Effects Of NCOA4-mediated Ferritinophagy In Inflammatory Responses In Periodontitis

[Background/Objective]Periodontitis is an inflammatory disease of the periodontium.Iron homeostasis plays a crucial role in the combat against pathogen invasion.Ferrous iron can trigger generous production of reactive oxygen species(ROS)by Fenton reaction.Excessive accumulation of ROS could break cellular homeostasis,resulting in oxidative stress and mitochondrial dysfunction.Most of imported iron was stored in the ferritin,excess iron is also can be pumped out of the cell by ferroportin(FPN,also known as SLC40A1).Nuclear receptor coactivator 4(NCOA4),a selective cargo receptor to deliver ferritin to lysosome may trigger release of ferritin-bound iron into the cytosol.The aim of the present study was to explore whether NCOA4-mediated ferritinophagy participated in the pathogenesis of periodontitis and its role in promoting the periodontal inflammation.[Methods]Inflamed and healthy periodontal tissues were harvested for immunobiological staining of ferritinophagy-related genes in the periodontal tissues,while real-time quantitative PCR(q PCR)was utilized to detect m RNA transcription.Periodontal ligament fibroblasts(PDLFs)were isolated and infected with Porphyromonase gingivalis at different multiplicity of infection(MOI=10,50,250)in vitro.m RNA transcription and protein expression of genes involved in the iron metabolism,including NCOA4,transferrin receptor 1(TFR1),ferroportin(SLC40A1), ferritin heavy chain and light chain were detected by q PCR and Western blot.Levels of labile iron pool and ROS production was detected by flow cytometry and confocal endoscopy.Small interference RNA was utilized to knock down NCOA4 to determined the role of NCOA4 in periodontitis.To explore the pathways related to changes in ferritinophagy,Western blot was utilized to detect the signaling pathways activated by P.gingivalis stimulation of PDLFS.After pretreatment with pathway inhibitors,q PCR and ELISA were used to detect levels of Interleukin-6(IL-6)and Monocyte chemoattractant protein-1(MCP-1)after P.gingivalis stimulation of PDLFs for 24 h.[Results]Elevated expression of NCOA4,ferritin heavy chain and light chain were observed in the diseased periodontal tissues.P.gingivalis infection at the MOI of 250 promoted expression of TFR1,NCOA4 and microtubule-associated protein 1-light chain 3 B(LC3B),enhanced levels of intracellular labile iron pool and ROS production.NCOA4 knockdown reduced ROS generation in PDLFs in response to P.gingivalis and mitigated production of proinflammatory MCP-1 and IL-6.P.gingivalis triggered activation of c-Jun N-terminal kinase(JNK)and p38mitogen-activated protein kinase signaling pathway.In addition,inhibitors of JNK,SP600125,and inhibitors of p38,SB203580 blocked NCOA4 and proinflammatory cytokine transcription.[Conclusion]NCOA4-ferritinophagy participated in the progress of periodontitis progression.P.gingvalis-triggered ferritinophagy aggravated production of ROS and inflammatory responses in PDLFS.Inhibiting the NCOA4 expression can reduce the inflammatory response.These findings suggest iron homeostasis plays an important role in the pathogenesis of periodontitis and will open new opportunities for developing therapeutic options to treat Periodontitis.