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中华口腔医学研究杂志(电子版) ›› 2016, Vol. 10 ›› Issue (01) : 11 -16. doi: 10.3877/cma.j.issn.1674-1366.2016.01.003

所属专题: 文献

基础研究

古曲抑菌素A对脂多糖诱导人牙髓细胞炎症反应的影响
湛敏康1, 王新璇1, 徐琼1,()   
  1. 1. 510055 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室
  • 收稿日期:2015-11-30 出版日期:2016-02-01
  • 通信作者: 徐琼

Effects of trichostatin A on the inflammation induced by lipopolysaccharide in human dental pulp cells

Minkang Zhan1, Xinxuan Wang1, Qiong Xu1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2015-11-30 Published:2016-02-01
  • Corresponding author: Qiong Xu
  • About author:
    Corresponding author: Xu Qiong, Email:
引用本文:

湛敏康, 王新璇, 徐琼. 古曲抑菌素A对脂多糖诱导人牙髓细胞炎症反应的影响[J]. 中华口腔医学研究杂志(电子版), 2016, 10(01): 11-16.

Minkang Zhan, Xinxuan Wang, Qiong Xu. Effects of trichostatin A on the inflammation induced by lipopolysaccharide in human dental pulp cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2016, 10(01): 11-16.

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(FIL-6 mRNA= 22.538,PIL-6 mRNA= 0.002;FIL-8 mRNA= 20.253,PIL-8 mRNA= 0.002;FIL-6蛋白= 9.327,PIL-6蛋白= 0.007;FIL-8蛋白= 9.6894,PIL-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(t30 min= 6.437,P30 min= 0.003;t60 min= 6.386,P60 min= 0.003;t120 min= 4.368,P120 min= 0.012)和IκB-α磷酸化程度(t15 min= 3.822,P15 min= 0.019;t30 min= 4.467,P30 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。

Objective

To investigate the effect of trichostatin A (TSA) , a histone deacetylases inhibitor (HDACi) , on the inflammation of human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS) .

Methods

HDPCs were cultured from human healthy tooth pulp. Three experimental groups were designed: blank control group, LPS (1 μg/ml) group and TSA (25 or 50 nmol/L) +LPS group. The mRNA and protein expression of pro-inflammatory cytokines (IL-6, IL-8) were assayed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and sandwich ELISA kit. The phosphorylation of main members in NF-κB signal pathway were examined by Western blot. The mRNA and protein secretion of IL-6 and IL-8 were evaluated by One-Way ANOVA, expressions of NF-κB signal pathway were analysed with t test.

Results

RT-qPCR and ELISA showed that mRNA and protein expression of IL-6 and IL-8 were all increased by LPS (1 μg/ml) , whereas their expression were significantly reduced by TSA (25 nmol/L) pretreatment in hDPCs (FIL-6 mRNA= 22.538, PIL-6 mRNA= 0.002; FIL-8 mRNA= 20.253, PIL-8 mRNA= 0.002; FIL-6= 9.327, PIL-6= 0.007; FIL-8= 9.6894, PIL-8= 0.011) . The p-IKKα/β, p-p65 and p-IκB-α were activated with the stimulation of LPS, while TSA pretreatment decreased LPS-induced up-regulation of p-IKKα/β (t30 min= 6.437, P30 min= 0.003; t60 min= 6.386, P60 min= 0.003; t120 min= 4.368, P120 min= 0.012) and p-IκB-α (t15 min= 3.822, P15 min= 0.019; t30 min= 4.467, P30 min= 0.011) .

Conclusions

TSA could reduce the expression of pro-inflammatory cytokines induced by LPS in hDPCs, and suppress the phosphorylation of IKKα/β and IκB-α, suggesting that TSA may inhibit the inflammation of dental pulp by de-activation of NF-κB signal pathway.

