炎症诱导酶在结直肠癌中的表达及其临床病理意义

目的 检测环氧化酶-2(cyclooxygenase-2,COX-2)和诱导型氮氧合酶(inducible nitric oxidesynthase,iNOS)在结直肠癌(colorectal carcinoma,CRC)中的表达,为研究炎症与CRC发生的关系提供理论依据.方法 应用流式细胞术和SP法免疫组化检测100例CRC及11例癌旁正常黏膜中COX-2和iNOS的表达,并结合临床病理因素进行分析.结果 免疫组化显示,COX-2在CRC中表达阳性率为76.00%,而正常黏膜中只有9.10%,差异有显著性(P<0.05).COX-2表达阳性率在CRC淋巴结转移组和无淋巴结转移组分别为87.50%(42/48)、65.38%(34/52),两组比较差异有显著性(P<0.05);FCM检测发现,高、中、低分化组平均FI值分别为2.47±1.41、2.70±1.08、3.01±1.26,各组COX-2的表达均高于正常黏膜组(1.00±0.28)(P<0.01);CRC淋巴结转移组(2.45±1.41)高于无淋巴结转移组(1.61±1.27)(P<0.05).CRC中iNOS表达阳性率(81.00%)高于正常黏膜(27.27%,P<0.05),有淋巴结转移组的阳性率(91.67%)高于无淋巴结转移组(71.15%,P<0.05).COX-2与iNOS表达呈正相关(P<0.05).结论 在CRC中COX-2和iNOS表达异常增高,在CRC中COX-2和iNOS的表达与淋巴结转移密切相关,可能在CRC的进展及转移中有重要作用.     

大肠腺癌中iNOS和COX-2的表达及意义

目的 探讨大肠腺癌中iNOS和COX-2的表达及其与大肠腺癌生物学行为的关系.方法 采用SP免疫组化方法检测78例大肠腺癌标本及33例癌旁正常肠黏膜组织中的iNOS和COX-2的表达,并对两指标表达与临床病理参数之间进行相关分析.结果 ① iNOS和COX-2在大肠腺癌中阳性率分别显著高于正常大肠黏膜阳性率(P<0.05);② iNOS、COX-2的表达与大肠癌淋巴结转移明显相关(P<0.05);③ iNOS和COX-2在大肠腺癌中的阳性表达与临床分期有关,在TNM分期中Ⅲ+Ⅳ期阳性表达率高于Ⅰ+Ⅱ期(P<0.05);④ 大肠腺癌组织中COX-2表达与iNOS表达之间存在明显相关性(P<0.05).结论 COX-2和iNOS过度表达参与了大肠腺癌的发生发展过程,并与其淋巴结转移和TNM分期有关;大肠腺癌中COX-2和iNOS的表达具有正向协同性.     

Hemostatic properties of a venomic protein in rodent dermal injuries

Hemostatic properties of a factor Xa-like protease (Q8009) from the Australian snake Pseudonaja textilis textilis were determined. In tail-tip transection and dermal incision (hind limb) models, reagents were applied with collagen matrix. Blood was collected on filter paper chads for 12 one-minute intervals or until hemostasis. Determination of blood loss was performed using the hematin content and reported as blood loss per minute and total blood lost. Results from the studies demonstrated that the addition of the protease Q8009 and collagen matrix significantly reduced the volume of blood loss and shortened the time-to-hemostasis. In the dermal incision model, Q8009 (100, 250 and 1000 microg/ml) plus collagen matrix significantly reduced (p<0.001) the volume of blood lost relative to Thrombin and shortened the time-to-hemostasis to 2.0 min compared to 4.77 min with Thrombin. In the tail-tip transection model when Q8009 was mixed with a collagen matrix there was no significant reduction in blood loss, when compared to Thrombin plus collagen matrix. However, when injured tail-tips were held in Q8009 (1000 microg/ml) solution, there was a significant reduction (p<0.001) in blood loss (5.88 microl) versus that of Thrombin at 58.0 mul, and time-to-hemostasis was reduced from 11 min with Thrombin to 3 min when the Q8009 solution was used. In these studies, topical application of the venomic protease Q8009 significantly reduced total blood loss with a shorter time-to-hemostasis relative to Thrombin.     

