Inhibition of connexin 43-mediated hemichannel activity promotes odontoblast differentiation of human dental pulp cells induced by lipopolysaccharide
PURPOSE:To investigate the role and mechanism of connexin 43(Cx43)in odontoblast differentiation of hu-man dental pulp cells(hDPCs)induced by lipopolysaccharide(LPS).METHODS:The maxillary first molar injury model of SD rats was established.The expression pattern of Cx43 in dental pulp repair after injury was detected by immunofluo-rescence(IF)staining.hDPCs was respectively stimulated with 0,1,10,100 and 1 000 ng/mL LPS for 6 h to screen the op-timal concentration,and then the expression of Cx43 was inhibited and overexpressed in hDPCs.Quantitative real-time PCR(qRT-PCR)and Western blot(WB)were used to detect the expression of Cx43 and dentin sialophosphoprotein(DSPP),dental matrix protein-1(DMP-1),osterix(Osx)and extracellular signal-regulated kinase(ERK)activity.Furthermore,hD-PCs were treated with specific Cx43 channel inhibitors to investigate the effect of Cx43-mediated channel activity in odontoblast differentiation of hDPCs,and to explore the role and mechanism of Cx43 in regulating odontoblast differentia-tion of hDPCs induced by LPS.Statistical analysis was performed with SPSS 26.0 software package.RESULTS:IF results showed that Cx43 was mainly expressed in the odontoblast layer in healthy dental pulp tissues.At 3-24 h after tooth in-jury,the expression of Cx43 decreased and then gradually increased to the normal level;from 3 days to 2 weeks after in-jury,the expression of Cx43 tended to be down-regulated which was in the odontoblast layer and pulp proper.The expres-sion of DSPP mRNA was significantly up-regulated in the hDPCs stimulated with 10 ng/mL LPS for 6 h(P＜0.01).Inhibi-tion of Cx43 significantly up-regulated the expression of DSPP,DMP-1 and Osx mRNA induced by LPS in hDPCs(P＜0.05),while overexpression of Cx43 obviously inhibited the expression of factors related to LPS-induced odontoblast differ-entiation(P＜0.01)and the fluorescence intensity of DSPP.10 ng/mL LPS activated ERK signal in hDPCs,and overexpres-sion of Cx43 significantly attenuated the activity of ERK signal induced by LPS(P＜0.01).Inhibition of Cx43-mediated hemichannel(HC)promoted mRNA expression of factors related to odontoblast differentiation in hDPCs and the activity of ERK signal induced by LPS(P＜0.05),while blocking Cx43-mediated gap junction channel(GJC)inhibited odontoblast dif-ferentiation.CONCLUSIONS:Cx43 participates in the regulation of dental pulp repair after injury,and its expression shows a downward trend as a whole.Inhibition of Cx43 or blocking of HC promotes LPS-induced ERK signal activity and odontoblast differentiation of hDPCs.
Human dental pulp cellsLipopolysaccharideConnexin 43Gap junction channelHemichannel
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