抑制连接蛋白43介导半通道活性促进脂多糖诱导的人牙髓细胞成牙本质分化

doi:10.19439/j.sjos.2024.01.004

摘要/Abstract

摘要： 目的: 探讨连接蛋白43（connexin 43,Cx43）在脂多糖（lipopolysaccharide,LPS）诱导的人牙髓细胞（human dental pulp cells,hDPCs）成牙本质分化过程中的作用和机制。方法: 建立SD大鼠上颌第一磨牙损伤模型,免疫荧光（immunofluorescence,IF）染色检测Cx43在牙髓组织损伤后修复中的表达模式变化。分别采用0、1、10、100和1 000 ng/mL LPS刺激hDPCs 6 h,筛选最适浓度,随后抑制和过表达hDPCs中Cx43的表达。实时定量PCR（qRT-PCR）及免疫印迹法检测Cx43和成牙本质分化相关因子牙本质涎磷蛋白（dentin sialophosphoprotein,DSPP）、牙本质基质蛋白1（dental matrix protein-1,DMP-1）、成骨相关转录因子（osterix,Osx）表达及细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）活性变化。进一步对hDPCs施以特异性Cx43通道抑制剂,检测Cx43介导的通道活性在hDPCs成牙本质分化中的作用,初步探讨Cx43调节LPS诱导的hDPCs 成牙本质分化的作用和机制。采用SPSS 26.0软件包对数据进行统计学处理。结果: IF结果显示,在健康牙髓组织中,Cx43主要表达于成牙本质细胞层,牙损伤3~24 h,Cx43表达减弱,随后逐渐上调,直至正常水平;损伤后3 天~2 周,表达呈下调趋势,并且表达于成牙本质细胞层和固有牙髓中。以10 ng/mL LPS刺激hDPCs 6 h,可显著上调DSPP的mRNA表达（P<0.01）。抑制Cx43,可显著上调hDPCs内LPS诱导的DSPP、DMP-1和Osx mRNA表达（P<0.05）;过表达Cx43,则显著抑制LPS诱导的成牙本质分化相关因子表达（P<0.01）和DSPP荧光强度。以10 ng/mL LPS激活hDPCs内ERK信号,过表达Cx43可显著减弱LPS诱导的ERK信号活性（P<0.01）。抑制Cx43介导的半通道,促进LPS诱导的hDPCs成牙本质分化相关因子mRNA表达和ERK信号活性（P<0.05）;而阻断Cx43介导的细胞间通道,则抑制成牙本质分化。结论: Cx43参与调控牙髓组织的损伤后修复,并且其表达整体呈下调趋势;抑制Cx43或阻断HC,可促进LPS诱导的ERK信号活性和hDPCs成牙本质分化。

关键词: 人牙髓细胞, 脂多糖, 连接蛋白43, 缝隙连接通道, 半通道

Abstract: PURPOSE: To investigate the role and mechanism of connexin 43（Cx43）in odontoblast differentiation of human 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 immunofluorescence(IF) staining. hDPCs was respectively stimulated with 0, 1, 10, 100 and 1 000 ng/mL LPS for 6 h to screen the optimal 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, hDPCs 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 differentiation 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 injury, the expression of Cx43 decreased and then gradually increased to the normal level; from 3 days to 2 weeks after injury, the expression of Cx43 tended to be down-regulated which was in the odontoblast layer and pulp proper. The expression of DSPP mRNA was significantly up-regulated in the hDPCs stimulated with 10 ng/mL LPS for 6 h(P<0.01). Inhibition 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 differentiation(P<0.01) and the fluorescence intensity of DSPP. 10 ng/mL LPS activated ERK signal in hDPCs, and overexpression 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 differentiation. 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.

Key words: Human dental pulp cells, Lipopolysaccharide, Connexin 43, Gap junction channel, Hemichannel

中图分类号:

R781

张安妮, 丁灿灿, 黄丽苹, 李适廷. 抑制连接蛋白43介导半通道活性促进脂多糖诱导的人牙髓细胞成牙本质分化[J]. 上海口腔医学, 2024, 33(1): 22-29.

ZHANG An-ni, DING Can-can, HUANG Li-ping, LI Shi-ting. Inhibition of connexin 43-mediated hemichannel activity promotes odontoblast differentiation of human dental pulp cells induced by lipopolysaccharide[J]. Shanghai Journal of Stomatology, 2024, 33(1): 22-29.

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