Data Sheet 1_LncRNA NRIR inhibits osteogenesis by promoting macrophage M1 polarization through RSAD2/NF-κB axis in peri-implantitis.pdf

IntroductionPeri-implantitis is an inflammatory condition affecting the hard and soft tissues surrounding osseointegrated implants, characterized by progressive alveolar bone destruction. The long non-coding RNA Negative Regulator of Interferon Response (lncRNA NRIR) is widely recognized as a biomarker for certain autoimmune diseases and participates in their pathogenesis. However, our previous studies revealed significant upregulation of NRIR in peri-implantitis, suggesting its potential role in peri-implantitis. In peri-implantitis lesions, there is often a substantial infiltration of M1 macrophages. Thus, this study investigated the regulatory role and underlying mechanisms of NRIR in macrophage polarization during peri-implantitis.
MethodsTranscriptome sequencing analysis revealed radical S-adenosyl methionine domain containing 2 (RSAD2) as an NRIR-interacting mRNA in macrophages. Using siRNA gene knockdown technology, we suppressed NRIR and RSAD2 expression in M1 macrophages derived from THP-1 cells. Subsequently, we employed RT-qPCR, Western blot, flow cytometry, and immunofluorescence staining to assess the levels of inflammatory cytokines and M1 macrophage-associated markers, aiming to elucidate the involvement of NRIR/RSAD2/NF-κB axis in macrophage polarization. Supernatants from NRIR-knockdown macrophages were collected to prepare the culture medium for bone marrow mesenchymal stem cells (BMSCs). The expression of osteogenic-related factors in BMSCs was evaluated through RT-qPCR, Western blot, Alkaline phosphatase (ALP) activity, and alizarin red S (ARS) staining. Furthermore, a rat peri-implantitis model was established, and the degree of peri-implant tissue inflammation and bone loss was assessed using micro-CT scanning and immunohistochemistry after treatment with various macrophage supernatants.
ResultsNRIR knockdown reduced RSAD2 expression and suppressed activation of the NF-κB pathway, consequently decreasing inflammatory cytokines and M1 macrophage-associated cytokine expression in THP-1 macrophages. Functionally, NRIR knockdown in macrophages promoted osteogenic differentiation of BMSCs. In vivo experiments showed that supernatants derived from NRIR-knockdown macrophages resulted in reduced inflammatory infiltration, diminished bone resorption, and increased expression of osteogenesis-related factors.
DiscussionThis study demonstrates that NRIR functions as a pro-inflammatory modulator in peri-implantitis by activating M1 macrophages through the RSAD2/NF-κB axis, providing novel insights into peri-implantitis pathogenesis that may inform future preventive and therapeutic strategies.

引言
种植体周围炎（Peri-implantitis）是一类累及骨整合种植体（osseointegrated implants）周围软硬组织的炎症性疾病，以进行性牙槽骨破坏为典型特征。长链非编码RNA（long non-coding RNA）干扰素应答负调控因子（Negative Regulator of Interferon Response，lncRNA NRIR）已被广泛认可为部分自身免疫性疾病（autoimmune diseases）的生物标志物，并参与此类疾病的发病进程。然而，我们既往的研究发现，NRIR在种植体周围炎组织中存在显著上调，提示其可能在种植体周围炎中发挥潜在调控作用。在种植体周围炎病灶（peri-implantitis lesions）中，常存在大量M1型巨噬细胞（M1 macrophages）浸润。因此，本研究旨在探讨NRIR在种植体周围炎巨噬细胞极化（macrophage polarization）中的调控作用及其潜在分子机制。

方法
转录组测序（Transcriptome sequencing）分析显示，含自由基S-腺苷甲硫氨酸结构域2（radical S-adenosyl methionine domain containing 2，RSAD2）是巨噬细胞中与NRIR相互作用的信使RNA（mRNA）分子。我们采用小干扰RNA基因沉默技术（siRNA gene knockdown），在THP-1细胞（THP-1 cells）诱导的M1型巨噬细胞中抑制NRIR与RSAD2的表达。随后，通过实时定量逆转录聚合酶链反应（RT-qPCR）、蛋白质印迹法（Western blot）、流式细胞术（flow cytometry）及免疫荧光染色（immunofluorescence staining）检测炎症因子（inflammatory cytokines）与M1型巨噬细胞相关标志物的表达水平，以阐明NRIR/RSAD2/核因子κB（NF-κB）信号轴在巨噬细胞极化中的调控作用。收集NRIR沉默巨噬细胞的上清液，制备骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）的培养液。通过RT-qPCR、Western blot、碱性磷酸酶（Alkaline phosphatase，ALP）活性检测及茜素红S染色（alizarin red S staining，ARS）评估BMSCs中成骨相关因子（osteogenic-related factors）的表达情况。此外，我们构建了大鼠种植体周围炎模型（rat peri-implantitis model），经不同巨噬细胞上清液处理后，采用显微计算机断层扫描（micro-CT scanning）与免疫组织化学染色（immunohistochemistry）评估种植体周围组织的炎症程度与骨丢失情况。

结果
在THP-1巨噬细胞中，沉默NRIR可降低RSAD2的表达并抑制核因子κB通路的激活，进而减少炎症因子与M1型巨噬细胞相关细胞因子的表达与分泌。功能实验显示，巨噬细胞中NRIR沉默可显著促进BMSCs的成骨分化（osteogenic differentiation）。体内动物实验表明，NRIR沉默巨噬细胞的上清液可减轻种植体周围组织的炎症浸润（inflammatory infiltration）、减少骨吸收（bone resorption），并上调成骨相关因子的表达水平。

讨论
本研究证实，NRIR可通过激活RSAD2/NF-κB信号轴介导M1型巨噬细胞极化，从而在种植体周围炎中充当促炎调节因子（pro-inflammatory modulator），为阐明种植体周围炎的发病机制提供了全新视角，可为未来的疾病预防与治疗策略提供潜在的理论依据。