Effect And Mechanism Of Connexin 43 On Autophagy And Odontoblast Differentiation Of Human Dental Pulp Cells Induced By Lipopolysaccharide

Objective: An inflammatory model was established by infecting human dental pulp cells(h DPCs)with lipopolysaccharide(LPS)to explore the association of connexin 43(Cx43)with autophagy,and its effect and mechanism on odontoblast differentiation of h DPCs.Methods: 1.The expression of Cx43 and LC3 in isolated teeth under different infection conditions was observed by IF.Autophagy-related factors with significant expression changes in pulpitis were screened by Microarray and verified by RT-PCR.2.h DPCs cultured by collagenase digestion method were used for follow-up experiments.h DPCs were stimulated with 10.0 μg/m L LPS for 0-24 h,and the expression of Cx43,autophagy-related factors ATG2,ATG4,ATG5,ATG12,ATG16,ULK1,BECN1,LC3 and odontoblast differentiation-related factors Runx2,Osterix,DSPP,DMP-1 and ALP mRNA were detected by RT-PCR to determine the best time of LPS action in the follow-up experiment.Cx43 gene-silencing lentiviral vector was constructed to interfere with Cx43 expression by h DPCs infection.LPS stimulation was used to simulate dental pulp infection in vitro.The expression of odontoblast differentiation factor was detected by RT-PCR and IF,and the expression of autophagy related factors was detected by RT-PCR and WB.3.si RNA-Cx43 of h DPCs were pretreated with 1 μM LYN-1604(ULK1 agonist)for 24 h,and then treated with LPS for 6 h.The expression of odontoblast differentiation-related factors was detected by RT-PCR and IF to clarify the mechanism of Cx43 regulation of odontoblast differentiation in h DPCs.h DPCs were pretreated with 100 μM Gap19(Cx43-mediated HC selective channel inhibitor)and 100 μM 18-α-glycyrrhetinic acid(Cx43-mediated GJC chemical channel inhibitor)for 1 h,followed by LPS stimulation for 6 h,respectively.The expression of factors related to odontoblast differentiation was detected by RT-PCR and WB to determine the pathway of Cx43 regulating the realization of odontoblast differentiation in h DPCs.Results: 1.The results of IF showed that the expression levels of Cx43 and LC3 were slightly up-regulated in superficial caries.With the aggravation of dental pulp infection,the expression of Cx43 gradually increased and significantly increased on the cell membrane,and the upregulated expression of LC3 resulted in obvious nuclear metastasis.The results of Microarray showed that the autophagy-related factors with obvious changes after dental pulp infection were ATG2,ATG4,ATG5,ATG12,ATG16,ULK1,BECN1 and LC3,which were consistent with the results of RT-PCR.2.The primary hDPCs were successfully cultured in vitro by collagenase digestion,and the 3rd-6th generation cells were selected for follow-up experiment.RT-PCR detection showed that when LPS stimulated h DPCs for 6 h,the expression of Cx43 and autophagy-related factors was the highest,and significantly inhibited odontoblast differentiation,so the follow-up experiment used 10.0μg/m L LPS stimulation for 6 h.Cx43 silenced lentivirus(MOI=40,5.0μg/m L Polybrene)transfected h DPCs,green fluorescence expression was observed after 48-72 h,and the expression of Cx43 in h DPCs was decreased by RT-PCR and WB.The results of RT-PCR and WB showed that inhibition of Cx43 expression significantly up-regulated the expression of odontoblast differentiation-related factors such as Runx2 and DSPP,and down-regulated the expression of autophagy-related factors such as ATG16 and LC3 under normal and LPS stimulation.IF results showed that Osterix was expressed in the nucleus,inhibition of Cx43 significantly enhanced the staining intensity of Osterix in the nucleus of normal cultured and LPS-stimulated h DPCs,while DSPP was mainly expressed in the cytoplasm,and the staining result was similar to Osterix.3.The results of RT-PCR and IF showed that autophagy stimulated by h DPCs pretreated with LYN-1604 enhanced the inhibitory effect of LPS on odontoblast differentiation and weakened the promoting effect of inhibiting Cx43 on cell differentiation.By pretreating h DPCs with Gap19 and 18-α-glycyrrhetinic acid,RT-PCR and WB showed that using Gap19 could reverse the inhibitory effect of LPS on the expression of odontoblast differentiation factors such as Runx2 and DSPP,which was consistent with the inhibition of Cx43 expression.However,using 18-α-glycyrrhetinic acid did not significantly up-regulated the expression of odontoblast differentiation factors such as Runx2 and DSPP,indicating that Cx43 reduced the level of h DPCs autophagy and promoted odontoblast differentiation by inhibiting its mediated HC activity.Conclusion: This study showed that both Cx43 and autophagy are involved in the process of dental pulp inflammation and repair.Stimulation of 10.0 μg/m L LPS for 6 h up-regulated the expression levels of Cx43 and autophagy-related factors in h DPCs,activated Cx43-mediated HC activity and autophagy reaction,and inhibited the odontoblast differentiation of h DPCs.Inhibition of Cx43 expression could down-regulate LPS-induced autophagy and promote the odontoblast differentiation of h DPCs.Cx43 decreased the autophagy level of h DPCs and promoted odontoblast differentiation by inhibiting its mediated HC activity,but not GJC.