Anti-inflammatory activity of PAS-1, a protein component of Ascaris suum

Background: Recently, we identified a 200 kDa protein (PAS-1) from Ascaris suum worms, that suppresses the humoral immune response. Here, the effect of PAS-1 on inflammatory leukocyte migration induced by bacterial lipopolysaccharide (LPS) was investigated.Methods: Cellular migration and cytokine release, stimulated by LPS or LPS+PAS-1, were analyzed in air pouches induced in the shaved back of BALB/c mice. Cytokines were determined by ELISA and RT-PCR on air pouch exudates and in vitro stimulated peritoneal macrophages.Results: The significant cellular influx induced by LPS, consisting predominantly of neutrophils, was highly suppressed in the presence of PAS-1, but not a non-related protein. PAS-1 led also to a marked reduction of TNF-, IL-1 and IL-6 levels in both LPS-stimulated air pouches and peritoneal macrophage cultures. In contrast, PAS-1 induced a significant increase of IL-10 and TGF- production.Conclusions: These results demonstrate that PAS-1 has a potent anti-inflammatory activity, probably due to the stimulation of regulatory cytokines in macrophages, thus leading to the inhibition of pro-inflammatory cytokine production.Received 2 June 2004; returned for revision 16 July 2004; accepted by A. Falus 7 September 2004     

Acacia ferruginea inhibits inflammation by regulating inflammatory iNOS and COX-2

Inflammation is a local defensive reaction of a host to cellular injury or infection. Prolonged inflammation can contribute to pathogenesis of many disorders. Identification of naturally occurring phytoconstituents that can suppress inflammatory mediators can lead to the discovery of anti-inflammatory therapeutics. Acacia ferruginea is used traditionally to treat numerous ailments including hemorrhage, irritable bowel syndrome and leprosy. The present study evaluated the anti-inflammatory activity of A. ferruginea extract against acute (carrageenan) and chronic (formaldehyde) inflammation in Balb/c mice. Pre-treatment with A. ferruginea extract (10?mg/kg BW) for 5 consecutive days via intraperitonial (IP) administration significantly inhibited subsequent induction of paw edema in both models; the effects were comparable to that of the standard drug indomethacin. The results also showed the A. ferruginea extract significantly inhibited nitric oxide (NO) synthesis and iNOS expression (as measured in serum), diminished inflammation in – and neutrophil infiltration to – the paw tissues and led to a reduction in the number of COX-2⁺ immunoreative cells (as evidenced by histologic and immunohistochemical analyses) in the paws relative to those in paws of mice that received the irritants only. Further, in vitro studies showed the extract could significantly scavenge free radicals generated as in DPPH and NO radical generating assays. Taken together, the results showed that A. ferruginea extract imparted potent anti-oxidant and -inflammatory effects, in part by maintaining oxidative homeostasis, inhibiting NO synthesis and suppressing iNOS and COX-2 expression and so could potentially be exploited as a potential plant-based medication against inflammatory disorders.     

Inhibition of cyclooxygenase-2 suppresses invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 and CD44

Expression of cyclooxygenase-2 (COX-2) in tumors is known to be associated with enhanced angiogenesis, suppression of host immunity, and tumor invasion. In the present study, human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 were used to evaluate the effects of NS-398, a selective inhibitor of COX-2, and COX-2 antisense oligonucleotide (COX-2 AS) on the invasion activity of OSCC cells. Matrigel invasion assay revealed that the invasiveness of NA and HSC-4 was suppressed by treatment with either NS-398 or COX-2 AS. These reagents down-regulated the secretion of matrix metalloproteinase-2 (MMP-2) to culture supernatant as well as the expression of MMP-2 mRNA and protein. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an activator of proMMP-2, was also down-regulated by treatment with these reagents. Furthermore, expression of CD44 on the surface of these cells was reduced by treatment with either NS-398 or COX-2 AS. In addition, MMP-2 antisense oligonucleotides reduced the expression of CD44 on the surface of both OSCC cell lines. These findings suggest that NS-398 and COX-2 AS suppress the invasiveness of OSCC cells via down-regulation of MMP-2 and CD44. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC patients.This revised version was published online in August 2005 with a corrected cover date.     

