Journal of Prevention and Treatment for Stomatological Diseases ›› 2017, Vol. 25 ›› Issue (7): 420-425.doi: 10.12016/j.issn.2096-1456.2017.07.003

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Role of TRAF6 in inflammatory responses of human osteoblast-like cells with Enterococcusfaecalis

Yu LU(), Chengxia LIU, Zhongjun LIU   

  1. Department of Endodontics, Stomatological Hospital, Southern Medical University, Guangzhou 510280, China
  • Received:2017-03-06 Revised:2017-04-26 Online:2017-07-20 Published:2018-09-03

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Abstract:

Objective Explore the role and status of tumor necrosis factor receptor-associated factor 6 (TRAF6) in the inflammatory response of human osteoblast-like cells MG63 which was triggered by Enterococcus faecalis (E. faecalis) and its lipoteichoic acid (LTA). Methods SiRNA technology was applied to silence the TRAF6 gene of MG63 cells, Using E.faecelis and its LTA to stimulate the silence MG63 cells with different hours. After that, using real-time PCR technology to detect toll-like receptor 2 (TLR2) and TRAF6 gene expression and using ELISA assay to detect proinflammatory cytokines interleukin-1β and TNF-alpha expression levels.Results When MG63 cells was infected by E. faecalis, its LTA, TLR2 and TRAF6 gene level has increased to varying degrees (P< 0.05); interleukin-1β and TNF-alpha expression was significantly higher (P< 0.05). When TRAF6 gene of MG63 cells was silenced by siRNA, pro-inflammatory cytokines interleukin-1β, interleukin-6, interleukin -8 and TNF-alpha expression decreased significantly (P< 0.05). Conclusion E. faecalis and its toxic components is identified by MG63 cells mainly through TLR2 receptors. The major virulence factor in periapical infections caused by E. faecalis is LTA.

Key words: E. faecalis, LTA, TLR2, Human osteoblast-like cells, TRAF6

CLC Number: 

  • R781.05

Table 1

Gene expression level of TLR2 and TRAF6 by E. faecalis and its LTA-infected MG63 cells x?± s,n=3"

分组 TLR2 TRAF6
刺激物 0 h 6 h t1值 P1值 24 h t2值 P2值 0 h 6 h t3值 P3值 24 h t4值 P4值
空白对照组 E. faecelis 1.00±0.14 11.26±1.00 2.92 0.002 12.11±0.52 2.92 0.0004 1.00±0.10 28.25±3.63 2.92 0.003 17.47±2.45 2.92 0.004
E. faecelis LTA 1.00±0.11 9.35±0.96 2.92 0.002 12.66±1.40 2.92 0.0024 1.00±0.26 36.62±3.13 2.92 0.001 26.52±4.73 2.92 0.006
转染对照组 E. faecelis 1.00±0.15 12.37±1.20 2.92 0.002 13.57±1.52 2.92 0.002 1.00±0.18 22.18±4.98 2.92 0.009 21.98±2.72 2.92 0.003
E. faecelis LTA 1.00±0.14 9.42±1.31 2.92 0.004 10.14±0.14 2.13 0.001 1.00±0.12 28.33±3.30 2.92 0.002 21.67±3.25 2.92 0.004
转染组 E. faecelis 1.00±0.16 10.35±1.73 2.92 0.006 6.17±1.25 2.92 0.005 1.00±0.21 1.03±0.23 2.13 0.431 1.17±0.13 2.35 0.166
E. faecelis LTA 1.00±0.33 14.56±2.39 2.92 0.01 10.67±0.94 2.92 0.002 1.00±0.12 0.98±0.12 2.13 0.449 1.04±0.24 2.13 0.408

Figure 1

The expression of TRAF6 protein in transfected MG63 cells with transfected TRAF6 siRNA"

Figure 2

The expression of TRAF6 protein in transfected MG63 cells after stimulated by E. faecalis and LTA"

Table 2

IL-1β and TNF-α secretion by E. faecalis and its LTA-infected MG63 cells x?± s,n=3,pg/mL"

组别 IL-1β TNF-α
0 h 8 h 24 h 0 h 8 h 24 h
E. faecelis E. faecelisLTA E. faecelis E. faecelisLTA E. faecelis E. faecelisLTA E. faecelis E. faecelisLTA E. faecelis E. faecelisLTA E. faecelis E. faecelisLTA
空白对照组 22.42±2.91 22.42±2.91 94.82±9.90 82.09±6.31 196.27±11.18 123.93±6.56 42.69±7.81 42.69±7.81 468.39±37.26 365.05±16.30 300.08±37.05 237.98±25.01
t1值 2.92 2.13 2.35 2.35 2.13 2.13 2.92 2.92
P1值 0.005 0.001 0.001 0.001 0.001 0.001 0.015 0.009
转染对照组 22.85±0.71 22.85±0.71 93.58±12.91 81.05±6.89 193.25±8.56 120.70±10.60 46.47±2.87 46.47±2.87 507.79±49.15 429.89±28.00 325.67±25.55 270.88±36.17
t2值 2.92 2.35 2.35 2.92 2.35 2.13 2.92 2.92
P2值 0.01 0.001 0.001 0.004 0.002 0.005 0.001 0.011
转染组 31.71±0.35 31.71±0.35 56.24±2.40 46.77±6.21 74.30±7.07 65.06±3.78 46.70±7.49 46.70±7.49 237.54±30.58 176.06±21.48 178.20±6.79 129.06±4.91
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