【作者】 李潇；

【导师】 杨丕山；

【作者基本信息】 山东大学 ， 口腔临床医学（专业学位）， 2018， 博士

【摘要】 牙周炎是口腔致病微生物诱发宿主免疫应答导致的慢性感染性疾病,微生物与宿主的相互作用决定了疾病的过程和进展。牙周组织炎症破坏的机制现尚不十分明确,目前认为口腔致病菌感染是牙周炎的始动因素,但牙周大多数软硬组织损伤主要是由于宿主对感染的免疫应答失调而引起,在此过程中,促炎细胞因子如IL-lβ、IL-6、TNF-α等大量释放,导致和进一步放大炎症反应。TNF-α被认为是牙周炎发病机制中的关键炎症因子。TNF-α可以通过激活多种相关炎性通路如NF-κB信号通路从而促进下游炎症因子的表达,继而导致炎性组织损伤。因此,对TNF-α及其相关通路的拮抗是研究者们关注的热点。目前尽管已有研究取得了一定进展,例如采用新型靶向抑制剂拮抗TNF-α以阻断其导致牙周组织破坏的途径,但效果并不理想。进一步研究牙周炎致病的分子机制,研究开发用于牙周炎辅助治疗的新策略是非常必要的。颗粒蛋白前体(progranulin,PGRN)是一种由593个氨基酸组成的自分泌生长因子。PGRN广泛表达于多种组织,尤其在快速增殖的上皮细胞、神经元、免疫细胞和脂肪细胞中大量表达,参与多种病理生理过程,如早期胚胎发生、组织修复、肿瘤发生及神经退行性疾病。近年来,PGRN在免疫、感染和炎症中的作用备受关注。作为TNF受体(TNFR)的新配体,PGRN通过靶向结合TNFR从而拮抗TNF-α功能而发挥抗炎作用。例如,在类风湿性关节炎小鼠模型中,给予人PGRN重组蛋白(rhPGRN)可显著减轻小鼠的炎症反应。此外,PGRN在炎性肠病、脂多糖诱导的急性肺部炎症和毒素诱导的脑损伤中均显示了抗炎和保护作用。然而,与普遍观察到的抗炎作用相反,PGRN在某些疾病模型如肥胖和胰岛素抵抗性糖尿病中表现出促炎作用。目前,PGRN在牙周炎中的作用及机制尚不清楚。因此,我们的研究旨在探讨PGRN的表达与临床慢性牙周炎疾病过程之间的关系,在实验性牙周炎大鼠模型中评估外源性重组PGRN蛋白潜在的抗炎作用。进一步采用细胞学实验,探究PGRN对人牙龈成纤维细胞炎症反应及相关信号通路的影响。材料和方法1 PGRN在慢性牙周炎中的表达1.1 PGRN在牙周炎患者牙龈组织中的表达研究对象分为慢性牙周炎组(n=15,平均年龄为37.8±6.9岁)及健康对照组(n=15;平均年龄为35.2±6.6岁)。慢性牙周炎组的牙龈组织取自需要进行牙周翻瓣术的炎症病损部位,健康对照组的牙龈组织取自需要行牙冠延长术的牙周健康部位。分别采用免疫组织化学染色法及RT-qPCR法对牙周组织中PGRN的表达进行检测。1.2龈沟液中PGRN水平的检测及PGRN/TNF-α摩尔比值分析选取30位慢性牙周炎患者及28位牙周健康志愿者为研究对象。分别在治疗前及牙周基础治疗后一个月收集取样位点的龈沟液标本。通过ELISA方法检测治疗前后取样位点龈沟液中PGRN,TNF-α和IL-1β的分泌水平,计算治疗前后PGRN/TNF-α的摩尔比值并进行统计学分析。2 rhPGRN在大鼠实验性牙周炎模型中的作用2.1大鼠实验性牙周炎模型的建立及PGRN检测采用丝线联合正畸结扎丝结扎双侧上颌第一磨牙的方法,建立大鼠实验性牙周炎模型,分别于实验的0天,10天及20天时处死大鼠,取上颌骨标本,HE染色后进行组织学分析;收集大鼠实验牙的新鲜牙龈组织,使用RT-qPCR检测牙龈组织中PGRN mRNA表达水平。2.2 rhPGRN对大鼠实验性牙周炎组织病损的影响为探究PGRN在牙周炎中的作用,我们观察了外源性rhPGRN对大鼠实验性牙周炎局部组织病损的影响。牙周炎大鼠每48小时注射rhPGRN或PBS至上颂第一磨牙的牙龈中,分别于注射后10天和20天处死大鼠。取上颂骨标本,HE染色后行组织学分析,观察牙周炎性细胞浸润及牙槽骨吸收的情况;TUNEL染色观察细胞凋亡情况。RT-qPCR和ELISA方法检测牙龈组织中炎性细胞因子TNF-α和IL-1β的表达。3 PGRN对人牙龈成纤维细胞(HGF-1)炎症反应的影响及分子机制3.1不同浓度Pg-LPS刺激对HGF-1炎性因子表达的影响HGF-1细胞培养于6孔细胞培养板中,分为3组:1)对照组;2)0.1μg/mL Pg-LPS 刺激组;3)1μg/mLPg-LPS 刺激组。于不同时间点(0、2、6、12、24 h),提取细胞总RNA,RT-qPCR检测炎性细胞因子TNF-α、IL-1β、IL-6和MCP-1在mRNA水平的变化。3.2 rhPGRN对Pg-LPS诱导HGF-1表达炎性因子的影响HGF-1细胞分别培养于6孔和96孔细胞培养板中,实验分组:1)对照组;2)Pg-LPS(1μg/mL)处理组;3)Pg-LPS + rhPGRN 共处理组:rhPGRN(500ng/mL)预处理HGF-1细胞2h,加入pg-LPS(1μg/mL)刺激。于不同时间点(0、2、6、12及24 h),分别提取细胞总RNA和收集细胞上清液,采用RT-qPCR检测上述炎性因子(TNF-α、IL-1β、IL-6 和 MCP-I)mRNA 的表达,ELISA 检测 TNF-α和IL-1β蛋白水平的表达。3.3 rhPGRN对Pg-LPS介导的NF-κB信号通路激活的影响HGF-1细胞培养于6孔细胞培养板中,实验分组同3.2。于不同时间点(0,5,15,30,60,120 min)提取细胞总蛋白,采用Western Blot方法检测NF-κB信号通路的相关蛋白(p-p65、p65、p-IκBα、IκBα)。结果1 PGRN在慢性牙周炎中的表达1.1 PGRN在慢性牙周炎患者的牙龈组织中表达增高免疫组化结果显示,PGRN在健康对照组牙龈标本中呈弱阳性表达。高倍镜下观察,发现PGRN主要分布于健康牙龈上皮细胞的细胞质中。在慢性牙周炎患者病损部位的牙龈样本中PGRN多呈中度或强阳性表达,PGRN染色强度及阳性细胞数目均较健康对照组明显增高。在高倍镜下观察,发现在牙周炎患者牙龈组织中,PGRN不仅分布于上皮细胞的细胞质中,还广泛分布于牙龈上皮的细胞外基质中。染色强度的相对定量分析显示两组之间PGRN的表达水平有统计学差异(p＜0.01)。RT-qPCR检测结果表明慢性牙周炎组PGRN的mRNA水平明显高于健康对照组(P<0.01)。1.2牙周基础治疗前后龈沟液中PGRN的检测及PGRN/TNF-α摩尔比值分析ELISA结果显示,在牙周基础治疗前,慢性牙周炎组龈沟液中的PGRN水平明显高于健康对照组(p<0.05)。而后,对慢性牙周炎组牙周治疗前后的PGRN和炎性细胞因子水平进行比较。结果显示,牙周基础治疗后一个月,慢性牙周炎组的牙周临床指征显著好转,龈沟液中炎性细胞因子TNF-α水平明显降低(p<0.01),PGRN水平也呈下降趋势(p<0.05),但PGRN/TNF-α的摩尔比值较治疗前的基线水平明显升高(P<0.01)。