Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts

Tamai R, Kiyoura Y, Sugiyama A. Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts. J Periodont Res 2011; 46: 13–20. © 2010 John Wiley & Sons A/S Background and Objective: Nitrogen‐containing bisphosphonates (NBPs) are widely used as anti‐bone‐resorptive drugs. However, use of NBPs results in inflammatory side‐effects, including jaw osteomyelitis. In the present study, we examined the effects of alendronate, a typical NBP, on cytokine production by human peripheral blood mononuclear cells (PBMCs) and gingival fibroblasts incubated with lipid A. Methods: The PBMCs and gingival fibroblasts were pretreated with or without alendronate for 24 h. Cells were then incubated in the presence or absence of lipid A for a further 24 h. Levels of secreted human interleukin (IL)‐1β, IL‐6, IL‐8 and monocyte chemoattractant protein‐1 (MCP‐1) in culture supernatants were measured by ELISA. We also examined nuclear factor‐κB (NF‐κB) activation in both types of cells by ELISA. Activation of Smad3 in the cells was assessed by flow cytometry. In addition, we performed an inhibition assay using SIS3, a specific inhibitor for Smad3. Results: Pretreatment of PBMCs with alendronate promoted lipid A‐induced production of IL‐1β and IL‐6, but decreased lipid A‐induced IL‐8 and MCP‐1 production. In human gingival fibroblasts, alendronate pretreatment increased lipid A‐induced production of IL‐6 and IL‐8, and increased NF‐κB activation in gingival fibroblasts but not PBMCs stimulated with lipid A. In contrast, alendronate activated Smad3 in both types of cells. Finally, SIS3 inhibited alendronate‐augmented IL‐6 and IL‐8 production by human gingival fibroblasts but up‐regulated alendronate‐decreased IL‐8 production by PBMCs. Conclusion: These results suggest that alendronate‐mediated changes in cytokine production by gingival fibroblasts occur via regulation of NF‐κB and Smad3 activity.     

Effect of implant surface material and roughness to the susceptibility of primary gingival fibroblasts to inflammatory stimuli

ObjectivesThe impact of the implant surface material and roughness on inflammatory processes in peri-implantitis is not entirely clear. Hence, we investigated how titanium and zirconia surfaces with different roughness influence the susceptibility of primary human gingival fibroblasts to different inflammatory stimuli.MethodsPrimary human gingival fibroblasts were isolated from 8 healthy individuals and cultured on following surfaces: smooth titanium machined surface (TiM), smooth zirconia machined surface (ZrM), moderately rough titanium surface (SLA), or moderately rough zirconia surface (ZLA). Subsequently, stimulation with one of the following stimuli was performed: Porphyromonas gingivalis lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, interleukin (IL)-1β. The resulting production of IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1 was measured by qPCR and ELISA.ResultsP. gingivalis LPS induced IL-6 and MCP-1 production was slightly higher on titanium surfaces compared to zirconia surfaces. IL-1β induced IL-6 production was not affected by any surface characteristic. The production of MCP-1 in response to IL-1β was higher on smooth compared to rough surfaces and was not affected by the material. The production of IL-6 and MCP-1 in response to TNF-α was most strongly affected by surface characteristics. Higher production of these cytokine was observed on smooth compared to rough surfaces and on titanium compared to zirconia surfaces. Surface characteristics had only minor effects on IL-8 production.SignificanceThe susceptibility of primary gingival fibroblasts to inflammation depends on various factors, such as surface material, surface roughness and the nature of inflammatory stimuli. All these factors might determine susceptibility to peri-implantitis.     

Hydrogen sulfide synergistically upregulates Porphyromonas gingivalis lipopolysaccharide-induced expression of IL-6 and IL-8 via NF-κB signalling in periodontal fibroblasts

Objectives

The periodontal pathogen Porphyromonas gingivalis produces hydrogen sulfide (H₂S). H₂S in the oral cavity is positively correlated with periodontitis but the mechanism by which H₂S contributes to periodontal diseases is obscure. We investigated the effect of H₂S in combination with P. gingivalis lipopolysaccharide (LPS) on expression of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 in periodontal fibroblasts and the underlying mechanism of action.

