Ebselen inhibits tumor necrosis factor-alpha-induced c-Jun N-terminal kinase activation and adhesion molecule expression in endothelial cells

Tumor necrosis factor-alpha (TNF-alpha) stimulates expression of endothelial cell (EC) genes that may promote atherosclerosis in part by an activation of mitogen-activated protein (MAP) kinases. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), a selenoorganic compound, is effective for acute ischemic stroke; however, its effect on EC has not yet been elucidated. We examined the effect of ebselen on TNF-alpha-induced MAP kinase activation and adhesion molecule expression in cultured human umbilical vein endothelial cells (HUVEC). Extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 were rapidly and significantly activated by TNF-alpha in HUVEC. TNF-alpha-induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 were not affected. Apoptosis signal-regulated kinase 1 (ASK1) was suggested to be involved in TNF-alpha-induced JNK activation because transfection of kinase-inactive ASK1 inhibited TNF-alpha-induced JNK activation. Ebselen inhibited TNF-alpha-induced TNF receptor-associated factor 2 (TRAF2)-ASK1 complex formation and phosphorylation of stress-activated protein kinase ERK kinase 1 (SEK1), which is an upstream signaling molecule of JNK. Finally, TNF-alpha-induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) activation and resultant intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions were inhibited by ebselen. Specific inhibitors for JNK and NF-kappaB also inhibited TNF-alpha-induced ICAM-1 and VCAM-1 expressions in HUVEC. These findings suggest that ebselen prevents TNF-alpha-induced EC activation through the inhibition of TRAF2-ASK1-SEK1 signaling pathway, which leads to JNK activation. Inhibition of JNK by ebselen may imply its usefulness for the prevention of atherosclerosis relevant to EC activation.     

大肠埃希菌脂多糖对高脂饮食动物血脂及炎性反应的影响

目的研究大肠埃希菌脂多糖对高脂饮食兔血脂和炎性反应的影响。方法给含0．5％胆固醇的饲料,3周后,分别在第4、8、12周采用耳动脉内、颈部、腹股沟处肌肉注射大肠埃希菌脂多糖（LPS）,并设立正常组和单纯高脂组。16周后观察兔的一般状态,取血清检查血脂六项、C-反应蛋白和TNF—α,取耳动脉、颈动脉、主动脉弓、胸主动脉、腹主动脉、髂动脉、肝脏,放置4％多聚甲醛中过夜,常规行HE染色,检查血管病变和相关脏器病变情况。结果单纯高脂组血清中胆固醇和LDL-C较正常组增加,复合模型组动物血清中胆固醇和LDL-C均明显高于单纯高脂组,单纯高脂组TNF-α较正常组高,复合模型组TNF-α比单纯高脂组高。病理显示主动脉弓变化明显,复合模型组内膜斑块弥漫,而单纯高脂组内膜只出现单个小斑块,单纯高脂组和复合模型组心脏病变区别不大,均见轻度水肿和小脂肪滴;单纯模型组肝脏细胞轻度水肿,而复合模型组肝脏脂肪滴明显。结论大肠埃希菌脂多糖加重了内膜斑块的形成,加剧了血脂代谢的紊乱和炎性反应。     

Exercise training increases membrane bound form of tumor necrosis factor-alpha receptors with decreases in the secretion of soluble forms of receptors in rat adipocytes

We examined the effects of exercise training (treadmill running over 9 weeks) on the ability of isolated adipocytes to secrete tumor necrosis factor-alpha (TNF-alpha) and type 1 soluble TNF receptor (sTNFR1) in vitro in Wistar rats. We also examined the effects of exercise training on the expression of membrane bound forms of type 1 TNF receptor (mTNFR1) in adipocyte crude membranes of the same rat subjects. Exercise training significantly increased the secretions of TNF-alpha from isolated adipocytes. Treatment with a cyclooxygenase inhibitor, either indomethacin (100 microM) or eicosatetraynoic acid (100 microM), significantly blocked the release of TNF-alpha from adipocytes in both exercise-trained rat group and sedentary control rat group, suggesting that some cyclooxygenase metabolite(s) acts as a ligand in TNF-alpha synthesis. Decreased amounts of TNF-alpha were found to be significantly greater in both exercise-trained rat group than in sedentary control rat group after incubation with inhibitors. Thus, the inhibitory effect of both indomethacin and eicosatetraynoic acid was significantly greater in adipocytes from exercise-trained rats. Both plasma sTNFR1 levels and adipocytes-derived sTNFR1 were found to be significantly less in the exercise-trained rat group. Western blot analysis revealed that exercise training remarkably increased the expressions of mTNFR1 in adipocyte crude membrane. Thus, exercise training enhanced the ability of isolated adipocytes to secrete TNF-alpha with reduced secretion of sTNFR1, and provoked the greater expressions of mTNFR1 in adipocyte crude membrane. These alterations may induce enhanced the autocrine effects of TNF-alpha within adipocytes in exercise-trained rats.     