Verapamil modulates LPS-induced cytokine production via inhibition of NF-kappa B activation in the liver

Objective: To investigate the effect of verapamil on Lipopolysaccharide (LPS)-induced cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10)] and nuclear factor kappa B (NF-κ B) in the liver. Methods and Materials: Adult male Sprague-Dawley rats were randomly divided into seven groups of eight rats each: control rats treated with saline (0.9 % NaCl); rats treated with saline and then challenged intraperitoneally with LPS (10 mg/kg); rats treated intraperitoneally with different levels of verapamil (1, 2.5, 5, 10 mg/kg) and then challenged with LPS (10 mg/kg); and rats treated only with verapamil (10 mg/kg). TNF-α, IL-6, IL-10 and NF-κ B in the liver tissues were investigated as well as the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) one hour after LPS injection. Results: LPS alone stimulated production of TNF-α, IL-6 and IL-10, and activated NF-κ B in the liver. Pretreatment with verapamil before LPS challenge reduced acute liver injury, down-regulated production of LPS-induced pro-inflammatory cytokines (TNF-α and IL-6), up-regulated production of anti-inflammatory cytokines (IL-10) and inhibited NF-κ B activation in the liver in a dose-dependent manner. Conclusion: Verapamil can attenuate acute liver injury by down-regulating the production of TNF-α and IL-6 and up-regulating IL-10 in the liver, possibly via inhibition of NF-κ B. Received 8 August 2005; returned for revision 25 September 2005; returned for final revision 18 November 2005; accepted by M. Katori 6 December 2005     

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。     

Quercetin inhibits expression of inflammatory cytokines through attenuation of NF-κB and p38 MAPK in HMC-1 human mast cell line

Objective and design: Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce production of pro-inflammatory cytokines with immune regulatory properties. We investigated the effect of quercetin on the expression of pro-inflammatory cytokines in human mast cell line, HMC-1. Methods: HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 (PMACI). Results: Quercetin decreased the gene expression and production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 in PMACI-stimulated HMC-1 cells. Quercetin attenuated PMACI-induced activation of NF-κB and p38 mitogen-activated protein kinase. Conclusion: Our study provides evidence that quercetin may suitable for the treatment of mast cell-derived allergic inflammatory diseases. Received 18 October 2006; returned for revision 26 November 2006; accepted by A. Falus 4 December 2006     

A synthetic hydroxypropenone inhibits nitric oxide,prostaglandin E2, and proinflammatory cytokine synthesis

HMP [3-(2-hydroxyphenyl)-1-(5-methyl-furan-2-y-l) propenone] was evaluated for its ability to inhibit the synthesis of major proinflammatory mediators and cytokines in interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-induced RAW 264.7 cells and phorbol myristate acetate (PMA)-differentiated/LPS-induced U937 cells. HMP suppressed the production of nitric oxide (NO) with significant inhibitory effects at doses as low as 0.78?μM (P?<?0.05). Prostaglandin E2 (PGE2) secretion was also inhibited at doses of 12.5?μM and above (P?<?0.01). The secretion of both TNF-α and IL-6 were only inhibited at the highest dose used (25?μM; P?<?0.001). IL-1β secretion was also inhibited from 12.5?μM onwards (P?<?0.01). This inhibition was demonstrated to be caused by down-regulation of inducible enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), without direct effect upon iNOS or COX-2 enzyme activity. HMP only inhibited iNOS (P?<?0.001) and IL-1β (P?<?0.05) gene expression at the highest tested concentration. HMP did not affect the secretion of chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) and the anti-inflammatory cytokine IL-10. The most striking effect of HMP was its NO inhibitory activity and therefore we conclude that HMP is a selective inhibitor of iNOS.     

三七皂苷Rg1抑制脂多糖诱导的小胶质细胞激活

目的 探讨三七皂苷Rg1(Rg1)对脂多糖(LPS)诱导的小胶质细胞系BV-2细胞炎性因子释放的抑制作用.方法 用LPS刺激BV-2细胞构建炎症模型,采用四甲基偶氮唑盐比色法检测Rg1对BV-2细胞活力的影响,免疫荧光染色和反转录PCR方法检测不同浓度Rg1(10、20、40μmol/L)对细胞炎性蛋白酶诱导型一氧化氮合酶(iNOS)和环氧合酶-2 (COX-2)、细胞炎性因子肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)、炎性信号分子NF-κB蛋白与mRNA的表达变化.结果 不同浓度的Rg1在转录水平和翻译水平上明显抑制了LPS诱导的细胞炎性蛋白酶iNOS和COX-2、细胞炎性因子TNF-α和IL-1β与炎性信号分子NF-κB的上调,并且iNOS、COX-2和NF-κB的表达呈剂量依赖性.结论 Rg1可通过调控LPS诱导的小胶质细胞系BV-2细胞炎性因子释放从而抑制小胶质细胞激活,发挥抗神经炎症的作用.     

