| Objective: Chronic apical periodontitis(CAP)was considered to be a result of inflammatory responses to infections from root canals,which induced granuloma formation and resorption of alveolar bone.Porphyromonas endodontalis(P.endodontalis)was a unique pathogens of pulp infection,its lipopolysaccharide(LPS)was a major virulence factor,a large number of proinflammatory cytokines such as interleukin(IL)-1,IL-6,IL-8 and tumor necrosis factor(TNF)which were secreted by the stimulating of P.endodontalis LPS palyed crucial roles in inflammatory responses.Proinflammatory cytokines were produced by P.endodontalis LPS-induced IL-6.The role of IL-6 was accelerated osteoclast differeatiation and supressed osteoblast activity.Osteoclast was activated by IL-6 which was directly binding to osteoclast surface receptors.Some previous findings reported that the secretion of proinflammatory cytokines coule be suppressed by miR-146a-5p through decreasing the expressions of tumor necrosis factor receptor-associated factor-6(TRAF6)and interleukin receptor-associated kinase-1(IRAK1).However,little information was available regarding the functional involvement of miR-146a-5p in the pathogenesis of inflammatory response in CAP.Whether miR-146a-5p played an anti-inflammatory role in P.endodontalis LPS-mediated osteoblastic inflammation was still unclear.Hairy and enhancer-of-split related with YRPW motif(Hey)is a member of the basic helix-loop-helix(b HLH)-type transcription factors,Hey2 had been confirmed to be induced by the Notch signaling pathway which played a key role in the cardiac development,angiogenesis,bone tissue differentiation,tooth development,pulp regeneration and repair after injury.In the early stage,bioinformatics was used to predict whether Hey2 might be a potential target gene of miR-146a-5p.The role of Hey2 in CAP and P.endodontalis LPS-induced osteoblastic inflammation was unknown.In conclusion,we intended to exam the levels of miR-146a-5p and Hey2 expression in CAP and discuss the possible relationships among miR-146 a,Hey2 and IL-6 in inflammatory responses of P.endodontalis LPS–treated MC3T3-E1 cells,to explore the functional roles of miR-146 a and Hey2 in CAP,as well as underlying mechanisms,to further investigate Hey2 as a transcriptional repressor regulatory mechanism of inflammation.Our study might provide a new insight into target treatment of CAP.Methods:1.Experimental methods1.P.endodontalis culture,P.endodontalis LPS extraction and identification.2.MC3T3-E1 cells,293 T cells culture.3.A total of 20 periapical tissue from CAP patients and 13 normal periodontal ligament tissue from clinically healthy volunteers were obtained after approving by the Ethics Committee and obtaining the informed consent of the patients.4.Hematoxylin-Eosin(H-E)staining was used to measure the extent of inflammatory infiltration in periapical tissues.5.Immunohistochemical(IHC)staining was used to detect Hey2 protein expression in periapical tissues.6.The MC3T3-E1 cells were transfected with miR-146a-5p mimics,inhibitor and Hey2 overexpression plasmid.7.The relative expression of miR-146a-5p,Hey2 and IL-6 m RNA in each group were detected by real-time reverse transcription-polymerase chain reaction(real-time RT-RCR).8.The level of IL-6 protein excretion were examed by Enzyme-like immunosorbent assay(ELISA).9.The level of Hey2 protein in each group were confirmed by Western blot.10.The binding of miR-146a-5p to Hey2 3’UTR was examined by dual luciferase activity assay in 293 T cells.11.Chromatin Immunoprecipitation(Ch IP)analysis was performed to assess whether Hey2 protein could be recruited to the IL-6 promoter in osteoblasts after 10μg/ml P.endodontalis LPS treatment for 24 h.2.Statistical analysis Each experiment was performed at least in triplicate.The collected data are presented as the mean ± standard deviation(SD)except for the gender of the patients using Mann-Whitney U test.Statistical analyses were performed using SPSS 18.0 software.The P-values were calculated using a one-way analysis of variance(ANOVA)or Dunnett’s t-test.A P value of < 0.05 was considered statistically significant.Results:1.Expression of miR-146-5p and Hey2 in periapical tissues(1)There was a large number of inflammatory cell infiltration in periapical lesions compared to normal periodontal tissues.(2)Hey2 were mainly located in nuclei and cytoplasm,Hey2 positive was observed in plasma cells,neutrophils and macrophages in periapical lesions.(3)Both the relative expression of miR-146-5p and Hey2 were significantly increased in the CAP group compared with healthy tissue.2.Negative correlation between miR-146a-5p and P.endodontalis LPS-induced IL-6expression in MC3T3-E1 cells(1)MC3T3-E1 cells were stimulated with different concentrations of P.endodontalis LPS for 24 h,miR-146-5p was statistically upregulated in a dose-dependent manner.Moreover,the expression of IL-6 was significantly increased,with the highest level by10 μg/m L P.endodontalis LPS.(2)MC3T3-E1 cells were treated with 10 μg/m L P.endodontalis LPS for different time,the results showed that the expressions of miR-146-5p and IL-6 were statistically raised,with the highest level at 10 h.(3)During the process of P.endodontalis LPS-induced osteoblastic inflammation,the expression of IL-6 was significantly decreased by miR-146 a mimics,while was significantly increased by miR-146 a inhibitor.3.Association between miR-146 a and Hey2(1)Hey2 expression was negatively regulated by miR-146-5p in P.endodontalis LPS-stimulated MC3T3-E1 cells.Overexpression of Hey2 was significantly decreased the expression of miR-146-5p.(2)Dual luciferase activity assay confirmed that miR-146a-5p could bind to specific site of Hey2 3’UTR,the relative luciferase activity of Hey2 3’UTR in WT was significantly decreased by overexpression of miR-146a-5p.4.Hey2 suppressed IL-6 expression induced by P.endodontalis LPS in osteoblasts(1)The expression of Hey2 was only significantly increased by 5,10 or 15 μg/m L P.endodontalis LPS treatment.Besides,the expression of Hey2 was significantly upregulated at 24 or 48 h after P.endodontalis LPS administration.(2)The expression of IL-6 was significantly decreased by overexpression of Hey2 compared to the control group under P.endodontalis LPS stimulation.(3)After 10 μg/m L of P.endodontalis LPS stimulation of osteoblasts for 24 h,the recruitment of Hey2 on the IL-6 promoter at-400~-200 bp was significantly increased.Conclusions:1.The expression of miR-146-5p and Hey2 were significantly increased in the CAP group compared with healthy tissue,miR-146-5p and Hey2 might be involved in the pathogenesis of CAP.2.P.endodontalis LPS-induced IL-6 expression was suppressed by miR-146a-5p,miR-146a-5p might played an anti-inflammatory role in inflammatory responses of P.endodontalis LPS–treated MC3T3-E1 cells.3.Mi R-146a-5p and Hey2 formed a mutual negative feedback loop,miR-146a-5p could bind to specific site of Hey2 3’UTR,Hey2 was a target gene of miR-146a-5p.4.The expression of Hey2 was significantly increased by P.endodontalis LPS,Hey2 suppressed IL-6 expression induced by P.endodontalis LPS in osteoblasts through binding to the promoter region of IL-6,Hey2 could function as transcriptional repressor of IL-6 gene in inflammatory responses. |
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