【作者】 田磊；

【导师】 孙秀梅；

【作者基本信息】 吉林大学 ， 口腔医学硕士（专业学位）， 2025， 硕士

【摘要】 研究目的:本研究旨在通过构建牙龈卟啉单胞菌脂多糖（Porphyromonas Gingivalis Lipopolysaccharides,P.g-LPS）刺激牙周膜细胞的体外炎症模型及结扎丝诱导的大鼠牙周炎体内炎症模型,评估去铁胺（Deferoxamine,DFO）对牙周炎（Periodontitis）的治疗效果,并初步探究其通过调控铁死亡（Ferroptosis）抑制牙周炎的分子机制。为DFO辅助治疗牙周炎提供理论依据,同时为牙周炎的药物治疗提供新思路。实验方法:于吉林大学口腔医院颌面外科门诊收集健康牙齿,通过酶消化法提取原代牙周膜细胞并传代培养,通过免疫荧光法检测波形蛋白（Vimentin）及角蛋白（Cytokeratin）,对牙周膜细胞进行鉴定。用不同浓度DFO（0μM、50μM、100μM和200μM）刺激牙周膜细胞,通过CCK-8和Calcein/PI实验筛选DFO的最适药物浓度。将细胞随机分为空白对照组、P.g-LPS组、低浓度治疗组和高浓度治疗组。通过RT-q PCR检测白细胞介素-6（Interleukin-6,IL-6）、肿瘤坏死因子（Tumor necrosis factor-α,TNF-α）和白细胞介素-1β（Interleukin-1β,IL-1β）的表达水平,评价DFO对牙周炎症的控制效果;通过检测各组细胞内Fe²⁺、活性氧（Reactive oxygen species,ROS）和丙二醛（Malondialdehyde,MDA）的表达水平,评估DFO对牙周膜细胞内氧化应激水平的影响及其对脂质过氧化物的清除能力。通过Western Blot检测铁死亡相关通路蛋白谷胱甘肽过氧化物酶4（Glutathione peroxidase 4,GPX4）和溶质载体家族7成员11（Solute carrier family7 member 11,SLC7A11）的蛋白表达水平,同时检测各组细胞中谷胱甘肽（Glutathione,GSH）的含量变化,初步探讨DFO能否通过调控SLC7A11/GPX4抑制铁死亡缓解牙周炎。用正畸结扎丝结扎大鼠双侧上颌第一磨牙牙颈部,构建牙周炎体内模型,建模成功后于大鼠双侧上颌第一磨牙牙龈处局部注射剂量为50mg/kg和100mg/kg的DFO溶液。给药14天后处死大鼠,取心、肝、脾、肺、肾进行生物安全性检测;取大鼠第一磨牙周围牙龈组织提取RNA,通过RT-q PCR检测炎症相关因子IL-6、TNF-α和IL-1β的m RNA的表达水平,评估DFO对牙周炎炎症反应的调控作用。取大鼠牙槽骨组织样本,利用Micro CT进行三维重建,定量分析釉质牙骨质界到牙槽嵴顶距离、骨小梁数量、骨体积分数、骨小梁厚度和骨小梁分离度等相关指标,评估牙槽骨改建情况。此外,制备石蜡切片进行HE染色,观察牙龈上皮组织中炎性细胞浸润程度。通过免疫组织化学染色法观察SLC7A11和GPX4蛋白的表达情况,评价DFO对铁死亡相关分子标志物的影响。实验结果:（1）体外实验结果显示:200μM DFO对牙周膜细胞表现出明显的细胞毒性,而50μM和100μM DFO处理组对细胞活力无显著影响;50μM和100μM DFO都能够有效抑制P.g-LPS诱导的牙周膜细胞炎症因子（TNF-α、IL-1β和IL-6）的表达水平（p<0.05）。P.g-LPS刺激后牙周膜细胞内的Fe²⁺、ROS和MDA的含量显著升高,DFO处理后Fe²⁺、ROS和MDA的含量降低,其中100μM DFO的效果更好;此外,P.g-LPS刺激的牙周膜细胞中SLC7A11、GPX4和GSH的蛋白含量下降,在DFO处理后SLC7A11、GPX4和GSH的蛋白水平升高。（2）体内实验结果显示:50 mg/kg和100 mg/kg剂量的DFO对大鼠心、肝、脾、肺、肾等主要脏器均未产生毒性作用;在大鼠牙周炎模型中可见,牙龈组织中有大量炎性细胞浸润、牙槽骨明显吸收,牙周组织中的GPX4和SLC7A11的蛋白表达降低;使用以上两种剂量的DFO处理后,能够减少牙周炎大鼠牙龈组织中炎性细胞,抑制牙槽骨的吸收;GPX4和SLC7A11的蛋白表达也明显增加。结论:（1）DFO能够有效降低牙周炎中的氧化应激水平,减少脂质过氧化物的积累,具有一定的抗炎作用。（2）DFO可通过调控SLC7A11/GPX4通路抑制铁死亡,保护牙周膜细胞及牙周组织,抑制牙周炎。更多还原

