l‐arginine‐dependent nitric oxide production of a murine macrophage‐like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg‐LPS). RAW264.7 cells were incubated with i) various concentrations of Pg‐LPS or Salmonella typhosa LPS (St‐LPS), ii) Pg‐LPS with or without l ‐arginine and/or N^G‐monomethyl‐l ‐arginine (NMMA), an arginine analog or iii) Pg‐LPS and interferon‐γ (IFN‐γ) with or without anti‐IFN‐γ antibodies or interleukin‐10 (IL‐10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg‐LPS, but was observed after stimulation with St‐LPS. Exogenous l ‐arginine restored the ability of Pg‐LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg‐LPS with exogenous l ‐arginine was abolished by NMMA. IFN‐γ induced independent NO production by Pg‐LPS‐stimulated macrophages and this stimulatory effect of IFN‐γ could be completely suppressed by anti‐IFN‐γ antibodies and IL‐10. These results suggest that Pg‐LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an l ‐arginine‐dependent mechanism which is itself independent of the action of IFN‐γ.     

Nitric oxide production by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could stimulate a murine macrophage cell line (RAW264.7 cells) to produce nitric oxide (NO). The cells were treated with LPS-A. actinomycetemcomitans or Escherichia coli LPS (LPS-Ec) for 24 h. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B and cytokines (IFN-gamma, TNF-alpha, IL-4 and IL-12) on the production of NO were also determined. The role of protein tyrosine kinase, protein kinase C and microtubulin organization on NO production were assessed by incubating RAW264.7 cells with genistein, bisindolylmaleide and colchicine prior to LPS-A. actinomycetemcomitans stimulation, respectively. NO levels from the culture supernatants were determined by the Griess reaction. The results showed that LPS-A. actinomycetemcomitans stimulated NO production by RAW264.7 cells in a dose-dependent manner, but was slightly less potent than LPS-Ec. NMMA and polymyxin B blocked the production of NO. IFN-gamma and IL-12 potentiated but IL-4 depressed NO production by LPS-A. actinomycetemcomitans-stimulated RAW264.7 cells. TNF-alpha had no effects on NO production. Genistein and bisindolylmalemaide, but not colchicine, reduced the production of NO in a dose-dependent mechanism. The results of the present study suggest that A. actinomycetemcomitans LPS, via the activation of protein tyrosine kinase and protein kinase C and the regulatory control of cytokines, stimulates NO production by murine macrophages.     

The effect of parachlorophenol and camphorated parachlorophenol on nitric oxide production by a murine macrophage cell line,RAW264.7

The aim of this study was to determine the effect of parachlorophenol (PCP) and camphorated parachlorophenol (CMCP) on nitric oxide (NO) production by a murine macrophage cell line, RAW264.7. The cells were incubated on plastic disks with either PCP or CMCP. Plastic adherent and nonadherent cells were subsequently stimulated with recombinant mouse IFN-gamma or bacterial lipopolysaccharide (LPS). Nitric oxide (NO) levels detected from the culture supernatants were determined by the Griess reaction. The results showed that PCP and CMCP diluted at 10(-1) but not at 10(-3) suppressed NO production by both plastic adherent and nonadherent cells, suggesting that both phenolic compounds may suppress NO production by murine macrophages in a dose-dependent manner.     

脂多糖刺激单核巨噬细胞培养上清液对成骨细胞成骨特性的影响

目的 探讨建立牙周炎症的体外实验模型的可行性,为后续进行牙周炎症状态下应力对牙槽骨改建的研究奠定基础.方法 以牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)脂多糖(lipopolysaccharide,LPS)Pg-LPS刺激小鼠单核巨噬细胞(RAW264.7)的培养上清液作用于体外培养的小...     

