【作者】 王丽娜；

【导师】 牛卫东；

【作者基本信息】 大连医科大学 ， 病原生物学， 2016， 博士

【摘要】 根尖周病是一种以细菌感染为主的免疫炎症反应。粪肠球菌(Enterococcus faecalis,Efaecalis)是继发性及难治性牙髓根尖周感染中常见致病菌。在根管再治疗病例中粪肠球菌的检出率从29%至77%不等。粪肠球菌具有多种毒力因子,包括细胞溶解素、粘附蛋白、明胶酶、脂磷壁酸(lipoteichoic acid,LTA)等,其中脂磷壁酸LTA是粪肠球菌主要致病因子之一,具有较强的免疫原性,是导致根尖周炎症的主要毒力因子。LTA可以耐受抗菌素和杀菌剂,引发一系列炎症因子的释放及成骨细胞凋亡。研究发现粪肠球菌LTA通过与小鼠巨噬细胞RAW264.7细胞膜上的TLR2相互作用,经磷酸化激活核因子-κB(NF-κB)信号通路促使炎性因子TNF-α和NO的分泌和释放,这可能是引起根尖周炎症及骨破坏的原因之一。固有免疫系统主要通过三类模式识别受体(PRR)来识别外来病原体,已经发现三类模式识别受体主要包括:RIG-I样受体(RLR)、Toll样受体(TLR)、和NOD样受体(NLR)。其中,RIG-I样受体主要识别病毒,而在细菌引起的根尖周病的免疫反应研究中,虽然多种证据表明Toll样受体通路与牙髓根尖周免疫炎症性疾病密切相关,但当Toll样受体通路被抑制后,仍有炎症存在,提示我们很可能有另一种通路在根尖周骨破坏中起作用,而NLR信号通路将是研究根尖周炎发病机制新的突破口。关于NLR信号通路,近年来的研究热点集中在IL-1β生成相关的NLRP3炎症小体(inflammasome)通路。NLRP3可高表达于复层上皮细胞及髓系免疫细胞中,提示其可能在机体抵御病原微生物的第一道防线中发挥关键作用。在受到外界刺激后,NLRP3通过寡聚化聚集大量的Pro-Caspase-1,从而形成NLRP3炎性体并促使Caspase-1活化,进而在天然免疫防御中促进Pro-IL-1β的切割成熟释放IL-1β,其中IL-1β是NLRP3炎症体活化的标志。有研究表明,相较于可逆性牙髓炎,在不可逆性牙髓炎中,NLRP3/Caspase-1表达明显增高。粪肠球菌作用于THP-1巨噬细胞,发现其可通过激活Caspase-1,刺激IL-1β分泌和细胞死亡来促进牙髓炎症的发生发展。薯蓣皂苷(Dioscin)天然存在于薯蓣科植物穿山龙(Dioscorea nipponica Makino)的根茎,俗称薯蓣皂素,是生产甾体激素类药物的重要基础原料,具有溶血、降血脂、抗菌、消炎、保肝、抑癌及治疗骨质疏松等作用。薯蓣皂苷可通过抑制LPS所激活的CK2、JNK、NF-κB及AP-1信号通路的核转移,最终抑制了活性氧(Reactive oxygen species,ROS),IL-1和IL-6等炎性因子的表达。中药对多种疾病均有较好的治疗效果,而薯蓣皂苷对NLRP3炎性体的作用还尚未见报道。本研究分体内实验和体外实验两部分。在体内实验部分中,首先检测了人慢性根尖周炎病变组织中NLRP3炎性体的表达情况,研究结果显示,NLRP3炎性体在根尖周病变组织的表达明显高于正常组织,说明NLRP3炎性体参与了人慢性根尖周炎的进展过程,为本课题的进一步研究奠定了基础。粪肠球菌是难治性根尖周炎的主要致病菌,因此我们建立了粪肠球菌感染大鼠的根尖周炎动物模型,探讨粪肠球菌对NLRP3炎性体的促活化作用,分析了炎性体随着根尖周病变的进程加剧炎症反应并引发根尖周骨破坏的作用。在体外实验部分,研究了粪肠球菌毒力因子LTA对NLRP3炎性体的活化机制,并探讨中药薯蓣皂苷在此过程中的抗炎作用以及该药物在LTA存在下的成骨功能,为临床难治性根尖周炎的治疗提供实验室基础。第一部分 NLRP3炎性体在根尖周炎中的表达及作用实验一 NLRP3炎性体在临床慢性根尖周炎病变组织中的表达目的:探讨临床慢性根尖周炎病变组织中NLRP3炎性体的表达情况,为本研究的深入展开奠定临床应用基础方法:收集因慢性根尖周炎需根尖外科手术治疗或拔牙患者的根尖周病变组织,通过Real-time qPCR及免疫组织化学的方法检测NLRP3炎性体mRNA及蛋白的表达情况。结果:NLRP3、Caspase-1及IL-1β在人慢性根尖周病变中的表达量明显高于正常对照组(p<0.05)。相关性分析结果显示,NLRP3与Caspase-1的表达有中度相关性,IL-1β的表达与NLRP3及Caspase-1为低度相关。结论:NLRP3炎性体参与了人的慢性根尖周炎症反应。实验二粪肠球菌感染的大鼠根尖周炎模型的建立及NLRP3炎性体在根尖周炎中的表达及作用目的:建立粪肠球菌感染的大鼠根尖周炎动物模型,观察和分析不同病变时期根尖周炎症反应及骨破坏情况,检测NLRP3、Caspase-1及IL-1β在大鼠根尖周病变组织的表达及可能作用,并分析NLRP3炎性体与根尖周骨破坏的关系。方法:粪肠球菌是难治性根尖周炎的主要致病菌,故将粪肠球菌封入大鼠的根管内建立大鼠根尖周炎的动物模型,封入PBS棉球作为实验对照组,通过Micro-CT检测根尖周骨破坏情况及计算根尖周骨破坏体积,验证动物模型是否成功建立,并用HE染色确认炎症进展的病理学变化。Real-time qPCR检测NLRP3炎性体mRNA的表达情况,免疫组织化学染色检测NLRP3、Caspase-1及IL-1β炎性体蛋白的定位及表达,并利用酶组织化学染色的方法检测破骨细胞的数量,分析破骨细胞与IL-1β阳性细胞表达的相关性,推测NLRP3炎性体在根尖周骨破坏中的可能作用。结果:(1)Micro-CT及HE染色的结果显示,成功的构建了粪肠球菌所致的大鼠根尖周炎动物模型,根尖周炎症在2周时为急性期,自3周开始转为慢性炎症反应。根尖周骨破坏从1周时开始至3周时破坏达到最大(p<0.05),4周时骨破坏体积同第3周。(2)NLRP3、Caspase-1及IL-1β的mRNA及蛋白在封菌组及未封菌组均有表达,但封菌组NLRP3炎性体的表达明显高于封入PBS组,炎性体的表达随着炎症进展过程的不同而有明显的变化,在2周炎症进展的急性期,所有因子的表达均至最高值(p<0.05)。(3)相关性分析结果显示,封菌组NLRP3与Caspase-1呈中度相关性,NLRP3与IL-1β、Caspase-1与IL-1β的表达呈高度相关,未封菌组均为中度相关。(4)破骨细胞的数量亦是在2周时达到高峰,封菌组高于未封菌组,破骨细胞数与IL-1β阳性细胞的表达呈中度相关性。结论:粪肠球菌所致的根尖周炎症反应过程中,NLRP3、Caspase-1及IL-1β表达明显增高,活化标志物IL-1β的高表达与破骨细胞的数量有着较强的相关性,提示NLRP3炎性体在根尖周病变的炎症反应及骨破坏过程中起到一定的作用。第二部分粪肠球菌脂磷壁酸LTA对NLRP3炎性体的活化作用及机制目的:检测粪肠球菌脂磷壁酸LTA能否直接活化NLRP3炎性体,并探讨其对NLRP3炎性体的活化机制。