诱导型一氧化氮合酶与环氧合酶-2在肿瘤组织中的共表达

诱导型一氧化氮合酶(iNOS)和环氧合酶-2(COX-2)均与肿瘤发生、生长、血管形成及转移有关。在正常生理条件下,两者在多数组织内均检测不到,但在受到各种病理因素的刺激后,两者均能被迅速诱导表达。在许多恶性肿瘤组织中,两者的表达一致上调,呈正相关,其联合表达与肿瘤的生物学行为密切相关。研究发现iNOS与COX-2有可能通过Wnt-β-catenin信号实现了信息交流和相互调控,并通过这种机制,共同在肿瘤的各种生物学行为中发挥作用。     

The chemokine receptor CCR7 is expressed on epithelium of non-inflamed gastric mucosa, Helicobacter pylori gastritis, gastric carcinoma and its precursor lesions and up-regulated by H. pylori

CCR7 chemokine-receptor expression on tumour cells of gastric carcinoma has been associated with lymph-node metastasis and is thought to play an important role in metastasis. However, so far it is unknown whether CCR7 is newly up-regulated on gastric carcinoma or already expressed in non-neoplastic gastric epithelium. Therefore, epithelial CCR7 expression was investigated in the process of gastric carcinogenesis: non-inflamed mucosa --Helicobacter pylori gastritis -- intestinal metaplasia/dysplasia -- gastric carcinoma. CCR7 was expressed by gastric epithelium in non-inflamed gastric mucosa (n = 5), H. pylori gastritis (n = 17), intestinal metaplasia (n = 10), dysplasia (n = 3) and on tumour cells in 20 of 24 patients with gastric carcinoma (13/14 intestinal-type; 7/10 diffuse-type) as tested by immunohistochemistry. As CCR7 expression by gastric epithelium was significantly stronger in H. pylori gastritis than in non-infected mucosa, the influence of H. pylori on CCR7 receptor expression of gastric epithelial cells was investigated by fluorescence activated cell sorter analysis. H. pylori strains up-regulated the CCR7 chemokine-receptor in CCR7-positive cell lines. No difference in CCR7 up-regulation between cag(+) and cag(-)H. pylori strains was found. Epithelial CCR7 up-regulation by H. pylori may alter the metastatic fate of gastric carcinoma. Additionally, CCR7 expression not only on gastric carcinoma, but also on non-neoplastic gastric epithelium, suggests a novel biological function.     

诱导型一氧化氮合酶在糖尿病性阴茎勃起功能障碍大鼠阴茎的表达

目的:探讨诱导型一氧化氮合酶(iNOS)在糖尿病性阴茎勃起功能障碍(ED)发病进程中的可能作用.方法:注射链脲佐菌素建立糖尿病大鼠模型,分别在注射8周和12周后观察阴茎勃起次数,取大鼠阴茎,用ABC免疫组织化学方法检测阴茎iNOS的分布与表达,免疫印迹检测阴茎iNOS蛋白量的变化.结果:糖尿病大鼠的阴茎勃起次数低于对照组,并随病程延长降低;与对照组比较,糖尿病组阴茎内iNOS阳性细胞数和平均光密度、iNOS蛋白量升高,并随病程延长而进行性升高.结论:糖尿病组大鼠阴茎iNOS表达的升高与ED相关.     

Anti-inflammatory effect of Mentha longifolia in lipopolysaccharide-stimulated macrophages: Reduction of nitric oxide production through inhibition of inducible nitric oxide synthase

