雌激素对牙周膜细胞促炎因子及OPG/RANKL表达的调控作用研究
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摘要
研究背景
骨质疏松(OP, osteoporosis)和牙周病(PD, periodontal disease)是世界范围内广泛发生的两类骨吸收性疾病。雌激素缺乏是绝经后妇女骨质疏松的重要致病因素之一,而骨质疏松则是牙周疾病与牙齿缺失的可能致病因素之一。临床研究表明绝经后妇女尽管口腔卫生状况没有改变,但循环雌激素水平降低时牙周病发病率明显增加。动物实验研究发现大鼠去势(OVX, ovariectomy)诱导的雌激素缺乏可引起下颌骨密度降低、下颌骨骨皮质变薄及牙槽骨吸收。而且,大量研究表明雌激素可能在慢性牙周疾病的发生、发展中具有重要意义。然而,雌激素影响牙周组织健康的确切分子机制目前尚不清楚。
牙周病是炎性骨吸收性疾病,其主要特征是牙龈炎与牙槽骨吸收。一般认为,牙周病的组织损伤是牙周病原体及其代谢产物,特别是脂多糖(LPS,lipopolysaccharide)诱导的宿主免疫反应引起的,免疫细胞及局部组织细胞(如牙龈成纤维细胞、牙周膜细胞)分泌炎性因子参与这一过程。牙周膜(PDL, periodontal ligament)位于牙槽骨与牙骨质之间,其可影响骨形成-骨吸收平衡,在维持牙周组织代谢平衡中具有重要作用。
Lerner指出雌激素缺乏可引起牙周组织骨改建失衡,即雌激素可能调控骨形成-骨吸收平衡而影响炎性诱导的牙周骨重建。以往,有关雌激素对牙周组织改建的影响的研究主要集中于雌激素对牙周膜细胞增殖及成骨能力的影响,而关于雌激素对牙周组织骨吸收的影响及相关作用机制的研究甚少。目前,雌激素缺乏引起的炎性因子表达改变,尤其是促炎因子IL-1、IL-6、TNF-α及破骨细胞形成相关因子OPG/RANKL表达水平改变对牙周组织的影响引起了学者们的广泛关注。
丝裂原激活蛋白激酶(MAPK,Mitogen-activated protein kinases)可分为细胞外信号调控激酶(ERK-1/2,extracellular signal-regulated kinase)、c-jun N末端激酶(JNK,c-jun N-terminal kinase)及p38 MAPK三部分。MAPK是介导细胞反应的重要信号系统,参与了增殖、分化及凋亡等细胞活动,在炎性反应调控中具有重要作用。研究表明,尽管表达水平各不相同,牙周膜细胞表达所有MAPKs信号分子,MAPKs信号途径在牙周炎性疾病的发生、发展中具有重要的调控作用。
研究目的
(1)雌激素是否影响牙周膜细胞促炎因子TNF-α、IL-1β和IL-6表达;
(2)雌激素是否影响牙周膜细胞OPG/RANKL表达;
(3) TNF-α、IL-1β和IL-6是否参与调控LPS、E2诱导RANKL、OPG表达;
(4) MAPKs信号途径否参与调控LPS、E2作用下牙周膜细胞促炎因子TNF-α、IL-1β和IL-6表达。
研究方法
(1)体外分离、培养(人)牙周膜细胞,以E. coli LPS(20μg/mL)为预刺激物诱导(人)牙周膜细胞促炎因子表达,加入不同浓度(10-7,10-8或10-9 M)的E2(17β-estradiol)培养6,12,24或72h。以未作任何处理的(人)牙周膜细胞为空白对照,特异性雌激素受体拮抗剂ICI 182,780处理组(10-6 M)作阴性对照。分别于6,12,24和72h收集细胞培养上清液,0.2%Txiton X-100裂解细胞,收集细胞裂解液。采用TRIzol试剂提取细胞总RNA,于-70℃保存备用。
RT-PCR、ELISA分别检测E. coli LPS、E2作用下牙周膜细胞中TNF-α、IL-6和IL-1βmRNA及Pr(Protein)表达改变。
(2) Real-time PCR、ELISA分别检测LPS、E2作用下牙周膜细胞OPG、RANKL mRNA及Pr表达改变。