人胃癌细胞中COX-2基因的表达及其生物学活性

目的 为研究环氧合酶－２（ｃｙｃｌｏｏｘｙｇｅｎａｓｅ－２,ＣＯＸ－２）或通过前列腺素（ｐｒｏｓｔａｇｌａｎｄｉｎ,ＰＧ）引起的肿瘤免疫机制,我们检测了人胃癌细胞中ＣＯＸ－２ｍＲＮＡ及蛋白质水平的表达,ＰＧＥ２的释放和ＣＯＸ－２酶活性的抑制剂消炎痛（ｉｎｄｏｍｅｔｈａｃｉｎ,Ｉｎｄｏ）对ＰＧＥ２４ 释放和肿瘤细胞生长的影响。方法 采用免疫细胞化学染色,ＲＴ－Ｐ 及ＭＴＴ比色试验方法研究人胃癌细胞Ｃ     

Concanavalin-A displays leishmanicidal activity by inducing ROS production in human peripheral blood mononuclear cells

Abstract

The context of the article: Leishmania amazonensis has a wide geographical distribution throughout South American countries and can cause self-healing to severe cases as mucocutaneous or visceral forms. Leishmaniasis presents a balance of inflammatory and anti-inflammatory cytokines which is responsible for promoting the activation of phagocytes, essential to control the infection and lead to tissue repair/resolution of the disease, respectively.

Results and discussion: Our model revealed that the treatment with Con-A was capable to stimulate human PBMC cells by increasing the phagocytic capacity and promoting parasite elimination. The pretreatment with Con-A promoted inflammatory (IFN-γ, TNF-α, IL-2 and IL-6) and anti-inflammatory (IL-4 and IL-10) cytokines production, increased the reactive oxygen species (ROS) sinthesys as well as the expression and presence of iNOS enzyme, but not nitric oxide production.

Conclusion: Based on the data obtained, it was possible to infer that Con-A induces the ROS production, responsible for eliminating parasites in addition to regulatory cytokines synthesis which are important for disease resolution.     

Distinct cytokine patterns exist in peripheral blood mononuclear cell cultures of patients with prostate cancer

In this study, we have concurrently assayed for IL-2, IL-4, IL-6, IL-10, TNF-alpha, and IFN-gamma in 24-h serum-free cultures of peripheral blood mononuclear cells (PBMC) obtained from seventeen patients with prostate cancer (CaP) per cytokine bead array analysis. The purpose of the study is to examine the nature of the cytokine profile operating among patients and to correlate with their physical, biochemical, and clinical parameters. Unstimulated PBMC cultures from patients with hormone-sensitive metastatic disease demonstrated elevated level of baseline TNF-alpha compared to patients with high-risk, locally advanced disease. Younger patients exhibited significantly higher levels of IL-4 and TNF-alpha compared to older patients following PHA stimulation. Similarly, significantly higher ratios of IFN-gamma/IL-4, IFN-gamma/IL-10, and IL-2/IL-4, a favorable type-1 cytokine pattern, were observed in patients with lower serum PSA compared to patients with high serum PSA. These results indicate the existence of distinct cytokine patterns among patients with CaP.     

Atherosclerosis and the protective role played by different proteins in apolipoprotein E-deficient mice

The aim of the present study was to analyze the early events in atherogenesis and the role of pro- or anti-atherosclerotic proteins in the development of atherosclerotic lesions. We used apolipoprotein E-deficient (E(0)) mice that spontaneously develop hypercholesterolemia and atherosclerotic lesions in the aorta in a time-dependent manner. Aortas of mice aged 6, 8, 10 and 12 weeks were examined to determine histopathological changes. In mice aged 8-12 weeks, developing atherosclerotic lesions were present in different regions of the aortas. These lesions protruded into the lumen of the vessel and showed lipid deposits, lipid-filled macrophages and extensive accumulation of collagen and elastic fibers throughout the entire arterial wall. A parallel immunohistochemical study included analysis of three proteins known to be involved in atherosclerosis, i.e. inducible nitric oxide synthase (iNOS, NOS2), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2). Increased immunolabelling of iNOS and VEGF accompanied atherosclerosis development in E(0) mice aged 8, 10 and 12 weeks. On the contrary, immunolabelling for MMP2 was negative in E(0) mice aged 10 and 12 weeks. Our results indicate morphological alterations in the Tunica intima and Tunica media of atherosclerotic aortas and possible protective roles for iNOS and VEGF proteins against atherosclerosis development. These data may be relevant for developing therapeutic strategies for atherosclerosis development.     

