Up-regulation of nucleotide-binding oligomerization domain 1 in inflamed human dental pulp

Introduction

The innate immune response is activated by recognition of microbial components through specific pattern recognition receptors including nucleotide-binding oligomerization domain (NOD)-like receptors. However, the regulation of NOD-1 in inflamed human dental pulp remains poorly understood. This study aimed to evaluate the expression of NOD-1 in healthy and inflamed human dental pulps. In addition, the secretion of chemokines induced by NOD-1 and the related signaling pathways were studied.

Methods

Samples of human dental pulp tissues were obtained from freshly extracted wisdom teeth. The protein localization of NOD-1 in the pulp tissues was detected by immunohistochemistry. In addition, human dental pulp fibroblasts were stimulated with NOD-1 agonist γ-D-glutamylmeso-diaminopimelic acid. Production of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) was determined by an enzyme-linked immunosorbent assay. The involvement of mitogen-activated protein kinase (MAPK) signaling pathways was examined by Western blot analysis, and the association of MAPK signaling with chemokine production was determined.

Results

The results demonstrated the expression of NOD-1 in normal dental pulp, and up-regulated NOD-1 expression was observed in inflamed dental pulp. On stimulation with NOD-1 agonist, production of IL-8 and MCP-1 was induced in a dose-dependent manner. Moreover, phosphorylation of p38 MAPK and Jun N-terminal kinase (JNK) was enhanced by stimulation of NOD-1. With the treatment of p38 MAPK and JNK inhibitors, the NOD-1–induced IL-8 production was suppressed.

Conclusions

In response to microbial invasion, the expression of NOD-1 can be regulated in a ligand-inducible manner. NOD-1 might participate in pulp inflammation through chemokine production via MAPK signaling pathways.     

Hinokitiol increases the angiogenic potential of dental pulp cells through ERK and p38MAPK activation and hypoxia-inducible factor-1α (HIF-1α) upregulation

Hinokitiol, a natural iron-chelating agent, is known to have diverse biological and pharmacological activities in various cell types. However, the effect of hinokitiol on dental pulp cells has not yet been reported. In this study, hinokitiol increases hypoxia-inducible factor-1α (HIF-1α) protein levels and vascular endothelial growth factor (VEGF) secretion in human dental pulp cells. The extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways are involved in hinokitiol-induced HIF-1α protein expression in dental pulp cells. Conditioned media from hinokitiol-treated pulp cells enhances angiogenesis in vitro and in vivo. Overall, these results show that hinokitiol promotes ERK and p38MAPK activation and HIF-1α-induced VEGF production, thus increasing the angiogenic potential of dental pulp cells.     

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.     

Regulation of TNF-alpha-induced IL-6 production in MG-63 human osteoblast-like cells

Tumor necrosis factor-alpha (TNF-alpha) stimulates osteoblast production of interleukin-6 (IL-6), an inflammatory cytokine implicated in osteoclastic bone resorption. Therefore, we tested the hypothesis that TNF-alpha-induced IL-6 production in MG-63 osteosarcoma cells occurs via the p38 mitogen-activated protein kinase (MAPK) pathway. TNF-alpha activated p38 MAPK and stimulated IL-6 secretion by MG-63 cells, and pre-incubation of cells with the p38 MAPK inhibitor abrogated TNF-alpha-dependent IL-6 secretion. Transfection of IL-6 full-length and 5-deletion gene promoter reporter constructs indicated that p38 MAPK activation by TNF-alpha enhanced IL-6 gene expression, and that the p38 MAPK-responsive region resided in the proximal 260-bp segment. Transfection of NFkappaB and C/EBPbeta-sensitive reporter promoter constructs demonstrated that NFkappaB activity was enhanced and that constitutive C/EBPbeta was inhibited by TNF-alpha, with both effects being p38 MAPK-dependent. In conclusion, although p38 MAPK activation by TNF-alpha stimulates IL-6 secretion by MG-63 cells, it has opposing effects on c/EBPbeta and NFkappaB activity.     

Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation of cultured human dental pulp cells by low-power gallium-aluminium-arsenic laser irradiation

AIM: To examine whether low-power laser irradiation (LPLI) promotes cellular proliferation of human dental pulp-derived fibroblast-like cells (dental pulp cells). METHODOLOGY: Dental pulp cells were obtained by primary culture of human dental pulp tissues from extracted third molar teeth. The phosphorylation of the mitogen-activated protein kinase (MAPK) family after LPLI of these cells was investigated by Western blotting. By using a specific MAPK/ERK kinase (MEK) inhibitor (PD098059), the specific effect of LPLI on the MAPK pathway was also investigated by Western blotting as described above. The incorporation of [3H]thymidine into the cells after LPLI was determined, and statistical analysis was performed by Wilcoxon signed-ranks test. RESULTS: Extracellular signal-regulated protein kinase (ERK) 1/2 was phosphorylated between 5 and 30 min after LPLI. Moreover, PD098059 inhibited LPLI-mediated ERK1/2 activation. LPLI did not affect p38 MAPK or c-Jun N-terminal kinase (JNK) phosphorylation. But LPLI did not stimulate [3H]thymidine incorporation into these cells. CONCLUSIONS: These results indicated that LPLI activated MAPK/ERK, a signal for proliferation, differentiation and survival, but did not activate the stress signals p38 MAPK and JNK in human dental pulp cells.     

Prostaglandin F2α-Induced Interleukin-8 Production in Human Dental Pulp Cells Is Associated With MEK/ERK Signaling

Prostaglandin F_2alpha (PGF_2α) and interleukin-1beta (IL-1β) levels are elevated in inflamed dental pulp. The roles of IL-1β and PGF_2α in the pathogenesis of pulpal inflammation await investigation. We found that IL-1β stimulated PGF_2α production of human dental pulp cells. IL-1β and PGF_2α (0.5–10 μmol/L) also induced IL-8 production and mRNA expression in pulp cells. Aspirin inhibited IL-1β–induced PGF_2α, but not IL-8 production. PGF_2α-induced IL-8 production and mRNA expression were inhibited by U0126 (an inhibitor of mitogen-activated protein kinase kinase [MEK1/2]) inhibitor), whereas SQ22536 (an adenylate cyclase inhibitor) enhanced this event. These results indicate that IL-1β–induced IL-8 production in pulp cells is not mainly via direct activation of cyclooxygenase and PGF_2α generation. PGF_2α-induced IL-8 production is possibly via activation of MEK/extracellular signal-regulated kinase signaling, but not by activation of adenylate cyclase. IL-1β and PGF_2α might involve the pathogenesis of pulpal inflammation via induction of IL-8 production.     

Leptin induces IL-6 and IL-8 expression through leptin receptor Ob-Rb in human dental pulp fibroblasts

Objective: Leptin, through binding to its special receptor (Ob-Rb), has potent effects on immunity and inflammation. This study aimed to investigate the expression of leptin receptor Ob-Rb in human dental pulp fibroblasts (HDPFs) and the effects of leptin on the production of proinflammatory cytokines of IL-6 and IL-8 by HDPFs.

Methods: Ob-Rb expression was determined by quantitative real-time PCR (real-time PCR), Western blot and immunofluorescence analyses in cultured HDPFs. Small interfering RNA (siRNA) was transfected into HDPFs to down-regulate the expression of Ob-Rb. Real-time PCR and enzyme-linked immunosorbent assay (ELISA) were used to determine the proinflammatory cytokines of IL-6 and IL-8 levels in leptin-stimulated HDPFs. The involved signalling pathways that mediate the leptin-stimulated production of proinflammatory cytokines were investigated using Western blot and specific signalling inhibitor analyses.

Results: The expression levels of Ob-Rb mRNA and protein were detected in HDPFs. Leptin could stimulate mRNA and protein expression of IL-6 and IL-8 in HDPFs in a concentration-dependent and time-dependent manner. Transfection with siRNA targeting Ob-Rb resulted in remarkable reduction of IL-6 and IL-8 expressions by HDPFs. In accordance with the enhanced expression of proinflammatory cytokines, leptin stimulation resulted in rapid phosphorylation of STAT3, p38 MAPK, ERK and Akt in HDPFs. Inhibiting JAK2/STAT3, p38 MAPK or PI3K/Akt substantially decreased leptin-induced IL-6 production, whereas blocking ERK and p38 MAPK substantially suppressed IL-8 production from leptin-stimulated HDPFs.

Conclusions: Leptin may up-regulate IL-6 and IL-8 production through binding with Ob-Rb in HDPFs via the activation of different intracellular signalling pathways.     