Abstract

Mentha longifolia is an aromatic plant used in flavoring and preserving foods and as an anti-inflammatory folk medicine remedy. The present study assessed the effects of M. longifolia extracts, including essential oil and crude methanol extract and its fractions (ethyl acetate, butanol and hexane), on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in lipopolysaccharide (LPS)-stimulated J774A.1 cells using real-time polymerase chain reaction (PCR). The cytotoxic effects of the extracts on the cells were examined and non-cytotoxic concentrations (<0.2?mg/ml) were used to examine their effects on NO production and iNOS mRNA expression. Only the hexane fraction that contained high levels of phenolic and flavonoid compounds at concentrations from 0.05–0.20?mg/ml significantly reduced NO production in LPS-stimulated cells (p?<?0.001). Real-time PCR analysis indicated the ability of this fraction at the same concentrations to significantly decrease iNOS as well as TNFα mRNA expression in the cells (p?<?0.001). All extracts were able to scavenge NO radicals in a concentration-dependent manner. At concentrations greater than 0.2?mg/ml, total radicals were 100% scavenged. In conclusion, M. longifolia possibly reduces NO secretion in macrophages by scavenging NO and inhibiting iNOS mRNA expression, and also decreases TNFα pro-inflammatory cytokine expression, thus showing its usefulness in the inflammatory disease process.     

Opposite effect of oxidative stress on inducible nitric oxide synthase and haem oxygenase-1 expression in intestinal inflammation: anti-inflammatory effect of carbon monoxide

Inducible nitric oxide synthase (iNOS) is expressed in intestinal epithelial cells (IEC) of patients with active inflammatory bowel disease (IBD) and in IEC of endotoxaemic rats. The induction of iNOS in IEC is an element of the NF-kappaB-mediated survival pathway. Haem oxygenase-1 (HO-1) is an AP-1-regulated gene that is induced by oxidative stress. The enzyme produces carbon monoxide (CO), which may attenuate the inflammatory response. The aim of this study was to investigate the regulation and interaction of iNOS and HO-1 in response to inflammation and oxidative stress. Male Wistar rats were treated with the thiol-modifying agent diethylmaleate (DEM) to induce oxidative stress and rendered endotoxaemic by LPS injection. Human colonic biopsies and the human colon carcinoma cell line DLD-1 were treated with DEM and the lipid peroxidation end-product 4-hydroxynonenal to induce oxidative stress and exposed to cytokine mix (CM) to mimic inflammation. In some experiments, cells were incubated with 250-400 ppm CO prior to and during stimulation with CM. HO-1 and iNOS expression was evaluated by RT-PCR, western blotting, and immunohistology. NF-kappaB activation was evaluated by EMSA. LPS induced iNOS but not HO-1 in epithelial cells of the ileum and colon. Oxidative stress strongly induced HO-1 in epithelial and inflammatory cells. Combined oxidative stress and endotoxaemia decreased iNOS expression but strongly induced HO-1 expression. Similarly, CM induced iNOS but not HO-1 in colonic biopsies and DLD-1 cells. Oxidative stress prevented iNOS induction in an NF-kappaB-dependent manner but increased HO-1 expression in CM-exposed DLD-1 cells. CO inhibited iNOS mRNA induction in CM-stimulated DLD-1 cells. These data demonstrate opposite regulation of iNOS and HO-1 in intestinal epithelial cells in response to cytokine exposure and oxidative stress. These findings suggest that iNOS (NF-kappaB driven) and HO-1 (AP-1 driven) represent mutually exclusive survival mechanisms in intestinal epithelial cells.     

Evaluation of the applicability of the gastric carcinoma risk index for intestinal type cancer in Japanese patients infected with Helicobacter pylori

The gastric carcinoma risk index is a histological criteria to Helicobacter pylori-infected patients with a high risk of gastric cancer. The aim of this study was to examine the applicability of this index for the intestinal-type gastric cancer in Japanese patients with H. pylori infection. In 55 patients with early intestinal-type gastric cancer and 69 control subjects, we calculated the gastric cancer risk index score by evaluating the grade of mononuclear cell (MNC) and polymorphonuclear cell (PMN) infiltration and the presence of intestinal metaplasia. The gastric cancer index score was significantly higher in patients with gastric cancer (P<0.01). The presence of intestinal metaplasia was significantly more frequent in cancer patients than in controls, while infiltration of MNCs or PMNs in the corpus was not different in the two groups. Within the gastric cancer risk index, the presence of intestinal metaplasia was the only criteria associated with the development of intestinal-type gastric cancer in Japan. The gastric cancer risk index may not be applicable to identify H. pylori-positive patients at high risk of developing intestinal-type gastric cancer in Japan. Received: 6 October 1999 / Accepted: 22 December 1999     

