LPS刺激牙龈成纤维细胞IL-6、IL-8的产生和膜表面受体CD14的表达

目的：观察牙龈卟啉菌和中间普氏菌LPS刺激人牙龈成纤维细胞合成IL-6、IL-8的能力，并了解LPS是否通过细胞表面膜受体mCD14介导信号传导。方法：4种浓度的LPS在不同时间段。分别作用于体外培养的人牙龈成纤维细胞。采用ELISA检测IL-6、IL-8含量的变化，同时利用逆转录聚合酶链反应，进一步观察IL-6、IL-8，CD14在mRNA水平的表达特征。结果：ELISA和RT-PCR的反应结果证实，受LPS的刺激，细胞合成和分泌细胞因子IL-6、IL-8的能力明显增强，但未检测到细胞表面膜受体mCD14mRNA的表达。结论：LPS用于牙龈成纤维细胞合成和分泌细胞因子没有通过膜受体mCD14的介导。     

Interleukin-10 inhibits expression of interleukin-6 and -8 mRNA in human dental pulp cell cultures via nuclear factor-kappaB deactivation

The effects of interleukin (IL)-10 on the expression of IL-6 and IL-8 mRNA in human dental pulp cell cultures were investigated by using the Northern blot analysis. On stimulation with Prevotella intermedia lipopolysaccharide (PiLPS), IL-10 was produced in peripheral blood but was not detected in human dental pulp cell culture supernatants. IL-10 inhibited IL-8 mRNA expression, which is normally stimulated by PiLPS, IL-1alpha, and tumor necrosis factor-alpha and inhibited IL-6 mRNA expression, which is normally stimulated by IL-1alpha. In addition, IL-10 inhibited the activation of nuclear factor-kappaB, which is normally induced by PiLPS. We conclude that IL-10 inhibits expression of IL-6 and IL-8 mRNA in dental pulp cell cultures by inhibiting the activation of nuclear factor-kappaB.     

古曲抑菌素A对脂多糖诱导人牙髓细胞炎症反应的影响

目的研究组蛋白去乙酰化酶抑制剂曲古抑菌素A（TSA）对脂多糖（LPS）诱导人牙髓细胞（hDPC）炎症反应的影响。 方法采用酶组织块法体外分离培养hPDC,按处理因素不同分为空白对照组、LPS组（1 μg/ml）、TSA（25或50 nmol/L预处理）+LPS（1 μg/ml）组,实时荧光定量聚合酶链反应（PCR）和ELISA检测促炎因子白细胞介素（IL）-6、IL-8 mRNA及蛋白表达量,Western blot检测NF-κB信号通路关键信号蛋白IKKα/β、p65及IκB-α磷酸化程度的变化。IL-6、IL-8 mRNA及蛋白表达采用方差分析,NF-κB信号通路Western blot结果采用独立样本t检验。 结果TSA（25 nmol/L）预处理能显著降低LPS刺激引起的IL-6、IL-8 mRNA与蛋白表达（F_{IL-6 mRNA}= 22.538,P_{IL-6 mRNA}= 0.002;F_{IL-8 mRNA}= 20.253,P_{IL-8 mRNA}= 0.002;F_IL-6蛋白= 9.327,P_IL-6蛋白= 0.007;F_IL-8蛋白= 9.6894,P_IL-8蛋白= 0.011）;LPS刺激能激活NF-κB信号通路中关键蛋白p-IKKα/β、p-p65和p-IκB-α的表达,而TSA预处理可降低IKKα/β（t_{30 min}= 6.437,P_{30 min}= 0.003;t_{60 min}= 6.386,P_{60 min}= 0.003;t_{120 min}= 4.368,P_{120 min}= 0.012）和IκB-α磷酸化程度（t_{15 min}= 3.822,P_{15 min}= 0.019;t_{30 min}= 4.467,P_{30 min}= 0.011）。 结论TSA可显著抑制LPS刺激下hDPC促炎因子的分泌,同时降低IKKα/β和IκB-α磷酸化程度,提示TSA可能通过降低NF-κB信号通路活性抑制牙髓炎症发展。     

Nicotine and lipopolysaccharide affect cytokine expression from gingival fibroblasts