【Abstract】 Objective:This study aims to evaluate the therapeutic efficacy of Deferoxamine(DFO)on periodontitis by constructing an in vitro inflammatory model of periodontal ligament cells stimulated by Porphyromonas gingivalis lipopolysaccharides(P.g-LPS)and an in vivo rat periodontitis model induced by ligature.Additionally,the molecular mechanism by which DFO inhibits periodontitis through the regulation of ferroptosis is preliminarily explored.This research provides a theoretical basis for the adjunctive use of DFO in periodontitis treatment and offers new insights into pharmacological interventions for periodontitis.Methods:1.Primary periodontal ligament cells were isolated from healthy teeth collected at the Department of Oral and Maxillofacial Surgery,Jilin University of Stomatology,using enzymatic digestion and subcultured.Cell identification was performed via immunofluorescence staining for Vimentin and Cytokeratin.Different concentrations of DFO(0μM,50μM,100μM,and 200μM)were applied to the cells,and the optimal concentration was determined using CCK-8 and Calcein/PI assays.2.Cells were random Ly divided into four groups:control,P.g-LPS,low-dose DFO treatment,and high-dose DFO treatment.The expression levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and interleukin-1β(IL-1β)were measured by RT-q PCR to evaluate the anti-inflammatory effects of DFO.Intracellular Fe2?,reactive oxygen species(ROS),and malondialdehyde(MDA)levels were assessed to determine the impact of DFO on oxidative stress and lipid peroxidation.3.Western blot was performed to detect the expression levels of ferroptosis-related proteins,including glutathione peroxidase 4(GPX4)and solute carrier family 7 member 11(SLC7A11).Glutathione(GSH)levels were also measured to preliminarily investigate whether DFO alleviates periodontitis by inhibiting ferroptosis via the SLC7A11/GPX4 pathway.4.A rat periodontitis model was established by ligating the bilateral maxillary first molars with orthodontic wires.After successful modeling,DFO solutions(50mg/kg and 100 mg/kg)were locally injected into the gingival tissue around the first molars.After 14 days,rats were euthanized,and major organs(heart,liver,spleen,lungs,and kidneys)were collected for biosafety evaluation.Gingival tissue RNA was extracted,and the m RNA levels of IL-6,TNF-α,and IL-1βwere quantified by RT-q PCR to assess the anti-inflammatory effects of DFO.Micro-CT was used for three-dimensional reconstruction of alveolar bone,and parameters such as cementoenamel junction to alveolar crest distance,trabecular number,bone volume fraction,trabecular thickness,and trabecular separation were analyzed to evaluate alveolar bone remodeling.Hematoxylin and eosin(HE)staining was performed on paraffin-embedded sections to observe inflammatory cell infiltration.Immunohistochemical staining was conducted to examine the expression of SLC7A11and GPX4 proteins,assessing the impact of DFO on ferroptosis-related molecular markers.Results:1.In vitro experiments demonstrated that 200μM DFO exhibited significant cytotoxicity,while 50μM and 100μM DFO had no notable effect on cell viability.Both 50μM and 100μM DFO effectively suppressed the expression of inflammatory cytokines(TNF-α,IL-1β,and IL-6)induced by P.g-LPS(p<0.05).P.g-LPS stimulation significantly increased intracellular Fe2?,ROS,and MDA levels,which were reduced by DFO treatment,with 100μM DFO showing superior efficacy.Additionally,P.g-LPS decreased the protein levels of SLC7A11,GPX4,and GSH,which were restored by DFO treatment.2.In vivo experiments revealed that DFO at doses of 50 mg/kg and 100 mg/kg caused no toxicity to major organs(heart,liver,spleen,lungs,and kidneys)in rats.In the periodontitis model,significant inflammatory cell infiltration and alveolar bone resorption were observed,accompanied by reduced expression of GPX4 and SLC7A11 in periodontal tissues.DFO treatment at both doses reduced inflammatory cell infiltration,inhibited alveolar bone resorption,and increased the expression of GPX4 and SLC7A11 proteins.Conclusion:1.DFO effectively reduces oxidative stress and lipid peroxidation in periodontitis,demonstrating significant anti-inflammatory properties.2.DFO inhibits ferroptosis by modulating the SLC7A11/GPX4 pathway,thereby protecting periodontal ligament cells and tissues and alleviating periodontitis.更多还原