Material and methods

Gingival fibroblasts (GFs) and periodontal ligament cells (PDLCs) were treated with different concentrations of the H₂S donor NaHS in the presence/absence of P. gingivalis LPS for different time periods. Expression of IL-6 and IL-8 was detected by real-time PCR and ELISA. The activity of nuclear factor-kappa B (NF-κB) signalling was investigated using western blotting, EMSA and pathway blockade assays.

Results

Real-time PCR and ELISA results showed that H₂S not only upregulated expression of IL-6 and IL-8 at mRNA and protein levels in a dose- and time-dependent manner, but also aggravated P. gingivalis LPS-induced expression of IL-6 and IL-8 in GFs and PDLCs. Western blotting and EMSA showed that NF-κB signalling was activated by NaHS, P. gingivalis LPS, and both, which was in accordance with the expression levels of IL-6 and IL-8 in GFs and PDLCs. These results were confirmed using a NF-κB pathway blockade assay.

Conclusions

H₂S synergistically upregulated P. gingivalis LPS-induced expression of IL-6 and IL-8 in GFs and PDLCs via activation of NF-κB signalling, which could promote the development of periodontitis.     

Anti-inflammatory activity of cannabinoid receptor 2 ligands in primary hPDL fibroblasts

ObjectivesApproximately 65 million adults in the US have periodontitis, causing tooth loss and decreased quality of life. Cannabinoids modulate immune responses, and endocannabinoids are prevalent during oral cavity inflammation. Targets for intervention in periodontal inflammation are cannabinoid type 1 and 2 receptors (CB1R, CB2R), particularly CB2R because its levels increase during inflammation. We previously demonstrated that SMM-189 (CB2R inverse agonist) decreased pro-inflammatory cytokine production in primary microglial cells. The hypothesis of this study was that cannabinoids anandamide (AEA), HU-308 (CB2R selective agonist), and SMM-189 decrease pro-inflammatory IL-6 and MCP-1 production by primary human periodontal ligament fibroblasts (hPDLFs) stimulated with P. gingivalis LPS, TNF-α, or IL-1β.DesignCytotoxic effects of cannabinoid compounds (10⁻⁴–10^−6.5 M), LPS (1–1000 ng/ml), TNFα (10 ng/ml) and IL-1β (1 ng/ml) were assessed by measuring effects on cellular dehydrogenase activity. IL-6 and MCP-1 production were measured using Mesoscale Discovery (MSD) Human Pro-Inflammatory IL-6 and MSD Human Chemokine MCP-1 kits and analyzed using MSD Sector 2400 machine.ResultsEC₅₀ values for AEA, SMM-189, and HU-308 were 16 μM, 13 μM, and 7.3 μM respectively. LPS (1 μg/ml), TNF-α (10 ng/ml), and IL-1β (1 ng/ml) increased IL-6 and MCP-1 production, which were inhibited by AEA, SMM-189, and HU-308. AEA alone significantly increased IL-6, but not MCP-1 levels, but the other cannabinoids alone had no effect.ConclusionThe effective inhibition of LPS, TNF-α, IL-1β stimulated IL-6 and MCP-1 production by CB2R ligands in hPDLFs suggests that targeting the endocannabinoid system may lead to development of novel drugs for periodontal therapy, aiding strategies to improve oral health.     