BACKGROUND: This in vitro study investigated the influence of nicotine, lipopolysaccharide (LPS), and a combination of both agents on cytokine expression from human gingival fibroblasts (HGFs). METHODS: HGFs were exposed for 48 hours to 250 microg/ml nicotine, 1 microg/ml Porphyromonas gingivalis LPS, or both. The expression of multiple cytokines was detected in the HGFs conditioned media using cytokine protein arrays. RESULTS: The untreated HGFs expressed several cytokines, which included relatively high levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1). They also expressed low levels of growth-regulated oncogene (GRO), IL-3, and IL-10. Nicotine had the greatest effect on the expression of GRO-alpha, IL-7, IL-10, and IL-15 compared to the untreated control. P. gingivalis LPS had the greatest effect on the expression of GRO-alpha; IL-7; IL-10; and RANTES (regulated on activation, normal T-cell expressed, and presumably secreted) compared to the untreated control. The combination of both agents had the biggest impact on the expression of GRO-alpha, IL-7, IL-10, IL-15, RANTES, and interferon-gamma (IFN-gamma) compared to the untreated control. CONCLUSION: HGFs exposed to nicotine, P. gingivalis LPS, or a combination of both agents increased the expression of multiple cytokines.     

脂多糖和转化生长因子-β1对牙髓细胞Toll样受体4的表达及信号通路的影响

目的研究牙髓炎症过程中,在促炎因子脂多糖（LPS）和抑炎因子转化生长因子-β1（TGF-β1）同时存在的情况下,牙髓细胞表面Toll样受体4（TLR4）的表达水平及相关信号分子的变化情况。方法LPS、TGF-β1作用于体外培养的牙髓细胞,用流式细胞术检测牙髓细胞表面TLR4的表达;实时荧光定量聚合酶链反应（real-time PCR）、Westernblot方法检测相关信号分子的表达水平,包括进化保守的Toll信号中介分子（ECSIT）和核转录因子-κB（NF-κB）;酶联免疫吸附试验（ELISA）检测前炎症因子白细胞介素-6（IL-6）的分泌水平;然后进一步通过real-time PCR法检测临床炎症牙髓组织中相应指标的变化。结果体外培养的牙髓细胞在LPS、TGF-β1共同作用下,细胞表面TLR4的表达水平没有明显变化,但是IL-6分泌增加,ECSIT表达增加,NF-κB入核增加。临床标本的real-time PCR结果表明：炎症状态下的牙髓组织中TGF-β1 mRNA表达增加,TLR4 mRNA表达没有明显变化,ECSIT及IL-6 mRNA表达增加。结论牙髓炎症发展过程中,虽然牙髓组织中TGF-β1表达增加,抑制细胞表面TLR4的表达,但TLR4的信号通路仍然被活化,主要机制可能是LPS引起信号分子ECSIT的活化,从而抑制TGF-β1信号通路的活化。     

miR-146a在人牙髓细胞中的表达及其作用研究

目的 检测miR-146a在正常及脂多糖（LPS）刺激的人牙髓细胞（hDPC）中的表达,探讨miR-146a对hDPC分泌炎性因子的影响及其机制.方法 （1）实时荧光定量聚合酶链反应（PCR）方法测定正常及LPS刺激的hDPC miR-146a的表达水平.（2） Lipofectamine 2000脂质体分别转染化学合成的miR-146a类似物（miR-146a mimics）、miR-146a抑制剂（miR-146a inhibitor）及其相应无关序列小RNA对照,转染24 h后1μg/ml LPS刺激hDPC,4h后实时荧光定量PCR检测白细胞介素6（IL-6）、IL-8 mRNA的表达,酶联免疫吸附试验（ELISA）检测细胞上清IL-6、IL-8的分泌;Western blot 法检测miR-146a下游靶标白细胞介素1受体相关激酶1（IRAK1）及肿瘤坏死因子受体相关因子6（TRAF6）的蛋白表达水平.两组比较采用独立样本t检验,多组比较采用方差分析.结果 （1）经LPS刺激的hDPC miR-146a表达水平高于正常hDPC（12 h：t=8.488,P=0.014;24 h：t=39.661,P＜0.001）.（2）转染miR-146a mimics的hDPC在1μg/mlLPS刺激下产生炎性因子IL-6、IL-8的水平低于阴性对照,差异有统计学意义（IL-6 PCR：P＜0.001,IL-6 ELISA：P＜0.001;IL-8 PCR：P＜0.001,IL-8ELISA：P=0.011）;过表达miR-146a后IRAK1及TRAF6蛋白水平明显下调（IRAK1：P=0.002; TRAF6：P＜ 0.001）.结论 miR-146a在LPS刺激的hDPC中表达上调,转染miR-146a可下调其靶基因IRAK1及TRAF6表达并降低IL-6、IL-8分泌,推测miR-146a参与牙hDPC的炎症反应调控.     