以上结果表明,牙周炎患者龈沟液中PGRN/TNF-α比值与患者疾病的严重程度呈现负相关,提示PGRN可能具有潜在的抗炎作用。2 rhPGRN在大鼠实验性牙周炎中的作用研究2.1大鼠实验性牙周炎模型的建立及PGRN检测组织病理学分析结果显示,上颂磨牙结扎术后10天,大鼠的牙龈上皮糜烂,结缔组织中有大量炎性细胞浸润。结扎术后20天,有明显的牙周袋形成,在上颂第一磨牙和第二磨牙的牙间隔区出现明显的牙槽骨吸收。说明,大鼠实验性牙周炎的建模成功。为观察内源性PGRN在大鼠实验性牙周炎组织中的表达状况,采用RT-qPCR检测大鼠牙周炎牙龈组织中PGRN的表达。结果显示,与结扎术前的基线水平相比,结扎术后10天大鼠牙龈组织中PGRN mRNA表达增加(p<0.05),术后20天的PGRN mRNA水平与术后10天基本一致。两组之间无统计学差异。2.2 rhPGRN对大鼠实验性牙周炎组织病损的影响2.2.1 rhPGRN减少实验性牙周炎大鼠的炎性细胞浸润组织病理学分析示,大鼠结扎术后10天,与牙周炎对照组相比,牙周炎/rhPGRN注射组的大鼠牙龈组织中,炎性细胞浸润和细胞外基质破坏的程度均明显降低(p<0.05)。结扎术后20天,牙周炎/rhPGRN注射组的炎性细胞浸润程度进一步下降。牙周组织炎性细胞计数的统计学分析进一步支持以上结论。2.2.2 rhPGRN抑制实验性牙周炎大鼠的牙槽骨吸收组织病理学结果显示,大鼠结扎术后10天,牙周炎对照组与牙周炎/rhPGRN注射组第一磨牙远中牙槽骨吸收的程度无明显差异。大鼠结扎术后20天,牙周炎对照组牙槽骨吸收的程度进一步加重,出现明显的牙槽骨嵴水平降低,然而牙周炎/rhPGRN注射组仅有轻度的牙槽骨破坏吸收,两组之间有显著统计学差异(p<0.05)。2.2.3 rhPGRN减少实验性牙周炎大鼠的牙周组织细胞凋亡TUNEL染色结果显示,大鼠结扎术后10天及20天,在牙周炎对照组牙龈乳头的上皮结缔组织中可见大量TUNEL荧光染色阳性的成纤维细胞。牙周炎/rhPGRN注射组仅观察到少量的TUNEL阳性细胞。对TUNEL阳性细胞进行计数,并行统计学分析,结果示在术后10天和20天两组之间均具有统计学差异(p<0.01)。以上结果说明,rhPGRN可减少牙周炎大鼠牙周组织中成纤维细胞的凋亡。2.2.4 rhPGRN降低实验性牙周炎大鼠牙周组织中促炎细胞因子的表达采用RT-qPCR和ELISA方法检测大鼠牙周炎牙龈组织中促炎细胞因子的表达。结果显示,大鼠结扎术后10天,牙周炎/rhPGRN注射组TNF-α的表达水平即较牙周炎对照组明显降低,统计学分析两组间差异显著(P<0.01)。术后20天,牙周炎/rhPGRN注射组TNF-α和IL-1β在mRNA及蛋白水平的表达均较牙周炎对照组有显著的下降(P<0.01)。3 rhPGRN对人牙龈成纤维细胞炎症反应的影响及分子机制3.1不同浓度Pg-LPS刺激对HGF-1炎性因子表达的影响RT-qPCR结果显示,1μg/mLPg-LPS可有效刺激HGF-1产生炎性细胞因子,TNF-α、IL-1β、1L-6和MCP-1在mRNA水平明显升高,与对照组细胞相比差异显著(p<0.01)。故采用1μg/mLPg-LPS用于后续实验。3.2 rhPGRN降低Pg-LPS诱导的HGF-1炎性因子表达RT-qPCR检测结果显示,与单独Pg-LPS处理组相比,Pg-LPS+rhPGRN共处理组炎性因子TNF-α、IL-1β、IL-6和MCP-1 mRNA表达降低(p<0.01),两组间差异显著。ELISA结果进一步验证,rhPGRN处理降低了 TNF-α和IL-1β蛋白水平的分泌(p<0.01)。以上结果证明,在牙周致病菌Pg-LPS诱导的牙周细胞炎症环境中,PGRN可有效降低人牙龈成纤维细胞中促炎细胞因子的产生与释放,从而发挥抗炎作用。3.3 rhPGRN抑制Pg-LPS诱导的NF-κB信号通路的激活为进一步探讨rhPGRN抑制Pg-LPS介导炎症反应的分子机制,我们检测了rhPGRN对Pg-LPS诱导的NF-κB信号通路活化的影响。Western Blot结果显示,Pg-LPS可激活HGF-1细胞中的NF-κB信号通路相关信号分子,磷酸化的NF-κB pp65以及pIκB-α蛋白表达增加。与单独Pg-LPS处理组相比,Pg-LPS+rhPGRN共处理组的NF-κB pp65和pIKB-α蛋白表达降低,说明重组PGRN在一定程度上抑制了 Pg-LPS介导的NF-κB信号通路的激活。结论1、慢性牙周炎患者牙龈组织及龈沟液中PGRN的表达水平明显升高。牙周炎患者行牙周基础治疗后,龈沟液中PGRN/TNF-α摩尔比值较治疗前升高且与疾病严重程度呈负相关。提示PGRN在慢性牙周炎中可能具有潜在的抗炎功效。2、局部注射重组PGRN可缓解大鼠实验性牙周炎的炎症反应,抑制牙槽骨丧失并减轻牙周组织的细胞凋亡。证明,PGRN对实验性牙周炎的炎症损伤具有保护作用。3、细胞学实验证明,重组PGRN能够抑制Pg-LPS诱导的HGF-1炎性因子表达,其机制与PGRN抑制了炎症环境中NF-κB信号通路的活化有关。综上所述,本研究通过临床和动物实验发现颗粒蛋白前体(PGRN)在牙周炎中具有抗炎和保护作用。细胞学实验证明,PGRN抑制炎性细胞因子的表达与其阻断NF-κB信号通路的活化有关。PGRN对牙周炎的保护作用及其机制尚需进一步的研究。更多还原

【Abstract】 Periodontitis is a chronic inflammatory disease characterized by the destruction of supporting soft and hard tissues of the teeth.Though the current understanding on periodontitis implicates microbial residents of the subgingival biofilm as the primary cause of the disease,the major periodontal destruction progresses from the hosts’inflammatory response to the bacterial challenge.The increased pro-inflammatory cytokines,such as interleukin-1 beta(IL-1β)and tumor necrosis factor alpha(TNF-a),significantly amplified the inflammatory response,produced the lytic enzymes and