PPARγ在脂多糖刺激牙周膜细胞中调控NF-κB信号通路的作用研究

目的:探讨PPARγ在牙周炎中调控作用机制。方法:组织块法培养健康人牙周膜细胞(human periodontal ligament cells, hPDLCs)并鉴定。细胞处理分为以下4组,A组:对照组;B组:脂多糖(lipopolysaccharide,LPS )刺激组;C组:罗格列酮对照组,即二甲基亚砜(dimethylsulfoxide, DMSO)处理组;D组:罗格列酮处理组。免疫印迹法检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptor γ,PPARγ)和核因子κB(nuclear factor κB, NF-κB) p65胞核、胞浆及总蛋白含量,细胞免疫荧光检测NF-κB p65表达部位。实时定量PCR和酶联免疫法检测白细胞介素1β(interleukin-1β,IL-1β)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)RNA和蛋白表达。结果:LPS刺激下,胞核PPARγ表达降低NF-κB p65表达升高,相应的IL-1β和TNF-α RNA及蛋白表达量均升高。同时加入罗格列酮后,胞核PPARγ表达升高NF-κB p65表达降低,且IL-1β和TNF-α RNA及蛋白表达量均降低。差异均有统计学意义(P<0.01)。结论:PPARγ通过下调NF-κB信号通路抑制脂多糖刺激下牙周膜细胞炎症因子RNA表达和蛋白分泌,进而调节牙周炎症反应。     

Tumor necrosis factor-α and interleukin-1β expression pathway induced by Streptococcus mutans in macrophage cell line RAW 264.7

Streptococcus mutans, a major etiological agent of dental caries, frequently causes systemic disease, such as subacute bacterial endocarditis, if it enters the bloodstream. In this study, the production pathways of the proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), induced by S. mutans in mouse macrophage were examined using a quantitative real-time polymerase chain reaction and an enzyme-linked immunosorbent assay. The S. mutans stimulated the expression of TNF-α and IL-1β mRNA at a multiplicity of infection of 1 : 100, which increased at 2 and 4 h, respectively, to 24 h. It also induced the production of high levels of the TNF-α and IL-1β proteins, which increased at 2 h and reached a peak at 4 and 24 h, respectively. Nuclear factor-κB (NF-κB) was activated and reached a maximum level 30 min after the S. mutans treatment. The expression of TNF-α and IL-1β mRNA and protein was suppressed by the treatment with pyrrolidine dithiocarbamate, an NF-κB inhibitor. The S. mutans-induced TNF-α expression was suppressed by the presence of SB203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, or SP600125, a Jun N-terminal kinase (JNK) MAP kinase inhibitor. On the other hand, IL-1β expression was inhibited by extracellular signal-regulated kinase (ERK)/p38/JNK MAP kinase inhibitor pretreatment. In addition, TNF-α production was suppressed more in the Toll-like receptor 2(-/-) (TLR2(-/-)) macrophages than in the TLR4(-/-) macrophages, whereas IL-1β production was suppressed more in the TLR4(-/-) macrophages than in the TLR2(-/-) macrophages. These results show that S. mutans stimulates the production of TNF-α and IL-1β in the mouse macrophage cell line, RAW 264.7, by activating ERK/p38/JNK, and NF-κB through TLR2 and TLR4, respectively.     

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。     

Blocking Proinflammatory Cytokine Release Modulates Peripheral Blood Mononuclear Cell Response to Porphyromonas gingivalis