Role of nitric oxide in hydroxyapatite-induced phagocytosis by murine macrophage cell line (RAW264.7)

The aim of this study was to determine the role of nitric oxide (NO) in hydroxyapatite (HA)-induced phagocytosis by a murine macrophage cell line (RAW264.7). The cells were incubated with HA particles at various incubation time and phagocytosis was assessed using phagocytic index (PI). NO production from the culture supernatants was determined by the Griess reagent. The inducible nitric oxide synthase (iNOS) expression was determined by Western blot. The particles were also incubated with cells pretreated with various concentrations of L-N(6)-(1-iminoethyl) lysine hydrochloride (L-NIL) or L-arginine. Latex beads were used as a control. Our results showed that macrophage phagocytosis induced by HA was higher than that induced by the beads. However, NO production by HA-stimulated cells was lower than that by bead-stimulated cells. iNOS expression in both bead- and HA-stimulated cells was observed expressed at 7, 15, 30, and 60 min. l-Arginine enhanced but l-NIL inhibited both phagocytosis and NO production by HA-stimulated cells. The results of the present study suggest that nitric oxide may play a crucial role in HA-induced phagocytosis by RAW264.7 cells.     

他米巴罗汀对牙龈卟啉单胞菌抗原刺激RAW264.7细胞的影响

目的 探讨他米巴罗汀（Tamibarotene,Am80）对经牙龈卟啉单胞菌（Porphyromonas gingivalis,P.gingivalis）抗原刺激后的小鼠单核巨噬细胞RAW264.7的影响。方法 采用P.gingivalis抗原刺激RAW264.7细胞,分别用不同浓度的Am80进行干预;相差显微镜下观察各组细胞生长情况;MTS法检测不同浓度Am80对抗原刺激RAW264.7细胞的增殖情况;酶联免疫吸附法（ELISA）检测细胞培养上清中抗炎因子IL-10的含量。结果 10 nmol/L Am80能够显著抑制P.gingivalis抗原刺激RAW264.7细胞的增殖分化（P<0.001）;Am80（10 nmol/L）致抗原刺激的细胞IL-10的分泌量显著增加（P<0.05）;倒置相差显微镜下观察显示Am80抑制抗原刺激细胞的增殖分化。结论 Am80（10 nmol/L）时可显著促进P.gingivalis抗原诱导小鼠巨噬细胞分泌抗炎因子IL-10,并可能由此抑制P.gingivalis抗原诱导的炎症反应。     

牙龈卟啉单胞菌脂多糖(Pg-LPS)对破骨细胞EphA2基因表达的调控

目的:研究小鼠单核巨噬细胞系RAW264.7在破骨分化过程中,Pg-LPS对破骨细胞EphA2表达的影响。方法:用终浓度10 mg/L的Pg-LPS 刺激RAW264.7 细胞后,分别在1、3、5 d,应用RT-PCR检测破骨细胞中EphA2基因和破骨细胞相关基因(破骨细胞内基质金属蛋白酶( MMP9)、ACP5、c-fos、组织蛋白酶K(CtsK)、NFATc1)的表达,并且通过酒石酸抗酸性磷酸酶(TRAP)染色观察实验组和对照组破骨细胞的分化成熟情况。结果:RT-PCR检测10 mg/L的Pg-LPS在第3天和5天,实验组比对照组EphA2基因表达分别增高2.4倍和1.2倍,两组之间存在显著差异(P<0.01);同时也能够促进破骨相关基因c-fos、NFATc1、CtsK、ACP5、MMP9的表达,实验组与对照组相比差异有显著性;TRAP染色结果显示:实验组比对照组的TRAP阳性多核细胞数目明显增多。结论:10 mg/L的Pg-LPS对小鼠单核巨噬细胞系RAW264.7,在破骨分化的中期和晚期均能够促进EphA2基因的表达,但是在破骨分化早期对EphA2基因的表达无明显作用。     

