【作者】 朴钰； 孙雪飞； 侯铁舟；

【Author】 Yu Piao;Xuefei Sun;Tiezhou Hou;Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University;Department of Cariology & Endodontics, College of Stomatology, Xi’an Jiaotong University,Xi’an Jiaotong University;

【机构】 陕西省颅颌面精准医学研究重点实验室； 西安交通大学口腔医院牙体牙髓病科；

【摘要】 目的 :组织酸化是炎症的主要特征之一,当牙髓炎发生时,组织受累区氧供不足,细胞进行无氧酵解,pH可下降至5.5-5.6,形成胞外酸化微环境。酸敏感离子通道（ASICs）是一类由细胞外酸化微环境所激活的阳离子通道,属于上皮钠通道/退化蛋白离子通道超家族,其中ASIC1a亚型受到胞外酸化刺激时,介导Ca2+内流,激活下游信号通路。在本研究中,我们旨在探讨ASIC1a是否促进牙髓炎症及其分子机制。方法:通过免疫荧光法（IF）或免疫组化法（IHC）检测人正常与牙髓炎组织和DPSCs中ASIC1a的表达。给予牙髓细胞不同时间点胞外pH 6.0酸刺激及相同时间下不同pH刺激,建立胞外酸化微环境,采用Western blotting,qRT-PCR和免疫荧光检测NF-κB通路激活情况,qRT-PCR检测炎症因子IL-6、TNF-α表达情况,验证胞外酸化是否诱导牙髓炎症。使用Ca²⁺荧光成像探究胞外酸化微环境对Ca²⁺内流的影响,进一步通过ASIC1a特异性抑制剂PcTx1,胞内Ca²⁺螯合剂BAPTA-AM与无钙Hanks液探索Ca²⁺内流在ASIC1a调控牙髓炎进展中的作用。使用Western blotting、q-PCR、免疫荧光实验探究在PcTx1, BAPTA-AM,CaN抑制剂作用下NF-κB,CaN,IL-6,TNF-α表达情况。结果:IF或IHC显示ASIC1a在牙髓炎牙髓组织内较人正常牙髓组织表达升高。胞外pH 6.0酸性微环境可显著促进IL-6,TNF-α表达,并激活NF-κB通路。Ca²⁺荧光成像实验显示胞外酸化微环境可诱发牙髓细胞ASIC1a离子通道开放及Ca²⁺内流。使用ASIC1a特异性抑制剂,胞内Ca²⁺螯合剂,CaN抑制剂后,可逆转胞外酸化诱导的NF-κB通路激活及IL-6,TNF-α表达升高。结论:综上所述,胞外酸化微环境通过ASIC1a介导的Ca²⁺/CaN/NF-κB通路促进牙髓炎症,本研究为不可复性牙髓炎持续性进展提供实验支持及理论依据。更多还原

【Abstract】 Aim: Tissue acidification is one of the main characteristics of inflammation. When pulpitis occurs,the oxygen supply in the affected area of the tissue is insufficient, the cells undergo anaerobic fermentation, and the pH can be reduced to 5.5-5.6, forming an extracellular acidification microenvironment. Acid-sensitive ion channels（ASICs） are a class of cation channels activated by the extracellular acidification microenvironment, belonging to the epithelial sodium channel/degenerate protein ion channel superfamily. When stimulated by extracellular acidification, ASIC1a subtype mediates Ca2+ influx and activates downstream signaling pathways.In this study, we aimed to investigate whether ASIC1a promotes pulpitis and its molecular mechanism.Methodology: The expression of ASIC1a in human normal and pulpitis tissues and DPSCs was detected by immunofluorescence（IF） or immunohistochemistry（IHC）. Dental pulp cells were given extracellular pH 6.0 acid stimulation at different time points and different pH stimulation at the same time to establish extracellular acidification microenvironment. Western blotting, qPCR and immunofluorescence were used to detect the activation of NF-κB pathway, and qPCR was used to detect the expression of inflammatory factors IL-6 and TNF-α. To verify whether extracellular acidification induces pulpitis. To investigate the influence of extracellular acidified microenvironments on the quality of Ca²⁺ inflow using Ca²⁺ fluorescence imaging and further explore the role of Ca²⁺ flow in the regulation of the progression of pulpitis by ASIC1a through the specific inhibitor PcTx1, the intracellular Ca²⁺ chelating agent BAPTA-AM and calcium-free Hanks fluid. Western blotting, q-PCR and immunofluorescence experiments were used to investigate the expression of NF-κB, CaN, IL-6 and TNF-α under the action of PcTx1,BAPTA-AM and CaN inhibitors.Results: The expression of ASIC1a in pulpitis pulp tissue was higher than that in normal pulp tissue. Extracellular pH 6.0 acidic microenvironment significantly promote the expression of IL-6,TNF-α, and activate the NF-κB pathway. Ca²⁺ fluorescence imaging experiments showed that extracellular acidification microenvironments induce the opening of ASIC1a ion channels and Ca²⁺ intravascular flow in dental pulp cells. The use of ASIC1a specific inhibitors, intracellular Ca²⁺ chelating agents and CaN inhibitors reverse extracellular acidification-induced activation of the NF-κB pathway and increased expression of IL-6 and TNF-α.Conclusions: In summary, extracellular acidification microenvironment promotes pulpitis through the ASIC1a-mediated Ca²⁺/CaN/NF-κB pathway. This study provides experimental support and theoretical basis for the sustainable development of irreversible pulpitis.更多还原