牙周炎患者自身组织核酸内NLRP3 mRNA表达水平测定

目的:通过对牙周炎患者自身组织核酸内炎性小体复合物NLRP3,NLRC4及其相关基因IL-18,IL-1β,Caspase-1等mRNA表达水平的检测,明确炎性小体NLRP3在牙周炎中具有调控作用。方法:采集翻瓣术中慢性牙周炎患者炎性牙周组织以及正畸患者健康牙拔除术获取的健康牙周组织,提取总RNA逆转录cDNA ,-20 ℃冻存备用。Real-time PCR检测NLRP3、NLRC4、IL-18、IL-1β、Caspase-1 mRNA的表达。结果:与健康组牙周组织相比较,牙周炎患者自身组织核酸内NLRP3、IL-18、IL-1β、Caspase-1 mRNA的表达水平明显增高;而NLRC4 mRNA的表达在两组间差异不显著。结论:NLRP3及其相关基因IL-18、IL-1β、Caspase-1 mRNA在牙周炎患者自身组织核酸内的高表达,提示NLRP3在牙周炎的发病机制中具有影响作用。     

NLRP3/Caspase-1炎性体通路在大鼠根尖周炎中表达的研究

目的:研究大鼠实验性根尖周炎中NLRP3/Caspase-1炎性体通路的表达。方法:30只大鼠双侧下颌第一磨牙开髓后封入PBS缓冲液棉球,玻璃离子封闭髓腔,分别于开髓后0、7、14、21d和28d处死大鼠,分离双侧下颌骨。组织学处理后HE染色观察根尖周组织炎症状况,免疫组织化学染色和实时荧光定量PCR分别检测NLRP3、Caspase-1和IL-1β的表达情况。结果:NLRP3、Caspase-1和IL-1β在炎性根尖周组织中均有表达;与对照组相比,三者表达水平均明显增高,差异有统计学意义(P<0.05);其中Caspase-1和IL-1β阳性细胞数与NLRP3阳性细胞数呈显著正相关(P<0.01)。结论:根尖周组织中存在着NLRP3/Caspase-1炎性体通路,提示该通路在根尖周组织固有免疫中可发挥重要作用。[关键词]炎性体NLRP3Caspase-1根尖周炎     

牙周炎和健康人牙周组织中细胞焦亡水平的比较研究

目的:研究慢性牙周炎和健康人的牙周组织中细胞焦亡水平是否存在差异。方法:分别纳入全身健康、无牙周病史、牙列完整的患者9例作为健康对照组，慢性牙周炎III～IV期患者9例作为牙周炎组。收集患者的牙周组织，免疫组化染色对牙龈组织中消皮素D(GSDMD)、半胱天冬酶-1(caspase-1)、NLRP3炎症小体、白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)的表达水平及分布进行定性分析；实时定量PCR检测牙龈组织和牙周膜组织中GSDMD、caspase-1、NLRP3、IL-1β及IL-18 mRNA的表达差异。结果:免疫组化染色显示牙周炎组的牙龈组织中GSDMD、caspase-1、NLRP3、IL-1β、IL-18的阳染色面积均大于健康组(P<0.05)。实时定量PCR检测显示，牙周炎组牙龈组织和牙周膜组织中GSDMD、caspase-1、NLRP3、IL-1β、IL-18 mRNA的表达量明显高于健康组(P<0.05)。结论:慢性牙周炎牙周组织的细胞焦亡水平高于健康人，提示细胞焦亡可能参与了牙周炎的发生发展。     

P38MAPK在滑膜细胞表达炎性因子中的作用

目的:研究p38丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)在脂多糖(LPS)诱导大鼠滑膜成纤维细胞(synovial fibroblast,SF)合成炎性因子IL-1β、MMP3中的作用.方法:采用酶消化法分离获得大鼠SF,体外培养并鉴定,分别作为空白对照组、与不同浓度LPS共培养、以SB203580预处理后与LPS共培养,24h后免疫化学染色、RT-PCR分别检测通路活化水平和炎性因子蛋白及mRNA表达量.结果:LPS可刺激SF使IL-1β、MMP3蛋白和mRNA表达上调,表达量随LPS浓度增高而增加(与对照组相比P＜0.05);且与LPS共培养后SF中p38 MAPK通路活化,阻断p38 MAPK后,炎性因子表达显著下调(P＜0.05).结论:LPS诱导SF高表达IL-1β、MMP3,p38 MAPK在其中发挥重要作用.     

