Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, College of Stomatology, Chongqing Medical University, Chongqing, China.
J Periodontal Res. 2020 Jan;55(1):125-140. doi: 10.1111/jre.12696. Epub 2019 Sep 21.
As a chronic infectious disease, periodontitis could lead to tooth and bone loss. Low-intensity pulsed ultrasound (LIPUS) is a safe, noninvasive treatment method to effectively inhibit inflammation and promote bone differentiation. However, the application of LIPUS in curing periodontitis is still rare. Our study aimed to explore the ability of LIPUS to inhibit inflammatory factors and promote the osteogenic differentiation capacity of human periodontal ligament cells (hPDLCs), and its underlying mechanism.
Human periodontal ligament cells were obtained and cultured from the premolar tissue samples for experiments. First, hPDLCs were treated for 24 hours using lipopolysaccharide (LPS) and then exposed to LIPUS (10 mW/cm , 30 mW/cm , 60 mW/cm , and 90 mW/cm ) to determine the appropriate intensity to inhibit expression of the inflammatory factors interleukin-6 (IL-6) and interleukin-8 (IL-8) expression. The expression of IL-6 and IL-8 was detected by real-time PCR and enzyme-linked immunosorbent assay. The safety of the most appropriate intensity of LIPUS was tested by a cell counting kit 8 test and an apoptosis assay. Then, LPS-induced hPDLCs were treated in osteogenic medium for 7-21 days with or without LIPUS (90 mW/cm , 30 min/d) stimulation. The osteogenic genes RUNX2, OPN, OSX, and OCN were measured by real-time PCR. Additionally, osteogenic differentiation capacity was determined using alkaline phosphatase (ALP) staining, ALP activity analysis, and Alizarin red staining. The activity of the nuclear factor-kappa B (NF-κB) signaling pathway was determined by western blotting, real-time PCR, immunofluorescence, and pathway blockade assays.
Lipopolysaccharide significantly upregulated the production and gene expression of IL-6 and IL-8, while LIPUS stimulation significantly inhibited IL-6 and IL-8 expression in an intensity-dependent manner. LIPUS (90 mW/cm ) was chosen as the most appropriate intensity, and there was no detrimental influence on cell proliferation and status with or without osteogenic medium. In addition, consecutive stimulation with LIPUS (90 mW/cm ) for 30 min/d for 7 days could also inhibit IL-6 and IL-8 gene expression, upregulate the expression of the osteogenesis-related genes RUNX2, OPN, OSX, and OCN, and promote osteogenic differentiation capacity in osteogenic medium in inflamed hPDLCs. The NF-κB signaling pathway was inhibited with LIPUS (90 mW/cm ) via inhibition of the phosphorylation of IκBα and the translocation of p65 into the nucleus in inflamed hPDLCs. Additional investigations of the NF-κB inhibitor, BAY 11-7082, revealed that LIPUS (90 mW/cm ) acted similarly to BAY 11-7802 to inhibit the NF-κB signaling pathway and increase osteogenesis-related genes and promote the osteogenic differentiation capacity of inflamed hPDLCs.
Low-intensity pulsed ultrasound (90 mW/cm ) stimulation could be a safe method to inhibit IL-6 and IL-8 in hPDLCs by inhibiting the NF-κB signaling pathway. The effect of LIPUS (90 mW/cm ) and BAY 11-7082 on LPS-induced inflammation demonstrated that both of these agents were capable of promoting osteogenesis-related gene expression and osteogenic differentiation in hPDLCs, suggesting that the effect of LIPUS on the promotion of osteogenic activity could be mediated in part through its ability to inhibit the NF-κB signal pathway. Hence, LIPUS could be a potential therapeutic method to cure periodontitis.
作为一种慢性传染病，牙周炎可导致牙齿和骨丢失。低强度脉冲超声（LIPUS）是一种安全、非侵入性的治疗方法，可有效抑制炎症并促进骨分化。然而，LIPUS 在治疗牙周炎中的应用仍很少见。我们的研究旨在探讨 LIPUS 抑制炎症因子和促进人牙周韧带细胞（hPDLC）成骨分化能力的作用及其潜在机制。
从前磨牙组织样本中获取并培养人牙周韧带细胞进行实验。首先，用脂多糖（LPS）处理 hPDLCs 24 小时，然后用 LIPUS（10 mW/cm 、30 mW/cm 、60 mW/cm 和 90 mW/cm ）处理，以确定抑制炎症因子白细胞介素-6（IL-6）和白细胞介素-8（IL-8）表达的合适强度。通过实时 PCR 和酶联免疫吸附试验检测 IL-6 和 IL-8 的表达。通过细胞计数试剂盒 8 试验和细胞凋亡试验测试最适强度 LIPUS 的安全性。然后，用 LPS 诱导 hPDLCs 在成骨培养基中培养 7-21 天，并用或不用 LIPUS（90 mW/cm 、30 分钟/d）刺激。通过实时 PCR 测量成骨基因 runt 相关转录因子 2（RUNX2）、骨桥蛋白（OPN）、成骨特异性转录因子 2（OSX）和骨钙素（OCN）的表达。此外，通过碱性磷酸酶（ALP）染色、ALP 活性分析和茜素红染色来测定成骨分化能力。通过 Western blot、实时 PCR、免疫荧光和通路阻断试验测定核因子-κB（NF-κB）信号通路的活性。
脂多糖显著上调 IL-6 和 IL-8 的产生和基因表达，而 LIPUS 刺激则呈强度依赖性显著抑制 IL-6 和 IL-8 的表达。选择 90 mW/cm 的 LIPUS 作为最合适的强度，无论是有无成骨培养基，都不会对细胞增殖和状态产生有害影响。此外，连续刺激 90 mW/cm 的 LIPUS 30 分钟/d 7 天也可以抑制 IL-6 和 IL-8 基因表达，上调成骨相关基因 RUNX2、OPN、OSX 和 OCN 的表达，并促进成骨培养基中炎症 hPDLC 的成骨分化能力。LIPUS（90 mW/cm ）通过抑制 IκBα 的磷酸化和 p65 向核内的易位抑制了 NF-κB 信号通路。对 NF-κB 抑制剂 BAY 11-7082 的进一步研究表明，LIPUS（90 mW/cm ）与 BAY 11-7802 相似，可抑制 NF-κB 信号通路，增加成骨相关基因，并促进炎症 hPDLC 的成骨分化能力。
低强度脉冲超声（90 mW/cm ）刺激可通过抑制 NF-κB 信号通路抑制 hPDLC 中的 IL-6 和 IL-8。LIPUS（90 mW/cm ）和 BAY 11-7082 对 LPS 诱导的炎症的作用表明，这两种药物都能促进 hPDLC 中成骨相关基因的表达和成骨分化，提示 LIPUS 对促进成骨活性的作用部分可能是通过其抑制 NF-κB 信号通路的能力介导的。因此，LIPUS 可能是治疗牙周炎的一种潜在治疗方法。