stimulated the production of chemokines.TNF-a is considered as a master regulator in the pathogenesis of periodontitis,participating in tissue destruction and osteoclastogenesis in periodontal diseases.Although few studies have made great progress in developing treatment strategies such as new classes of inhibitors that target TNF-a or lipoxygenase to block the pathways responsible for periodontal tissue breakdown,the effect is far from ideal.Further study on the mechanisms of the molecular basis involved in periodontal diseases is essential to identify new targets and develop new techniques and strategies for the prevention and treatment of these diseases.Progranulin(PGRN),also known as GRN-epithelin precursor,is a 593-amino-acid autocrine growth factor and is abundantly expressed in epithelial cells,neurons,immune cells,and adipocytes.PGRN plays a critical role in multiple pathophysiological processes,including early embryogenesis,tissue repair,neurodegeneration and tumorigenesis.Recent studies have highlighted the importance of PGRN in immunity,infection,and inflammation.In particular,as a novel ligand of TNF receptors(TNFRs),PGRN plays an anti-inflammatory role by targeting TNFRs and antagonizing TNF-a function.For example,administration of recombinant human PGRN(rhPGRN)significantly alleviated inflammatory responses in mouse models of rheumatoid arthritis.In addition,PGRN has a protective role in inflammatory bowel diseases,lipopolysaccharide(LPS)-induced acute pulmonary inflammation and toxin-induced brain injury.In contrast to the commonly observed anti-inflammatory effect,PGRN exhibited pro-inflammatory effects in some disease models,such as obesity and insulin-resistant diabetes.The role of PGRN in periodontitis still remains unclear.Considering the inflammatory nature of the disease,our study aimed to investigate the relationship between PGRN expression and disease process in patients with chronic periodontitis(CP),as well as assess the potential use of exogenous PGRN as a therapeutic agent for CP in an experimental periodontitis rat model.Further cytology experiments were performed to explore the role of PGRN on inflammatory response and related signaling pathways in human gingival fibroblasts.MATERIALS AND METHODS1 The expression of PGRN in patients of chronic periodontitis(CP)1.1 PGRN expression in gingival tissuesGingival biopsies were obtained from CP patients(n=15;mean age 37.8±6.9 years)and healthy controls(n=15;mean age 35.2+6.6 years).Gingival biopsies of CP patients were obtained by performing periodontal flap surgery at the periodontitis-affected sites.Normal gingival biopsies were collected during crown-lengthening procedures from periodontally healthy sites.Expressions of PGRN in gingival biopsies were assessed by RT-qPCR and immunohistochemistry,respectively.1.2 PGRN levels and PGRN/TNF-a molar ratio in gingival