方法:1、选用粪肠球菌主要毒力因子LTA作为唯一刺激因素,作用于小鼠巨噬细胞RAW264.7 细胞上,运用 Real-time qPCR、Western blot 及 ELISA 方法检测 NLRP3炎性体相关因子mRNA及蛋白的表达情况。2、用NF-κB抑制剂BAY11-7082抑制NF-κB的核转位,检测LTA对NLRP3的活化是否借助NF-κB信号通路。为了检测抑制剂的抑制效率,通过免疫荧光染色来检测NF-κB的表达情况。3、用还原型烟酰胺腺嘌呤二核苷酸磷酸酶(nicotinamide adenine dinueleotide phosphate,NADPH)氧化酶抑制剂DPI抑制ROS的产生,检测LTA对NLRP3的活化是否通过ROS的产生而实现。为了检测抑制剂的抑制效率,荧光染色及流式细胞仪检测ROS的表达情况。结果:1、LTA可以直接活化小鼠巨噬细胞RAW264.7细胞的NLRP3炎性体。LTA作用于细胞2小时,可以促进NLRP3、Caspase-1及活化的IL-1β蛋白的高表达,24小时后NLRP3及Caspase-1蛋白的表达与2小时无明显差异,IL-1β蛋白的表达24小时高于2小时。NLRP3 mRNA的表达在第1天时最低(p<0.05),3天与5天时的表达差异无统计学意义(p>0.05),Caspase-1 mRNA在1、3、5天的表达无明显差异,IL-1β mRNA的表达在3天时达到峰值(p<0.05)。2、NF-κB抑制剂可以有效的抑制NF-κB P65的核转位,而一旦NF-κB信号通路被抑制,NLRP3、Caspase-1及IL-1β的蛋白及mRNA的表达均明显降低。3、DPI可以有效的抑制ROS的产生,ROS的表达被抑制后,NLRP3、Caspase-1及IL-1β的蛋白及mRNA的表达亦明显降低。结论:LTA是粪肠球菌活化NLRP3炎性体的毒力因子,LTA不依赖于外源性ATP的作用,能直接激活小鼠巨噬细胞系RAW264.7的NLRP3炎性体相关蛋白及mRNA的表达,主要借助NF-κB信号通路的活化及ROS的产生。第三部分中药薯蓣皂苷对NLRP3炎性体的抑制作用及促进骨形成的实验研究目的:检测中药薯蓣皂苷是否可以抑制粪肠球菌LTA所诱导的NLRP3炎性体的活化,及其在LTA存在下能否促进骨形成,为临床寻找合适的根尖周炎治疗药物提供实验室基础。方法:1、选用中药薯蓣皂苷作为实验药物,作用于小鼠巨噬细胞RAW264.7,用Real-time qPCR的方法检测NLRP3炎性体相关因子mRNA的表达,Western blot及ELISA方法检测NLRP3炎性体蛋白的表达情况。通过免疫荧光染色来检测薯蓣皂苷对NF-κB的表达情况,荧光染色及流式细胞仪检测其对ROS的表达情况。2、将中药薯蓣皂苷作用于小鼠胚胎成骨前体细胞MC3T3-E1,检测在LTA存在下薯蓣皂苷对细胞形态的影响,并用Real-time qPCR的方法检测成骨相关因子ALP、Runx2及OCN的mRNA表达情况,通过茜素红染色检测矿化结节的形成情况。结果:1、Real-time qPCR、Western blot及ELISA实验的结果显示,中药薯蓣皂苷在LTA存在下,可以明显降低NLRP3、Caspase-1及IL-1β的mRNA及蛋白的表达。免疫荧光染色及流式细胞仪的检测结果证实,其对NLRP3的抑制主要是通过抑制NF-κB的活化及ROS的释放而实现。2、中药薯蓣皂苷具有较强促进骨形成的作用,当药物作用于成骨细胞前体细胞的第3天,早期成骨因子基因ALP及Runx2的表达即高于正常对照组(p<0.05)。随着作用时间的延长,ALP及Runx2的表达也逐渐增高(p<0.05)。骨形成后期的标志物OCN在第7天开始表达高于正常对照组(p<0.05),在14天时的表达最高(p<0.05),当诱导延长至21天时,形成了较多的矿化结节,说明骨形成已经出现。结论:(1)薯蓣皂苷可以明显的抑制NLRP3炎性体的表达。其机制是通过抑制了 NF-κB信号通路及ROS的释放而实现。(2)薯蓣皂苷具有较强的促进骨形成的作用。(3)薯蓣皂苷可以作为临床难治性牙髓根尖周病治疗的候选药物。更多还原

【Abstract】 Periapical periodontitis was an immune inflammatory response which was primarily inflected by bacteria.Enterococcus faecalis(E.faecalis)is one of the common pathogenic bacteria with prevalence ranging from 29%to 77%in the endodontic periradicular lesions.E.faecalis has multiple virulence factors,including,protein adhesion,gelatinase,lipoteichoic acid(LTA)and so on.LTA,one of the main pathogenic factors,has a strong immunogenicity and is the main virulence factor.LTA can tolerate antibiotics and fungicides,triggering a series of inflammatory cytokine release and osteoblast apoptosis.The study found that E.faecalis LTA can interact with TLR2 of murine macrophage cell RAW264.7,then activated the NF-κB signaling pathway to promote the secretion of inflammatory cytokines TNF-a and NO,which may be one of the reasons causing periapical inflammation and bone destruction.As the first line of defense,innate immunity recognizes foreign pathogens by three kinds of pattern recognization receptors(PRR).Now three kinds of PRRs found mainly include the RIG-I like receptor(RLR),toll-like receptor(TLR),and NOD-like receptor(NLR).RLR mainly identify the virus,on the