Nitric oxide production by a human osteoblast cell line stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide

Background/aim:  Human osteoblasts induced by inflammatory stimuli express an inducible nitric oxide synthase (iNOS). The aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans lipopolysaccharide stimulates the production of nitric oxide (NO) by a human osteoblast-like cell line (HOS cells).
Methods:  Cells were stimulated directly with A. actinomycetemcomitans lipopolysaccharide or pretreated with the following l -NIL (an iNOS inhibitor), anti-CD14, Toll-like receptor 2 (TLR2), or TLR4 antibody before stimulation with A. actinomycetemcomitans lipopolysaccharide. The role of the cyclic nucleotides was assessed by pretreating the cells with the following; ODQ (a guanylyl cyclase inhibitor); SQ22536 (an adenylyl cyclase inhibitor); db-cAMP (a cyclic adenosine monophosphate analog); br-cGMP (a cyclic guanosine monophosphate analog); forskolin (an adenylyl cyclase activator), IBMX [a non-specific phosphodiesterase (PDE) inhibitor], or KT5720 [a protein kinase A (PKA) inhibitor]. The cells were also preincubated with genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], BPB [a phospholipase A2 (PLA2) inhibitor], and NDGA (a lipoxygenase inhibitor). The iNOS activity and nitrite production in the cell cultures were determined spectrophotometrically.
Results:  The results showed that A. actinomycetemcomitans lipopolysaccharide stimulated both iNOS activity and nitrite production by HOS cells; this was reduced by l -NIL, anti-CD14, or anti-TLR4 antibody, SQ22536, KT5720, genistein, bisindolylmaleimde, BPB, and NDGA, but was enhanced by db-cAMP, IBMX, and forskolin.
Conclusion:  These results therefore suggest that A. actinomycetemcomitans lipopolysaccharide may induce the production of NO by HOS cells via a CD14–TLR4 molecule complex, a cAMP–PKA pathway, as well as by a PTK, PKC, PLA2, and lipoxygenase-dependent mechanism.     

Salivary nitric oxide levels in inflammatory periodontal disease - a case-control and interventional study

To cite this article: Int J Dent Hygiene 10 , 2012; 67–73
DOI: 10.1111/j.1601‐5037.2011.00508.x
Parwani SR, Chitnis PJ, Parwani RN. Salivary nitric oxide levels in inflammatory periodontal disease – A case‐control and interventional study. Abstract: Background: Biochemical markers of inflammatory periodontal disease present in saliva can partially determine the extent of periodontal disease. Furthermore, collection of salivary constituents is a simple and non‐invasive procedure. Nitric oxide (NO) has been linked to etiopathogenesis of inflammatory periodontal disease and is expressed in saliva. This study was conducted with the objective of estimating salivary NO levels in inflammatory periodontal diseases (gingivitis and periodontitis) and comparing these levels with control subjects. A re‐assessment of these levels was also made after providing appropriate treatment with a view to ascertain its diagnostic and prognostic values. Methods: This was a case–control as well as an interventional study including a total of 90 (30 control, 30 gingivitis and 30 periodontitis) subjects. Saliva samples were collected from each subject, and NO levels were assayed by Griess reaction. Results: NO levels were increased significantly in gingivitis and periodontitis subjects as compared with controls. There was a statistically significant decrease in the NO levels in each study group after the healing period (corresponding to the reduced clinical signs of inflammation). Our study also correlated probing pocket depths with salivary NO levels in periodontitis group where we found a positive correlation between the two. Conclusion: Salivary NO levels can be utilized as a good indicator of the inflammatory status of the periodontium, and evaluating its levels in saliva by Griess reaction on a photoelectric colorimeter is a reliable, accurate and faster method to estimate the level of inflammation in periodontal tissues.     

Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthases and inhibiting endothelial nitric oxide synthases

Sun W, Wu J, Lin L, Huang Y, Chen Q, Ji Y. Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthases and inhibiting endothelial nitric oxide synthases. J Periodont Res 2010; 45: 381–388. © 2010 The Authors. Journal compilation © 2010 Blackwell Munksgaard Background and Objective: The purpose of this study was to examine the ability of Porphyromonas gingivalis to invade human umbilical vein endothelial cells (HUVECs) and to study the effects of P. gingivalis ATCC 33277 on the production of nitric oxide (NO) and on the expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in HUVECs. We attempted to throw light on the pathway of damage to endothelial function induced by P. gingivalis ATCC 33277. Material and Methods: P. gingivalis ATCC 33277 was cultured anaerobically, and HUVECs were treated with P. gingivalis ATCC 33277 at multiplicities of infection of 1:10 or 1:100 for 4, 8, 12 and 24 h. HUVECs were observed using an inverted microscope and transmission electron microscopy. NO production was assayed through measuring the accumulation of nitrite in culture supernatants. Expression of both iNOS and eNOS proteins was investigated through western blotting. Results: It was found that P. gingivalis ATCC 33277 can adhere to HUVECs by fimbriae, invade into HUVECs and exist in the cytoplasm and vacuoles. P. gingivalis ATCC 33277 can induce iNOS and inhibit eNOS expression, and stimulate the release of NO without any additional stimulant. Conclusion: Our study provides evidence that P. gingivalis ATCC 33277 can invade HUVECs, and the ability of P. gingivalis ATCC 33277 to promote the production of NO may be important in endothelial dysfunction, suggesting that P. gingivalis ATCC 33277may be one of the pathogens responsible for atherosclerosis.     

Establishment of a cell line with phenotypes of chondrocyte from a human osteogenic sarcoma of the mandible

We have succeeded in transplanting a human osteogenic sarcoma of the mandible into athymic mice. The transplanted tumor showed marked chondrogenesis and mineralization. Recently, a cell line (USAC) with phenotypes of chondrocyte has been established from the transplanted tumor. USAC cells were stellate or spindle-shaped in sparse culture, but polygonal or spherical at sub-confluency to confluency. In long-term culture, the cells were condensed and calcified nodules were formed. Production of types I, II and X collagen were detected by immunohistochemical staining and Western blot analysis. Type I collagen was strongly expressed in the stellate or spindle-shaped cells. Although type II collagen was usually present in all cells during culture, it was strongly stained in polygonal cells at confluency. Type X collagen was seen in large polygonal cells around calcified nodules. Marked [35S]-sulfate uptake and metachromasia were seen at the confluent stage and in the nodule. The cells around the nodules were positive for alkaline phosphatase, and the center of the nodules was stained with alizarin red. The potentiality of cartilage formation was confirmed by in vivo experiments using a diffusion chamber in athymic mice. These observations indicate that USAC cells maintain characteristics of chondrocyte progenitor cells and thus may serve as a useful model to study the sequential events of chondrogenesis and the process of morbid endochondral calcification. This experiment also demonstrated that transplantation of tumor tissue into athymic mice is a convenient strategy for establishment of a cell line.     

诱导型一氧化氮合酶在颌骨肉瘤中的表达及意义

目的：研究诱导型一氧化氮合酶（iNOS）在颁骨肉瘤中的表达情况及其和肿瘤血管形成、临床病理特征之间的关系。方法：应用S-P免疫组织化学法检测iNOS和CDB4在颌骨肉瘤中的表达。结果：颌骨肉瘤中存在iNOS的过度表达;iNOS表达与微血管密度有关;iNOS表达与颌骨肉瘤大小、病理分级、临床分期、初／复发等临床病理特征有关,与肿瘤的转移无关。结论：iNOS有促进颌骨肉瘤中肿瘤血管形成的作用,iNOS是肿瘤治疗的一个重要靶点。     