Mangiferin inhibits lipopolysaccharide-induced production of interleukin-6 in human oral epithelial cells by suppressing toll-like receptor signaling

ObjectiveOral epithelial cells have currently been found to play an important role in inflammatory modulation in periodontitis. Mangiferin is a natural glucosylxanthone with anti-inflammatory activity. The aim of this study was to investigate the regulatory effect of mangiferin on lipopolysaccharide (LPS)-induced production of proinflammatory cytokine interleukin-6 (IL-6) in oral epithelial cells and the underlying mechanisms.DesignThe levels of LPS-induced IL-6 production in OKF6/TERT-2 oral keratinocytes were detected using enzyme-linked immunosorbent assay (ELISA). The expression of Toll-like receptor (TLR) 2 and TLR4 was determined using western blot analysis. And the phosphorylation of TLR downstream nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) was examined using cell-based protein phosphorylation ELISA kits.ResultsWe found that mangiferin reduced LPS-upregulated IL-6 production in OKF6/TERT-2 cells. Additionally, mangiferin inhibited LPS-induced TLR2 and TLR4 overexpression, and suppressed the phosphorylation of NF-κB, p38 MAPK and JNK. Moreover, mangiferin repressed IL-6 production and TLR signaling activation in a dose-dependent manner after 24 h treatment.ConclusionsMangiferin decreases LPS-induced production of IL-6 in human oral epithelial cells by suppressing TLR signaling, and this glucosylxanthone may have potential for the treatment of periodontitis.     

Signaling pathways regulating IL-1alpha-induced COX-2 expression

Interleukin-1alpha(IL-1alpha) stimulates the production of prostaglandin E(2) (PGE(2)) in odontogenic keratocyst fibroblasts. However, the signaling pathways remain obscure. In this study, we investigated IL-1alphasignaling pathways that regulate cyclooxygenase-2 (COX-2) expression in odontogenic keratocyst fibroblasts. IL-1alphaincreased the expression of COX-2 mRNA and protein, and PGE(2) secretion in the fibroblasts. IL-1alphaincreased the phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). PD-98059, SB-203580, SP-600125, and PDTC-which are inhibitors of ERK1/2, p38, JNK, and nuclear factor-kappaB (NF-kappaB), respectively-attenuated the IL-1alpha-induced COX-2 mRNA expression and activated protein kinase C PGE(2) secretion. IL-1alpha(PKC), and PKC inhibitor staurosporine inhibited IL-1alpha-induced phosphorylation of ERK1/2, p38, and JNK, and decreased IL-1alpha-induced COX-2 mRNA expression. Thus, in odontogenic keratocyst fibroblasts, IL-1alphamay stimulate COX-2 expression both through the PKC-dependent activation of ERK1/2, p38, and JNK signaling pathways, and through the NF-kappaB cascade.     

牙髓卟啉单胞菌内毒素诱导成骨细胞表达炎症因子的信号通路研究

目的检测细胞外信号调节激酶（ERK）1/2和p38丝裂原活化蛋白激酶抑制剂对牙髓卟啉单胞菌内毒素（LPS）诱导成骨细胞白细胞介素（IL）-1β mRNA和IL-6 mRNA的影响,探讨根尖周病变牙槽骨吸收的可能病理机制。方法成骨细胞MG-63经PD98059和SB203580预处理1 h后,加入牙髓卟啉单胞菌LPS作用6 h,应用逆转录聚合酶链反应（RT-PCR）检测IL-1β mRNA和IL-6 mRNA的表达水平。结果PD98059预处理后,牙髓卟啉单胞菌LPS诱导MG-63表达IL-1β mRNA的水平下降。SB203580预处理后,牙髓卟啉单胞菌LPS诱导MG-63表达IL-1β mRNA和IL-6 mRNA水平均下降。结论牙髓卟啉单胞菌LPS诱导MG-63细胞表达IL-1β mRNA依赖ERK1/2和p38MAPK信号转导通路,表达IL-6 mRNA依赖p38MAPK信号转导通路。     

Dental pulp fibroblasts contain target cells for lysophosphatidic Acid

Lysophosphatidic acid (LPA) is a locally produced bioactive phospholipid which is involved in tissue repair. The objective of this study was to determine whether dental pulp tissue also responds to the phospholipid. Effects of LPA on proliferation, differentiation, and mitogen-activated protein kinase (MAPK) signaling of dental pulp fibroblasts (DPF) were examined in vitro. We report that DPF express LPA receptors LPA1, LPA2, and LPA3 and respond to the ligand with increased mitogenic activity. Involvement of extracellular signal-regulated kinase, p38 MAPK, and c-Jun NH(2)-terminal kinase in LPA signaling could be demonstrated by use of specific inhibitors and detection of the phosphorylation status of the kinases. An increased mitogenic activity paralleled a decreased number of alkaline-phosphatase-positive cells and expression levels of dentin sialophosphoprotein and osteocalcin. Together, these results suggest that dental pulp fibroblasts can respond to LPA, a process that may play a role in pulp tissue repair.     

小檗碱对牙髓干细胞MAPK/ERK信号通路及成牙本质向分化的影响研究

目的 探讨小檗碱对牙髓干细胞(dental pulp stem cells,DPSCs)成牙本质向分化及p38丝裂原活化蛋白激酶(MAPK)/细胞外调节蛋白激酶(ERK)信号通路的影响.方法 采用酶消化法提取DPSCs,将培养鉴定的DPSCs传代培养至第3～5代,用于以下实验.实验分为对照组(正常培养DPSCs)、矿化...     