Capsular polysaccharide from Actinobacillus actinomycetemcomitans inhibits IL-6 and IL-8 production in human gingival fibroblast

We previously reported that a capsular polysaccharide (CP) from Actinobacillus actinomycetemcomitans Y4 induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures. However, the effects of A. actinomycetemcomitans Y4 CP on human gingival fibroblasts (HGF) are still unclear. The present study was undertaken to test the hypothesis that A. actinomycetemcomitans Y4 CP alters the production of inflammatory cytokines, such as interleukin-6 (IL-6) and IL-8 by HGF. When HGF were cultured with various concentrations of Y4 CP for 24 h, IL-6 and IL-8 production decreased in a concentration-dependent manner. Y4 CP (100 microg/ml) suppressed the release of IL-6 from 9.09 +/- 0.08 ng/ml to 0.34 +/- 0.21 ng/ml (P < 0.01) and IL-8 production decreased from 3.76 +/- 0.03 ng/ml to 0.09 +/- 0.01 ng/ml (P < 0.01). Y4 CP suppressed 70-80% of the release of IL-6 and IL-8 from HGF stimulated with Y4 lipopolysaccharide (LPS), too. Interestingly, anti-A. actinomycetemcomitans Y4 CP completely inhibited the effect of A. actinomycetemcomitans Y4 CP on IL-6 and IL-8 production from HGF. These results indicate that Y4 CP inhibits the release of IL-6 and IL-8 from HGF, suggesting that A. actinomycetemcomitans Y4 modulates the inflammatory response in periodontitis. Remarkably, this inhibitory effect was reversed by specific anti-A. actinomycetemcomitans Y4 CP suggesting an important relationship between the organism and the humoral host response.     

Lipopolysaccharide from Actinobacillus actinomycetemcomitans stimulates production of interleukin-1beta, tumor necrosis factor-alpha, interleukin-6 and interleukin-1 receptor antagonist in human whole blood

Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) is supposed to be an important etiological agent in localized juvenile periodontitis (LJP). We have studied the effect of lipopolysaccharide (LPS) extracted from these periodontopathogenic bacteria on synthesis of the proinflammatory cytokines, interleukin-1beta(IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) in human whole blood. LPS from A. actinomycetemcomitans in concentrations > or =1 ng/ml induced a significant production of all these proinflammatory cytokines, whereas LPS from Escherichia coli (E. coli), strain 026:B6 had to be added in concentrations > or =1 microg/ml to obtain a similar effect. Similarly, LPS from A. actinomycetemcomitans > or =0.1 ng/ml resulted in production of IL-1ra, while LPS from E. coli 026:B6 had to be added at > or =10 ng/ml to obtain similar effects. It has been suggested that the ratio between production of proinflammatory and anti-inflammatory cytokines may influence the outcome of periodontal diseases. Other in vitro and in vivo studies have, however, indicated that very large excesses (100-1000 times) of IL-1ra compared to IL-1beta are required to shift the IL-1ra:IL-1beta ratio in favor of an inhibition of IL-1 bioactivity. In our ex vivo system, we found that stimulation with extremely low doses of A. actinomycetemcomitans LPS (0.1-1 ng/ml) resulted in IL-1ra production solely, without concomitant production of IL-1beta, the excess of IL-1ra over IL-1beta peaking at 1 ng/ml, which accordingly should suggest that LPS from A. actinomycetemcomitans primarily has proinflammatory effects.     

Effect of calcium hydroxide-treated Prevotella nigrescens on the gene expression of matrix metalloproteinase and its inhibitor in MG63 cells

The purpose of this study was to monitor the expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) produced by an osteoblastic cell line MG63 stimulated with Prevotella nigrescens lipopolysaccharides (LPS), and to compare the level of secretion before and after the P. nigrescens LPS was treated with calcium hydroxide [Ca(OH)2]. The underlying hypothesis is that the balance between MMP and TIMP secretion is the key to an understanding of the host degradative pathways involved in the pathogenesis of bacterial derived pulpal and periapical diseases. Confluent monolayers of MG63 human osteosarcoma cells were exposed to varying concentrations of P. nigrescens or Escherichia coli LPS. Alternately, confluent cultures were exposed to 10 microg/ml of bacterial LPS pretreated with Ca(OH)2 (12.5 mg/ml) for 72 hours. At the end of the experimental period, total RNA was extracted and real-time quantitative polymerase chain reaction (PCR) was performed for MMP-1, TIMP-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results showed that the expression of MMP-1 mRNA was low and invariant for the experimental period in the negative controls. However, exposure to P. nigrescens LPS increased expression after 48 hours. Expression of TIMP-1 mRNA was highly increased at 24 and 48 hours with lower concentrations of LPS in contrast to a suppression with a concentration of 10 microg/ml. Treatment of P. nigrescens LPS with Ca(OH)2 resulted in a down-regulation of MMP-1, whereas pretreated E. coli LPS demonstrated no stimulatory activity for MMP-1 gene expression. Both types of LPS when pretreated with Ca(OH)2 induced slightly up-regulated expression of TIMP-1.     

