| BackgroundBoth osteoporosis and periodontal disease are bone-resorptive diseases. Estrogen deficiency is believed to be one of the major causes of post-menopausal osteoporosis. Osteoporosis is considered as one of the risk factors for periodontal disease and tooth loss. Clinical observations in post-menopausal women have confirmed an increased prevalence of periodontal disease with lower estrogen level, even when oral hygiene remained unchanged. Animal experiments with ovariectomized (OVX) rats also demonstrated that estrogen deficiency may result in low mineral density of mandible, thin cortex of mandibular body, and resorption of alveolar bone. Furthermore, a number of studies suggested that estrogen may have an important role in chronic inflammatory periodontal diseases. However, the etiology of estrogen-associated periodontal diseases remains an enigma, partly because the precise effects of estrogen on periodontal tissues at molecular level are not yet known.Periodontitis is a chronic inflammatory disease characterized by gingival inflammation and alveolar bone resorption. It is generally accepted that much of the periodontal tissue destruction observed in periodontal disease is host mediated through inflammatory cytokines by local tissues and immune cells in response to the bacterial flora and its products/metabolites, especially lipopolysaccharide (LPS). The periodontal ligament (PDL), which lies between alveolar bone and cementum, plays a vital role in maintaining the homeostasis of periodontal tissues by affecting coordinated balance of bone-forming and bone-resorbing.A report reviewed by Lerner has demonstrated that estrogen deficiency can provoke an imbalance in the remodeling sequence of periodontal tissues. Previous studies mainly focused on whether estrogen affects the bone formation capability of PDL cells, such as the production of osteocalcin. However, the effects of estrogen on bone resorption in periodontium have been less explored. Estrogen deficiency-triggered changes in inflammatory cytokines are emerging as a common theme that may have a significant impact on bone resorption in periodontal tissues. The inflammatory cytokines that have attracted the most attention are TNF-α, IL-1β, IL-6, OPG and RANKL.Aims(1) To explore the modulatory effects of estrogen on expression of pro-inflammatory cytokines such as TNF-α, IL-1βand IL-6 in the hPDL cells (2) To assess the modulatory effect of estrogen on OPG and RANKL expression in the hPDL cell.(3) To investigate whether estrogen modulate LPS-induced OPG and RANKL expression through the induction of TNF-α, IL-1βand IL-6 in the hPDL cell.(4) To determine whether estrogen modulate LPS-induced pro-inflammatory cytokines biosynthesis via multiple mitogen-activated protein kinase pathways in the hPDL cells.Methods(1) The hPDL cells were stimulated by E. coli LPS (Sigma, 20μg/ml)) with or without estrogen (17β-estradiol, E2, Sigma) (10-7, 10-8 or 10-9 M) for 6, 12, 24, 48 and 72 hours. The untreated hPDL cells served as normal control. To confirm the specificity of the estrogen effects, we employed ICI 182,780 (Tocris Cookson, 10-6 M), a specific estrogen receptor antagonist, as negative control. At specific times, culture media were collected. And the hPDL cells of all treatment groups were lysed in 0.2% Triton X-100 and the cell lysates were collected and centrifugated. The cell-free supernatants were stored at -70°C until being assayed.TNF-α, IL-1βand IL-6 mRNA induction was detected via reverse transcription-polymerase chain reaction (RT-PCR), while protein levels were quantified via enzyme-linked immunosorbent assays (ELISA).(2) To investigate the effects of E2 on expression of OPG and RANKL at both mRNA and protein level, quantitative Real-Time PCR and ELISA was performed.(3) The effects of anti TNF-α, anti IL-1βand sIL-6R on mRNA expression of OPG and RANKL induced by E2 and LPS were determined using Real-Time PCR.(4) The effects of biochemical inhibitors of ERK, JNKand p38 MAPK on pro-inflammatory cytokines TNF-α, IL-1βand IL-6 secretion were detected by ELISA.Results(1) In our study, it was found that E2 had little effect on the spontaneous expression of TNF-α, IL-1βand IL-6 by hPDL cells. Our results showed that E. coli LPS can induce the expression of TNF-α, IL-1βand IL-6 at both mRNA and protein level by hPDL cells, whereas co-treatment of E2 significantly suppressed LPS-stimulated production of TNF-α, IL-1βand IL-6 in hPDL cells in adose-dependent manner.(2) In this study, we observed that LPS up-regulated RANKL as well as OPG expression, though the OPG-inducing effect was not significant. We also found that E2 time-dependently increased OPG expression and attenuated the RANKL-inducing effects of LPS, however, no detectable effect on RANKL expression was observed with treatment of E2 alone. The LPS-induced decrease of OPG/RANKL ratio in hPDL cells was completely reversed by E2.(3) Our results demonstrated that neutralizing antibodies for these cytokines effectively inhibited LPS-induced OPG and RANKL expression while they did not abolish the induction of OPG by E2.(4) Our study showed that blocking JNK MAPK with SP600125 reduced LPS-induced TNF-αexpression. All MAPK inhibitors significantly reduced the IL-1βprotein levels induced by LPS. Our results also indicate that both PD98059 and SP600125 may suppress induction of IL-6 by LPS, whereas SB203580 had little effect on LPS-stimulated IL-6 biosynthesis.Conclusions(1) It was suggested that E2 may not alter the ability of hPDL cells to produce pro-inflammatory cytokines, but that it may modify the stimulatory effect of LPS on pro-inflammatory cytokines in hPDL cells.(2) Our study suggested that the LPS-induced decrease of OPG/RANKL ratio in hPDL cells was completely reversed by E2. Therefore, we proposed that E2 may influence the progression of periodontal disease via altering the ratio of OPG to RANKL in hPDL cells.(3) According to our study, we speculate that estrogen may modulate OPG expression in a manner independent of TNF-α, IL-1βand IL-6, although LPS may induce OPG and RANKL expression in a manner dependent on upstream inflammatory stimulators.(4) Our results indicated that, though the MAPK signaling pathways involved in the induction of pro-inflammatory cytokines varied among TNF-α, IL-1βand IL-6, induction of TNF-α, IL-1βand IL-6 by E. coli LPS requires signaling through ERK, JNK and p38 MAPK in hPDL cells. It is possible that E2 may, at least in part, modulate LPS-induced pro-inflammatory cytokines expression through multiple mitogen-activated protein kinase pathways in periodontal ligament cells.
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