crevicular fluid(GCF)Thirty CP patients and 28 healthy controls were recruited.GCF samples were collected from the gingival crevice of the study sites,at the baseline and one month after non-surgical periodontal treatment.The levels of PGRN,TNF-a,and 1L-1β in the GCF before and after non-surgical periodontal treatment were quantified by ELISA.Then,the molar ratio of PGRN/TNF-a was calculated.2 The role of rhPGRN in an experimental periodontitis rat model2.1 Establish of an experimental periodontitis model in rat and detection of PGRNExperimental periodontitis was induced by ligature placement and six rats were respectively sacrificed with an overdose anesthetic at 0,10 and 20 days.Maxillary specimens with teeth and periodontal tissues were collected.HE staining was conducted for morphological analysis.Expressions of PGRN levels in gingival tissues of rat were detected by RT-qPCR.2.2 The role of rhPGRN in an experimental periodontitis rat modelTo directly investigate the effects of PGRN in periodontitis,recombinant human PGRN(rhPGRN)or its vehicle was injected into the gingiva of rats ’with ligature-induced experimental periodontitis every 48h.Local inflammatory cell infiltration and alveolar bone loss were assessed by morphological analysis.Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)assay in histological sections.And the expression levels of TNF-a and IL-1β in the gingiva were determined by RT-qPCR and ELISA.3 Effects of rhPGRN and its molecular mechanism on Pg-LPS-induced inflammatory response in human gingival fibroblasts(HGF-1)3.1 Inflammatory response in HGF-1 cells stimulated by different dosages of Pg-LPSAfter cultured in 6-well plates,the HGF-1 cells were stimulated with Pg-LPS(0.1μg/mL)or Pg-LPS(1μg/mL)for 0,2,6,12 and 24 h.The mRNA expression of inflammatory cytokines(TNF-α IL-1β IL-6 and MCP-1)were evaluated by RT-qPCR.3.2 Effects of rhPGRN on Pg-LPS-induced inflammatory response in HGF-1 cellsAfter cultured in 6-well and 96-well plates,the HGF-1 cells were pre-treated with rhPGRN(500 ng/mL)or PBS 2 h before Pg-LPS(1μg/mL)was added.The total RNA was isolated at 0,2,6,12 and 24 h after Pg-LPS treatment.The mRNA expression of inflammatory cytokines(TNF-α、1L-1β.IL-6 and MCP-1)were evaluated by RT-qPCR and the protein expression of TNF-a and IL-1β were evaluated by ELISA.3.3 Effects of rhPGRN on Pg-LPS-induced NF-kB activationAfter cultured in 6-well plates,the HGF-1 cells were pre-treated with rhPGRN(500 ng/mL)or PBS 2 h before Pg-LPS(1μg/mL)was added.The total protein was isolated at 0,5,15,30,60 and 120 min after Pg-LPS treatment.Western Blot was used to detect the level of the NF-kB p65,phospho-p65,IκBa and phospho-IKBa.Results1 The expression of PGRN in patients of chronic periodontitis1.1 PGRN is highly expressed in gingival tissues of patients with CPOn immunohistochemistry,PGRN staining in gingival specimens of CP