Regulation of hyaluronan synthase gene expression in human periodontal ligament cells by tumour necrosis factor-alpha, interleukin-1beta and interferon-gamma

Accumulation and fragmentation of hyaluronic (HA) accompanies the inflammatory changes in the periodontium and gingival crevicular fluid are involved in periodontitis, but the mechanism for this is unknown. Recently, three human hyaluronan-synthase (HAS1, 2, and 3) genes have been cloned and characterised as synthesising hyaluronans of different molecular weights. Both HAS1 and HAS2 synthesise high molecular-weight HA, whereas HAS3 produces lower molecular weight HA. In the present study the regulation of HAS genes by cytokines in cultured human periodontal ligament (PDL) cells was investigated using a novel real-time fluorescence polymerase chain reaction detection system. Human PDL cells derived from premolars were cultured with or without tumour necrosing factor (TNF)-alpha (1-100 ng/ml), interleukin (IL)-1beta (0.1-10 ng/ml) and interferon (IFN)-gamma (1-100 ng/ml). Expression of HAS mRNA was assessed in cultured cells treated with these cytokines for 0-24 h. The expression of HAS2 mRNA was enhanced about 4.5- and 2.2-fold at maximum after 3-h stimulation with 10 ng/ml TNF-alpha and 1 ng/ml IL-1beta, respectively, whereas IFN-gamma exerted little effect on HAS2 or HAS3 mRNA expression during the experiment. Expression of HAS3 mRNA was increased by about 14- and 10-fold after 3-h stimulation with 10 ng/ml TNF-alpha and 1 ng/ml IL-1beta, respectively. These results suggest that TNF-alpha and IL-1beta regulate HAS expression, and consequently may result in an accumulation of HA and an increase in HA of a lower molecular-weight.     

TLR4 mediates LPS-induced VEGF expression in odontoblasts

Lipopolysaccharide (LPS) from gram-negative bacteria cell walls such as Prevotella intermedia and Escherichia coli induce vascular endothelial growth factor (VEGF) expression in odontoblasts, but not in undifferentiated dental pulp cells. CD14 and TLR4 are responsible for LPS signaling in macrophages, but their expression levels and function in dental pulp cells are unknown. We showed here that murine odontoblast-like cells (MDPC-23) express CD14 and TLR4 by immunohistochemistry and flow cytometry. In contrast, undifferentiated dental pulp cells (OD-21) presented low or no expression of these two receptors. MDPC-23 cells showed CD14 and TLR4 up-regulation upon exposure to LPS, as determined by real time PCR. Dominant negative murine TLR4 (DN-mTLR4) transfected MDPC-23 cells did not show upregulated VEGF expression in response to LPS stimulation. These results demonstrate that odontoblast-like cells express CD14 and TLR4, and that LPS-induced VEGF expression is mediated, at least in part, by TLR4 signaling.     

Lipopolysaccharide from Actinobacillus actinomycetemcomitans stimulates macrophages to produce interleukin-1 and tumor necrosis factor mRNA and protein

Actinobacillus actinomycetemcomitans is associated with periodontal disease in children and adults. We report that low concentrations of lipopolysaccharide (LPS) from A. actinomycetemcomitans stimulated human macrophages to increase dramatically their accumulation of mRNA coding for interleukin-1 alpha (IL-1 alpha), IL-1 beta as well as tumor necrosis factor (TNF). Protein levels of IL-1 and TNF alpha also increased. Levels of these mRNAs increased by 4-5 fold as compared with unstimulated macrophages when these cells were cultured with as little as 2 ng/ml LPS from A. actinomycetemcomitans. Polymyxin binds and blocks the action of LPS; polymyxin inhibited the ability of LPS from A. actinomycetemcomitans to increase levels of IL-1 beta mRNA. The LPS of A. actinomycetemcomitans stimulated increased levels of IL-1 beta mRNA in the presence of cycloheximide, showing that stimulation by this LPS did not require new synthesis of protein. Furthermore, dexamethasone inhibited the ability of LPS from A. actinomycetemcomitans to stimulate the accumulation of mRNA coding for IL-1 beta. A. actinomycetemcomitans is an invasive microorganism of the gingiva; high intragingival numbers correlate with sites undergoing local destruction of the periodontium. IL-1 alpha, IL-1 beta, and TNF are potent monokines that mediate inflammation and resorption of bone. Out studies suggest that macrophages migrating to these gingival sites of A. actinomycetemcomitans infection will be stimulated by LPS of A. actinomycetemcomitans to produce IL-1 alpha, IL-1 beta and TNF. These cytokines will mediate gingival inflammation and stimulate resorption of alveolar bone.