诱导型一氧化氮合酶基因转染对人舌癌细胞体内致瘤力的影响

目的研究外源性基因诱导型一氧化氮合酶(iNOS)转染人舌癌细胞系Tca8113后,对肿瘤细胞生物学特性以及裸鼠体内致瘤力的影响。方法采用脂质体转染法将真核表达载体pCMV-iNOS/Tca转染入舌癌细胞系Tca8113,得到高表达iNOS的细胞系pCMV-iNOS/Tca,转染空白质粒的细胞系pCMV-Tca,并以Western blot方法鉴定转染效果。四甲基偶氮唑蓝(MTT)比色绘制生长曲线;流式细胞仪检测细胞周期分布;通过裸鼠成瘤实验,比较转染前后体内成瘤能力的差别。结果与未转染组和转染空白载体组比较,pCMV-iNOS/Tca组的细胞生长速度比Tca组快(P>0.05),细胞周期中G2/M和S期比例增加(P<0.05)。体内成瘤能力试验结果显示,pCMV-Tca、pCMV-iNOS/Tca组和Tca组平均瘤重分别为(1.1±0.24)g、(2.5±0.48)g和(1.3±0.32)g,pCMV-iNOS/Tca组的成瘤力高于pCMV-Tca和Tca组(P<0.01)。结论转染iNOS基因后,Tca8113细胞具有更强的体外增殖能力和体内致瘤能力。     

口腔扁平苔癣患者组织中一氧化氮及一氧化氮合酶的检测

目的：了解一氧化氮（NO0及一氧化氮合酶（NOS）在口腔扁平苔藓（OLP）发病中的作用。方法：选择OLP患者（其中糜烂型12例，非糜烂型18例）及健康献血员各30例，分别检测局部病变组织和正常对照组织中NO含量（硝酸还原酶法）和NOS活性（化学比色法），所得数据采用配对t检验进行统计学处理。结果：NO值，OLP组高于正常对照组（P＜0．05），糜烂型虽高于非糜烂型（P＞0．05），但无统计学差异；NOS，OLP组明显高于正常对照组（P＜0．01），糜烂型也明显高于非糜烂型（P＜0．01）。结论：在OLP发生发展的不同阶段，NO和NOS呈动态变化，过多的NO含量及NOS活性增加与OLP发生发展有关。本研究提示，NO生成抑制剂可作为一种新型药物，用于辅助治疗OLP。     

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.     

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). RAW 264.7 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 NG-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma 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-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW 264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.     

Effects of an inducible nitric oxide synthase inhibitor on experimentally induced rat pulpitis

Nitric oxide (NO) is a biological effector molecule involved in a large variety of reactions and, as synthesized by inducible nitric oxide synthase (iNOS), has many important roles in inflammatory conditions. This study aimed to evaluate the anti-inflammatory effects of an iNOS-specific inhibitor, N-(3-(aminomethyl)benzyl)acetamidine (1400W), on experimentally induced rat pulpitis in the upper incisors of 6-wk-old male Wistar rats. 1400W (1 mg kg(-1)), the non-specific NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg kg(-1)), or sterile saline (control) were administered before the application of lipopolysaccharide (LPS). Rats were killed 3, 6, 9, 12, 24, 48, and 72 h after LPS application, and immunocompetent cells were detected immunohistochemically. The numbers of granulocytes infiltrating into the pulp were significantly depressed in the 1400W group compared with the saline and L-NAME groups. The kinetics of the macrophages and Ia(+) cells in the 1400W group were similar to those in the L-NAME group, while the maximum numbers in both groups were significantly reduced compared with those in the saline group. These results suggest that NO may be responsible for the infiltration of immunocompetent cells in the progress of pulpitis, and that 1400W is a promising candidate for controlling pulpal inflammatory reactions.     