Interleukin-18 reduces expression of cardiac tumor necrosis factor-alpha and atrial natriuretic peptide in a murine model of viral myocarditis

Heart failure is generally believed to begin with myocyte damage caused by a variety of insults, including ischemia, toxin or myocardial infection. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), has been hypothesized to play a pathogenetic role in the transition from compensated to decompensated heart failure. Interleukin-18 (IL-18), a recently cloned cytokine synthesized by Kupffer cells, activates macrophages. We examined the therapeutic effect of IL-18 on the modulation of TNF-alpha gene expression in failing heart in a murine model of heart failure caused by viral myocarditis. The heart weight (HW)/ body weight (BW) ratio in IL-18 treated mice 7 days after viral inoculation was significantly lower (P<0.01) than in the untreated controls. Myocardial necrosis and inflammatory cell infiltration were significantly lower in IL-18 treated mice than untreated mice 5 and 7 days after inoculation. The expression of TNF-alpha mRNA in the myocardium was significantly lower on days 5 and 7 in IL-18 treated mice than in infected untreated mice. We conclude that concurrent systemic administration of IL-18 is beneficial in mice with myocarditis, and may be mediated through reduced expression of TNF-alpha in the heart.     

Molecular heterogeneity of tumor endothelium

Tumors depend on a vascular supply for their growth. Tumor blood vessels, which are derived from normal tissue vasculature, display a markedly abnormal phenotype. Tumor endothelial properties are highly varied in space and time. An important goal is to delineate the range of phenotypes in tumor endothelium and to identify tumor endothelial-specific molecular signatures. This information should lead to a more complete understanding of the mechanisms of tumor growth, the discovery of new therapeutic targets, and the development of biomarkers for diagnosis and surveillance. The goals of this review are to outline recent advances in dissecting tumor endothelial-cell-specific gene expression, to address mechanisms of phenotypic heterogeneity in tumor vascular beds, and to discuss the therapeutic implications of these findings.     

睾酮对内皮细胞妊娠相关血浆蛋白-A mRNA表达的影响

目的:观察睾酮对内皮细胞妊娠相关血浆蛋白-A(PAPP-A)m RNA表达的影响。方法:人原代脐静脉内皮细胞,选择第3~4代生长状态良好的细胞用于实验。实验分组:1空白对照组;2肿瘤坏死因子(TNF)-α(终浓度100μg/L)培养细胞组;3TNF-α(终浓度100μg/L)及睾酮1×10-8mol/L培养细胞组;4睾酮1×10-8mol/L培养细胞组。实验结束后收集各组细胞,用RT-PCR方法检测各组细胞PAPP-A m RNA表达水平。结果:TNF-α作用2 h后,内皮细胞中PAPP-A m RNA表达水平升高(P0.05),且随TNF-α作用时间的延长,PAPP-A表达逐渐升高,在16小时达高峰;睾酮作用后,PAPP-A m RNA表达水平较TNF-α组明显降低(P0.01)。结论:TNF-α上调内皮细胞PAPP-A m RNA的表达,而睾酮抑制了TNF-α对PAPP-A分泌增加的刺激作用。     

Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells

Taraxacum officinale (TO) has been frequently used as a remedy for women's disease (e.g. breast and uterus cancer) and disorders of the liver and gallbladder. Several earlier studies have indicated that TO exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we investigated the effect of TO on the cytotoxicity and production of cytokines in human hepatoma cell line, Hep G2. Our results show that TO decreased the cell viability by 26%, and significantly increased the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1alpha production compared with media control (about 1.6-fold for TNF-alpha, and 2.4-fold for IL-1alpha, P < 0.05). Also, TO strongly induced apoptosis of Hep G2 cells as determined by flow cytometry. Increased amounts of TNF-alpha and IL-1alpha contributed to TO-induced apoptosis. Anti-TNF-alpha and IL-1alpha antibodies almost abolished it. These results suggest that TO induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.     