表1 实时荧光定量PCR引物序列
图1 CCK8法检测不同浓度TSA对人牙髓细胞增殖活性的影响
图2 实时荧光定量PCR检测IL-6、IL-8 mRNA表达水平
图3 双抗体夹心ELISA法检测细胞上清IL-6、IL-8蛋白含量
图4 Western blot检测NF-κB信号通路IKKα/β、p65和IκB-α蛋白磷酸化程度变化
[1]
Vianna ME, Horz HP, Conrads G,et al. Effect of root canal procedures on endotoxins and endodontic pathogens[J]. Oral Microbiol Immunol,2007,22(6):411-418.
[2]
He W, Qu T, Yu Q,et al. LPS induces IL-8 expression through TLR4,MyD88,NF-kappaB and MAPK pathways in human dental pulp stem cells[J]. Int Endod J,2013,46(2):128-136.
[3]
Chang J, Zhang C, Tani-Ishii N,et al. NF-kappaB activation in human dental pulp stem cells by TNF and LPS[J]. J Dent Res,2005,84(11):994-998.
[4]
Fleiss B, Nilsson MK, Blomgren K,et al. Neuroprotection by the histone deacetylase inhibitor trichostatin A in a model of lipopolysaccharide-sensitised neonatal hypoxic-ischaemic brain injury[J]. J Neuroinflammation,2012(9):70.
[5]
Elaut G, Rogiers V, Vanhaecke T. The pharmaceutical potential of histone deacetylase inhibitors[J]. Curr Pharm Des,2007,13(25):2584-2620.
[6]
Marks PA. Histone deacetylase inhibitors:a chemical genetics approach to understanding cellular functions[J]. Biochim Biophys Acta,2010,1799(10-12):717-725.
[7]
Thangavel J, Samanta S, Rajasingh S,et al. Epigenetic modifiers reduce inflammation and modulate macrophage phenotype during endotoxemia-induced acute lung injury[J]. J Cell Sci,2015,128(16):3094-3105.
[8]
Cho JS, Kang JH, Han IH,et al. Antiallergic effects of trichostatin a in a murine model of allergic rhinitis[J]. Clin Exp Otorhinolaryngol,2015,8(3):243-249.
[9]
Tsaprouni LG, Ito K, Adcock IM,et al. Suppression of lipopolysaccharide- and tumour necrosis factor-alpha-induced interleukin (IL)-8 expression by glucocorticoids involves changes in IL-8 promoter acetylation[J]. Clin Exp Immunol,2007,150(1):151-157.
[10]
Duncan HF, Smith AJ, Fleming GJ,et al. Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells[J]. J Endod,2012,38(3):339-345.
[11]
Duncan HF, Smith AJ, Fleming GJ,et al. Histone deacetylase inhibitors epigenetically promote reparative events in primary dental pulp cells[J]. Exp Cell Res,2013,319(10):1534-1543.
[12]
Khan AU, Krishnamurthy S. Histone modifications as key regulators of transcription[J]. Front Biosci,2005(10):866-872.
[13]
Qiao Y, Giannopoulou EG, Chan CH,et al. Synergistic activation of inflammatory cytokine genes by interferon-γ-induced chromatin remodeling and toll-like receptor signaling[J]. Immunity,2013,39(3):454-469.
[14]
Kim ES, Lee JK. Histone deacetylase inhibitors decrease the antigen presenting activity of murine bone marrow derived dendritic cells[J]. Cell Immunol,2010,262(1):52-57.
[15]
Doherty R, O′Farrelly C, Meade KG. Epigenetic regulation of the innate immune response to LPS in bovine peripheral blood mononuclear cells(PBMC)[J]. Vet Immunol Immunopathol,2013,154(3-4):102-110.
[16]
Ghizzoni M, Haisma HJ, Maarsingh H,et al. Histone acetyltransferases are crucial regulators in NF-κB mediated inflammation[J]. Drug Discov Today,2011,16(11-12):504-511.
[17]
Wollebo HS, Bellizzi A, Cossari DH,et al. Epigenetic regulation of polyomavirus JC involves acetylation of specific lysine residues in NF-κB p65[J]. J Neurovirol,2015,21(6):679-687.
[18]
Ziesché E, Kettner-Buhrow D, Weber A,et al. The coactivator role of histone deacetylase 3 in IL-1-signaling involves deacetylation of p65 NF-κB[J]. Nucleic Acids Res,2013,41(1):90-109.
[19]
Sato T, Kotake D, Hiratsuka M,et al. Enhancement of inflammatory protein expression and nuclear factor κB(NF-κB)activity by trichostatin A(TSA)in OP9 preadipocytes[J]. PLoS One,2013,8(3):e59702.
[20]
He X, Wei Z, Zhou E,et al. Baicalein attenuates inflammatory responses by suppressing TLR4 mediated NF-κB and MAPK signaling pathways in LPS-induced mastitis in mice[J]. Int Immunopharmacol,2015,28(1):470-476.
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