Zeste基因增强子人类同源物2抑制剂GSK343调节巨噬细胞分化的作用

目的 研究Zeste基因增强子人类同源物2（EZH2）抑制剂GSK343调节巨噬细胞亚群的分化,探讨EZH2在牙周炎中潜在的治疗作用。方法 将巨噬细胞RAW264.7分为4组：空白组（A组）、对照组（B组）、内毒素（LPS）刺激组（C组）、LPS+GSK343组（D组）。细胞经培养及相应处理后,利用免疫印迹和酶联免疫吸附试验检测其表型生物学标志变化,包括肿瘤坏死因子-α（TNF-α）、诱导型一氧化氮合酶（iNOS）、白细胞介素-10（IL-10）和精氨酸酶-1（Arg-1）。利用大肠杆菌吞噬试验检测巨噬细胞RAW264.7在不同条件下对大肠杆菌的吞噬作用。结果 LPS可以诱导RAW264.7产生M1表型生物标志（TNF-α和iNOS表达增加）,在加入EZH2抑制GSK343后,IL-10和Arg-1表达升高,提示EZH2抑制剂GSK343可以诱导RAW264.7细胞由M1型向M2型转化;RAW264.7细胞具有吞噬大肠杆菌的作用,加入LPS的条件下吞噬大肠杆菌作用加强,而EZH2抑制剂GSK343可以调节RAW264.7细胞对大肠杆菌的吞噬作用。结论 EZH2抑制剂GSK343可以调节巨噬细胞的分化,在牙周炎的治疗中可能具有潜在作用。     

Kaempferol Inhibits P. intermedia Lipopolysaccharide‐Induced Production of Nitric Oxide Through Translational Regulation in Murine Macrophages: Critical Role of Heme Oxygenase‐1‐Mediated ROS Reduction

Background: Nitric oxide (NO) could be a potential target for the development of new therapeutic approaches to the treatment of periodontal disease because this molecule plays a significant role in the tissue destruction observed in periodontitis. In this study, the authors investigate the effect of kaempferol on the production of NO by murine macrophage‐like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and try to determine the underlying mechanisms of action. Methods: NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Real‐time polymerase chain reaction was performed to quantify inducible NO synthase (iNOS) and heme oxygenase‐1 (HO‐1) mRNA expression. iNOS and HO‐1 protein expression and phosphorylation of c‐Jun N‐terminal kinase and p38 were characterized via immunoblot analysis. Reactive oxygen species (ROS) production was measured using the redox‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate. Results: Kaempferol significantly inhibited NO production and expression of iNOS protein in P. intermedia LPS‐stimulated RAW246.7 cells without affecting iNOS mRNA expression. Kaempferol upregulated HO‐1 expression in LPS‐activated cells. Inhibition of HO‐1 activity by tin protoporphyrin IX (SnPP) abolished the suppressive effect of kaempferol on NO production. In addition, kaempferol significantly attenuated P. intermedia LPS‐induced increase of intracellular ROS, and SnPP blocked this reduction. Treatment with antioxidants downregulated the production of LPS‐induced NO. Conclusions: Kaempferol inhibits NO production and iNOS protein expression in P. intermedia LPS‐stimulated RAW264.7 cells at the translational level via HO‐1‐mediated ROS reduction and could be an efficient modulator of host response in the treatment of periodontal disease.     

DHA对牙龈卟啉单胞菌脂多糖诱导巨噬细胞炎症的抑制作用

目的 探讨二十二碳六烯酸(DHA)对牙龈卟啉单胞菌(P.g)脂多糖(LPS)诱导巨噬细胞炎症的抑制作用.方法 用CCK-8方法检测DHA对P.g-LPS诱导的小鼠单核/巨噬细胞系RAW264.7细胞的毒性.将P.g-LPS与RAW264.7细胞共培养24 h后,更换含有DHA的培养基培养24 h,实时荧光逆转录定量PC...     