Association of Helicobacter pylori-dependent gastritis with gastric carcinomas in young Japanese patients: histopathological comparison of diffuse and intestinal type cancer cases

AIMS: The causal relationship of H. pylori gastric colonization with gastric cancer development has not as yet been fully elucidated. The prevalence of H. pylori infection increases with age in the asymptomatic population in Japan, and reaches a high plateau in those older than 40 years. The objective of this study was to assess the link between H. pylori and gastric carcinomas in patients younger than 40 years. METHODS AND RESULTS: Detection of H. pylori and assessment of background mucosa based on the Sydney system was performed histopathologically for 40 Japanese gastric cancer cases younger than 40 years and compared with 40 age- and sex-matched controls. H. pylori infection in gastric mucosa was detected significantly more frequently (P < 0.001) in patients with cancer (29/40; 72.5%) than in controls (11/40; 27.5%). Additionally, by histopathological comparison between intestinal (18 cases) and diffuse (70 cases) types of young gastric cancer patients, mucosal atrophy and intestinal metaplasia were found to coexist with acute and chronic inflammation in the background mucosa of both intestinal and diffuse types, being significantly more prevalent than in young controls. CONCLUSIONS: As well as the high prevalence of H. pylori in young subjects with gastric cancer, it is clear that persistent infection induces mucosal damage, resulting in atrophy and intestinal metaplasia. Thus, acute/chronic gastritis could play an essential role in the early development of neoplasia in the stomach.     

缺氧诱导因子-1在缺氧诱导一氧化氮合成酶基因表达中的作用

目的 研究缺氧诱导因子-1(HIF-1)在一氧化氮合成酶基因缺氧诱导反应中的作用。方法 体外合成具有HIF-1特异结合位点的DNA片段(红细胞生成素3'-增强子片段),借助脂质体,转入培养的鼠主动脉内皮细胞和肺微血管细胞,用半定量RT-PCR方法测定诱导型一氧化氮合成酶(iNOS)mRNA。结果 (1)大鼠主动脉内皮细胞、肺微血管内皮细胞在常氧下培养,有iNOS基因表达;(2)缺氧能诱导这两种细胞iNOS基因表达增加;(3)野生型EPO3'-增强子片段能阻断缺氧对内皮细胞iNOS基因表达的诱导作用,而突变片段则无此作用。结论 在iNOS基因序列中,可能存在EPO3'-端增强子片段,其参与内皮细胞的缺氧反应。     

Expression of cyclooxygenase-2, alpha 1-acid-glycoprotein and inducible nitric oxide synthase in the developing lesions of murine leprosy

Murine leprosy is a chronic disease of the mouse, the most popular animal model used in biomedical investigation, which is caused by Mycobacterium lepraemurium (MLM) whose characteristic lesion is the macrophage-made granuloma. From onset to the end of the disease, the granuloma undergoes changes that gradually transform the environment into a more appropriate milieu for the growth of M. lepraemurium. The mechanisms that participate in the formation and maturation of the murine leprosy granulomas are not completely understood; however, microbial and host-factors are believed to participate in their formation. In this study, we analysed the role of various pro-inflammatory and anti-inflammatory proteins in granulomas of murine leprosy after 21 weeks of infection. We assessed the expression of cyclooxygenase-2 (COX-2), alpha acid-glycoprotein (AGP), and inducible nitric oxide synthase (iNOS) at sequential stages of infection. We also looked for the nitric-oxide nitrosylation product, nitrotyrosine (NT) in the granulomatous lesions of murine leprosy. We found that a pro-inflammatory environment predominates in the early granulomas while an anti-inflammatory environment predominates in late granulomas. No obvious signs of bacillary destruction were observed during the entire period of infection, but nitrosylation products and cell alterations were observed in granulomas in the advanced stages of disease. The change from a pro-inflammatory to an anti-inflammatory environment, which is probably driven by the bacillus itself, results in a more conducive environment for both bacillus replication and the disease progression.     

