Role And Mechanism Of Proganulin In Chronic Periondontitis

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-1?)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-1? 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-1? 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(0.1?g/mL)or Pg-LPS(1?g/mL)for 0,2,6,12 and 24 h.The mRNA expression of inflammatory cytokines(TNF-? IL-1? 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(1?g/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-??1L-1?.IL-6 and MCP-1)were evaluated by RT-qPCR and the protein expression of TNF-a and IL-1? 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(1?g/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,I?Ba 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-1? 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-? 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-1?,IL-6 and MCP-1)were significantly increased in the 1?g/mL Pg-LPS-stimulated HGF-1 cells compared with control cells(p<0.01).Therefore,1?g/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-1?,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-1? 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-?B 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-?B pathway.Pg-LPS activated NF-?B signaling pathway by increasing phosphorylation of p65 and I?B? 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-?B 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-?B 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.