(3)为研究TNF-α、IL-1β和IL-6对LPS、E2诱导的牙周膜细胞RANKL、OPG表达调控作用,我们在培养基内加入TNF-α、IL-1β中和性抗体(anti TNF-α,1μg/mL; anti IL-1β, 1μg/mL)或IL-6可溶性受体(sIL-6R, 200 ng/mL),并于12,24和48h分别收集细胞,-70℃保存待检。Real-time PCR检测TNF-α、IL-1β中和性抗体及IL-6可溶性受体对牙周膜细胞OPG、RANKL mRNA表达的影响。
(4)为了解丝裂原激活蛋白激酶(MAPKs)信号途径在牙周膜细胞促炎因子表达调控中的作用,于LPS、E2处理前15分钟分别向培养基内加入MAPKs信号途径生化抑制子PD98059(10μM)、SP600125(10μM)及SB203580(10μM)。细胞处理6h后收集细胞培养上清液,-70℃保存待检.以探讨MAPKs信号系统ERK、JNK及P38α在LPS诱导的促炎因子TNF-α、IL-1β和IL-6表达上调中的调控作用。ELISA检测MAPKs信号途径生化抑制子PD98059、SP600125及SB203580对LPS、E2作用下牙周膜细胞促炎因子TNF-α、IL-1β和IL-6表达的调控作用。
实验结果
(1)与对照组相比,E2对牙周膜细胞促炎因子TNF-α、IL-1β和IL-6自发性表达几乎无影响。E. coli LPS对促炎因子TNF-α、IL-6和IL-1βmRNA、Pr分泌具有显著促进作用,而E2可抑制LPS诱导的TNF-α、IL-6和IL-1βmRNA表达及Pr分泌,且E2对LPS诱导促炎因子表达的抑制作用具有剂量依赖性及时间依赖性。
(2)与对照组相比,LPS可显著促进牙周膜细胞RANKL mRNA、Pr表达,同时对OPG表达也有一定的促进作用;E2可有效抑制LPS诱导RANKL表达;而E2单独处理可显著促进OPG表达增强,但对牙周膜细胞RANKL表达几乎无影响。
(3) TNF-α、IL-1β中和性抗体及IL-6可溶性受体均可有效抑制LPS诱导的RANKL、OPG mRNA表达上调,该效应在细胞处理24～48h较明显。TNF-α、IL-1β中和性抗体及IL-6可溶性受体对E2诱导的OPG mRNA表达几乎无影响。
(4)①对TNF-α而言,SP600125(JNK生化抑制子)可有效抑制LPS的诱导效应,而PD98059(ERK生化抑制子)及SB203580(P38α生化抑制子)对LPS诱导的TNF-α分泌作用不明显;②对IL-1β而言,PD98059、SP600125及SB203580均可抑制LPS诱导的IL-1β,尤以SP600125最为显著;③对IL-6而言,PD98059和SP600125可抑制LPS诱导的IL-6表达上调,而SB203580作用不甚显著。
结论:
(1) E2可能并不改变牙周膜细胞促炎因子表达能力,但可调控LPS对牙周膜细胞促炎因子表达的刺激作用。
(2)E2可完全逆转LPS作用引起的OPG/RANKL比率降低。因此,雌激素可改变牙周膜细胞OPG/RANKL比率而影响牙周病的进展。
(3)LPS对RANKL、OPG表达的调控作用是通过上游促炎因子表达依赖性途径实现的,而E2诱导OPG表达上调的作用与促炎因子TNF-α、IL-1β和IL-6无关。
(4)尽管对促炎因子TNF-α、IL-1β和IL-6表达的调控机制不尽相同,但MAPKs信号系统参与了牙周膜细胞促炎因子表达调控。即LPS可激活多条MAPKs信号通路,以不同的激活途径调控牙周膜细胞TNF-α、IL-1β和IL-6表达。E2对LPS诱导的促炎因子表达具有显著的抑制作用,而MAPK信号系统参与了LPS诱导的促炎因子表达,因而我们推测E2可能,至少部分地,经MAPK信号途径抑制了LPS诱导的促炎因子表达。
Background
Both 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.