Background: Chronic periodontitis (CP) is an inflammatory disease in which cytokines play a major role in the progression of disease. Anti‐inflammatory cytokines (interleukin 4 [IL‐4] and IL‐10) were reported to be absent or reduced in diseased periodontal tissues, suggesting an imbalance between the proinflammatory and anti‐inflammatory mediators. This study tests the hypothesis that there is cellular crosstalk mediated by proinflammatory and anti‐inflammatory cytokines and that blocking proinflammatory cytokine (tumor necrosis factor‐α [TNF‐α] and IL‐1) production will enhance anti‐inflammatory cytokine (IL‐4 and IL‐10) production from peripheral blood mononuclear cells (PBMCs) in response to Porphyromonas gingivalis. Methods: PBMCs were isolated from individuals diagnosed with CP or healthy individuals and cultured for 24 hours. Concanavalin A (ConA) was used as an activator of lymphocyte function. Live and heat‐killed P. gingivalis or lipopolysaccharide from P. gingivalis were used as the bacterial stimulants. TNF‐α and IL‐1 production was neutralized by specific antibodies against TNF‐α and IL‐1α or IL‐β. Culture supernatants were evaluated by enzyme‐linked immunosorbent assay for TNF‐α, IL‐1β, IL‐4, and IL‐10 production. Results: Live P. gingivalis did not result in any significant IL‐10 or IL‐4 release, whereas heat‐killed P. gingivalis led to a significant increase in IL‐10 levels compared with unstimulated or live P. gingivalis–stimulated cells from both healthy individuals or those with CP. Overall, PBMCs from patients with CP produced significantly lower IL‐10 in response to ConA and P. gingivalis, suggesting chronic suppression of the anti‐inflammatory cytokine production. Blocking the proinflammatory cytokine response did not result in any substantial change in IL‐10 or IL‐4 response to live P. gingivalis. Blocking the proinflammatory cytokine response restored IL‐10 production by cells from CP in response to P. gingivalis lipopolysaccharide. Conclusions: These findings suggest that PBMCs from patients with CP have suppressed anti‐inflammatory cytokine production that can, in part, be restored by neutralizing proinflammatory cytokines. Monocytes are an important source of IL‐10 production, and monocyte‐derived IL‐10 might play a regulatory role in the pathogenesis of CP.     

Exposure to Porphyromonas gingivalis LPS during macrophage polarisation leads to diminished inflammatory cytokine production

ObjectiveThe objective of the present study was to determine the effects of concurrent LPS and cytokine priming, reflective of the in vivo milieu, on macrophage production of key periodontitis associated cytokines TNF, IL-1β and IL-6.DesignTHP-1 cells were pre-treated with combinations of Porphyromonas gingivalis and Escherichia coli lipopolysaccharide (LPS), concurrently with polarising cytokines IFNγ and IL-4, or PMA as a non-polarised control. Production of key periodontitis associated cytokines in response to subsequent LPS challenge were measured by enzyme − linked immunosorbent assay.ResultsCompared with cells incubated with IFNγ or IL-4 alone in the “polarisation” phase, macrophages that were incubated with LPS during the first 24 h displayed a down-regulation of TNF and IL-1β production upon secondary LPS treatment in the “activation” phase. In all three macrophage populations (M0, M1 and M2), pre-treatment with P. gingivalis LPS during the polarisation process led to a significant decrease in TNF production in response to subsequent activation by LPS (p = 0.007, p = 0.002 and p = 0.004, respectively). Pre-treatment with E. coli LPS also led to a significant down-regulation in TNF production in all three macrophage populations (p < 0.001). Furthermore, the presence of E. coli LPS during polarisation also led to the down-regulation of IL-1β in the M1 population (p < 0.001), whereas there was no measurable effect on IL-1β production in M0 or M2 macrophages. There was no significant effect on IL-6 production.ConclusionsMacrophages become refractory to further LPS challenge, whereby production of key periodontitis associated cytokines TNF and IL-1β is reduced after exposure to LPS during the polarisation phase, even in the presence of inflammatory polarising cytokines. This diminished cytokine response may lead to the reduced ability to clear infection and transition to chronic inflammation seen in periodontitis.     

The induction expression of human β-defensins in gingival epithelial cells and fibroblasts

ObjectiveThe gingival epithelial cells and fibroblasts can produce antimicrobial peptides when stimulated by inflammatory cytokines. The purpose of the present study was to test whether gingival keratinocytes and gingival fibroblasts respond differently to inflammatory cytokine activation. This will enable us to understand the chronic inflammatory response in the process of periodontal disease.DesignGingival keratinocytes and fibroblasts were isolated and treated with different concentrations of IL-1β and quantitative real-time PCR was performed to evaluate the induced expressions of hBD-1, hBD-2 and hBD-3. The induced response was compared between the gingival epithelial cells and fibroblasts. The inhibitors of p38 protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were applied to explore the molecular mechanism during the induction of hBDs in both cells.ResultsThe results showed that the hBDs expressions were found to be induced by different concentrations of IL-1β, but with several differences between gingival epithelial cells and fibroblasts. The hBDs mRNA expression in gingival fibroblasts was more sensitive compared with keratinocytes to different concentrations of IL-1β. The hBD-1 and hBD-3 expressions in these two cells were down-regulated by IL-1β and hBD-2 expression was up-regulated. The inflammatory cytokine IL-1β had dual effect on hBDs expression.ConclusionsThe gingival epithelial cells and fibroblasts respond differently to the inflammatory cytokine IL-1β which indicated different roles played by the two cells in the host defense. The dual effect of IL-1β on hBDs expression may contribute to the defensins down-regulation in periodontal disease.     

