The Role Of Ckip-1 In Regulating Cementoblast Mineralization Under Inflammation

Part.Ⅰ:Ckip-1 Regulated P.gingivalis-stimulated Cementoblast Mineralization NegativelyObjectiveThis part aimed to investigate the regulatory effect of Casein kinase 2 interacting protein 1(Ckip-1)on cementum formation and cementoblast mineralization,as well as it’s regulatory effect on cementoblast mineralization under P.gingivalis stimulation.MethodsCementum-dentine complex and root canal width of the mandibular first molars in wild type(WT)mice and Ckip-1 knockout(KO;Ckip-1-/-)mice were observed by Micro-CT.Cementum thickness was observed by HE staining.The expression of Osterix in cementoblasts was detected by immunohistochemistry and tissue immunofluorescence staining.Ckip-1 and mineralization-related markers were screened from the transcriptome sequencing data of mineralized mouse cementoblast cell line OCCM-30 for heat map analysis.The expression of mineralization-related markers,Ckip-1 and IL-6 in OCCM-30 induced for different days or stmulated with different MOIs of Pg was detected.RT-qPCR,Western blotting,ALP and alizarin red stainings were used to detect the mineralization of OCCM-30 stimulated with Pg or Pg-LPS.Ckip-1-silenced or overexpressed OCCM-30 was constructed to study the role of Ckip-1 in OCCM-30 mineralization with or without Pg/Pg-LPS stimulation,and proliferation.The changes of various signaling pathways were detected based on the above situation.Specific inhibitors were applied to confirm the involvement of p38,Erkl/2 and Akt signaling pathways.Results1.Ckip-1-/-mice showed thicker cementum-dentin complex and narrower root canal,more cellular cementum,higher expression of Osterix in cementoblasts in the mandibular first molars.2.The expression of Ckip-1 decreased during OCCM-30 mineralization and increased after Pg stimulation.Ckip-1 silencing promoted OCCM-30 mineralization and proliferation,while Ckip-1 overexpressing suppressed OCCM-30 mineralization.3.Pg and Pg-LPS could inhibit the expression of mineralization-related markers in OCCM-30,and inhibit the ALP activity and mineralized nodule formation ability.Higher mineralization in sh-Ckip-1 OCCM-30 group under Pg or Pg-LPS stimulation was also observed.4.p38,Akt,and Wnt signaling pathways in OCCM-30 were activated,while Erk1/2 signaling pathway was inhibited after Ckip-1 silencing.Contrary results were found in Ckip-1-overexpressed OCCM-30.Ckip-1 silencing-promoted mineralization was weakened when p38 and Akt signaling pathways were inhibited.Ckip-1 silencing-promoted mineralization was strengthened when Erk1/2 signaling pathway was inhibited.ConclusionCkip-1 could negatively regulate cementum formation and cementoblast mineralization.Ckip-1 silencing could promote cementoblast mineralization and alleviate Pgsuppressed cementoblast mineralization through MAPK and Akt signaling pathways.Part.Ⅱ:M2 Macrophages Alleviated P.gingivalis-suppressed Cementoblast MineralizationObjectiveMacrophages are the major cells of the immunoinflammatory microenvironment.This part aimed to study the chemotaxis of M2 macrophages towards Pg-stimulated cementoblasts,and the effects of M2 macrophages on the mineralization of cementoblast stimulated with Pg.MethodsM2 macrophages(Raw264.7)were induced by IL-4,and identified.The chemotaxis of M2 macrophages towards the supernatant from Pg-stimulated OCCM-30 was detected by crystal violet staining.RT-qPCR,Western blotting,and ELISA were used to detect the CCL2 expression and release in Pg-stimulated OCCM-30.CCR2 expression in M2 macrophages was detected by RT-qPCR.CCL2 expression in cementoblasts of apical periodontitis(AP)mice was detected by immunohistochemistry.The chemotaxis of M2 macrophages towards the CCL2-containing medium was also detected.The effects of M2 macrophages on OCCM-30 mineralization with or without Pg stimulation were evaluated by conditional medium(CM)and transwell coculture methods.The changes of p38 signaling pathway were detected based on the above two methods.Specific inhibitor was applied to confirm the involvement of p38 signaling pathway.Results1.After IL-4 induction,the expression of M2 polarization-related markers increased,and the expression of M1 polarization-related markers decreased.M2 macrophages had a better chemotaxis to Pg-stimulated OCCM-30.CCL2 expression and release increased under Pg stimulation.CCL2 expression increased in cementoblasts in vivo under inflammatory condition.CCR2 expression increased in M2 macrophages.M2 macrophages had a better chemotaxis to the medium containing CCL2.2.M2 macrophage supernatant and M2 macrophage could promote the expression of mineralization-related markers in OCCM-30 with or without Pg stimulation.3.p38 signaling pathway could be activated by M2 macrophages supernatant and M2 macrophages under Pg stimulation,and the expression of mineralization-related markers decreased after the inhibition of p38 signaling pathway.ConclusionPg could induce the release of CCL2 from cementoblasts.M2 macrophage possessed chemotaxis to Pg-stimulated cementoblasts through the CCL2/CCR2 axis,and alleviated Pg-suppressed cementoblast mineralization through p38 signaling pathway.Part.