patients was more intense than in normal gingiva,indicating enhanced PGRN expression in both cytoplasm of epithelial cells and the extracellular matrix.Relative quantification of staining intensity showed significant difference between the two groups(p<0.01).RT-qPCR analysis further confirmed that the mRNA level of PGRN was higher in CP lesions than in normal gingiva(p<0.01).1.2 PGRN levels and PGRN/TNF-a molar ratio in GCFThe levels of PGRN were significantly higher in CP patients than in healthy controls at baseline(p<0.05).With the decline of periodontal clinical indexes,the molar ratio of PGRN to TNF-a in GCF at one month after non-surgical treatment was significantly higher than at baseline(p<0.01).2 The role of rhPGRN in an experimental periodontitis rat model2.1 Establish of an experimental periodontitis rat model and detection of PGRNHistopathologic analysis at 10 days after the ligation demonstrated that there was an obvious epithelial cells erosion coupled with an intense inflammatory cell infiltration throughout the connective tissue.At 20 days after the ligation,an obvious alveolar bone loss was observed in the interproximal area between the first and second maxillary molars.RT-qPCR analysis indicated that there was a significant increase of PGRN level in gingival tissues of rat at 10 days after ligation when compared with baseline(p<0.05).And there was no significant difference between the levels of PGRN at 10 and 20 days.2.2 The role of rhPGRN in an experimental periodontitis rat model2.2.1 rhPGRN inhibites inflammatory cell infiltration in rats with experimental periodontitis(EP)Histopathologic analysis of the samples at 10 days after the first injection demonstrated that the EP/rhPGRN group had a significant reduction in the degree of inflammatory infiltration and extracellular matrix destruction as compared with the EP/vehicle group(p<0.05).At 20 days after the first injection,the histologic features were similar to those observed on day 10.Quantitative analysis further confirmed that the number of inflammatory cells was significantly decreased in the EP/rhPGRN group when compared with the EP/vehicle group at both time points.2.2.2 rhPGRN reduces bone loss in rats with EPHistomorphometric analysis revealed no significant difference of alveolar bone loss,as indicated by the linear distance from CEJ to ABC,between the EP/rhPGRN group and the EP/vehicle group on day 10(p>0.05).However,the EP/rhPGRN group showed significantly reduced alveolar bone loss when compared with the EP/vehicle group on day 20(p<0.05).2.2.3 rhPGRN attenuates cell apoptosis in rats with EPApoptosis was evaluated as the number of TUNEL+ cells above the interproximal alveolar bone on days 10 and 20.The number of TUNEL+ cells was significantly lower in the EP/rhPGRN group when compared with the EP/vehicle group at both time points(p<0.01).2.2.4 rhPGRN