Signalling mechanisms involved in the induction of inducible nitric oxide synthase by Lactobacillus rhamnosus GG,endotoxin, and lipoteichoic acid

Background and Aims: Probiotic Lactobacillus rhamnosus GG (Lactobacillus GG) has been found beneficial in the treatment of viral and antibiotic-associated diarrhea. Recently, it has also been shown to induce nitric oxide (NO) production, and have some other immunostimulatory effects. The aim of the present study was to investigate the mechanisms involved in the induction of inducible nitric oxide synthase (iNOS) and NO production by Lactobacillus GG. Methods and Results: iNOS expression and NO production induced by Lactobacillus GG, lipopolysaccharide (LPS), and lipoteichoic acid (LTA) was abrogated by NOS inhibitors L-NMMA and 1400W, by a protein synthesis inhibitor cycloheximide, by a tyrosine kinase inhibitor genistein and by a NF-B inhibitor pyrrolidinedithiocarbamate (PDTC) in J774 macrophages. Polymyxin B inhibited NO production induced by LPS, but did not inhibit Lactobacillus GG induced NO production. _P42/44 MAP-kinase inhibitor PD98059, dexamethasone and cyclosporine A inhibited partially iNOS protein expression and NO formation in LactobacillusGG, LPS and LTA treated cells. Ro 31-8220 (protein kinase C inhibitor) and SB203580 (p38 MAP-kinase inhibitor) had only a minor effect on NO production. Conclusions: Lactobacillus GG induced NO production through iNOS pathway and the mechanisms mediating that process were very similar with those involved in LPS and LTA induced NO synthesis.     

The Glu298Asp variant of the endothelial nitric oxide synthase gene is associated with an increased risk for abruptio placentae in pre-eclampsia

Attempts to define a pre-eclampsia susceptibility profile have been hampered by the wide clinical spectrum of the condition and the complex genetics underlying it. Genes that modulate blood pressure, fluid homeostasis and placental vascular development have been considered plausible candidates. Among these are the angiotensinogen (AGT) gene variant Met235Threo, which has been associated with pre-eclampsia and the endothelial nitric oxide synthase (eNOS) polymorphism Glu298Asp, which has been associated with both pre-eclampsia and abruptio placentae, a condition that often co-exists with pre-eclampsia. The aim of this study was to investigate a potential association between these gene variants and pre-eclampsia with and without abruptio placentae in a South African patient group. Fifty primigravidas with early onset, severe pre-eclampsia, 50 women presenting primarily with abruptio placentae (whether associated with pre-eclampsia or not) and a control panel of 50 healthy pregnant women constituted the study groups. The Met235Threo and Glu298Asp variants were characterised by polymerase chain reaction and restriction enzyme analysis. No association was demonstrated between the M235T variant of the AGT gene and pre-eclampsia or abruptio placentae. In contrast, the combined frequency of the eNOS variant genotypes (GT and TT) was significantly higher in the abruptio placentae group (49%) than the control group (21%) (p=0.006). Furthermore, in the pre-eclampsia patients who subsequently developed abruptio placentae, the eNOS GT genotype emerged as a major risk factor for the development of abruptio placentae (p<0.0001). These data suggest that the presence of a Glu298Asp eNOS variant may pre-dispose a pre-eclamptic woman to develop abruptio placentae or that it is a marker for predisposition.     