骨质疏松(OP, osteoporosis)和牙周病(PD, periodontal disease)是世界范围内广泛发生的两类骨吸收性疾病。雌激素缺乏是绝经后妇女骨质疏松的重要致病因素之一,而骨质疏松则是牙周疾病与牙齿缺失的可能致病因素之一。临床研究表明绝经后妇女尽管口腔卫生状况没有改变,但循环雌激素水平降低时牙周病发病率明显增加。动物实验研究发现大鼠去势(OVX, ovariectomy)诱导的雌激素缺乏可引起下颌骨密度降低、下颌骨骨皮质变薄及牙槽骨吸收。而且,大量研究表明雌激素可能在慢性牙周疾病的发生、发展中具有重要意义。然而,雌激素影响牙周组织健康的确切分子机制目前尚不清楚。
牙周病是炎性骨吸收性疾病,其主要特征是牙龈炎与牙槽骨吸收。一般认为,牙周病的组织损伤是牙周病原体及其代谢产物,特别是脂多糖(LPS,lipopolysaccharide)诱导的宿主免疫反应引起的,免疫细胞及局部组织细胞(如牙龈成纤维细胞、牙周膜细胞)分泌炎性因子参与这一过程。牙周膜(PDL, periodontal ligament)位于牙槽骨与牙骨质之间,其可影响骨形成-骨吸收平衡,在维持牙周组织代谢平衡中具有重要作用。
Lerner指出雌激素缺乏可引起牙周组织骨改建失衡,即雌激素可能调控骨形成-骨吸收平衡而影响炎性诱导的牙周骨重建。以往,有关雌激素对牙周组织改建的影响的研究主要集中于雌激素对牙周膜细胞增殖及成骨能力的影响,而关于雌激素对牙周组织骨吸收的影响及相关作用机制的研究甚少。目前,雌激素缺乏引起的炎性因子表达改变,尤其是促炎因子IL-1、IL-6、TNF-α及破骨细胞形成相关因子OPG/RANKL表达水平改变对牙周组织的影响引起了学者们的广泛关注。
丝裂原激活蛋白激酶(MAPK,Mitogen-activated protein kinases)可分为细胞外信号调控激酶(ERK-1/2,extracellular signal-regulated kinase)、c-jun N末端激酶(JNK,c-jun N-terminal kinase)及p38 MAPK三部分。MAPK是介导细胞反应的重要信号系统,参与了增殖、分化及凋亡等细胞活动,在炎性反应调控中具有重要作用。研究表明,尽管表达水平各不相同,牙周膜细胞表达所有MAPKs信号分子,MAPKs信号途径在牙周炎性疾病的发生、发展中具有重要的调控作用。
研究目的
(1)雌激素是否影响牙周膜细胞促炎因子TNF-α、IL-1β和IL-6表达;
(2)雌激素是否影响牙周膜细胞OPG/RANKL表达;
(3) TNF-α、IL-1β和IL-6是否参与调控LPS、E2诱导RANKL、OPG表达;
(4) MAPKs信号途径否参与调控LPS、E2作用下牙周膜细胞促炎因子TNF-α、IL-1β和IL-6表达。
研究方法
(1)体外分离、培养(人)牙周膜细胞,以E. coli LPS(20μg/mL)为预刺激物诱导(人)牙周膜细胞促炎因子表达,加入不同浓度(10-7,10-8或10-9 M)的E2(17β-estradiol)培养6,12,24或72h。以未作任何处理的(人)牙周膜细胞为空白对照,特异性雌激素受体拮抗剂ICI 182,780处理组(10-6 M)作阴性对照。分别于6,12,24和72h收集细胞培养上清液,0.2%Txiton X-100裂解细胞,收集细胞裂解液。采用TRIzol试剂提取细胞总RNA,于-70℃保存备用。
RT-PCR、ELISA分别检测E. coli LPS、E2作用下牙周膜细胞中TNF-α、IL-6和IL-1βmRNA及Pr(Protein)表达改变。
(2) Real-time PCR、ELISA分别检测LPS、E2作用下牙周膜细胞OPG、RANKL mRNA及Pr表达改变。