IL-1β与IL-10mRNA在牙龈组织中的表达

目的:检测IL-1β与IL-10mRNA在正常牙龈及牙周炎患者牙龈组织中的表达,探讨二者与炎症的关系及内源性抗炎因子IL-10对致炎因子IL-1β是否有拮抗作用。方法:采集14例成人牙周炎患者炎症区牙龈组织,6例正常牙龈组织,利用逆转录-聚合酶链反应检测其中IL-1β与IL-10mRNA的表达及其强弱程度。结果:成人牙周炎组牙龈组织IL-1βmRNA的阳性表达率为92.86%,IL-10mRNA阳性表达率为71.43%,二者与正常对照组相比均有显著性差异;两者之间无明显相关性;两者与临床指标间亦无明显相关性。结论:致炎性和抗炎性细胞因子均可在牙龈组织中表达;机体自身IL-10水平不足以完全拮抗IL-1β活性;IL-10对致炎性细胞因子IL-1β的调控作用有待进一步研究。     

NLRP3及其在口腔疾病中的研究进展

NLRP3受体是天然免疫系统细胞浆型模式识别受体中核苷酸结合寡聚化结构域样受体(NLR)家族的一个重要成员,在机体固有免疫应答中发挥独特的功能,其特点是可以通过形成复杂的蛋白质复合体——炎性体,介导炎症反应和细胞死亡。NLRP3炎性体在多种疾病和炎症反应中发挥重要作用,下面就NLRP3受体的结构、功能及其在口腔疾病中的作用作一综述。     

重视和规范牙周基础治疗的必要性

作为慢性感染性疾病,无论是局限于牙龈组织的菌斑性牙龈病,还是破坏牙周支持组织的各类牙周炎,都是以菌斑微生物为主要病因.20世纪60至70年代的研究表明,去除龈上和龈下细菌沉积物是牙周治疗的首要环节;80年代后的研究则认为通过龈下机械性治疗使得微生物转向非致病菌或益生菌,可以大量改善临床症状.迄今为止,以清除和控制菌斑微生物为核心内容的牙周基础治疗是控制牙周疾病的主要方法[1].     

巴戟天多糖通过上调沉默信息调节因子1抑制炎性牙周膜细胞NOD样受体热蛋白结构域相关蛋白3的表达及活性

目的 探讨炎性微环境下巴戟天多糖（MOP）对牙周膜细胞沉默信息调节因子1(SIRT1)及NOD样受体热蛋白结构域相关蛋白3(NLRP3)表达的影响。方法 将30只大鼠随机分为对照组（n=6）和模型组（n=24），模型组采用正畸丝结扎法建立牙周炎模型，3周后每组各处死6只大鼠，Micro-CT检测确认建模成功。剩余模型组大鼠随机分为牙周炎自然恢复组、生理盐水（NS）组和MOP组。MOP组于大鼠左上颌第一磨牙腭侧注射MOP [200 mg/（kg·3d）,50μL，持续4周]，NS组注射等体积NS，牙周炎自然恢复组不做任何处理。取大鼠左上颌骨组织，采用苏木精-伊红（HE）染色观察牙周膜细胞病理改变，免疫组织化学检测SIRT1、NLRP3表达量。体外培养人牙周膜成纤维细胞（hPDLFs）,CCK-8检测MOP对细胞活性的影响。将第4代细胞分为对照组、炎症组（10μg/mL脂多糖）及实验组（5μmol/L MOP,5μmol/L MOP+10μg/mL脂多糖）。利用实时定量聚合酶链反应（qRT-PCR）及Western blot检测SIRT1和NLRP3表达变化，免疫沉淀及酶联免疫法（ELIS...     