study of immune response of periapical disease caused by bacteria,although more evidence show that Toll-like receptor pathway and endodontic immune inflammatory disease has a closely related,but when Toll-like receptor pathway is inhibited,there is still the presence of inflammation,suggesting that another pathway may plays a role in periapical bone resorption,while NLR signaling pathways would be the new breakthrough.The researches on NLR signaling pathways focus on IL-1β-generation-related NLRP3 inflammasome which has high expression in stratified epithelium cells and myeloid cells,indicating that it may play a key role in the body’s first line of defense against pathogenic microorganisms.In response to external stimuli,by oligomerization,NLRP3 gathers a large number of Pro-Caspase-1,forms NLRP3 inflammasome and activates Caspase-1,and then promotes the Pro-IL-1βexpression,which are prerequisites for IL-1β secretion,IL-1βis a sign of activation of NLRP3 inflammasome in the innate immune defense,then causes the inflammation.Compared with reversible pulpitis,in irreversible pulpitis,NLRP3/Caspase 1 has significantly high erexpression.When used the E.faecalis act on macrophages THP-1,and found that E.faecalis can induce Caspase-1 activation and Pro-IL-1βexpression,then lead to overproduction of IL-1βand cause exaggerated pulpitis.So we speculate that NLRP3 inflammasome signaling pathways will be the future new direction to research the pathogenesis of periapical periodontitis.Dioscin is a natural product present in the rootstocks of dioscorea nipponica makino,commonly known as diosgenin,is an important basic raw material for the production of steroid hormone drug.Many researches have shown that dioscin has lots of medicinal effects,such as hepatoprotective properties,treatment of osteoporosis,anti-inflammatory,lipid-lowering,anti-tumor.Studies have demonstrated that Dioscin suppressed the expression of Reactive oxygen species(ROS),IL-1 and IL-6 by LPS-induced were through downregulated of CK2,JNK,NF-κB and AP-1 activation.However,the effects of dioscin on NLRP3 inflammasome is yet unknown.The present study divided into the in vivo and in vitro experiments.In the vivo experiments part,we first examined the expression of NLRP3 inflammasome in human chronic periapical inflammatory lesions,the results show that NLRP3 inflammasome expression was significantly higher than in normal tissues,indicating NLRP3 inflammasome involved in the progression of human chronic periapical inflammatory,which provided the basis for the further research.Enterococcus faecalis is the main pathogens in the refractory apical periodontitis,so we enclosed the E.faecalis sealed into the root canal to build the rat periapical periodontitis model,and to explore whether E.faecalis can activate the NLRP3 inflammasome and whether the inflammasome involved in the body in the progress of periapical inflammation and periapical