Endothelial Dysfunction in Tristetraprolin-deficient Mice Is Not Caused by Enhanced Tumor Necrosis Factor-α Expression

Cardiovascular events are important co-morbidities in patients with chronic inflammatory diseases like rheumatoid arthritis. Tristetraprolin (TTP) regulates pro-inflammatory processes through mRNA destabilization and therefore TTP-deficient mice (TTP^−/− mice) develop a chronic inflammation resembling human rheumatoid arthritis. We used this mouse model to evaluate molecular signaling pathways contributing to the enhanced atherosclerotic risk in chronic inflammatory diseases. In the aorta of TTP^−/− mice we observed elevated mRNA expression of known TTP targets like tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α, as well as of other pro-atherosclerotic mediators, like Calgranulin A, Cathepsin S, and Osteopontin. Independent of cholesterol levels TTP^−/− mice showed a significant reduction of acetylcholine-induced, nitric oxide-mediated vasorelaxation. The endothelial dysfunction in TTP^−/− mice was associated with increased levels of reactive oxygen and nitrogen species (RONS), indicating an enhanced nitric oxide inactivation by RONS in the TTP^−/− animals. The altered RONS generation correlates with increased expression of NADPH oxidase 2 (Nox2) resulting from enhanced Nox2 mRNA stability. Although TNF-α is believed to be a central mediator of inflammation-driven atherosclerosis, genetic inactivation of TNF-α neither improved endothelial function nor normalized Nox2 expression or RONS production in TTP^−/− animals. Systemic inflammation caused by TTP deficiency leads to endothelial dysfunction. This process is independent of cholesterol and not mediated by TNF-α solely. Thus, other mediators, which need to be identified, contribute to enhanced cardiovascular risk in chronic inflammatory diseases.     

Overexpression of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) in HepG2 cells leads to increased hyaluronan synthesis and cell proliferation by up-regulation of cyclin D1 in AKT-dependent pathway

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.     

Targeted Disruption of Ig-Hepta/Gpr116 Causes Emphysema-like Symptoms That Are Associated with Alveolar Macrophage Activation

Ig-Hepta/GPR116 is a member of the G protein-coupled receptor family predominantly expressed in the alveolar type II epithelial cells of the lung. Previous studies have shown that Ig-Hepta is essential for lung surfactant homeostasis, and loss of its function results in high accumulation of surfactant lipids and proteins in the alveolar space. Ig-Hepta knock-out (Ig-Hepta^−/−) mice also exhibit emphysema-like symptoms, including accumulation of foamy alveolar macrophages (AMs), but its pathogenic mechanism is unknown. Here, we show that the bronchoalveolar lavage fluid obtained from Ig-Hepta^−/− mice contains high levels of inflammatory mediators, lipid hydroperoxides, and matrix metalloproteinases (MMPs), which are produced by AMs. Accumulation of reactive oxygen species was observed in the AMs of Ig-Hepta^−/− mice in an age-dependent manner. In addition, nuclear factor-κB (NF-κB) is activated and translocated into the nuclei of the AMs of Ig-Hepta^−/− mice. Release of MMP-2 and MMP-9 from the AMs was strongly inhibited by treatment with inhibitors of oxidants and NF-κB. We also found that the level of monocyte chemotactic protein-1 is increased in the embryonic lungs of Ig-Hepta^−/− mice at 18.5 days postcoitum, when AMs are not accumulated and activated. These results suggest that Ig-Hepta plays an important role in regulating macrophage immune responses, and its deficiency leads to local inflammation in the lung, where AMs produce excessive amounts of reactive oxygen species and up-regulate MMPs through the NF-κB signaling pathway.     

NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μM) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.     

Non-thermal Plasma Activates Human Keratinocytes by Stimulation of Antioxidant and Phase II Pathways

Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.     

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Enhances Activation of p38 and ERK MAPKs,Increasing Interleukin-6 Synthesis in Airway Epithelial Cells Exposed to Pseudomonas aeruginosa

In cystic fibrosis (CF), the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRΔF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRΔF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRΔF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRΔF508 mutation in airway epithelial cells contributes to increase inflammation of the airways.     