T helper cells from aggressive periodontitis patients produce higher levels of interleukin-1 beta and interleukin-6 in interaction with Porphyromonas gingivalis

Objective

In this study, we analyzed the production of Interleukin-1 beta (IL-1β) and IL-6 by activated CD4+ cells obtained from aggressive periodontitis (AgP) patients in comparison with healthy subjects (HC).

Materials and methods

CD4+ cells were automatically separated from lymphocytes obtained from peripheral blood of patients with AgP and healthy controls. Cells were activated for 4, 8, and 24 h with three different stimuli: anti-CD3/anti-CD28, phytohemagglutinin (PHA), and Porphyromonas gingivalis (P. gingivalis) outer membrane protein (OMP). Protein levels were measured in supernatants of activated CD4+ cells by a bead-based immunoassay (CBA). In addition, serum antibodies against P. gingivalis were determined. Data were analyzed using U test (p?Results T helper cells of AgP patients activated with P. gingivalis OMP produced higher levels of IL-1β and IL-6 in comparison with healthy controls (p?P. gingivalis.

Conclusion

In view of these results, it is possible to conclude that P. gingivalis contributes to the pathogenesis of AgP by inducing high levels of pro-inflammatory cytokines such as IL-1β and IL-6 by peripheral CD4+ T helper cells.

Clinical relevance

In accordance with the clinical parameters and the immunological data, we suggest that full-mouth disinfection with adjunctive systemic antibiotics might be the anti-infectious non-surgical periodontal treatment of choice in this type of patients. Microbiological analyses at the beginning and at the end of the periodontal treatment are recommended. However, it is necessary to verify these data in longitudinal clinical studies.     

Porphyromonas gingivalis infection enhances Th17 responses for development of atherosclerosis

Objectives

Porphyromonas gingivalis has been shown to associate with the development of atherosclerosis. Recent studies indicate that IL-17-producing T helper 17 (Th17) cells have been correlated with the emergence of atherosclerosis. Therefore, we investigated whether the Th17 cell response and expression of Th17-related molecules, in contrast with Th1- and Treg cells, are enhanced by P. gingivalis-challenge in Apolipoprotein E knockout (ApoE KO) mice.

Design

Five mice were intravenously injected with P. gingivalis three times a week for 3 weeks and killed at 15 weeks of age. The proximal aorta lesion area, flow cytometry analysis and IL-17, IL-10, IFN-γ, and IL-1β levels in splenic cultures, and expression of Th17-related molecules in spleen and hearts were examined.

Results

P. gingivalis-challenge showed notable accumulation of atherosclerotic plaques by Oil Red O-staining in ApoE KO mice. Intracellular cytokine staining revealed that significantly elevated CD4⁺ interleukin (IL)-17A⁺ T cells and slightly increased CD4⁺ Foxp3⁺ T cells was recognized in spleen cells of P. gingivalis-challenged mice compared with those from non-infected mice. P. gingivalis-challenge significantly increased IL-17 and IL-1β production and RORγt expression in splenic cells. Furthermore, the expression of Th17-related genes such as IL-6, TGF-β, RORγt and STAT3 were elevated in splenic cells as well as heart tissue of P. gingivalis-challenged mice.

Conclusion

These results suggest that P. gingivalis infection may enhance pro-inflammatory Th17 cell responses in lesion areas and spleen, thereby accelerating atherosclerosis.     