牙龈卟啉单胞菌脂多糖对髓系细胞触发受体的激活作用

目的:研究牙龈卟啉单胞菌(Pg)脂多糖(LPS)对巨噬细胞中髓系细胞触发受体-1(TREM-1)和-2的激活作用。方法:分离培养小鼠腹腔巨噬细胞,并以1 mg/L的Pg-LPS刺激。采用实时荧光定量PCR技术检测TLR-2、TLR-4、TREM-1和TREM-2转录水平的变化,流式细胞术检测蛋白水平的变化,酶联免疫反应检测培养液中肿瘤坏死因子-α(TNF-α)和巨噬细胞炎症蛋白-lα(MIP-lα)水平的变化。 结果:Pg-LPS刺激巨噬细胞2 h后,巨噬细胞内TLR-2和TREM-1的转录水平均表达上调;流式细胞检测显示24 h后,细胞内TREM-1和TLR-2水平显著上调;TLR-4和TREM-2在实验中未见显著变化;Pg-LPS刺激巨噬细胞24 h后,TNF-α和MIP-1α水平显著上升组;TREM-1的抑制物LP-17多肽显著抑制了Pg-LPS的炎症刺激作用。结论:TLR-2和TREM-1参与了巨噬细胞应答Pg-LPS的先天免疫过程。     

脂多糖刺激单核细胞上清对成骨细胞Runx2/Cbfα1表达影响

目的　观察牙龈卟啉菌脂多糖(Pg-LPS)刺激的小鼠单核巨噬细胞RAW264.7培养上清对成骨细胞Runx2/Cbfα1表达的影响。 方法　收集经100 ng/ml Pg-LPS刺激的单核巨噬细胞RAW264.7培养24 h后上清,以20%的稀释浓度的分别作用于MC3T3-E1细胞1、6、12、24、48、72 h后,采用半定量RT-PCR与Western-Blot检测细胞Runx2/Cbfα1在基因与蛋白水平上的差异性表达。 结果　成骨细胞MC3T3-E1在20%稀释浓度的培养上清刺激后,半定量RT-PCR法发现Runx2/Cbfα1基因表达显著受抑制,且呈时间依赖性,72 h降到最低; Western-Blot检测显示细胞Runx2/Cbfαa1蛋白表达6 h后开始下降,72 h降至最低。 结论　Pg-LPS刺激的小鼠单核巨噬细胞RAW264.7培养上清能抑制小鼠成骨细胞Runx2/Cbfα1的表达且与时间呈一定相关。     

Effect of iRoot SP and mineral trioxide aggregate (MTA) on the viability and polarization of macrophages

ObjectiveThis study was performed to investigate the effect of iRoot SP and mineral trioxide aggregate (MTA) on the viability and polarization of macrophages.MethodsThe effect of iRoot SP and MTA on the viability of RAW 264.7 macrophages was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 1 and 2 days of culture. The gene expression levels of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), interleukin 12p40 (IL-12p40) were measured by quantitative real time polymerase chain reaction (qRT-PCR) after stimulation of the RAW 264.7 macrophages with iRoot SP and MTA. The expression levels of CD11c and CD206 in RAW 264.7 macrophages were examined by immunofluorescence and flow cytometry after stimulation with iRoot SP and MTA. The data were analyzed by one-way analysis of variance and the Tukey test.ResultsBoth iRoot SP and MTA were non-toxic to the RAW 264.7 macrophages. The use of iRoot SP and MTA increased the expression of IL-1β, TNF-α, IL-10, IL-12p40 on the first day of culture and could promote macrophage M1 and M2 polarization.ConclusionsMTA and iRoot SP have good biocompatibility with macrophages, and they induced both M1 and M2 polarization of the RAW 264.7 macrophages.     