Biochemical analysis of matrix metalloproteinase activation of chemokines CCL15 and CCL23 and increased glycosaminoglycan binding of CCL16

Leukocyte migration and activation is orchestrated by chemokines, the cleavage of which modulates their activity and glycosaminoglycan binding and thus their roles in inflammation and immunity. Early research identified proteolysis as a means of both activating or inactivating CXC chemokines and inactivating CC chemokines. Recent evidence has shown activating cleavages of the monocyte chemoattractants CCL15 and CCL23 by incubation with synovial fluid, although the responsible proteases could not be identified. Herein we show that CCL15 is processed in human synovial fluid by matrix metalloproteinases (MMPs) and serine proteases. Furthermore, a family-wide investigation of MMP processing of all 14 monocyte-directed CC chemokines revealed that each is precisely cleaved by one or more MMPs. By MALDI-TOF-MS, 149 cleavage sites were sequenced including the first reported instance of CCL1, CCL16, and CCL17 proteolysis. Full-length CCL15-(1-92) and CCL23-(1-99) were cleaved within their unique 31 and 32-amino acid residue extended amino termini, respectively. Unlike other CCL chemokines that lose activity and become receptor antagonists upon MMP cleavage, the prominent MMP-processed products CCL15-(25-92, 28-92) and CCL23-(26-99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays. MMP processing of CCL16-(1-97) in its extended carboxyl terminus yields two products, CCL16-(8-77) and CCL16-(8-85), with both showing unexpected enhanced glycosaminoglycan binding. Hence, our study reveals for the first time that MMPs activate the long amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation.     

Pulmonary biocompatibility assessment of inhaled single-wall and multiwall carbon nanotubes in BALB/c mice

With the widespread application of carbon nanotubes (CNTs) in diverse commercial processes, scientists are now concerned about the potential health risk of occupational exposures. In this study, CNT-induced pulmonary toxicity was investigated by exposing BALB/c mice to aerosolized single-wall (SW) CNT and multiwall (MW) CNT (5 μg/g of mice) for 7 consecutive days in a nose-only exposure system. Microscopic studies showed that inhaled CNTs were homogeneously distributed in the mouse lung. The total number of bronchoalveolar lavage polymorphonuclear leukocytes recovered from the mice exposed to SWCNT and MWCNT (1.2 × 10(6) ± 0.52 and 9.87 × 10(5) ± 1.45; respectively) was significantly greater than control mice (5.46 × 10(5) ± 0.78). Rapid development of pulmonary fibrosis in mice that inhaled CNT was also confirmed by significant increases in the collagen level. The lactate dehydrogenase levels were increased nearly 2- and 2.4-fold in mice that inhaled SWCNT and MWCNT, respectively, as compared with control mice. In addition, exposure of CNTs to mice showed a significant (p < 0.05) reduction of antioxidants (glutathione, superoxide dismutase, and catalase) and induction of oxidants (myloperoxidase, oxidative stress, and lipid peroxidation) compared with control. Apoptosis-related proteins such as caspase-3 and -8 activities were also significantly increased in mice that inhaled CNT than in control mice. Together, this study shows that inhaled CNTs induce inflammation, fibrosis, alteration of oxidant and antioxidant levels, and induction of apoptosis-related proteins in the lung tissues to trigger cell death.     

An efficient one-pot synthesis of thiochromeno[3,4-d]pyrimidines derivatives: Inducing ROS dependent antibacterial and anti-biofilm activities

An efficient synthesis of thiochromeno[3,4-d]pyrimidine derivatives has been achieved successfully via a one-pot three-component reaction of thiochrome-4-one, aromatic aldehyde and thiourea in the presence of 1-butyl-3-methyl imidazolium hydrogen sulphate [Bmim]HSO₄. This new protocol has the advantages of environmental friendliness, high yields, short reaction times, and convenient operation. Furthermore, among all the tested derivatives, compounds 4b and 4c exhibited promising antibacterial, minimum bactericidal concentration and anti-biofilm activities against Staphylococcus aureus MTCC 96, Staphylococcus aureus MLS16 MTCC 2940 and Bacillus subtilis MTCC 121. The compound 4c also showed promising intracellular ROS accumulation in Staphylococcus aureus MLS16 MTCC 2940 comparable to that of ciprofloxacin resulting in apoptotic cell death of the bacterium.     