唇癌VEGF和NOS的表达与癌细胞增殖关系的研究

目的 :研究唇癌组织血管内皮生长因子 (VEGF)、诱导型一氧化氮合酶 (iNOS)、内皮型一氧化氮合酶(eNOS)的表达与癌细胞增殖的相关性。方法 :应用免疫组化方法检测 42例唇癌手术切除标本VEGF、iNOS、eNOS和增殖细胞核抗原 (PCNA)的分布及表达。结果 :① 2 6例 ( 61.90 % )唇癌组织表达VEGF ,2 8例 ( 66.67% )表达iNOS ,3 1例 ( 73 .81% )表达eNOS ;②VEGF与iNOS的表达具有明显相关性 ,VEGF与eNOS的表达无明显相关性 ;③表达VEGF的唇癌PCNA标记指数 (PLI)明显高于不表达VEGF的唇癌 ;表达iNOS的唇癌PLI明显高于不表达iNOS的唇癌。表达eNOS的唇癌PLI与不表达eNOS的唇癌无显著性差异 (P >0 .0 5 )。结论 :①VEGF与iNOS的表达具有明显相关性 ,说明iNOS在VEGF的生成和发挥作用过程中起重要作用 ,②PLI随着VEGF和iNOS表达的增加而增加 ,说明二者对唇癌细胞增殖具有促进作用。     

Type II nitric oxide synthase (NOS2) expression correlates with lymph node status in oral squamous cell carcinoma

In tumour biology, nitric oxide (NO) has a complex array of concentration-dependent actions, including both inhibitory and promoting effects. It is thought that the levels of NO found in many human cancers lead to enhanced angiogenesis and tumour dissemination. In the current study, we assessed the immunohistochemical expression of the enzyme type II nitric oxide synthase (NOS2) in 41 cases of oral squamous cell carcinoma and correlated the findings with lymph node status. A significant relationship was found between NOS2 expression and lymph node metastasis (P<0.0002). Furthermore, lymph node metastasis correlated with the degree and intensity of staining seen (P<0.001). No correlation was found between the size of the primary tumour, degree of tumour differentiation or smoking status and NOS2 staining. Western blotting confirmed NOS2 protein expression in select cases. As with many other human tumours, NOS2 is not a ubiquitous finding in oral cancer. Its expression may be of value in assessing lymph node status prior to surgery, and it represents a target for possible therapeutic manipulation.     

口腔恶性黑色素瘤中血管内皮生长因子和一氧化氮合酶的表达与癌细胞增殖的关系

目的研究口腔恶性黑色素瘤组织血管内皮生长因子(VEGF)、诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)的表达与癌细胞增殖的相关性。方法应用免疫组化方法检测11例口腔恶性黑色素瘤手术切除标本VEGF、iNOS、eNOS和增殖细胞核抗原(PCNA)的分布及表达。结果11例中7例(63.64%)瘤组织表达VEGF,6例(54.55%)表达iNOS,9例(81.82%)表达eNOS,VEGF与iNOS的表达具有相关性,而与eNOS的表达无相关性;表达VEGF的口腔恶性黑色素瘤PCNA标记指数(PLI)高于不表达VEGF者;表达iNOS的口腔恶性黑色素瘤PLI高于不表达iNOS者。表达与不表达eNOS的口腔恶性黑色素瘤PLI差异无显著性(P>0.05)。结论iNOS、PLI对口腔恶性黑色素瘤细胞增殖具有促进作用。     

一氧化氮合酶在犬下颌骨牵张成骨过程中的表达和意义

目的研究一氧化氮合酶（NOS）在犬下颌骨牵张成骨及骨创伤修复过程的表达和意义。方法28只犬随机分成牵张组和直接延长组各12只及正常对照组4只,用免疫组化法检测牵张第6天、牵张后固定2周和8周NOS表达水平的变化。结果牵张第6天牵张组可见牵开区组织内炎性细胞浸润,血管周围和间质内较多红细胞漏出。牵张及固定早期,牵张组和直接延长组诱导型NOS（iNOS）与内皮型NOS（eNOS）阳性表达均明显高于正常对照组,直接延长组的iNOS和eNOS阳性表达低于牵张组,差异均有统计学意义（P<0.05）;牵张后固定8周iNOS和eNOS阳性表达3组间比较差异无统计学意义（P>0.05）。结论NOS在牵张早期表达升高,结合在牵张早期组织内红细胞漏出,提示牵张成骨过程中存在某种程度的微创伤,这种微创伤可能是牵张成骨的重要启动因素之一。     

