Preliminary Study On The Regulatory Effect And Mechanism Of Resolvin D1 On The LPS-induced Inflammation In Dental Pulp Cells

Objective: Pulpitis is one of the most common oral diseases.Excessive or uncontrolled inflammation of the dental pulp could result in the chronic inflammation and even pulp necrosis.Recent studies demonstrate that the resolution of inflammation is an active process that occurs via the biosynthesis of endogenous antiinflammatory mediators rather than a passive phase.Resolvin D1（RvD1）is known as an endogenous pro-resolving lipid mediator which possesses anti-inflammatory and pro-resolving properties.In this study,we aimed to investigate the effect of RvD1 on the inflammation induced by lipopolysaccharide（LPS）in human dental pulp cells（DPCs）.Materials and Methods:（1）DPCs were extracted from normal premolars and third molars with informed consent and were identified by immunofluorescence assay.Different concentrations of LPS were used to induce the inflammation of the DPCs.（2）The regulatory effect of RvD1 in pulpitis induced by LPS in hypoxia was investigated.The effects of RvD1 on the proliferation and viability of DPCs were measured by CCK8.The effect of RvD1 on inflammatory molecules was assessed by ELISA,quantitative real-time RT-PCR（q PCR）and Western blot.The total Superoxide Dismutase（SOD）activities of DPCs were monitored under a hypoxic condition.（3）Further experiments were performed to explore if RvD1 effected through the FPR2/ALX.The expression of FPR2/ALX was measured by immunofluorescence assay and Western blot（WB）.The effect of PBP（the specific inhibitor for the FPR2/ALX）on the proliferation and viability of DPCs were measured by CCK8.After FPR2/ALX blocked,ELISA was performed to measure the concentrations of secreted pro-inflammatory cytokines of the DPCs,and the gene expression of IL-6and IL-1β was measured by q PCR.The expression of p-NF-κB p65 and p-p38 was examined by WB.Results:（1）The results of the immunofluorescence assay showed positive staining for vimentin and negative staining for cytokeratin,which demonstrated DPCs derived from the ecto-mesenchymal tissue.（2）RvD1 reduced the gene expression and secretion of inflammatory cytokines（IL-6 and IL-1β）in DPCs induced by LPS.The total SOD increased after treated by RvD1 in the presence or absence of LPS in the hypoxic environment.（3）FPR2/ALX were expressed in the DPCs and there was higher expression when DPCs was stimulated by LPS.The anti-inflammatory effect of RvD1 was suppressed when FPR2/ALX was blocked by PBP since the m RNA level and supernatant concentration of IL‐1β,IL‐6 increased compared to the LPS + RvD1 group.The expression of p-NF-κB and p-p38 showed no statistical differences among the LPS group,RvD1 + LPS group and RvD1 + LPS + PBP group.Conclusion: The application of RvD1 could protect DPCs from the inflammatory condition induced by LPS in hypoxia through a FPR2/ALX receptor-dependent manner and stimulate an antioxidant response of DPCs.