自噬抑制肿瘤坏死因子α诱导人胎盘胎儿来源间充质干细胞发生凋亡

目的:探讨TNF-α诱导人胎盘胎儿来源间充质干细胞(hfPMSCs)发生凋亡和自噬的作用,以及自噬对细胞凋亡的调控作用。方法:利用流式细胞术检测无血清培养hfPMSCs中CD73、CD90、CD105、CD14、CD34、CD45的表达;用终浓度20μg/L的TNF-α处理hfPMSCs 24h,以未处理细胞作为对照组。Annexin V/PI双染色检测TNF-α对hfPMSCs凋亡程度的影响;分别提取各组总蛋白,Western blot检测自噬标志基因LC3Ⅰ/Ⅱ的表达;利用mRFP-GFP-LC3腺病毒感染细胞,观察胞内点状聚集形成的情况;利用Atg5干扰慢病毒(si-Atg5)及阴性对照慢病毒(si-NC)感染hfPMSCs,Annexin V/PI双染色检测TNF-α对慢病毒感染后hfPMSCs凋亡程度的影响。结果:所培养细胞具有典型的MSCs形态,呈CD73~+CD90~+CD105~+/CD14~-CD34~-CD45~-细胞; Annexin V/PI染色结果显示,TNF-α作用24 h后,hfPMSCs凋亡数和凋亡率均高于对照组(P 0. 05);Western blot检测自噬标志蛋白表达结果表明,TNF-α可增加LC3Ⅱ的表达(P 0. 05);荧光共聚焦显微境观察到TNF-α可显著提高细胞中的点状聚集。利用si-Atg5感染细胞,抑制hfPMSCs自噬的发生,与对照慢病毒si-NC感染细胞比较,可显著促进TNF-α诱导hfPMSCs凋亡的发生(P 0. 05)。结论:TNF-α诱导的自噬抑制人胎盘胎儿来源MSCs凋亡的发生,具有一定的保护性作用。     

TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity

Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor-alpha converting enzyme (TACE) processing/activation. Using the furin-deficient LoVo cells, as well as the furin-proficient synoviocytes and HT1080 cells expressing the furin inhibitor alpha(1)-PDX, we demonstrate that furin activity alone is not sufficient for effective maturation and activation of the TACE enzyme. Data from in vitro and in vivo cleavage assays indicate that PACE-4, PC5/PC6, PC1 and PC2 can directly cleave the TACE protein and/or peptide. PC inhibition in macrophages reduced the release of soluble TNF-alpha from transmembrane pro-TNF-alpha. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation.     

Prolactin increases tumor necrosis factor alpha expression in peripheral CD14 monocytes of patients with rheumatoid arthritis

Tumor necrosis factor (TNF)-α is one of the major proinflammatory mediators of rheumatic arthritis (RA); the regulatory factors for TNF-α release is not fully understood. This study aims to investigate the role of prolactin receptor (PRLR) activation in regulating the expression and release of TNF-α from CD14⁺ monocytes. The results showed that the expression of PRLR was detectable in CD14⁺ monocytes of healthy subjects, which was markedly increased in RA patients. Exposure to PRL in the culture increased the expression and release of TNF-α from CD14⁺ monocytes, which was abolished by the PRLR gene silencing or blocking the mitogen activated protein (MAPK) pathway. We conclude that exposure to PRL increases TNF-α release from CD14⁺ monocytes of RA patients, which can be abolished by PRLR gene silencing or treating with MAPK inhibitor.     

The role of TNF-alpha in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk

The recent insight that inflammation contributes to the development of atherosclerosis and type 2 diabetes mellitus constitutes a major breakthrough in understanding the mechanisms underlying these conditions. In addition, it opens the way for new therapeutic approaches that might eventually decrease the prevalence of these public health problems. Tumor necrosis factor-alpha (TNF-alpha) has been shown to play a key role in these processes and thus might be a potential therapeutic target. Increased concentrations of TNF-alpha are found in acute and chronic inflammatory conditions (e.g., trauma, sepsis, infection, rheumatoid arthritis), in which a shift toward a proatherogenic lipid profile and impaired glucose tolerance occurs. Although therapeutic blockade of TNF-alpha worsens the prognosis in patients with abscesses and granulomatous infections, this strategy is highly beneficial in the case of chronic inflammatory conditions, including rheumatoid arthritis. Current investigations assessing the impact of anti-TNF agents on intermediary metabolism suggest that TNF-alpha blockade may improve insulin resistance and lipid profiles in patients with chronic inflammatory diseases.     