bone resorption in rats.In the vitro experiments aim to detect the activation mechanism of the virulence factor of Enterococcus faecalis LTA on NLRP3 inflammasome,and explore the role of traditional Chinese medicine Dioscin in this process,as well as the drugs’bone formation function in the presence of LTA,and provide the laboratory basis for the clinical refractory periapical treatment.Specific studies were as follows:Part 1:Expression and role of NLRP3 inflammasome in periapical periodontitis Experiment 1:Expression of NLRP3 inflammasome in clinical chronic periapical periodontitisObjective:To investigate the expression of NLRP3 inflammasome in clinical chronic periapical periodontitis,and provided the clinical application basis for next research.Methods:We collected the apical lesions of chronic periapical periodontitis in patients who need surgery or tooth extraction,we used the method of Real-time qPCR and immunochemistry to detecte the expression of NLRP3 inflammasome.Results:NLRP3,Caspase-1 and IL-1βin human periapical periodontitis expression was significantly higher than the control group(p<0.05).Correlation analysis showed that,NLRP3 and Caspase-1 expression are associated with a modest,IL-1βand the expression of Caspase-1 and NLRP3 had low correlation.Conclusion:NLRP3 inflammasomes involved in human chronic periapical inflammation.Experiment 2:Establish the E.faecalis infected rat periapical periodontitis model and investigate the expression and role of NLRP3 inflammasomesObjective:Establish the E.faecalis infected rat periapical periodontitis model,observed and analyzed the inflammation and bone resorption during different pathological periapical lesions.Detected the expression of NLRP3,Caspase-1 and IL-1βin rat periapical lesions,and analyze the possible role and the relationship between periapical bone resorption and NLRP3 inflammasomes.Methods:E.faecalis is a major pathogen for the refractory periapical,so we enclosed the E.faecalis sealed into the root canal to build the rat periapical periodontitis model,and enclosed PBS cotton as experimental controls.Micro-CT and HE staining was confirmed the progression of inflammation,immunohistochemical to detect the protein localization and expression of NLRP3,and through Real-time qPCR to detect the mRNA of NLRP3,Caspase-1 and the IL-1β,while using an enzyme staining method to detect the osteoclasts expression,analysis the relationship of osteoclast cells with IL-1β,suggesting the possible role of NLRP3 inflammasome in the bone resorption of periapical periodontitis.Results:(1)Micro-CT and HE staining showed that we successfully constructed E.faecalis induced rat periapical animal model,periapical inflammation in 2 weeks was the acute phase,3 weeks was the beginning of chronic inflammation.The areas of periapical bone resorption from 1 week,and reached the maximum at 3 weeks(p<0.05),at 4 weeks the area with the 3 weeks.