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.     

HDAC5-Mediated Acetylation of p100 Suppresses Its Processing

IntroductionPeriodontitis is a condition involving chronic inflammation in the gums, periodontal ligaments, cementum, and alveolar bone. Nuclear factor-κB (NF-κB) activation is the prominent mediator of inflammation and osteoclast differentiation. The role of histone deacetylase 5 (HDAC5) in periodontitis development and NF-κB regulation is not fully understood.MethodsWe used primary mouse bone marrow–derived osteoclast cultures in vitro and a mouse model of chronic periodontists (CPD) treated with the HDAC4/5 inhibitor LMK-235. Real-time polymerase chain reaction, micro computed tomography, flow cytometry, western blot, and immunoprecipitation were used to study proinflammatory cytokines, NF-κB activation, HDAC5 activity, and the interaction of HDAC5 with NF-κB p100.ResultsLMK-235, a selective inhibitor of HDAC4 and HDAC5, reduced osteoclast marker gene expression (Cstk, Acp5, and Calcr) and tartrate-resistant acid phosphatase activity in primary osteoclast cultures. LMK-235 reduced the increase in cementoenamel junction–alveolar bone crest distance, inflammatory cell infiltration of gingival tissues, and expression levels of interleukin (IL)-1β, tumor necrosis factor alpha, IL-6, and IL-23a, indicating an ameliorative effect on CPD. Immunoprecipitation experiments have further confirmed p100–HDAC5 interaction, acetylation levels of p100, and NF-κB activation.ConclusionsThese results indicate that HDAC5 binds and deacetylates p100, leading to its activation, increased proinflammatory cytokine production, gingival infiltration, and osteoclast differentiation, thus promoting alveolar bone resorption. HDAC5 inhibition is therefore a potentially promising therapeutic strategy for the treatment of periodontitis.     

炎症因子通过核因子κB通路促进口腔鳞状细胞癌转移的体外研究

目的 探讨炎症因子与核因子κB(nuclear factor kappa B,NF-κB)信号转导通路在口腔鳞状细胞癌转移中的意义.方法 应用口腔鳞状细胞癌低转移细胞系Tca8113和高转移细胞系Tb为研究对象,通过蛋白质印迹法和荧光素酶报告基因法检测口腔鳞状细胞癌细胞系中NF-κB通路活性.用NF-κB抑制因子α(inhibitor of kappa B alpha,IκBa)抑制质粒第32、36位丝氨酸磷酸化位点被丙氨酸替代的质粒(pBabe-SR-IκBa)和NF-κB通路抑制剂吡咯烷二硫代氨基甲酸盐(pyrolidinedithiecar bamate,PDTC)抑制信号转导通路活性,并用侵袭小室实验(TransweH)检测口腔鳞状细胞癌高转移系Tb细胞侵袭能力的变化.此外,通过酶联免疫吸附法检测pBabe-SR-IκBα和PDTC抑制信号转导通路后,肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)、白介素(IL)-1a、IL-6、IL-8和粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)等炎症因子的分泌.结果 蛋白质印迹法检测显示:高转移Tb细胞中磷酸化IκBα和磷酸化p65的表达量明显高于低转移细胞系Tea8113,分别为Tea8113细胞的3.19倍和6.81倍.荧光素酶(luciferase)报告基因结果显示,Tb细胞NF-κB的启动子活性为Tca8113的2.12倍(P<0.01),并对TNF-α更为敏感.转染pBabe-SR-IκBα和应用PDTC抑制NF-κB通路后,对Tb细胞的体外侵袭能力抑制率分别为21.9%和69.3%.此外,酶联免疫吸附试验法显示通路抑制后,TNF-α、IL-1α、IL-6、IL-8和GM-CSF等炎症因子的分泌也明显降低.结论 TNF-α、IL-1α、IL-6、IL-8和GM-CSF等炎症因子可能通过NF-κB通路促进口腔鳞状细胞癌细胞转移.