Regulation And Mechanism Of The Combination Of Lipoxin A4 And Resolvin E1 On Pulpitis

Background:Pulpitis is a common disease in oral clinic.It is the key prerequisite for vital pulp preservation when inflammatory response is under control,but there is still no effective method.Pro-resolving is a relatively new strategy to regulate inflammation.Our previous study has found that resolvin E1(RvE1),one of specialized pro-resolving mediators(SPMs),could reduce pulpitis infiltration to a certain extent,but cannot decrease the inflammation level to that of normal pulp.The combined application of SPMs is an effective means to improve its function of anti-inflammatory and proresolving.Therefore,it is worth further studying how to select optimal SPMs for combined application in pulpitis and exploring underlying mechanism.Dental pulp fibroblasts(DPFs)are the most abundant and widely distributed cells in the pulp tissue,which play an important role in regulating inflammatory response by secreting a large number of pro-inflammatory cytokines and changing the local microenvironment.Nuclear factor kappa B(NF-κB)signaling pathway is the most important pathway for pro-inflammatory factor production,in which nuclear translocation,phosphorylation and acetylation of NF-κB subunit p65 are the key steps of its activation.The inhibition of NF-κB signaling pathway is an important part of preventing the inflammatory cascade and avoiding the continuous infiltration of inflammation.However,the regulation of NF-κB signaling pathway by combined application of SPMs is still unclear.Acetylation of p65 is the precondition for its stable binding to DNA and transcription of downstream target genes,and deacetylation results in the separation of activated p65 from DNA,loss of transcriptional activity,and gradual dephosphorylation of p65,returning to the cytoplasm to bind to IκB,thus inhibiting the activation of NF-κB signaling pathway.The acetylation of p65 is directly regulated by the deacetylases SIRT1 and SIRT6,but the regulatory role of SIRT7 and its specific mechanism remain unclear.Therefore,the present study aimed to determine whether the combined application of SPMs can further promote the resolution of pulpitis by in vitro and in vivo experiments.Meanwhile,the effect and mechanism of SPMs combination on NF-κB signal pathway was explored through LPS-induced DPFs in vitro.Our study may enrich the theoretical basis for the pulpitis and provide potential therapeutic targets and strategies for vital pulp preservation.Objective:1.To screen the optimal combination of SPMs for inflammatory inhibition in LPSinduced DPFs,and to detect the regulation effect of combined application of RvE1 and lipoxin A4(LXA4)on LPS-induced DPFs.2.To investigate the expression changes of SIRTs and the role of SIRT7,as well as the functions of specific receptors,Chem R23 and ALX,in resolution mediated by RvE1 and LXA4 combination.3.To obseve the therapeutic effect of RvE1 and LXA4 combination through a rat pulpitis model in vivo,and to explore the key regulatory molecules in pulpitis resolution by RNA-sequencing(RNA-seq).Methods:1.DPFs were isolated and cultured in vitro,and were pretreated with RvE1,LXA4,PD1 and Ma R1,respectively,followed by inflammatory stimulation with 1 μg/m L LPS.Quantitative real-time polymerase chain reaction(q PCR)was used to detect RELA m RNA expression to determine the best combination.2.LPS-induced DPFs were pretreated with RvE1 and LXA4 alone or simultaneously,and the m RNA levels of RELA and its downstream inflammasome and proinflammatory factors were detected by q PCR.Western blot was used to detect the corresponding protein expressions,the amount of p65 in nucleus and cytoplasm respectively,as well as the phosphorylation and acetylation levels of p65 in the total protein.3.The m RNA and protein levels of SIRT1,SIRT6 and SIRT7 were detected in DPFs after LPS-inducing,RvE1 and LXA4 treatment alone and combined application.The double immunofluorescence labeling of SIRT7 with p-p65 or AC-p65 was employed.After silencing SIRT7,RELA m RNA expression was detected by q PCR.The amount of p65 in nucleus and cytoplasm respectively,as well as the phosphorylation and acetylation levels of p65 were detected by wastern blotting and double immunofluorescence labeling.Meanwhile,the m RNA and protein levels of SIRT1 and SIRT6 were detected.4.The m RNA