脂多糖刺激牙龈成纤维细胞产生白细胞介素11、6的研究

目的观察牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）、伴放线放线杆菌（Actinobacillus actinomycetemcomitans，Aa）、大肠杆菌（Escherichia coli，Ec）的脂多糖（lipopolysacchrides，LPS）刺激人牙龈成纤维细胞（human gingival fibroblasts，HGF）合成白细胞介素（interleukin，IL）-11和IL-6的能力，并了解内源性前列腺素是否介导IL-11和IL-6的产生。方法将3种浓度的LPS（0．1、1、10mg／L）分别作用于体外培养的HGF 24h，另外在10mg／L的LPS中加入10^-6 moL／L的吲哚美辛后再分别刺激HGF 24h，ELISA法检测HGF上清液中IL-11和IL-6含量的变化。结果Aa-，Ec-LPS（10mg／L）和Pg-LPS（1、10mg／L）刺激HGF后IL-11水平显著提高；Pg-，Ec-LPS（0．1、1、10mg／L）和Aa-LPS（1、10ms／L）刺激HGF产生IL-6的水平明显增高；这些作用均受到吲哚美辛的抑制作用。结论LPS使HGF产生IL-11和IL-6的水平明显增加，并受到内源性前列腺素的正向调控。     

低强度脉冲式超声波在脂多糖诱导的RAW264.7巨噬细胞分化中的抗炎和抗氧化作用

目的研究低强度脉冲式超声波（LIPUS）对RAW264.7巨噬细胞极化的影响及相关分子机制。 方法100 ng/mL脂多糖（LPS）和10 ng/mL白细胞介素（IL）-4诱导巨噬细胞RAW264.7分别向M1和M2型极化，45 mW/cm²强度LIPUS对巨噬细胞处理25 min。采用流式细胞术检测巨噬细胞氧化应激活性氧（ROS）水平、M1分化标志物CD80和CD11b，以及M2分化标志物CD163的表达水平。采用反转录聚合酶链反应（RT-PCR）技术检测CD80、CD11b、核转录因子κB（NF-κB）p65、肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-1β和IL-6的mRNA水平。采用Western blot技术检测细胞p65、p-p65、TNF-α、IL-1β和IL-6表达水平。采用流式细胞术检测细胞培养上清TNF和IL-6表达水平。 结果LIPUS可明显减少LPS诱导的RAW264.7细胞ROS水平。LPS诱导后，RAW264.7细胞M1分化标志物CD80和CD11b表达和转录水平均上调；LIPUS可抑制LPS对RAW264.7细胞向M1的诱导，差异具有统计学意义。并且，LIPUS可促进IL-4诱导的巨噬细胞M2分化标志物CD163表达。LPS诱导的RAW264.7细胞炎症因子NF-κB p65、TNF-α、IL-1β和IL-6 mRNA水平上调，LIPUS可下调这些炎症因子的mRNA水平，差异均具有统计学意义。LIPUS抑制了LPS对RAW264.7细胞因子蛋白p65和p-p65、IL-1β、TNF-α和IL-6蛋白表达上调的作用。胰酶可以通过激活ROS-NF-κB通路，回复LIPUS促进巨噬细胞M1分化的作用。 结论LIPUS可通过ROS-NF-κB抑制RAW264.7向巨噬细胞M1型分化，促进RAW264.7向巨噬细胞M2型分化。氧化应激和炎症因子表达水平被抑制。LIPUS可能在牙周疾病中起到抑制氧化和炎症的作用，从而发挥对牙周疾病的治疗功能。     

重组人β防御素3对牙龈卟啉单胞菌脂多糖致炎作用的影响

目的:以牙龈卟啉单胞菌脂多糖(P.g-LPS)诱发载脂蛋白E基因敲除(ApoE-/-)小鼠和RAW264.7小鼠单核巨噬细胞的急性炎症反应,并应用重组人β防御素3(rhBD3),观察其对炎症的干预效果。方法:20周龄雄性ApoE-/-小鼠随机均分为PBS对照组、P.g-LPS组和P.g-LPS+rhBD3组,分别经腹腔注射给药2h后,检测血清中不同炎症指标(MCP-1、TNF-α、IL-6、IL-1β和NO)的水平。同时,以rhBD3干预P.g-LPS对RAW264.7细胞的致炎作用,分别检测培养上清和细胞中炎症指标的水平及其mRNA的相对表达量。结果:经P.g-LPS刺激后,ApoE-/-小鼠血清MCP-1、TNF-α、IL-6和NO的水平较对照组显著上调;而同时应用rhBD3能明显降低MCP-1、TNF-α和NO表达量。P.g-LPS能显著上调RAW264.7细胞培养上清中TNF-α和NO的水平以及细胞中TNF-α和IL-6的mRNA相对表达量,rhBD3对此具有抑制作用。结论:rhBD3对P.g-LPS在体内、外诱导的急性炎症具有一定的抑制效应,可能在牙周炎与高脂血症的相互作用中发挥免疫调节作用。     