颜面皮肤癌VEGF和NOS的表达与癌细胞增殖关系的研究

目的:研究颜面皮肤癌组织血管内皮生长因子(VEGF)、诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)的表达与癌细胞增殖的相关性.方法:应用免疫组化方法检测47例颜面皮肤癌手术切除标本VEGF、iNOS、eNOS和增殖细胞核抗原(PCNA)的分布及表达.结果:①32例(68.09%)颜面皮肤癌组织表达VEGF,30例(63.83%)表达iNOS;35例(74.47%)表达eNOS.②VEGF与iNOS的表达具有明显相关性,VEGF与eNOS的表达无明显相关性.③表达VEGF的颜面皮肤癌PCNA标记指数(PLI)明显高于不表达VEGF的颜面皮肤癌;表达iNOS的颜面皮肤癌PLI明显高于不表达iNOS的颜面皮肤癌.表达eNOS的颜面皮肤癌PLI与不表达eNOS的颜面皮肤癌无显著性差异(P＞0.05).结论:①VEGF与iNOS的表达具有明显相关性,说明iNOS在VEGF的生成和发挥作用过程中起重要作用;②PLI随着VEGF和iNOS表达的增加而增加;说明二者对颜面皮肤癌细胞增殖具有促进作用.     

一氧化氮对小鼠原代成骨细胞的增殖及分化调控

探讨一氧化氮（nitric oxide，NO）对小鼠原代成骨细胞的矿化调控作用。方法：分离培养小鼠原代成骨细胞，给予外源性NO供体S-亚硝基N-乙酰基青霉胺（SNAP）2周，检测成骨细胞增殖活性和碱性磷酸酶活性。Von Kossa方法检测成骨细胞矿化结节形成情况，免疫组化方法检测骨唾液酸蛋白（BSP）表达。结果：NO对成骨细胞增殖具有双向作用：在β-甘油磷酸钠加抗坏血酸存在下，72h内在一个比较宽的浓度范围（10^-2μmol／L～10^-1μmol／L）NO供体SNAP呈剂量依赖性地刺激成骨细胞样细胞生长，高于10^-2μmol／LSNAP则产生抑制作用。和β-甘油磷酸钠加Ascorbic acid作用组相比，SNAP可明显增加碱性磷酸酶活性，Von Kossa检测可观察到细胞间钙盐沉积，两组药物都能促使成骨样细胞骨唾液酸蛋白表达，而SNAP单独应用无明显矿化作用。结论：适量浓度一氧化氮，以β-甘油磷酸钠和抗坏血酸为介质，可加速小鼠原代成骨细胞增殖分化，并可能通过调控骨唾液酸蛋白表达刺激骨钙化。     

Biological and morphological characteristics of a transplanted cartilaginous tumor derived from a human osteogenic sarcoma

A cartilaginous tumor derived from a human osteogenic sarcoma of the mandible has been maintained by serial passage to nude mice. Tumor growth was multi-lobular. Radiopaque spots were seen scattered throughout the tumor at three months after transplantation. Both light and electron microscopic examination at three months revealed that the tumor contained cartilaginous cells at various stages of differentiation. There was metachromasia throughout tumor lobules except in the marginal region. Von Kossa staining was positive in the central region. Ultrastructural study identified four subtypes of chondrocytic cells of a neoplastic nature. In the extracellular matrix around hypertrophic cells, matrix vesicles were observed with mineral deposits. Alkaline phosphatase was found on the plasma membrane and Golgi complexes of hypertrophic cells, and on matrix vesicles. Thus cell lineage and the manner of calcification of the transplanted tumor were similar to those of epiphyseal growth cartilage.