疱疹病毒和牙周致病菌在牙周炎中相互作用的研究进展

疱疹病毒和牙周致病菌的相互作用影响着牙周炎的发生发展,二者共同感染是牙周病发病的主要病因.在牙周炎患者的龈下菌斑中,病毒的检出率远较健康者高.疱疹病毒和牙周致病菌二者可在牙周病发病的多个阶段内相互影响:病毒促进细菌的黏附和定植、牙周致病菌促进病毒的活化,二者共同改变宿主的免疫反应.在牙周病发病过程中,牙周致病菌的黏附定植、疱疹病毒的活化以及宿主的免疫抑制机制等,可相对科学合理地解释牙周病的发作区域、进展速度和严重程度等特征.如果疱疹病毒介导的分子旁路与牙周炎导致的系统性疾病的生物联系被证实,那么传统的以清除细菌为目的的治疗方法对于牙周炎治疗的效果将是有限的,而将抗病毒药物和龈下刮治和根面平整术相结合的治疗方法可能在治疗和预防牙周病及其复发方面更加有效.疱疹病毒-牙周致病菌模型不仅使人们对牙周炎的发病机制有了更深的认识,也为牙周病治疗指出了新的方向.     

细胞自噬和炎症反应的相互调控与牙周炎

细胞自噬在真核细胞中广泛存在,其通路可将细胞内衰老损伤的蛋白质和细胞器等细胞成分运送至溶酶体进行降解、清除并循环利用降解后的营养物质。炎症反应是机体应答组织损伤和病原微生物感染等有害刺激的一种保护性反应,过度的炎症反应会导致组织损伤和疾病;而自噬可通过降解DNA、活性氧族等内源性刺激来抑制炎性小体聚集,降解白细胞介素（IL）-1β前体来抑制IL-1β等促炎因子的分泌。牙周致病菌的毒力因子参与牙周组织的破坏,通过其自身携带或释放的脂多糖、肽聚糖和细菌DNA等与宿主细胞的Toll样受体（TLR）等相互作用诱发组织局部炎性细胞浸润和释放炎症因子,导致牙周炎。在牙周局部组织中,病原相关分子模式和损伤相关分子模式通过与TRL或核苷酸结合寡聚化结构域蛋白样受体相互作用,在激活先天免疫反应时诱发自噬,而自噬同时可通过负向调控TLR信号来影响炎症反应。本文就自噬与炎症反应的相互调控作用和自噬与牙周炎的相关性等研究进展作一综述,旨在揭示牙周炎等炎症性疾病的发病机制,为其治疗探索新的途径。     

Hyperglucose Contributes to Periodontitis: Involvement of the NLRP3 Pathway by Engaging the Innate Immunity of Oral Gingival Epithelium

Background: The NLRP3 inflammasome is essentially a family of intracellular innate immune sensors that can respond to bacterial challenge and initiate early host immunity responses. However, the involvement and possible molecular mechanism of the NLRP3 pathway in the context of chronic periodontitis (CP) and diabetes mellitus have yet to be fully elucidated. Methods: Gingival tissues were collected from patients with CP and/or type 2 diabetes mellitus (T2DM), and the expression of NLRP3 and interleukin (IL)‐1β was analyzed by immunohistochemistry. To explore the possible molecular mechanism, human gingival epithelial cells (HGECs) were established in vitro and challenged with lipopolysaccharide (LPS) and/or high glucose. High extracellular K⁺ was applied as an inhibitor of NLRP3. The NLRP3 pathway was analyzed by immunocytochemistry and quantitative polymerase chain reaction. Results: Compared with control individuals, NLRP3 and IL‐1β were significantly upregulated in oral gingival epithelium of patients with CP and/or T2DM (P <0.05). The expression of NLRP3 was significantly upregulated in HGECs when stimulated in vitro by LPS or high glucose (P = 0.00). The simultaneous stimulation of LPS and high glucose contributed to significant upregulation of NLRP3 expression versus LPS or high glucose alone (P = 0.00). Although expression of caspase 1 and IL‐1β protein were increased in HGECs when stimulated by LPS, they were partially inhibited after the NLRP3 was successfully blocked. Conclusion: For patients with T2DM and CP, hyperglycemic status may exacerbate the inflammation state of gingival tissue by activating the NLRP3 pathway, and this abnormal host inflammatory response may contribute to further tissue breakdown.     