and protein expressions of SIRT1,SIRT6 and SIRT7 in DPFs were detected by inhibiting Chem R23 and ALX.The m RNA level of RELA,the amount of p65 in nucleus and cytoplasm respectively,as well as the phosphorylation and acetylation levels of p65 were detected,and co-localization of SIRT7 with p-p65 or Acp65 was observed by double immunofluorescence labeling.Meanwhile,m RNA and protein levels of inflammasome and pro-inflammatory factors were detected.5.A pulpitis model of rat anterior was established,and pulp capping was performed by adding RvE1 and LXA4 alone or in combination.HE and immunohistochemical staining were used to evaluate the infiltration level of pulpitis,the formation of ectopic mineralization tissues and the expression of pro-inflammatory factors.Co-localization of SIRT7 with p-p65 or Ac-p65 was observed by double immunofluorescence labeling.6.A pulpitis model of rat molars was established.The samples were collected 1,3and 7 days after pulp exposure,and RNA was extracted for RNA-seq.The differentially expressed genes were screened for GO enrichment analysis and KEGG enrichment analysis,and the expression of SIRTs was verified.Results:1.Both RvE1,LXA4,PD1 and Ma R1 reduced RELA m RNA expression in LPSinduced DPFs,and RvE1 or LXA4 showed a greater inhibition than other twos.Hence,the combination of RvE1 and LXA4 was adopted in the subsequent experiments.2.Combination of RvE1 and LXA4 significantly reduced RELA m RNA and downstream NLRP3,Caspase1,IL-1β,IL-18,IL-6,TNF-α,CCL2,CCL7,ICAM-1 and VCAM-1,and protein levels of NLRP3,caspase1,IL-1β and IL-18.Meanwhile,their combined usage down-regulated the ratio of p65 in nucleus,and phosphorylation and acetylation levels of p65.3.Combination of RvE1 and LXA4 up-regulated the expression levels of SIRT1,SIRT6 and SIRT7 in LPS-induced DPFs,and SIRT7 was co-located with p-p65 and Acp65 in nucleus.After silencing SIRT7,the ratio of p65 in nucleus,phosphorylation and acetylation levels of p65 were significantly increased,and the m RNA and protein expression levels of SIRT1 and SIRT6 were decreased as well.4.After Chem R23 and ALX inhibition,the m RNA and protein expression levels of SIRT1,SIRT6 and SIRT7 were significantly decreased,RELA m RNA expression,the proportion of p65 in nucleus,phosphorylation and acetylation levels of p65 were significantly up-regulated,and expressions of NLRP3,caspase1,IL-1β and IL-18 were also increased.5.Combination of RvE1 and LXA4 completely reversed the immune cell infiltration in the pulpitis of rat anterior and avoided the formation of ectopic calcification.In line with in vitro results,the combination of RvE1 and LXA4 up-regulated SIRT7 expression,inhibited the phosphorylation and acetylation of p65,and decreased the protein expression levels of inflammatory markers NLRP3,caspase1,IL-1β and IL-18.6.Results of RNA-seq showed that the enrichment of NF-κB signaling pathway increased during pulpitis infiltration,and the gene transcription levels of SIRTs decreased to a certain extent.Conclusion:1.Combination of RvE1 and LXA4 significantly inhibited the NF-κB signaling pathway in LPS-induced DPFs,including inhibiting the nuclear translocation,phosphorylation and acetylation of p65,and thus inhibiting the transcription,maturation and secretion of downstream pro-inflammatory factors.In a rat pulpitis model,combination of RvE1 and LXA4 significantly reduced inflammatory response,avoided the formation of ectopic calcification inside pulp,reduced expressions of proinflammatory factors,and improved the pulp microenvironment,so that promoted the resolution of pulpitis and the recovery of tissue homeostasis.2.Combination of RvE1 and LXA4 up-regulated the expressions of SIRT1,SIRT6 and SIRT7,and the increased SIRT7 expression is one of the important mechanisms in pulpitis resolution.Silencing SIRT7 significantly weakened the inhibitory effect of RvE1 and LXA4 on NF-κB signaling pathway,and decreased the expressions of SIRT1 and SIRT6.The expression pattern of SIRT1-7 genes in inflammatory pulp tissues was not completely the same.3.The pro-resolving function of RvE1 and LXA4 combination was mediated by Chem R23 and ALX.Inhibition of Chem R23 or ALX weakened the effects of RvE1 and LXA4 combination on NF-κB signaling pathway activation and pro-inflammatory factor expression,while inhibition of Chem R23 and ALX reversed their pro-resolving function.