Zeste同源蛋白2增强子通过调节巨噬细胞趋化影响牙髓炎症反应

Enhancer of zeste homolog 2 affects dental pulp inflammation by regulating macrophage chemotaxis

目的:探讨表观遗传调控子zeste同源蛋白2增强子(enhancer of zeste homolog 2,EZH2)在牙髓炎过程中的表达变化及其对巨噬细胞的趋化作用。方法:以10 g/L脂多糖(lipopolysaccharide,LPS)刺激大鼠牙髓,建立大鼠牙髓炎模型。采用免疫组织化学染色检测牙髓炎进展过程中EZH2的表达变化,采用免疫荧光双染法检测EZH2与CD68的表达及两者的共定位,利用CCK-8细胞增殖-毒性试剂盒检测不同浓度(1、10、20、40、100μg/L)EZH2重组蛋白对人牙髓细胞(human dental pulp cells,hDPCs)及人白血病单核细胞系(human leukaemia-derived monocytic cell line,THP-1)细胞增殖的影响,从而筛选出EZH2重组蛋白刺激hDPCs及THP-1细胞的适宜浓度。采用Transwell迁移实验检测EZH2重组蛋白处理的hDPCs上清液对THP-1细胞迁移作用的影响。结果:HE染色结果表明,在LPS诱导的大鼠牙髓炎模型中,随着LPS刺激时间延长,牙髓炎症反应逐步加重。免疫组织化学结果显示,在LPS诱导牙髓炎8 h内,EZH2表达下降,但在刺激1、3、7 d后,EZH2表达随刺激时间延长逐步上调。与对照组相比,LPS刺激大鼠牙髓3 d时EZH2与CD68表达显著升高,并且可以检测到两者在巨噬细胞中的共表达。CCK-8结果提示,EZH2重组蛋白刺激hDPCs及THP-1细胞的适宜浓度为20μg/L。与对照组上清液相比,加入EZH2重组蛋白刺激hDPCs后的上清液可以显著促进巨噬细胞趋化。结论:EZH2参与了牙髓炎发展过程,并促进巨噬细胞的趋化,提示EZH2在牙髓炎发展过程中有重要调控作用。

Objective:To investigate the expression changes of the epigenetic regulator enhancer of zeste homolog 2(EZH2)during pulp inflammation and the effect of EZH2 on macrophages migration.Methods:Rat dental pulp was stimulated with 10 g/L lipopolysaccharide(LPS)to establish a model of rat pulpitis at different stages of inflammation.Immunohistochemical staining was used to detect the expression changes of EZH2 during the progression of pulp inflammation.Immunofluorescence double stain-ing was used to detect the expression of EZH2,CD68 and their colocalization.To screen the appropriate concentration of EZH2 recombinant protein to stimulate hDPCs and human leukaemia-derived monocytic cell line(THP-1)cells,the effects of different concentrations(1,10,20,40,and 100μg/L)of EZH2 recombinant protein on proliferation of human dental pulp cells(hDPCs)and human monocyte cell line THP-1 were detected by cell counting kit-8(CCK-8).Transwell migration assay was used to detect the effect of supernatants of hDPCs treated with EZH2 recombinant protein on the migration of THP-1 cells.Results:HE staining results showed that in the model of rat pulp inflammation induced by LPS,with the prolongation of LPS stimulation,the inflammation response of pulp gradually increased.Immunohistochemical results showed that EZH2 expression decreased within 8 h of LPS-induced dental pulp inflammation;but after 1,3,and 7 d of stimulation,EZH2 expression gradually increased with the extension of the stimulation time.As for the normal rat dental pulp tissue,the positive expression of EZH2 was scattered in the odontoblast cell layer and the pulp proper.Compared with the control group,LPS stimulated the expression of EZH2 and CD68 in the infected dental pulp,and the colocalization of EZH2 and CD68 could be detected in macrophages.The results of CCK-8 suggested that the appropriate concentration of EZH2 recombinant protein to stimulate hDPCs and THP-1 cells was 20μg/L.Transwell cell migration assay confirmed that compared with the supernatant of EZH2 untreated HDPCs group,the supernatant of EZH2-treated hDPCs significantly promoted macrophage chemotaxis.Conclusion:EZH2 is involved in the development of pulpitis and promotes the chemotaxis of macrophages,which suggests that EZH2 may play an important regulatory role in the development of pulp inflammation.