Ⅲ:Ckip-1 Silencing-induced M2 Macrophages Facilitated Cementoblast MineralizationObjectiveCkip-1 could regulate multiple cell functions and biological processes.This part aimed to investigate whether Ckip-1 could also regulate M2 macrophage polarization under Pg stimulation,and further promote cementoblast mineralization.MethodsCkip-1-silenced macrophages(sh-Ckip-1)and its’ controls(sh-NC)were constructed by lentivirus infection method,and identified.The expression of polarization-related markers in sh-NC and sh-Ckip-1 macrophages was detected by Western blotting.M1 and M2 polarization of sh-NC and sh-Ckip-1 macrophages were induced by LPS plus IFN-y,and IL-4,respectively.The expression of polarization-related markers was detected by RT-qPCR and Western blotting.Pg was used to stimulate normal macrophages or sh-NC and sh-Ckip-1 macrophages.The expression of polarizationrelated markers was also detected by RT-qPCR and Western blotting.The effects of shCkip-1 macrophages on OCCM-30 mineralization were evaluated by CM and transwell coculture methods.The expression of mineralation-related markers was detected by RTqPCR and Western blotting,and ALP activity was detected by ALP staining.Results1.Green fluorescence indicated good transfection efficiency.Ckip-1 expression in shCkip-1 macrophages significantly decreased.Higher CD206 expression and lower IL-6 expression in Ckip-1-silenced macrophages was detected.2.Lower expression M1 polarization-related markers in sh-Ckip-1 macrophages under M1 polarization stimulation,and higher expression of M2 polarizationrelated markers in sh-Ckip-1 macrophages under M2 polarization stimulation was detected when compared to their controls.3.Pg could induce increased expression of M1 polarization-related markers and decreased expression of M2 polarization-related markers in macrophages.The expression of Ml polarization-related markers in sh-Ckip-1 macrophages decreased under Pg stimulation when compared to the control group.4.sh-Ckip-1 macrophage supernatant and sh-Ckip-1 macrophage could promote the expression of mineralization-related markers and the ALP activity of OCCM-30.ConclusionCkip-1 silencing could promote M2 macrophage polarization,and facilitate the transformation of Pg-stimulated macrophages from M1 phenotype to M2 phenotype.Meanwhile,Ckip-1 silencing-induced M2 macrophage also promoted cementoblast mineralization.Part.Ⅳ:Exosomal Transfer of Ckip-1 Silencing-induced M2 Macrophage-derived Let-7f-5p Alleviated P.gingivalis-suppressed Cementoblast MineralizationObjectiveWhether M2 macrophages promoted cementum regeneration through exosomes remain unknown.This part aimed to investigate the effect of Ckip-1 silencing-induced M2 macrophage-derived exosomes on cementoblast mineralization under Pg stimulation and its potential mechanism.MethodsExosomes(M0-EXO,M2-EXO)derived from M0 and M2 macrophages and exosomes(sh-NC-EXO,sh-Ckip-1-EXO)derived from sh-NC and sh-Ckip-1 macrophages were extracted by ultracentrifugation.Protein markers,morphology and size of exosomes were identified.The endocytosis of Dil-labeled exosomes by OCCM-30 was observed.The mineralization of OCCM-30 induced with different exosomes was detected by RTqPCR,Western blotting,and ALP staining.Pg-stimulated OCCM-30 was induced with sh-NC-EXO or sh-Ckip-1-EXO.The mineralization of OCCM-30 and IL-1β expression were also detected.The expression of Let-7 miRNAs and si-Ckip-1 in sh-NC and shCkip-1 macrophages,sh-NC-EXO and sh-Ckip-1-EXO and exosome-stimulated OCCM were detected by RT-qPCR.Macrophages transfected with FAM-Oligo were cocultured with OCCM-30 in a transwell system.The green fluorescence in OCCM-30 was identified.The Let-7 miRNAs were screened from the miRNA sequencing data of OCCM-30 induced for different days for heat map analysis.Let-7f-5p Mimic or Inhibitor were applied to evaluate the role of Let-7f-5p in OCCM-30 mineralization.The target gene of Let-7f-5p was predicted by databases.The Ckip-1 expression in OCCM-30 stimualted with Let-7f-5p Mimic or Inhibitor,or sh-Ckip-1-EXO was detected.Dual-luciferase reporter assay was used to determine whether Let-7f-5p could target Ckip-1.Results1.The expression of the characteristic markers of macrophage(F4/80,CD206)and markers of exosome(TSG-101,CD63,HSP90)was positive,and the expression of Calnexin was negative in exosomes.The cup-like exosomes with an average size of 140 nm was identified.These exosomes could be uptaken by OCCM-30.2.M2-EXO and sh-Ckip-1-EXO could promote the expression of mineralizationrelated markers and the ALP activity of OCCM-30.sh-Ckip-1-EXO could alleviate Pg-suppressed mineralization-related marker expression and the ALP activity of OCCM-30,as well as Pg-induced IL-1β expression.3.The expression of Let-7 miRNAs was increased in sh-Ckip-1 macrophages,shCkip-1-EXO and sh-Ckip-1-EXO-stimulated OCCM-30 when compared to their own controls.FAM-Oligo transfected into macrophages could be transferred to OCCM-30 in the transwell co-culture system.4.Heat map showed that most Let-7 miRNAs gradually increased during OCCM-30 mineralization.Let-7f-5p Mimic promoted the expression of mineralization-related markers and the ALP activity of OCCM-30,while Let-7f-5p Inhibitor inhibited the expression of mineralization-related markers and the ALP activity of OCCM-30.5.TargetScan and miRBase database predicted that Let-7f-5p might target Ckip-1.Let7f-5p and sh-Ckip-1-EXO could silence the expression of Ckip-1 in OCCM-30.Let7f-5p was further verified to target Ckip-1.Conclusionsh-Ckip-1-EXO could promote cementoblast mineralization and alleviate Pg-suppresed cementoblast mineralization by delivering Let-7f-5p targeting Ckip-1.