(2)NLRP3,Caspase-1 and IL-1β protein and mRNA were expressed in every group,but the expression of NLRP3 inflammasome sealing bacteria group was significantly higher than enclosed PBS group,the expression of NLRP3 inflammasomes was different as inflammatory processes,the significant changes during the acute phase of 2 weeks,the expression of all of the factors are to the highest value(p<0.05).(3)Correlation analysis showed that,NLRP3 and Caspase-1 was associated with a modest,the expression of NLRP3 and IL-1β,Caspase-1 and IL-1βhas highly correlated in E.faecalis+group,and in E.faecalis-group the relationship between the three groups was moderate correlation.(4)Osteoclast expression also reached the peak at 2 weeks,sealing bacteria group was significantly higher than enclosed PBS group,the expression of osteoclast-positive cells with IL-1βshowed a moderate correlation.Conclusion:In the periapical periodontitis caused by E.faecalis,the expression of NLRP3,Caspase-1 and IL-1βsignificantly increased,suggested that the E.faecalis can activate of the NLRP3 inflammasome,the high expression of IL-1β and the expression of osteoclasts has a strong correlation.Part 2.The activation mechanism of NLRP3 inflammasome by E.faecalis LTAObjective:Detected the E.faecalis LTA whether the virulence factor for E.faecalis activate the NLRP3 inflammasome,and to explore the activation mechanism of NLRP3 inflammasome.Methods:1.We chose the major virulence factor of E.faecalis LTA as the only stimulus actingon murine macrophage cells RAW264.7,using Real-time qPCR,Western blot and ELISA methods to detect the expression of NLRP3-associated inflammatory cytokine mRNA and protein.2.We used the NF-κB inhibitor BAY 11-7082 inhibiting NF-κB signaling pathway to detect NLRP3 inflammasome activation mechanism by LTA whether by means of NF-κB signaling pathway.In order to confirm the inhibitors suppression efficiency,we used immunofluorescence staining to detect the expression of NF-κB.3.We used the nicotinamide adenine dinue leotide phosphate(NADPH)oxidase inhibitor DPI inhibited the expression of ROS to verify LTA for NLRP3 activation mechanism.In order to confirm the inhibitors suppression efficiency,we used staining and flow cytometry to detect ROS.Results:1.LTA can directly activate NLRP3 inflammasome without the need of ATP.LTA stimulate the murine macrophage two hours,it can promote the high expression of protein of NLRP3,Caspase-1 and IL-1β,protein expression had no significant difference in 2 hours after 24 hours.NLRP3 mRNA expression at day 1 was the minimum(p<0.05),3 days and 5 days was not statistically significant(p>0.05).The mRNA expression of Caspase-1 had no significant difference between 1,3,5 days,while the mRNA expression of IL-1βat the peak in 3 days(p<0.05).2.NF-κB inhibitors can effectively inhibit NF-κB P65 nuclear transfer,and once NF-κB signaling pathway is inhibited,the mRNA and protein expression of