Salivary Levels of NLRP3 Inflammasome‐Related Proteins as Potential Biomarkers of Periodontal Clinical Status

Background: Emerging evidence suggests that activation of inflammasomes plays a central mechanism in pathogenesis of periodontitis. This study aims to compare salivary levels of nod‐like receptor family pyrin domain containing protein (NLRP) 3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), cysteine aspartase (caspase)‐1, and interleukin (IL)‐1β from individuals with aggressive (AgP) or chronic periodontitis (CP) and healthy controls (HC), as well as elucidate its association with periodontal clinical status. Methods: Saliva samples from individuals with CP (n = 75), AgP (n = 20), and HC (n = 69) were collected. Periodontal status was assessed by measurement of probing depth, clinical attachment level, and extent and severity of disease. Salivary levels of analytes were analyzed by enzyme‐linked immunosorbent assay. Association between biomarkers with CP or AgP was analyzed using multivariate binary logistic regression models. Results: Significantly higher levels of NLRP3, ASC, and IL‐1β were detected in periodontitis groups in comparison to the periodontally HC group. However, no significant differences were observed for caspase‐1 levels between clinical groups, and only NLRP3 salivary concentration was significantly higher in AgP compared with CP patients. Also, positive significant correlations among NLRP3, ASC, and IL‐1β salivary concentrations and clinical parameters were observed. Logistic regression analyses revealed a strong/independent association of NLRP3, ASC, and IL‐1β salivary levels with CP and AgP. Conclusion: Although the concentration of caspase‐1 in saliva samples makes its determination useless for detection of periodontal disease and/or its severity, salivary levels of NLRP3, ASC, and IL‐1β may act as strong/independent indicators of amount and extent of periodontal breakdown in both CP and AgP and could potentially be used for prevention and therapy of this group of diseases.     

Macrophages Play a Key Role in the Obesity‐Induced Periodontal Innate Immune Dysfunction via Nucleotide‐Binding Oligomerization Domain‐Like Receptor Protein 3 Pathway

Background: Obesity is associated with infiltration of macrophages into adipose tissue. However, effects of obesity on macrophage infiltration and activation in periodontal tissues with periodontitis are still to be elucidated. Methods: A diet‐induced obesity 16‐week mouse model was constructed, and periodontitis was induced by periodontal ligation for 10 days. The model consisted of periodontitis (P) and control (C) groups, with high fat (HF) and normal (N) diet conditions. Bone loss (BL) was analyzed by microcomputed tomography. In periodontal tissues, immunohistochemical staining and quantitative polymerase chain reaction (qPCR) detected expressions of: 1) nucleotide‐binding oligomerization domain‐like receptor protein 3 (NLRP3) pathway; 2) macrophage‐specific marker (F4/80); and 3) macrophage chemotactic protein 1 (MCP1). Bone marrow‐derived macrophages (BMDMs) from the mouse model were stimulated by Porphyromonas gingivalis lipopolysaccharide (LPS) in vitro (NC/NC + LPS: BMDMs from NC group without/with LPS stimulation; HFC/HFC + LPS: BMDMs from HFC group without/with LPS stimulation). Expressions of NLRP3 pathway in BMDMs were detected by immunocytochemical staining and qPCR. Results: BL increased significantly with periodontitis (NC versus NP; HFC versus HFP) and obesity (NP versus HFP). Expressions of NLRP3 pathway were significantly elevated in gingival tissues with periodontitis (NC versus NP; HFC versus HFP), but not with obesity (NC versus HFC; NP versus HFP). F4/80 and MCP1 expressions were significantly upregulated in gingival tissues with periodontitis (NC versus NP; HFC versus HFP) but significantly downregulated in the context of obesity (NP versus HFP). In vitro, NLRP3 pathway expressions were significantly upregulated in BMDMs after LPS stimulation (NC + LPS versus NC; HFC + LPS versus HFC), but significantly downregulated in HFC groups (HFC versus NC; HFC + LPS versus NC + LPS). Conclusion: Obesity may paralyze innate immune response of periodontium via attenuating infiltration and activation of macrophages and further aggravate periodontal disease.     

