY294002 的预处理和共孵育减弱了 PgLPS 诱导的 HDPCs 中 IL-6 和 IL-8 mRNA 的表达。较高浓度的 PgLPS 抑制了 HDPCs 中 ALP 的 mRNA 表达，但不抑制骨钙素和骨粘连蛋白的 mRNA 表达。
牙龈卟啉单胞菌通过调节 PI3K/Akt 信号通路，刺激 IL-6 和 IL-8 mRNA/蛋白表达和分泌，导致 HDPCs 牙髓炎症。这些结果有助于理解牙髓炎症和可能的牙髓炎症诊断和治疗的生物标志物。