胃癌组织中COX-2表达及其与微血管和微淋巴管密度的关系

目的 研究胃癌组织中COX-2表达的临床病理学意义,同时探讨COX-2与微血管密度(MVD)和微淋巴管密度(MLD)的关系.方法 采用免疫组化EnVision两步法,检测46例胃癌中COX-2的表达,同时标记CD34和D2-40,分别检测胃癌组织中微血管和微淋巴管密度.结果 46例胃癌中COX-2表达的阳性率为80.43%(37/46),COX-2的表达与患者性别、浸润深度无关(P>0.05),而与肿瘤大小、组织学分期、淋巴结转移和临床分期等临床病理参数密切相关(P<0.05);COX-2表达阳性组的平均生存时间明显低于表达阴性组(P<0.05);COX-2表达阳性组的MVD和MLD明显高于COX-2表达阴性组(P<0.05).结论COX-2可能通过促进肿瘤的血管和淋巴管形成而参与了胃癌细胞的浸润转移,导致胃癌患者预后不良.     

Oleic acid reduces lipopolysaccharide-induced expression of iNOS and COX-2 in BV2 murine microglial cells: Possible involvement of reactive oxygen species,p38 MAPK,and IKK/NF-κB signaling pathways

Microglia are the major cells involved in neuroinflammation resulting in brain tissue damage during infection and neurodegenerative diseases. In this study, we examined the effects of the monounsaturated fatty acid oleic acid (OA) on LPS-induced proinflammatory mediators production and the mechanisms involved in BV2 microglia. OA inhibited LPS-induced expression of iNOS and COX-2 as well as production of NO and prostaglandin E2. We showed that OA blocked LPS-induced NF-κB activation and phosphorylation of inhibitor κB kinase (IKK). We also showed that OA inhibited LPS-induced phosphorylation of Akt and p38 MAPK, but not that of ERK. Finally, we showed that OA reduced reactive oxygen species (ROS) accumulation and an anti-oxidant N-acetylcysteine inhibited NF-κB transactivation and phosphorylation of IKK and Akt in LPS-stimulated BV2 cells. Taken together, our results suggest that OA shows an anti-inflammatory effect by inhibiting ROS, p38 MAPK, and Akt/IKK/NF-κB signaling pathways in LPS-stimulated BV2 microglia.     

Niflumic acid exhibits anti-tumor activity in nasopharyngeal carcinoma cells through affecting the expression of ERK1/2 and the activity of MMP2 and MMP9

Niflumic acid (NFA) was known to inhibit cell proliferation or migration in several types of cancer. However, the function of NFA in human nasopharyngeal carcinoma (NPC) cells was not clarified. The proliferation of NPC cell line CNE-2Z cells with NFA treatment was detected using the cell counting kit-8 method and transwell assay was employed to assess the effect of NFA on the CNE-2Z cell migration and invasion. The activity of MMP2 and MMP9 was detected by Gelatin Zymography. Cell cycle distribution and apoptosis were detected using flow cytometry. In vitro pull-down assay, western blot, and computational technique were applied to investigate the NFA regulating signaling pathway. Our results indicated that the growth capacity and colony formation potential of CNE-2Z cells in soft agar were significantly suppressed by treatment with NFA. NFA inhibited the proliferation of CNE-2Z cells in a concentration and time-dependent manner. NFA exerted an S phase arrest on the CNE-2Z cells in a concentration-dependent manner, while promoting apoptosis in a dose-dependent manner. Migration and invasion potential of CNE-2Z cells were decreased by NFA treatment in vitro. In vitro pull-down assay and molecular modeling indicated that NFA directly bound with early respond kinase 1 (ERK1). Finally, the anti-tumor effect of NFA was suggested to be mediated by inhibiting early respond kinases (ERK) expression and the MMP2 and MMP9 activities. NFA has proliferation-inhibiting, invasion-suppressing, cell cycle-blocking and apoptosis-promoting effects on CNE-2Z cells through regulation of ERK/MAPK and our results indicates that NFA may serve as a candidate of anticancer drug for NPC.