背景:牙周炎是一种由细菌斑块感染引起的慢性炎症性疾病。虽然激活转录因子1(ATF1)在各种人类疾病中的参与已被广泛探索,其在牙周炎中的具体作用尚不清楚。本研究旨在阐明ATF1在牙周炎中的表达和生物学功能。
方法:从临床样品中获得原代人牙周膜细胞(hPDLCs)并随后进行表征。用牙龈卟啉单胞菌脂多糖(LPS,10μg/mL),hPDLC用ATF1载体或siRNA进行转染。通过实时定量聚合酶链反应(RT-qPCR)和蛋白质印迹测定评估LPS处理的hPDLC或转染的细胞中ATF1的表达水平。炎症因子,包括白细胞介素-6(IL-6),白细胞介素-8(IL-8),肿瘤坏死因子-α(TNF-α),和白细胞介素-1β(IL-1β),使用酶联免疫吸附测定(ELISA)定量。成骨蛋白的评估,如runt相关转录因子2(Runx2),骨桥蛋白(OPN),和骨保护素(OPG),以及非典型核因子-κB(NF-κB)途径相关蛋白(p65,p-p65,IkBα,p-IKBα),使用蛋白质印迹测定法进行。采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)测定和流式细胞术测定来检测细胞活力。
结果:LPS诱导炎症反应并阻碍hPDLCs的成骨分化(p<0.05,p<0.01)。此外,在LPS刺激的hPDLCs中,ATF1沉默增强了细胞增殖并抑制了细胞凋亡(p<0.05,p<0.01)。ATF1沉默不仅抑制了炎症反应,而且促进了LPS刺激的hPDLCs的成骨分化(p<0.05,p<0.01)。重要的是,ATF1沉默能有效阻断LPS诱导的NF-κB信号通路的激活(p<0.05,p<0.01,p<0.001)。
结论:ATF1成为一种有希望的治疗选择,抑制hPDLCs成骨分化,通过抑制NF-κB信号通路的磷酸化来减轻炎症反应。
BACKGROUND: Periodontitis is a chronic inflammatory disease resulting from bacterial plaque infection. While the involvement of activating transcription factor 1 (ATF1) has been extensively explored in various human diseases, its specific role in periodontitis remains unclear. This study aims to elucidate the expression and biological function of ATF1 in the context of periodontitis.
METHODS: Primary human periodontal ligament cells (hPDLCs) were procured from clinical samples and subsequently characterized. Following treatment with P. gingivalis lipopolysaccharide (LPS, 10 μg/mL), hPDLCs underwent transfection with either ATF1 vector or siRNA. The expression levels of ATF1 in LPS-treated hPDLCs or transfected cells were evaluated through real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Inflammatory factors, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1β), were quantified using Enzyme-linked Immunosorbent Assay (ELISA). The assessment of osteogenic proteins, such as runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG), as well as noncanonical nuclear factor-kappaB (NF-κB) pathway-related proteins (p65, p-p65, IkBα, p-IkBα), was conducted using western blot assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry assays were employed to detect cell viability.
RESULTS: LPS induced an inflammatory response and hindered the osteogenic differentiation of hPDLCs (p < 0.05, p < 0.01). Furthermore, ATF1 silencing enhanced cell proliferation and suppressed apoptosis in LPS-stimulated hPDLCs (p < 0.05, p < 0.01). ATF1 silencing not only restrained the inflammatory response but also promoted the osteogenic differentiation of LPS-stimulated hPDLCs (p < 0.05, p < 0.01). Importantly, ATF1 silencing effectively blocked the LPS-induced activation of the NF-κB signaling pathway (p < 0.05, p < 0.01, p < 0.001).
CONCLUSIONS: ATF1 emerges as a promising treatment option, inhibiting the osteogenic differentiation of hPDLCs and mitigating the inflammatory response by preventing the phosphorylation of the NF-κB signaling pathway.