Impact Of Resolvin D1 On The Inflammatory Phenotype Of Periodontal Ligament Cell Response To Hypoxia

Objective: Periodontal ligament cells（PDLCs）are critical for wound healing and tissue regeneration of periodontal diseases.Hypoxia is an important factor affecting periodontal disease,and one of the etiologies of plateau periodontal disease.Within an inflammatory periodontal pocket,a hypoxic environment can aggravate periodontal inflammation,where PDLCs response to the inflammation would change.Resolvin D1（Rv D1）is an endogenous lipid mediator,which can impact intracellular inflammatory pathways of periodontal/oral cells and promote periodontal regeneration.It is not clear how hypoxia and Rv D1 impact the inflammatory responses of lipopolysaccharide（LPS）-stimulated PDLCs.Therefore,this study aimed to test the hypothesis: Rv D1 could reverse hypoxia-induced changes in LPS-stimulated PDLCs.Methods: Human PDLCs were cultured from periodontal tissues and characterized by immunofluorescence staining of vimentin and cytokeratin.3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide（MTT）assay and enzyme-linked immunosorbent assay（ELISA）were applied to determine the optimal concentration of LPS for establishment of inflammatory PDLCs phenotype.To examine the effects of Rv D1 and hypoxia on the inflammatory responses of PDLCs,protein levels and gene expressions of inflammatory cytokines and signal transduction molecules were measured by ELISA,western blotting（WB）,and real-time quantitative reverse transcription PCR（real-time q RT-PCR）.Alizarin red S staining,real-time q RT-PCR and WB were employed to study the effects of Rv D1 and hypoxia on the osteogenic differentiation of inflammatory PDLCs phenotype.Results: Cells crawled out of the periodontal tissue radially,showed a typical spindle shape of fibroblastic morphology and stained positively for vimentin while negatively for keratin.Treatment of 0.1 μg/m L LPS on PDLCs could stimulate expression of interleukin-1β（IL-1β）with no cytotoxicity.It was found that hypoxia increases the expression of hypoxia inducible factor-1α（HIF-1α）at the protien level（p<0.05）and upregulates the expression of inflammatory factors at the gene level（p<0.05）.Rv D1 reduced the expression of IL-1β（p<0.05）in PDLCs under hypoxia both at the protein and RNA levels.There were increases in the expression of p38mitogen-activated protein kinase（p38 MAPK,p<0.01）and protein kinase B（Akt,p<0.05）in response to Rv D1.Also,a significantly higher density of calcified nodules was observed after treatment with Rv D1 for 21 days under hypoxia.Conclusion: Hypoxia up-regulated the inflammatory level of PDLCs.Rv D1 can reduce under-hypoxia-induced pro-inflammatory cytokines in inflammatory cells phenotype.Also,Rv D1 promotes the calcium nodules in PDLCs,possibly by affecting the p38 MAPK signaling pathway through Akt and HIF-1α.