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
作者：陈筑, 宿凌恺
Author：Zhu Chen, Lingkai Su
收稿日期：2016-05-12 年卷（期）页码：2017,35(2):139-139-144
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：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,
Key words：paeonol,Porphyromonas gingivalis,bone marrow-derived macrophage,osteoclast,inflammatory,differentiation,
基金项目：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]
中文摘要
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。
英文摘要
ObjectiveThis 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 withPorphyromonas gingivalis(P. gingivalis). This work also aims to investigate the underlying mechanisms of these abilities.MethodsBMM culture was treated with different paeonol concentrations at for 1 h and then stimulated withP. gingivalisfor 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 withP. 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.ResultsPaeonol was nontoxic to BMM within a range of 10-50 μmol·L-1. Flow cytometry showed that paeonol inhibited PD-L1 expression inP. 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 byP. gingivalis-induced BMM (P<0 .01). trap staining revealed that paenol treatment inhibited the differentiation ofP. gingivalis-induced BMM into osteoclasts. Western blot results suggested that paeonol decreased the expression of TRAP and RANK in BMM.ConclusionPaeonol dose-dependently inhibited the excretion of the inflammatory factors TNF-α, IL-1β, and IL-6 byP. gingivalis-induced BMM in a dose-dependent manner. Moreover, paenol treatment prevented the differentiation ofP. gingivalis-induced BMM differentiation into osteoclasts.
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