Effects of cytokines on Porphyromonas gingivalis-induced opsonophagocytosis of a murine macrophage cell line

A murine macrophage cell line was incubated with opsonized Porphyromonas gingivalis and various concentrations of rIFN-gamma, rIL-4 and rIL-10. The number of phagocyted cells were microscopically assessed. The results showed that IFN-gamma upregulated macrophage phagocytosis to this periodontopathogen, and the upregulatory effects was abolished by anti-IFN-gamma antibodies. Neither IL-4 nor IL-10 had any effects on opsonophagocytosis by this cell line. These results suggest that up-regulatory functions of IFN-gamma on phagocytic activities of macrophages may play a crucial role in the induction of immune response to P. gingivalis.     

牙龈卟啉单胞菌脂多糖对高脂血症兔腹腔巨噬细胞炎症相关因子基因表达的影响

目的 比较牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis on lipopolysaccharide,Pg-LPS)对健康和高脂血症兔腹腔巨噬细胞炎症相关因子基因表达的影响.方法 将健康新西兰兔12只随机分为2组,分别给予普通饲养喂养(健康兔)和高脂饲料喂养,6周后建立高脂血症模型(高脂兔).采用腹腔灌洗法分别获得健康和高脂兔巨噬细胞,将健康和高脂兔巨噬细胞均随机分为对照组、Pg-LPS组(1μg/mL)及阳性对照组大肠杆菌-脂多糖(Escherichia,E.coli-LPS)组(1μg/mL),24 h后采用实时PCR法分别检测各组巨噬细胞中C-反应蛋白(C-reaction protein,CRP)、白介素1β(interleukin-1β,IL-1β)、白介素6(interleukin-6,IL-6)、白介素8(interleukin-8,IL-8)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)mRNA的表达水平.结果 高脂兔对照组巨噬细胞的CRP、IL-1β、IL-6、IL-8、TNF-α水平高于健康兔对照组巨噬细胞;相较于对照组,高脂兔和健康兔Pg-LPS组巨噬细胞的CRP、IL-1β、IL-6、IL-8、TNF-α表达均升高,且高脂兔Pg-LPS组巨噬细胞以上因子的表达水平高于健康兔Pg-LPS巨噬细胞(P<0.05).结论 Pg-LPS可增强高脂血症兔巨噬细胞炎症相关因子mRNA的表达.     

糖皮质激素对口腔扁平苔藓辅助性T细胞平衡的影响

目的　明确口腔扁平苔藓 (OLP)患者的Th1 /Th2免疫应答模式,探讨糖皮质激素对OLP患者辅助性T细胞平衡的影响。方法　密度梯度离心法分离OLP患者和对照组外周血单个核细胞,分别用植物血凝素(PHA)和地塞米松刺激OLP患者外周血单个核细胞,采用酶联免疫吸附法,检测培养上清液中干扰素γ(IFN γ)和白细胞介素 4(IL 4)的含量;应用半定量逆转录聚合酶链反应技术,检测培养细胞中IFN γ和IL 4mRNA的表达水平。结果　外周血单个核细胞经PHA诱导培养,OLP患者组IFN γ的水平低于健康对照组 (P<0 05),IL 4的水平高于对照组,但差异无统计学意义(P>0 05),IFN γ/IL 4的比值低于对照组 (P<0 05 )。地塞米松可抑制IFN γ和IL 4的水平(P<0 01),且对IFN γ的抑制作用较IL 4更为显著。结论　OLP患者存在Th1 /Th2的平衡失调,为Th2占优势的免疫应答,糖皮质激素对OLP的Th1 /Th2细胞因子均有抑制作用。