Tissue pH and temperature regulate pulpal nociceptors

The TRPV1 receptor acts as a sensor for environmental changes in pH and temperature. Since many nociceptors express TRPV1, it is possible that local tissue-cooling may inhibit nociceptor activity via reduction of TRPV1 activation. The present study used isolated superfused rat dental pulp to test the hypothesis that capsaicin receptors are activated in inflamed tissue, as measured by alterations in neuropeptide release. We tested the hypothesis that alterations in the tissue temperature and pH of isolated superfused rat dental pulp regulate capsaicin-induced release of calcitonin gene-related peptide (CGRP). Application of capsaicin with increased proton concentration (i.e., lowered pH) produced a nearly two-fold increase in peak immunoreactive CGRP release, as compared with capsaicin applied at a pH of 7.4. Reduction in tissue temperature from 37 degrees C to 26 degrees C completely blocked the capsaicin effect. The study indicates that environmental stimuli regulate the activity of capsaicin-sensitive neurons innervating dental pulp, and these factors may be significant clinically in the development and amelioration of dental pain.     

Modulatory Roles of Interferon-γ through Indoleamine 2, 3-dioxygenase Induction in Innate Immune Response of Dental Pulp Cells

Introduction

Marked infiltration of inflammatory cells such as activated T cells producing interferon-γ (IFN-γ) is observed in severe pulpitis. However, the roles of IFN-γ in the innate immune response of dental pulp have not been reported. Indoleamine 2, 3-dioxygenase (IDO) is a regulator of immune responses, and the IDO expression is induced by IFN-γ in many cells whose expression in dental pulp is unknown. The purpose of this study was to determine the role of IFN-γ in the immune response through microbial pattern recognition receptors (PRRs) such as Toll-like receptors or nucleotide-binding oligomerization domain–like receptors on the production of proinflammatory cytokines such as CXCL10 and interleukin (IL)-6 and the expression of IDO in cultured human dental pulp cells (HDPCs).

Methods

HDPCs were established from explant cultures of healthy pulp tissues. CXCL10 and IL-6 production was determined using enzyme-linked immunosorbent assay. Confirmation of IDO localization in dental pulp tissues was examined using immunohistochemistry. IDO expression in HDPCs was analyzed by immunoblot.

Results

IFN-γ significantly up-regulated CXCL10 and IL-6 production in the HDPCs stimulated with ligands for PRRs in a concentration-dependent manner. The expression of IDO was detected in inflamed pulp tissue. In addition, IFN-γ in combination with the PRR ligands enhanced IDO expression in HDPCs compared with IFN-γ alone. Moreover, CXCL10 production in IFN-γ–stimulated HDPCs was inhibited by an IDO inhibitor.

Conclusions

This study showed the synergistic effects by IFN-γ on cytokine production and IDO expression in HDPCs, suggesting that IFN-γ may modulate the innate immune response of dental pulp.     

IGF-1对体外培养髁突软骨细胞增殖与凋亡的影响

目的：：研究IGF-1对IL-1β诱导的颞下颌关节软骨细胞增殖与凋亡的影响。方法：组织块培养法分离培养人髁突软骨细胞,通过细胞形态观察和免疫细胞化学染色进行鉴定。将培养的细胞分为：对照组、IL-1β组(10μg/L)、 IL-1β+IGF-1组(0、1、10、50、100μg/L), MTT法检测各组细胞增殖能力变化,流式细胞仪检测细胞周期分布以及细胞凋亡情况,Western blot检测各组细胞中凋亡相关因子Bcl-2、Bax和Caspase-3以及p38 MAPK/NF-κB蛋白表达变化。结果：人髁突软骨细胞生长状态良好,甲苯胺蓝染色胞质呈深蓝色,II型胶原呈阳性表达。与对照组比较,IL-1β组细胞增殖能力、Bcl-2/Bax比值明显降低,早凋与晚凋细胞百分数、Caspase-3、p38 MAPK/NF-κB蛋白表达均明显增加;与IL-1β组比较,1~100μg/L IGF-1预处理组细胞增殖能力、Bcl-2/Bax比值逐渐上升(P<0.05),细胞凋亡率、Caspase-3、p38 MAPK /NF-κB蛋白表达则逐渐下调(P<0.05),均呈现一定的浓度依赖性。结论：IGF-1可抑制IL-1β诱导的髁突软骨细胞凋亡并减轻p38 MAPK/NF-κB的活化。