外源性ATP对牙龈成纤维细胞NLRP3炎症小体活化的影响

目的: 观察外源性三磷酸腺苷（ATP）对牙龈卟啉单胞菌(P.gingivalis)和热灭活牙龈卟啉单胞菌(HP.gingivalis)感染的人牙龈成纤维细胞NLRP3 炎症小体（inflammasome）的活化以及下游因子IL-1β分泌的影响。方法: 组织块法体外培养人牙龈成纤维细胞（hGFs）获取原代细胞,经5 mmol/L ATP预处理,用100 MOI P.gingivalis、100 MOI HP.gingivalis体外刺激hGFs,实时定量PCR检测NLRP3、ASC、Caspase-1、IL-1β的基因表达;Western免疫印迹技术检测细胞内NLRP3、Caspase-1和IL-1β蛋白的表达;ELISA法检测白细胞介素1β（IL-1β）的分泌。采用Graphpad prism 6软件包对数据进行t检验或单因素方差分析。结果: 与对照组相比,P.gingivalis 下调NLRP3 、ASC mRNA,上调IL-1β基因表达,下调NLRP3 、IL-1β胞内蛋白水平。HP.gingivalis 诱导NLRP3 、IL-1β、ASC基因和胞内蛋白的表达,P.gingivalis或者 HP.gingivalis单独刺激对Caspase-1 mRNA水平及IL-1β分泌均无影响。ATP/P.gingivalis或ATP/HP.gingivalis 共刺激均明显上调NLRP3、ASC、Caspase-1和IL-1β基因及胞内蛋白水平,增加上清液中IL-1β的分泌水平。结论: 外源性ATP可调控牙周主要致病菌P.gingivalis感染的人牙龈成纤维细胞 NLRP3炎症小体的激活,介导炎症因子IL-1β的成熟与分泌。     

Activation of NLRP3 inflammasome in macrophages by mycoplasmal lipoproteins and lipopeptides

The NLRP3 inflammasome, an intracellular sensor consisting of the nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain containing 3 (NLRP3), the adaptor protein apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain (ASC), and procaspase‐1, plays critical roles in host defense against microbial pathogens by inducing production of interleukin‐1β (IL‐1β) and IL‐18. Mycoplasma salivarium and Mycoplasma pneumoniae cells activated murine bone marrow‐derived macrophages (BMMs) to induce production of IL‐1α, IL‐1β, and IL‐18. The IL‐1β production‐inducing activities of these mycoplasmas toward BMMs from Toll‐like receptor 2 (TLR2)‐deficient mice were significantly attenuated compared with those from C57BL/6 mice (B6BMMs). This result suggests the possibility that their lipoproteins as TLR2 agonists are involved in the activity. Lipoproteins of M. salivarium and M. pneumoniae (MsLP and MpLP), and the M. salivarium‐derived lipopeptide FSL‐1 induced IL‐1β production by B6BMMs, but not by BMMs from caspase‐1‐, NLRP3‐ or ASC‐deficient mice. The activities of MsLP and MpLP were not downregulated by the proteinase K treatment, suggesting that the active sites are their N‐terminal lipopeptide moieties. B6BMMs internalized the mycoplasmal N‐terminal lipopeptide FSL‐1 at least 30 min after incubation, FSL‐1‐containing endosomes started to fuse with the lysosomes around 2 hours, and then FSL‐1 translocated into the cytosol from LAMP‐1⁺ endosomes. The artificial delivery of FSL‐1 into the cytosol of B6BMMs drastically enhanced the IL‐1β production‐inducing activity. FSL‐1 as well as the representative NLRP3 inflammasome activator nigericin induced the NLRP3/ASC speck, but FSL‐1 located in a compartment different from the NLRP3/ASC speck.