Differences in donor CXCR4 expression levels are correlated with functional capacity and therapeutic outcome of angiogenic treatment with endothelial colony forming cells

CXCR4 expression is important for cell migration and recruitment, suggesting that the expression levels of CXCR4 may be correlated with functional activity of implanted cells for therapeutic neovascularization. Here, we examined differences between umbilical cord blood (CB) donors in the CXCR4 levels of endothelial colony forming cells (ECFCs), which are a subtype of endothelial progenitor cells (EPCs). We investigated the relationships between CXCR4 expression level and SDF-1α-induced vascular properties in vitro, and their in vivo contributions to neovascularization. We found that ECFCs isolated from different donors showed differences in CXCR4 expression that were linearly correlated with SDF-1α-induced migratory capacity. ECFCs with high CXCR4 expression showed enhanced ERK and Akt activation in response to SDF-1α. In addition, SDF-1α-induced migration and ERK1/2, Akt, and eNOS activation were reduced by AMD3100, a CXCR4-specific peptide antagonist, or by siRNA-CXCR4. Administration of high-CXCR4-expressing ECFCs resulted in a significant increase in therapeutic potential for blood flow recovery, tissue healing and capillary density compared to low-CXCR4-expressing ECFCs in hindlimb ischemia. Taken together, the functional differences among ECFCs derived from different donors depended on the level of CXCR4 expression, suggesting that CXCR4 expression levels in ECFCs could be a predictive marker for success of ECFC-based angiogenic therapy.     

Characterization of rat and human Kupffer cells after cryopreservation

Kupffer cells (KC) are the resident macrophages of the liver and represent about 80% of the total fixed macrophage population. They are involved in disease states such as endotoxin shock, alcoholic liver diseases and other toxic-induced liver injury. They release physiologically active substances such as eicosanoids and inflammatory cytokines (IL-1, IL-6, TNFalpha), and produce free radical species. Thus, KC are attractive targets for anti-inflammatory therapies and potential candidates responsible for differences in inflammation in liver disease seen between different individuals. However, to perform parallel in vitro experiments with KC from different donors a suitable method for conservation of KC would be necessary. Therefore, the present study evaluated, whether rat and human KC can be frozen, stored and recovered without losing their functional integrity. Rat and human KC were isolated and either cultured under standard conditions (fresh KC) or cryopreserved in special freezing medium (cryopreserved KC). At least 24 h later, cryopreserved KC were thawed, brought into suspension and seeded in the same density as fresh cells for subsequent experiments. Viability of cultured KC was analyzed by trypan blue exclusion. LPS (or PBS as control) stimulation was performed at different time points and cytokine release was analyzed with IL-6 and TNFalpha ELISAs, respectively. Phagocytic capacity was investigated by using a specific phagocytosis assay and FACS analysis. The recovery rate after thawing was around 57% for rat and around 65% for human cryopreserved KC. The results indicate, that KC can successfully be cryopreserved with an adequate recovery rate of viable cells. The properties of fresh and frozen KC can also be compared after thawing. Freshly isolated and cryopreserved cultured KC showed near-normal morphology and did not differ in the cultivation profiles over a period of 72 h. One to three days after seeding, frozen rat or human KC also retained inducible functions such as the production of TNFalpha or IL-6 after LPS challenge. Finally, regardless if they were cryopreserved or not, no differences in the phagocytic activities of the cells were obtained. Taken together, it is concluded that cryopreservation of KC does not change the physiological characteristics of the cells in vitro. Therefore, the method used here for cryopreservation of especially human KC allows the accumulation of KC from several donors for parallel in vitro experiments.     

Activation of PI3K-Akt-GSK3beta pathway mediates hepatocyte growth factor inhibition of RANTES expression in renal tubular epithelial cells

Hepatocyte growth factor (HGF) was recently reported to ameliorate renal inflammation in a rat model of chronic renal failure. HGF exerted its action through suppression of RANTES expression in renal tubules. In the present study, we utilized an in vitro model of human kidney proximal tubule epithelial cells (HKC) to elucidate the mechanisms of RANTES suppression by HGF. HGF significantly suppressed basal and TNF-alpha-induced mRNA and protein expression of RANTES in a time and dose dependent fashion. HGF elicited PI3K-Akt activation and inhibited GSK3, a downstream transducer of PI3K-Akt, by inhibitory phosphorylation at Ser-9. When the PI3K-Akt pathway was blocked by wortmannin, HGF inhibition of RANTES was abrogated, demonstrating that the PI3K-Akt pathway is necessary for HGF action. In addition, specific inhibition of GSK3 activity by lithium ion suppressed basal and TNF-alpha-induced RANTES expression, reminiscent of the action of HGF. To further investigate the role of GSK3 in modulating RANTES expression, we examined the effect of forced expression of wild type GSK3beta or an uninhibitable mutant GSK3beta, in which the regulatory Ser-9 residue is changed to alanine (S9A-GSK3beta) in HKC. Overexpression of wild type GSK3beta did not alter the inhibitory action of HGF on RANTES. In contrast, expression of S9A-GSK3beta abolished HGF inhibition of basal and TNF-alpha stimulated RANTES expression. These findings suggest that PI3K-Akt activation and subsequent inhibitory phosphorylation of GSK3beta are required for HGF-induced suppression of RANTES in HKC.     