NLRP3,Caspase-1 and IL-1βwere significantly reduced.3.DPI can effectively inhibit the production of ROS,once the expression of ROS is suppressed,the mRNA and protein expression of NLRP3,Caspase-1 and IL-1βwere also decreased significantly.Conclusion:LTA was the virulence factor for E.faecalis activate the NLRP3 inflammasome,E.faecalis LTA does not depend on the exogenous ATP,can directly activate the protein and mRNA of NLRP3 inflammasome expression in mouse macrophage cell line RAW264.7,mainly through the activation of NF-κB signal pathway and ROS production.Part 3.Experimental Study of Dioscin on the activation of NLRP3 inflammasome stimulated by LTA and the effected on bone formation Objective:We explored whether the medicine Dioscin can inhibit the activation of NLRP3 inflammasome by LTA,as well as the drugs’ bone formation function in the presence of LTAm and provide the laboratory basis for the clinical refractory periapical treatment.Methods:1.We used the traditional Chinese medicine Dioscin as the experiment drug to acted on the RAW264.7,Real-time qPCR method to detect the expression of NLRP3 inflammasome mRNA,Western blot and ELISA methods to detect the expression of NLRP3 inflammasome protein.By immunofluorescence staining to detect Dioscin on NF-κB expression,staining and flow cytometry for expression of ROS.2.Dioscin acted on the mouse embryonic osteoblast precursor cells MC3T3-E1,Real-time qPCR method to detect the expression of osteogenic factor ALP、Runx2 and OCN mRNA,and detected the formation of mineralized nodules by alizarin red staining.Results:1.Results of Real-time qPCR and Western blot showed that even with the presence of LTA,traditional chinese medicine Dioscin,can significantly reduce the expression of NLRP3,Caspase-1 and IL-1βmRNA and protein.Test results immunofluorescence staining and flow cytometry confirmed that the inhibition mechanism of Dioscin mainly through the inhibiting activation of NF-κB and ROS released.2.Chinese medicine dioscin has a strong role to promote the bone formation,when the drug acts on osteoblast precursor cells in the third days,the expression of early osteogenic factor Runx2 and ALP is higher than the normal control group(p<0.05).With the extension of time,the expression of Runx2 and ALP has gradually increased(p<0.05).Bone formation markers OCN in the 7th day was higher than the normal control group(p<0.05),the highest expression at 14 days(p<0.05),while inducing extended to 21 days,the formation of a greater amount of mineralization knotsection,indicating bone formation has occurred.Conclusion:(1)Dioscin NLRP3 can significantly inhibit the expression of NLRP3 inflammasome,and the mechanism is by inhibiting the activation of NF-κB signaling pathway and the release of ROS.(2)Dioscin has a strong role to promote bone formation.(3)Dioscin can be used as a drug candidates for the clinical refractory endodontic disease treatment.更多还原