【作者】 王小禾； 吴泽钰； 赵今；

【Author】 Xiaohe Wang;Zeyu Wu;Jin Zhao;Department of Cariology and Endodontics,The First Affiliated Hospital of Xinjiang Medical University (The Affiliated Stomatology Hospital of Xinjiang Medical University);Stomatology Disease Institute of Xinjiang Uyghur Autonomous Region;

【机构】 新疆医科大学第一附属医院(附属口腔医院)牙体牙髓病科； 新疆维吾尔自治区口腔医学研究所；

【摘要】 目的:牙髓炎(Pulptis)是当前最常见的口腔疾病之一,是导致牙髓发生炎症、坏死等不可逆损伤进而引起牙体缺损和缺失的常见原因,其已造成巨大的社会和经济负担。牙髓炎的发病机制尚未完全阐明,且目前缺乏准确、高效的诊断技术。因此,迫切需要探索牙髓炎诊断的潜在生物标志物和分子机制。方法:我们收集了5名牙髓炎患者与5名健康患者的牙髓组织,利用转录组学分析并鉴定差异表达基因(DEG)(P<0.05,|logFC|>1)。构建了DEG的蛋白质-蛋白质相互作用(PPI)网络使用PPI网络的拓扑学参数和CytoHubba插件分析确定了关键功能的基因子网络。并进一步对DEGs进行基因本体论(GO)富集和京都基因和基因组百科全书(KEGG)富集分析。此外,我们还采用体外低氧牙髓细胞(DPSCs)模型来验证与牙髓炎相关的潜在生物标志物,细胞功能和信号通路。结果:共鉴定出1351个差异表达基因,GO富集分析和KEGG信号通路分析表明,DEGs与immune system process,signaling receptor binding,plasma membrane,Mitophagy–animal,NF-kappa B signaling pathway,Autophagy–animal和Phagosome等显着相关。从PPI网络中鉴定出的15大枢纽基因主要为:PTPRC,CD8A,CD86,CD80,ITGAM,CD19,SELL,CTLA4,FCGR3A,CD69,TNF,FCGR2B,PRF1,CD44和IL7RC。体外细胞模型中,与常氧组相比,低氧环境下DPSCs线粒体膜电位(MMP)下降,IL-6、IL-1β、NF-κB及TNFα等炎症因子的表达上调。DPSCs中自噬体和自噬溶酶体数量明显增加,HIF-1α和LC3B在低氧环境下表达增加,且DPSCs中自噬体和自噬溶酶体数量明显增加。同时,WB结果进一步验证,低氧环境下DPSCs中炎症相关因子TNFα、NF-κB的表达上调,并且自噬相关蛋白LC3B-II表达增加、p62表达减少。提示低氧能引起细胞炎症反应及自噬现象的发生。同时,在加入3-MA抑制自噬后,炎症因子TNF-α、NF-κB的蛋白表达进一步增加。表明自噬可能是牙髓炎中DPSCs抵抗低氧和炎症的重要途径。结论:我们的研究发现了牙髓炎中与自噬相关的生物标志物,并证明了这些标志物在牙髓炎的发展过程中起到了重要作用,这将为研究牙髓炎的发病机制提供新的见解,并可能对其早期诊断和治疗干预具有广阔的应用价值。更多还原

【Abstract】 Objective:Inflammation of the dental pulp,also known as pulpitis,is one of the most common oral diseases and causes a huge social and economic burden.The assessment of the severity of pulpitis can guide the treatment plan,and early diagnosis is crucial to improve the prognosis of patients with pulpitis.However,the traditional assessment method of pulpitis severity has many limitations.It is identified by transcriptomics analysis and then verified by in vitro experiments,which has higher accuracy.Methods:In order to screen potential biomarkers related to pulpitis,we collected the pulp tissues of 5 pulpitis patients and 5 healthy controls,and used transcriptomic analysis to identify differen-tially expressed genes(DEGs)(P<0.05,|logFC|>1).We then constructed a protein-protein inter-action(PPI) network of DEGs and identified key functional gene subnetworks using topological parameters of the PPI network and CytoHubba plugin analysis.Gene Ontology(GO) enrichment analysis was used to identify the biological process(BP),cellular component(CC)and molecular function(MF) of the DEGs,and was also used to explore the biological function of the gene and identify key modules.Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis was used to identify signaling pathways of genes.In addition,we also established a dental pulp stem cell(DPSCs) model in vitro to verify the promising biomarkers,cell functions and signaling pathways screened above.The hypoxia model of DPSCs was used to identify autophagy and mitochondrial damage by MDC and JC-1 staining,respectively.The expression of HIF-1α and LC3B was detected by immunofluorescence(IF) technique.The expression and function of the above markers were verified by Western blot(WB)and enzyme-linked immunosorbent assay(ELISA).Results:A total of 1351 DEGs were identified,including 867 up-regulated genes and 484 down-regulated genes in the pulp tissues of pulpitis compared with healthy controls.GO enrichment analysis and KEGG signaling pathway analysis showed that DEGs were significantly related to immune system process,signaling receptor binding,plasma membrane,Mitophagy– animal,NF-kappa B signaling pathway,Autophagy – animal and Phagosome,etc.The 15 hub genes identified from the PPI network are mainly:PTPRC,CD8A,CD86,CD80,ITGAM,CD19,SELL,CTLA4,FCGR3A,CD69,TNF,FCGR2B,PRF1,CD44 and IL7RC.MDC staining found that autophagosomes in-creased in DPSCs under hypoxia,while JC-1 staining found that hypoxia caused mitochondrial damage.IF staining revealed an increased expression of autophagy-related gene LC3B.WB found that hypoxia can increase the expression of LC3-II and decrease the expression of p62 in DPSCs,increase cell autophagy,and reduce the inflammatory response at the same time.ELISA also confirmed the above results.Conclusions:Our results identified autophagy-related biomarkers in pulpitis and played an important role in the development of pulpitis,providing new insights into the pathogenesis and treatment of pulpitis.更多还原