内毒素诱导大鼠急性肺损伤模型的动态研究

目的:建立内毒素诱导大鼠急性肺损伤的模型并筛选出敏感检测指标。方法:90只wistar大鼠随机分为10组,其中9组以气管内滴注内毒素(Lipopolysaccharide,LPS)建立大鼠急性肺损伤(Acute lung injury,ALI)模型,另一组作为空白对照组。观察造模8、12、24、36、48、72、96、120 h后的肺泡灌洗液(BALF)中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)水平、肺组织的病理形态学变化、12h造模组与空白组的BALF中的多形核白细胞(PMN)百分比和蛋白浓度。结果:造模后BALF中的TNF-α、IL-6的浓度随时间延长显著升高且均在24 h达到峰值(P0.01);肺组织病理损伤也逐渐加重,12 h已出现明显的肺泡损伤、肺水肿、炎性细胞浸润等病变;12 h模型组BALF中PMN百分比和蛋白浓度较空白对照组显著增加(P0.01)。结论:在该实验条件下,气管内滴注LPS 8 mg/kg,12 h后即可建立ALI模型,可通过检测BALF中的TNF-α、IL-6浓度及肺组织的病变程度等指标进行模型评价。     

Role of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways in regulating replication of Penicillium marneffei in human macrophages

Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To elucidate the mechanisms involved, we investigated the role of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) pathways in cytokine expression, phagosome–lysosome fusion and replication of P. marneffei in P. marneffei-infected human macrophages. Analysis of both ERK1/2 and p38 showed rapid phosphorylation in response to P. marneffei. Using specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that ERK1/2 and p38 were essential for P. marneffei-induced tumor necrosis factor-α production, whereas p38, but not that of ERK, was essential for IL-10 production. Furthermore, the presence of PD98059 always decreased phagosomal acidification and maturation and increased intracellular multiplication of P. marneffei, whereas the use of SB203580 always increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that a proper balance of between ERK1/2 and p38 may play an important role in controlling the replication of P. marneffei. Our findings further indicate a novel therapeutic avenue for treating P. marneffei by stimulating ERK1/2 or activating ERK1/2-dependent mechanisms.     

Relationships between cobalamin,epidermal growth factor,and normal prions in the myelin maintenance of central nervous system

Cobalamin (Cbl), epidermal growth factor (EGF), and prions (PrPs) are key molecules for myelin maintenance in the central and peripheral nervous systems. Cbl and EGF increase normal prion (PrP^C) synthesis and PrP^C levels in rat spinal cord (SC) and elsewhere. Cbl deficiency increases PrP^C levels in rat SC and cerebrospinal fluid (CSF), and decreases PrP^C-mRNA levels in rat SC. The administration of anti-octapeptide repeat PrP^C region antibodies (Abs) to Cbl-deficient (Cbl-D) rats prevents SC myelin lesions and a local increase in tumor necrosis factor (TNF)-α levels, whereas anti-TNF-α Abs prevent SC myelin lesions and the increase in SC and CSF PrP^C levels. As it is known that both Cbl and EGF regulate SC PrP^C synthesis independently, and that Cbl regulates SC EGF synthesis, EGF may play both Cbl-independent and Cbl-dependent roles. When Cbl-D rats undergo Cbl replacement therapy, SC PrP^C levels are similar to those observed in Cbl-D rats. In rat frontal cortex (which is marginally affected by Cbl deficiency in histological terms), Cbl deficiency decreases PrP^C levels and the increase induced by Cbl replacement leads to their normalization. Increased nerve PrP^C levels are detected in the myelin lesions of the peripheral neuropathy of Cbl-D rats, and CSF PrP^C levels are also increased in Cbl-D patients (but not in patients with Cbl-unrelated neurological diseases). Various common steps in the downstream signaling pathway of Cbl, EGF, and PrP^C underlines the close relationship between the three molecules in keeping myelin normal.