丹皮酚对牙龈卟啉单胞菌诱导骨髓来源巨噬细胞功能的影响

doi:10.7518/hxkq.2017.02.006

华西口腔医学杂志 ›› 2017, Vol. 35 ›› Issue (2): 139-144.doi: 10.7518/hxkq.2017.02.006

丹皮酚对牙龈卟啉单胞菌诱导骨髓来源巨噬细胞功能的影响

陈筑¹(), 宿凌恺²()

1.贵阳市口腔医院牙体牙髓病科,遵义医学院附属贵阳口腔医院,贵阳 550002
2.浙江大学医学院附属口腔医院牙体牙髓病科,杭州 310000

收稿日期:2016-05-12 修回日期:2017-01-18 出版日期:2017-04-01 发布日期:2017-04-01
作者简介:
陈筑,副主任医师,博士,E-mail：17784810646@163.com
基金资助:
贵阳市高层次创新型青年科技人才培养计划[筑教发（2-012HK）209-50号];西部地区人才培养出国特别项目[（2013）5044号]

Effects of paeonol on the function of bone marrow-derived macrophage from Porphyromonas gingivalis-induced mice

Zhu Chen¹(), Lingkai Su²()

1. Dept. of Conservative Dentistry and Endodontics, Guiyang Hospital of Stomatology, Affiliated Guiyang Hospital of Stomatology, Zunyi Medical University, Guiyang 550002, China
2. Dept. of Conservative Dentistry and Endodontics, Stomatology Hospital Affiliated to Zhejiang University of Medicine, Hangzhou 310000, China

Received:2016-05-12 Revised:2017-01-18 Online:2017-04-01 Published:2017-04-01
Supported by:
Innovative Talents Program of Guiyang City [(2012HK)209-50];;Special Project of Talent Cultivation in the Western Region [(2013)5044].

摘要/Abstract

摘要：

目的观察丹皮酚对牙龈卟啉单胞菌作用下对体外培养的小鼠骨髓来源巨噬细胞（BMM）炎性因子分泌及向破骨细胞分化能力的影响,并探讨其作用机制。方法体外分离获得小鼠生长良好的BMM,加入不同浓度丹皮酚溶液处理1 h,再加入牙龈卟啉单胞菌进行24 h诱导刺激。采用流式细胞术检测炎症相关蛋白程序性死亡分子配体1（PD-L1）的表达量,采用酶联免疫吸附试验（ELISA）检测细胞上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）及白细胞介素-6（IL-6）水平;在核因子κB受体活化因子配体（RANKL）和巨噬细胞集落刺激因子（M-CSF）刺激后的BMM中加入不同浓度的丹皮酚溶液处理1 h后,加入牙龈卟啉单胞菌刺激,采用抗酒石酸性磷酸酶（TRAP）染色观察破骨细胞形成情况,Western blot检测破骨细胞形成相关蛋白TRAP和核因子κB受体活化因子（RANK）的表达。结果丹皮酚在10~50 μmol·L^-1的范围内,对BMM无毒性作用。流式细胞术结果提示丹皮酚可以抑制牙龈卟啉单胞菌诱导PD-L1的表达,并存在剂量依赖效应。ELISA实验证明丹皮酚能以剂量依赖性方式抑制BMM释放TNF-α、IL-1β及IL-6（P<0.01）。牙龈卟啉单胞菌可以明显诱导BMM分化形成破骨细胞;TRAP染色显示丹皮酚各浓度组能抑制BMM向破骨细胞分化;Western blot结果显示丹皮酚抑制TRAP和RANK表达且存在剂量依赖效应。结论丹皮酚可以剂量依赖方式抑制牙龈卟啉单胞菌诱导的BMM炎性因子TNF-α、IL-1β及IL-6的分泌及其向破骨细胞分化的能力。

关键词: 丹皮酚, 牙龈卟啉单胞菌, 骨髓来源巨噬细胞, 破骨细胞, 炎症, 分化

Abstract:

Objective This work aims to examine the effects of paeonol treatment on the ability of bone marrow-derived macrophage (BMM) to excrete inflammatory factors and to differentiate into osteoclasts upon induction with Porphyromonas gingivalis (P. gingivalis). This work also aims to investigate the underlying mechanisms of these abilities. Methods BMM culture was treated with different paeonol concentrations at for 1 h and then stimulated with P. gingivalis for 24 h before programmed death-ligand 1 (PD-L1) was quantified with flow cytometry. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were detected by enzyme-linked immunosor-bent assay (ELISA). The BMM culture was treated with the receptor activator for nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), and then with paeonol for 1 h prior to induction with P. gingivalis. Then, osteoclast formation was assessed using tartrate resis-tant acid phosphatase (TRAP) staining. The osteoclast-related proteins TRAP and receptor activator of nuclear factor-κB (RANK) were quantified by Western blotting. Results Paeonol was nontoxic to BMM within a range of 10-50 μmol·L^-1. Flow cytometry showed that paeonol inhibited PD-L1 expression in P. gingivalis-induced BMM in a dose-dependent manner. ELISA indicated that paeonol dose-dependently inhibited the excretion of TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM (P<0.01). TRAP staining revealed that paenol treatment inhibited the differentiation of P. gingivalis-induced BMM into osteoclasts. Western blot results suggested that paeonol decreased the expression of TRAP and RANK in BMM. Conclusion Paeonol dose-dependently inhibited the excretion of the inflammatory factors TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM in a dose-dependent manner. Moreover, paenol treatment prevented the differentiation of P. gingivalis-induced BMM differentiation into osteoclasts.

Key words: paeonol, Porphyromonas gingivalis, bone marrow-derived macrophage, osteoclast, inflammatory, differentiation

中图分类号:

R781.4

陈筑, 宿凌恺. 丹皮酚对牙龈卟啉单胞菌诱导骨髓来源巨噬细胞功能的影响[J]. 华西口腔医学杂志, 2017, 35(2): 139-144.

Zhu Chen, Lingkai Su. Effects of paeonol on the function of bone marrow-derived macrophage from Porphyromonas gingivalis-induced mice[J]. West China Journal of Stomatology, 2017, 35(2): 139-144.

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参考文献 15

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组别	IL-1β	IL-6	TNF-α
对照组	41.86±10.010	556.7±27.850	27.03±13.050
10 μmol·L^-1丹皮酚对照组	36.51±9.590	526.0±2.960	16.88±6.095
30 μmol·L^-1丹皮酚对照组	54.41±8.326	481.2±4.462	6.55±0.608
50 μmol·L^-1丹皮酚对照组	189.80±56.230	447.2±3.256	14.93±1.994
阳性刺激组	906.20±22.930	7 172.0±25.000	1 319.00±27.370
牙龈卟啉单胞菌+10 μmol·L^-1丹皮酚组	34.48±26.310^***	4 977.0±72.160^**	621.10±85.850^***
牙龈卟啉单胞菌+30 μmol·L^-1丹皮酚组	48.09±12.380^***	2 637.0±138.200^***	267.10±10.670^***
牙龈卟啉单胞菌+50 μmol·L^-1丹皮酚组	53.61±3.067^***	872.8±26.610^***	277.90±53.680^***

丹皮酚对牙龈卟啉单胞菌诱导骨髓来源巨噬细胞功能的影响

Effects of paeonol on the function of bone marrow-derived macrophage from Porphyromonas gingivalis-induced mice

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