reduces pro-inflammatory cytokine levels in rats with EPThe EP/vehicle group demonstrated a significant increase in both the mRNA and protein levels of TNF-a and IL-1β on days 10 and 20 when compared with the control group(p<0.01).rhPGRN administration significantly reduced the production of these pro-inflammatory cytokines,especially TNF-α when compared with the EP/vehicle group at both time points(p<0.01).3.Effects of rhPGRN and its molecular mechanism on the Pg-LPS-induced inflammatory response in HGF-1 cells3.1 The expression of inflammatory cytokines stimulated by different dosages of Pg-LPS in HGF-1 cellsAccording to RT-qPCR analysis,the mRNA levels of inflammatory cytokines(TNF-a,IL-1β,IL-6 and MCP-1)were significantly increased in the 1μg/mL Pg-LPS-stimulated HGF-1 cells compared with control cells(p<0.01).Therefore,1μg/mL Pg-LPS was choosed to be used in subsequent experiments.3.2 rhPGRN inhibites overexpression of inflammatory cytokines in Pg-LPS-stimulated HGF-1 cellsThe mRNA expression of TNF-a,IL-1β,IL-6 and MCP-1 in the HGF-1 cells co-treatmented with Pg-LPS and rhPGRN was significantly decreased compared with the HGF-1 cells with Pg-LPS treated alone,as proved by RT-qPCR(p<0.01).Furthermore,reduction of TNF-a and IL-1β levels in rhPGRN treated HGF-1 cells are confirmed by ELISA analysis(p<0.01).Above results suggest that PGRN can effectively reduce the production of pro-inflammatory cytokines in Pg-LPS-stimulated human gingival fibroblasts.3.3 rhPGRN attenuates activation of NF-κB in Pg-LPS-stimulated HGF-1 cellsTo further explore the molecular mechanism of rhPGRN inhibiting the Pg-LPS-mediated inflammatory response,we observed the effects of rhPGRN on NF-κB pathway.Pg-LPS activated NF-κB signaling pathway by increasing phosphorylation of p65 and IκBα in HGF-1 cells.However,the escalation of phospho-p65 and phospho-lKBa were attenuated by rhPGRN treatment.The results suggest that rhPGRN blocks the activation of Pg-LPS-mediated NF-αB signaling pathway to some extent,thereby reducing the expression of the flammatory factors regulated by this pathway.Conclusions1.PGRN is highly expressed in the gingiva and GCF of patients with CP.The molar ratio of PGRN to TNF-a in GCF of CP patients is negatively associated with the disease severity.2.Local delivery of rhPGRN to rats with EP reduces gingival inflammatory response,inhibites alveolar bone loss and alleviates cell apoptosis of periodontal tissue.These results demonstrate that PGRN plays a protective role in the inflammatory injury of experimental periodontitis.3.rhPGRN can reduce Pg-LPS-induced inflammatory cytokines production via blocking NF-αB pathways in HGF-1 cells.Our results provided new insights into the potential protective role of PGRN in periodontitis,which may benefit in the clinical preventive and therapeutic Strategy for periodontal-associated inflammation.更多还原