Data Sheet 2_LncRNA NRIR inhibits osteogenesis by promoting macrophage M1 polarization through RSAD2/NF-κB axis in peri-implantitis.docx

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主要特征。长链非编码RNA NRIR（long non-coding RNA Negative Regulator of Interferon Response，lncRNA NRIR）被广泛认为是部分自身免疫性疾病的生物标志物，并参与其发病过程。然而，本课题组前期研究发现，种植体周围炎组织中NRIR表达显著上调，提示其可能在种植体周围炎中发挥潜在作用。种植体周围炎病灶中常存在大量M1巨噬细胞浸润，因此本研究探讨了NRIR在种植体周围炎进程中对巨噬细胞极化的调控作用及其潜在机制。

**方法**：转录组测序分析显示，含自由基S-腺苷甲硫氨酸结构域蛋白2（radical S-adenosyl methionine domain containing 2，RSAD2）是巨噬细胞中与NRIR相互作用的mRNA。本研究采用小干扰RNA（small interfering RNA，siRNA）基因敲低技术，在THP-1细胞来源的M1巨噬细胞中抑制NRIR与RSAD2的表达。随后，本研究采用实时荧光定量聚合酶链式反应（real-time quantitative polymerase chain reaction，RT-qPCR）、蛋白质印迹（Western blot）、流式细胞术及免疫荧光染色，检测炎症因子与M1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）通路在巨噬细胞极化中的作用。收集NRIR敲低巨噬细胞的上清液，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（alizarin red S，ARS）染色，评估BMSCs中成骨相关因子的表达水平。此外，本研究构建了大鼠种植体周围炎模型，经不同巨噬细胞上清液处理后，采用显微CT（micro-CT）扫描及免疫组织化学技术评估种植体周围组织炎症程度与骨丢失情况。

**结果**：敲低NRIR可降低RSAD2的表达并抑制NF-κB通路的激活，进而减少THP-1巨噬细胞中炎症因子与M1巨噬细胞相关细胞因子的表达。功能层面，巨噬细胞中NRIR敲低可促进BMSCs的成骨分化。体内实验显示，NRIR敲低巨噬细胞来源的上清液可减轻炎症浸润、减少骨吸收，并上调成骨相关因子的表达。

**讨论**：本研究证实，NRIR可通过RSAD2/NF-κB通路激活M1巨噬细胞，在种植体周围炎中发挥促炎调控作用，为种植体周围炎的发病机制提供了新的见解，可为未来的预防与治疗策略提供参考。