Constitutive NADPH oxidase 4 activity resides in the composition of the B-loop and the penultimate C terminus

Redox regulation of signaling molecules contributes critically to propagation of intracellular signals. The main source providing reactive oxygen species (ROS) for these physiological processes are activated NADPH oxidases (Nox/Duox family). In a pathophysiological context, some NADPH oxidase complexes produce large amounts of ROS either as part of the antimicrobial immune defense or as pathologic oxidative stress in many chronic diseases. Thus, understanding the switch from a dormant, inactive conformation to the active state of these enzymes will aid the development of inhibitors. As exogenously expressed Nox4 represents the only constitutively active enzyme in this family, analysis of structural determinants that permit this active conformation was undertaken. Our focus was directed toward a cell-based analysis of the first intracellular loop, the B-loop, and the C-terminus, two regions of Nox family enzymes that are essential for electron transfer. Mutagenesis of the B-loop identified several unique residues and a polybasic motif that contribute to the catalytic activity of Nox4. By using a multifaceted approach, including Nox4-Nox2 chimeras, mutagenesis, and insertion of Nox2 domains, we show here that the penultimate 22 amino acids of Nox4 are involved in constitutive ROS generation. The appropriate spacing of the C-terminal Nox4 sequence may cooperate with a discrete arginine-based interaction site in the B-loop, providing an intrinsically active interface that could not be disrupted by peptides derived from the Nox4 C-terminus. These results indicate that accessibility for a Nox4-specific peptide inhibitor might be difficult to achieve in vivo.     

Vitellogenin Recognizes Cell Damage through Membrane Binding and Shields Living Cells from Reactive Oxygen Species

Large lipid transfer proteins are involved in lipid transportation and diverse other molecular processes. These serum proteins include vitellogenins, which are egg yolk precursors and pathogen pattern recognition receptors, and apolipoprotein B, which is an anti-inflammatory cholesterol carrier. In the honey bee, vitellogenin acts as an antioxidant, and elevated vitellogenin titer is linked to prolonged life span in this animal. Here, we show that vitellogenin has cell and membrane binding activity and that it binds preferentially to dead and damaged cells. Vitellogenin binds directly to phosphatidylcholine liposomes and with higher affinity to liposomes containing phosphatidylserine, a lipid of the inner leaflet of cell membranes that is exposed in damaged cells. Vitellogenin binding to live cells, furthermore, improves cell oxidative stress tolerance. This study can shed more light on why large lipid transfer proteins have a well conserved α-helical domain, because we locate the lipid bilayer-binding ability of vitellogenin largely to this region. We suggest that recognition of cell damage and oxidation shield properties are two mechanisms that allow vitellogenin to extend honey bee life span.     

Angiotensin-II and MARCKS: A HYDROGEN PEROXIDE- AND RAC1-DEPENDENT SIGNALING PATHWAY IN VASCULAR ENDOTHELIUM

MARCKS is an actin-binding protein that modulates vascular endothelial cell migration and cytoskeleton signaling (Kalwa, H., and Michel, T. (2011) J. Biol. Chem. 286, 2320-2330). Angiotensin-II is a vasoactive peptide implicated in vascular physiology as well as pathophysiology; the pathways connecting angiotensin-II and cytoskeletal remodeling are incompletely understood. Here we show that MARCKS is expressed in intact arterial preparations, with prominent staining of the endothelium. In endothelial cells, angiotensin-II-promoted MARCKS phosphorylation is abrogated by PEG-catalase, implicating endogenous H(2)O(2) in the angiotensin-II response. Studies using the H(2)O(2) biosensor HyPer2 reveal that angiotensin-II promotes increases in intracellular H(2)O(2). We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is attenuated by PEG-catalase. siRNA-mediated Rac1 knockdown blocks angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP(2)) biosensor revealed that angiotensin-II PIP(2) regulation depends on MARCKS and H(2)O(2). siRNA-mediated knockdown of MARCKS or Rac1 attenuates receptor-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. These studies establish a critical role for H(2)O(2) in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is critically dependent on MARCKS, Rac1, and c-Abl.     

Ursolic acid, a pentacyclin triterpene, potentiates TRAIL-induced apoptosis through p53-independent up-regulation of death receptors: evidence for the role of reactive oxygen species and JNK

Discovery of the molecular targets of traditional medicine and its chemical footprints can validate the use of such medicine. In the present report, we investigated the effect of ursolic acid (UA), a pentacyclic triterpenoid found in rosemary and holy basil, on apoptosis induced by TRAIL. We found that UA potentiated TRAIL-induced apoptosis in cancer cells. In addition, UA also sensitized TRAIL-resistant cancer cells to the cytokine. When we investigated the mechanism, we found that UA down-regulated cell survival proteins and induced the cell surface expression of both TRAIL receptors, death receptors 4 and 5 (DR4 and -5). Induction of receptors by UA occurred independently of cell type. Gene silencing of either receptor by small interfering RNA reduced the apoptosis induced by UA and the effect of TRAIL. In addition, UA also decreased the expression of decoy receptor 2 (DcR2) but not DcR1. Induction of DRs was independent of p53 because UA induced DR4 and DR5 in HCT116 p53(-/-) cells. Induction of DRs, however, was dependent on JNK because UA induced JNK, and its pharmacologic inhibition abolished the induction of the receptors. The down-regulation of survival proteins and up-regulation of the DRs required reactive oxygen species (ROS) because UA induced ROS, and its quenching abolished the effect of the terpene. Also, potentiation of TRAIL-induced apoptosis by UA was significantly reduced by both ROS quenchers and JNK inhibitor. In addition, UA was also found to induce the expression of DRs, down-regulate cell survival proteins, and activate JNK in orthotopically implanted human colorectal cancer in a nude mouse model. Overall, our results showed that UA potentiates TRAIL-induced apoptosis through activation of ROS and JNK-mediated up-regulation of DRs and down-regulation of DcR2 and cell survival proteins.     

Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H₂O₂ was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H₂O₂ to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H₂O₂ on kinase activity. Similar to the results obtained with H₂O₂-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H₂O₂ are increased. These results demonstrate that physiologically relevant concentrations of H₂O₂ can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.     

Inhibitors of catalase-amyloid interactions protect cells from beta-amyloid-induced oxidative stress and toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.     

Mitochondrial dysfunction and oxidative stress contribute to the pathogenesis of spinocerebellar ataxia type 12 (SCA12)

Spinal cerebellar ataxia type 12 (SCA12) has been attributed to the elevated expression of ppp2r2b. To better elucidate the pathomechanism of the neuronal disorder and to search for a pharmacological treatment, Drosophila models of SCA12 were generated by overexpression of a human ppp2r2b and its Drosophila homolog tws. Ectopic expression of ppp2r2b or tws caused various pathological features, including neurodegeneration, apoptosis, and shortened life span. More detailed analysis revealed that elevated ppp2r2b and tws induced fission of mitochondria accompanied by increases in cytosolic reactive oxygen species (ROS), cytochrome c, and caspase 3 activity. Transmission electron microscopy revealed that fragmented mitochondria with disrupted cristae were engulfed by autophagosomes in photoreceptor neurons of flies overexpressing tws. Additionally, transgenic flies were more susceptible to oxidative injury induced by paraquat. By contrast, ectopic Drosophila Sod2 expression and antioxidant treatment reduced ROS and caspase 3 activity and extended the life span of the SCA12 fly model. In summary, our study demonstrates that oxidative stress induced by mitochondrial dysfunction plays a causal role in SCA12, and reduction of ROS is a potential therapeutic intervention for this neuropathy.     

肿瘤微环境中ROS介导IgG表达对膀胱癌细胞增殖和侵袭能力的影响

摘要 目的：探讨肿瘤微环境（TME）中活性氧（ROS）介导免疫球蛋白G（IgG）表达对膀胱癌EJ细胞增殖、迁移和侵袭能力的影响。方法：临床收集的18例膀胱癌患者样本,通过Western blot法检测膀胱癌和癌旁正常组织样本中IgG表达量。利用免疫荧光染色（IF）技术分别对膀胱癌组织和癌旁正常组织中ROS和IgG分子进行共定位和相对定量分析。将活性氧清除剂N-乙酰基-L-半胱氨酸（NAC）加入膀胱癌细胞EJ中,实验分为3组：空白组（EJ细胞）、阴性对照组（EJ+PBS）、实验组（EJ+PBS+NAC）,10 mM NAC药物处理48小时后,运用DHE-ROS荧光探针技术和Western blot实验检测药物NAC对ROS和IgG相对表达水平的影响;通过克隆集落形成实验、划痕实验、Transwell实验检测去除ROS后对膀胱癌细胞增殖、迁移和侵袭的影响。结果：人体膀胱癌组织中ROS和IgG分子表达水平显著高于癌旁正常组织（P<0.001）;荧光显微镜显示膀胱肿瘤组织中正常膀胱尿路上皮细胞组织被肿瘤细胞严重破坏,结构紊乱不规则,IgG和ROS表达水平均升高,而癌旁组织膀胱尿路上皮组织的结构均匀规则;NAC药物处理EJ细胞后,与空白组和阴性对照组相比ROS和IgG表达显著降低,同时实验组细胞的增殖、迁移和侵袭能力明显下降（P<0.01）。结论：ROS和IgG在临床膀胱癌组织和体外膀胱癌细胞株EJ中均显著高表达,在肿瘤微环境中ROS通过调控IgG表达,从而促进膀胱癌细胞的增殖、迁移、侵袭。ROS和IgG可能成为膀胱癌早期诊断和生物治疗的临床新靶点。