Antagonistic effects of IL-4 and interferon-gamma (IFN-γ) on inducible nitric oxide synthase expression in bovine macrophages exposed to Gram-positive bacteria

Cytokine-mediated modulation of nitric oxide (NO) production by bacteria-stimulated bovine macrophages was studied. When Salmonella dublin, as a prototypic Gram-negative organism, was used, NO generation was barely enhanced by recombinant bovine and ovine IFN-γ, but was suppressed by IL-4. Salmonella dublin-induced NO generation was not influenced by a panel of nine other cytokines. The panel included IL-1, tumour necrosis factor (TNF) and IFN-α, which are active in a similar mouse macrophage model. The tested cytokines were either homologous or known to interact with bovine cytokine receptors. Recombinant bovine and ovine IFN-γ were the only cytokines which strongly enhanced NO synthesis by macrophages exposed to the Gram-positive organism, Listeria monocytogenes. Listeria-induced NO generation was strongly suppressed by recombinant human and bovine IL-4, but not by IL-10 and transforming-growth-factor-beta. Thus, two cytokines characterizing a Th1 and a Th2 response up- and down-regulate, respectively, bacteria-induced NO generation in bovine macrophages, whereas nine other cytokines had little activity in this regard. This modulation was reflected in changes in the steady state levels of mRNA coding for inducible nitric oxide synthase. Combinations of IFN-γ and IL-4 suggested that the relative proportion of these cytokines determined whether bacteria-induced NO generation was up- or down-regulated. At saturating IL-4 concentrations, stimulation of bacteria-induced NO generation in macrophages by T cell supernatants was solely dependent on IFN-γ. This was shown by antibody neutralization experiments and by a close correlation between the capacity of supernatants to stimulate NO generation and the IFN-γ content, as determined by immunoassay.     

Expression of cytokeratins typical for ductal and squamous differentiation in the human stomach: an immunohistochemical study of normal foveolar epithelium, Helicobacter pylori gastritis and intestinal metaplasia

The expression of the cytokeratins (CK) 1, 5, 6, 7, 10, 13 and 14 was studied immunohistochemically in gastric biopsies from both the antrum and the body of 70 patients. Normal gastric foveolar epithelium (9 cases) Helicobacter pylori gastritis (23) and intestinal metaplasia (38) were examined. Positive staining results for CK 1, 5, 10 and 14 were not observed using the 34βE12 antibody. With antibodies to CK 5/6, 7 and 13 some, but not all cases, were immunoreactive. Predominantly positive staining included less than 10% of the cells and was always restricted to the tips and the juxtaluminal areas of the foveolae. No difference was seen between the antrum and the body. Comparing normal gastric mucosa with gastritis and intestinal metaplasia, cases positive for CK 5/6 were observed less frequently in intestinal metaplasia types II and III compared to the other groups. CK 7 was expressed exclusively in intestinal metaplasia. CK 13 was seen in all groups of specimens. Thus, cytokeratins typical for ductal structures (CK 7) and squamous epithelia (CK 5/6, CK 13) can be regarded as an inconstant, but not unusual observation in the gastric mucosa. Their expression may be controlled by both differentiation-related as well as environmental factors.     

脑室内注射BDNF抗体对大鼠海马NOS表达的影响

探讨脑室内注射脑源性神经营养因子 (BDNF)抗体阻断内源性BDNF对大鼠海马一氧化氮合酶 (NOS)阳性神经元的影响。脑室内注射BDNF抗体一周后 ,采用Morris水迷宫进行行为检测 ;并用NADPH 黄递酶组化染色方法观察海马NOS阳性神经元数目的变化。与对照组相比 ,实验组大鼠空间学习和记忆能力明显下降 (P <0 0 1) ;实验组大鼠海马CA1区NOS阳性神经元数目 (38 37± 5 2 3)明显少于对照组 (4 9 5 3± 5 74 ) (P <0 0 1) ;实验组DG区NOS阳性神经元数目 (4 8 77± 5 5 1)明显少于对照组 (6 0 4 0± 7 39) (P <0 0 1)。脑室内注射BDNF抗体可导致大鼠空间学习记忆能力下降 ,海马NOS阳性神经元数目减少 ,提示BDNF对学习和记忆的影响可能与海马NOS阳性神经元数目的变化有关