(3)为研究TNF-α、IL-1β和IL-6对LPS、E2诱导的牙周膜细胞RANKL、OPG表达调控作用,我们在培养基内加入TNF-α、IL-1β中和性抗体(anti TNF-α,1μg/mL; anti IL-1β, 1μg/mL)或IL-6可溶性受体(sIL-6R, 200 ng/mL),并于12,24和48h分别收集细胞,-70℃保存待检。Real-time PCR检测TNF-α、IL-1β中和性抗体及IL-6可溶性受体对牙周膜细胞OPG、RANKL mRNA表达的影响。
(4)为了解丝裂原激活蛋白激酶(MAPKs)信号途径在牙周膜细胞促炎因子表达调控中的作用,于LPS、E2处理前15分钟分别向培养基内加入MAPKs信号途径生化抑制子PD98059(10μM)、SP600125(10μM)及SB203580(10μM)。细胞处理6h后收集细胞培养上清液,-70℃保存待检.以探讨MAPKs信号系统ERK、JNK及P38α在LPS诱导的促炎因子TNF-α、IL-1β和IL-6表达上调中的调控作用。ELISA检测MAPKs信号途径生化抑制子PD98059、SP600125及SB203580对LPS、E2作用下牙周膜细胞促炎因子TNF-α、IL-1β和IL-6表达的调控作用。
实验结果
(1)与对照组相比,E2对牙周膜细胞促炎因子TNF-α、IL-1β和IL-6自发性表达几乎无影响。E. coli LPS对促炎因子TNF-α、IL-6和IL-1βmRNA、Pr分泌具有显著促进作用,而E2可抑制LPS诱导的TNF-α、IL-6和IL-1βmRNA表达及Pr分泌,且E2对LPS诱导促炎因子表达的抑制作用具有剂量依赖性及时间依赖性。
(2)与对照组相比,LPS可显著促进牙周膜细胞RANKL mRNA、Pr表达,同时对OPG表达也有一定的促进作用;E2可有效抑制LPS诱导RANKL表达;而E2单独处理可显著促进OPG表达增强,但对牙周膜细胞RANKL表达几乎无影响。
(3) TNF-α、IL-1β中和性抗体及IL-6可溶性受体均可有效抑制LPS诱导的RANKL、OPG mRNA表达上调,该效应在细胞处理24～48h较明显。TNF-α、IL-1β中和性抗体及IL-6可溶性受体对E2诱导的OPG mRNA表达几乎无影响。
(4)①对TNF-α而言,SP600125(JNK生化抑制子)可有效抑制LPS的诱导效应,而PD98059(ERK生化抑制子)及SB203580(P38α生化抑制子)对LPS诱导的TNF-α分泌作用不明显;②对IL-1β而言,PD98059、SP600125及SB203580均可抑制LPS诱导的IL-1β,尤以SP600125最为显著;③对IL-6而言,PD98059和SP600125可抑制LPS诱导的IL-6表达上调,而SB203580作用不甚显著。
结论:
(1) E2可能并不改变牙周膜细胞促炎因子表达能力,但可调控LPS对牙周膜细胞促炎因子表达的刺激作用。
(2)E2可完全逆转LPS作用引起的OPG/RANKL比率降低。因此,雌激素可改变牙周膜细胞OPG/RANKL比率而影响牙周病的进展。
(3)LPS对RANKL、OPG表达的调控作用是通过上游促炎因子表达依赖性途径实现的,而E2诱导OPG表达上调的作用与促炎因子TNF-α、IL-1β和IL-6无关。
(4)尽管对促炎因子TNF-α、IL-1β和IL-6表达的调控机制不尽相同,但MAPKs信号系统参与了牙周膜细胞促炎因子表达调控。即LPS可激活多条MAPKs信号通路,以不同的激活途径调控牙周膜细胞TNF-α、IL-1β和IL-6表达。E2对LPS诱导的促炎因子表达具有显著的抑制作用,而MAPK信号系统参与了LPS诱导的促炎因子表达,因而我们推测E2可能,至少部分地,经MAPK信号途径抑制了LPS诱导的促炎因子表达。
Background
Both 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.
引文
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