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Journal of Peking University(Health Sciences) ›› 2020, Vol. 52 ›› Issue (1): 18-23. doi: 10.19723/j.issn.1671-167X.2020.01.003

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

Ying-yi CHEN,Zi-qi HU,Tian-qian HUI(),He LIU()   

  1. Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2019-10-10 Online:2020-02-18 Published:2020-02-20
  • Contact: Tian-qian HUI,He LIU E-mail:676266821@qq.com;heliu69@126.com
  • Supported by:
    Supported by the National Natural Science Foundation of China(81800959);Postdoctoral Fund of Peking University School and Hospital of Stomatology(YS0203)

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Abstract:

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 100 μg/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 20 μg/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.

Key words: Enhancer of zeste homolog 2, Pulpitis, Macrophages, Chemotaxis

CLC Number: 

  • R781.31

Figure 1

HE staining of rat pulp stimulated by LPS for different time A, HE staining of control group dental pulp; B and C, HE staining of the dental pulp after stimulated by LPS for 2 h or 8 h; D-F, HE staining of the dental pulp after stimulated by LPS for 1, 3, 7 d. n=3 per group."

Figure 2

EZH2 immunohistochemical staining of rat pulp stimulated by LPS for different time A, immunohistochemical (IHC) staining of the control group; B and C, EZH2 expression decreased after dental pulp stimulated by LPS for 2 h or 8 h; D-E, IHC staining of LPS stimulated the rat dental pulp for 1, 3 and 7 days. The expression of EZH2 was gradually up-regulated with the stimulation time. n=3 per group. Arrows indicate positive staining."

Figure 3

EZH2 and CD68 double immunofluorescence staining A-D, histological results of normal dental pulp; E-H, histological results of infected dental pulp; A and E, HE staining; B and F, expression of EZH2 (in red) was increased in infected dental pulp; C and G, expression of CD68 (in green) was also increased in infected dental pulp; D and H, colocalization of EZH2 and CD68 (in yellow) could be observed in both normal and infected dental pulp. n=3 per group. Arrows indicate positive staining."

Figure 4

CCK-8 cell proliferation-toxicity test A, THP-1 cells were cultured in media with different concentrations (1, 10, 20, 40 μg/L) of EZH2 recombinant protein. Cells were measured for proliferation by CCK-8; B, hDPCs were cultured in media with different concentrations (1, 10, 20, 40, 100 μg/L) of EZH2 recombinant protein. Cells were measured for proliferation by CCK-8. * P<0.05, versus control. Error bars represent the x ? ±s. "

Figure 5

Transwell cell migration assay A, positive control; B, negative control; C, supernatant of EZH2-treated hDPCs; D, supernatant of EZH2 untreated hDPCs. n=3 per group."

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