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1.
Abstract Clinical observations and epidemiological surveys indicated that the prevalence of hypertension and heart diseases is increased in cold regions or during winter. Cold exposure increased NADPH oxidase gp91(phox) protein expression in heart, kidneys, and aorta in rats. The aim of this study was to investigate if RNA interference (RNAi) silencing of gp91(phox) would attenuate cold-induced hypertension and cardiovascular and renal damage. The recombinant adeno-associated virus serotype 2 (AAV-2) vector carrying gp91(phox)-shRNA (gp91-shRNA) was constructed for inhibiting gp91(phox) protein expression in cold-exposed rats. Blood pressure (BP) was monitored using a telemetry system. BP was increased in the Control-shRNA and PBS groups within 1 week of exposure to moderate cold (5°C) and reached a plateau after 7 weeks. The cold-induced increase in BP was attenuated significantly by intravenous delivery of gp91-shRNA (1.25×10(10) particles/rat, 0.5?mL). One single dose of gp91-shRNA controlled hypertension for up to 10 weeks. In addition, gp91-shRNA reversed cold-induced vascular dysfunction. gp91-shRNA abolished the cold-induced up-regulation of gp91(phox) protein expression in heart, kidneys, and aorta, confirming effective silencing of gp91(phox). The cold-induced increases in NADPH oxidase activity and superoxide production were eliminated by silencing of gp91(phox), suggesting that the cold-induced up-regulation of NADPH oxidase activity may be attributed to the increased gp91(phox) protein expression. RNAi silencing of gp91(phox) abolished cold-induced cardiac and renal hypertrophy and attenuated aortic, coronary, and renal remodeling. The up-regulation of gp91(phox) may play a critical role in cold-induced cardiovascular dysfunction and organ damage. AAV delivery of gp91-shRNA may be a new and effective therapeutic approach for cold-related cardiovascular disorders. 相似文献
2.
Teixeira CE Priviero FB Webb RC 《The Journal of pharmacology and experimental therapeutics》2007,322(3):1093-1102
We aimed to characterize the relaxation induced by the soluble guanylyl cyclase (sGC) stimulator 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and its pharmacological interactions with nitric oxide (NO) in the corpus cavernosum (CC) from wild-type (WT), endothelial nitric-oxide synthase (eNOS)(-/-), and neuronal (n)NOS(-/-) mice. The effect of BAY 41-2272 on superoxide formation and NADPH oxidase expression was also investigated. Tissues were mounted in myographs for isometric force recording. Enzyme immunoassay kits were used for cGMP determination. sGC activity was determined in the supernatant fractions of the cavernosal samples by the conversion of GTP to cGMP. Superoxide formation and expression of NADPH oxidase subunits were studied using the reduction of ferricytochrome c and Western blot analysis, respectively. BAY 41-2272 (0.01-10 microM) relaxed CC with pEC(50) values of 6.36 +/- 0.07 (WT), 6.27 +/- 0.06 (nNOS(-/-)), and 5.88 +/- 0.07 (eNOS(-/-)). The relaxations were accompanied by increases in cGMP levels. N(omega)-Nitro-L-arginine methyl ester inhibited BAY 41-2272-evoked responses in CC from WT and nNOS(-/-), but not eNOS(-/-).1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one reduced and sildenafil potentiated the relaxations induced by BAY 41-2272 in all groups. BAY 41-2272 enhanced NO (endogenous and exogenous)-induced relaxations in a concentration-dependent manner. Expression and activity of sGC was similar among the different groups. Superoxide formation was reduced by BAY 41-2272 (0.1-1 microM). The compound also inhibited p22(phox) and gp91(phox) expression induced by 9,11-dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F(2 alpha (U46619). Our results demonstrated that sGC activation in the penis by BAY 41-2272 directly or via enhancement of NO effects may provide a novel treatment for erectile dysfunction, particularly in the event of an increased intrapenile oxidative stress. 相似文献
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Mastroeni P Vazquez-Torres A Fang FC Xu Y Khan S Hormaeche CE Dougan G 《The Journal of experimental medicine》2000,192(2):237-248
The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91phox(-/)-, iNOS(-/)-, and congenic wild-type mice. Although both gp91phox(-/)- and iNOS(-/)- mice demonstrated increased susceptibility to infection with S. typhimurium compared with wild-type mice, the kinetics of bacterial replication were dramatically different in the gp91phox(-/)- and iNOS(-/)- mouse strains. Greater bacterial numbers were present in the spleens and livers of gp91phox(-/)- mice compared with C57BL/6 controls as early as day 1 of infection, and all of the gp91phox(-/)- mice succumbed to infection within 5 d. In contrast, an increased bacterial burden was detected within reticuloendothelial organs of iNOS(-/)- mice only beyond the first week of infection. Influx of inflammatory CD11b(+) cells, granuloma formation, and serum interferon gamma levels were unimpaired in iNOS(-/)- mice, but the iNOS-deficient granulomas were unable to limit bacterial replication. The NADPH phagocye oxidase and iNOS are both required for host resistance to wild-type Salmonella, but appear to operate principally at different stages of infection. 相似文献
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Magi S Castaldo P Carrieri G Scorziello A Di Renzo G Amoroso S 《The Journal of pharmacology and experimental therapeutics》2005,315(1):291-296
In SH-SY5Y, a human neuroblastoma cell line, Aroclor 1254 (A1254), induced a dose-dependent (10-50 microg/ml) intracellular calcium concentration ([Ca2+]i) increase. Two rather specific sodium-calcium (Na+-Ca2+) exchanger (NCX) inhibitors, bepridil (10 microM) and KB-R7943 [2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate] (10 microM), reduced A1254-induced [Ca2+]i increase. A 24-h exposure to 30 microg/ml A1254 caused remarkable SH-SY5Y neuroblastoma cell damage. It is noteworthy that both bepridil and KB-R7943 counteracted A1254-induced neuronal injury. These results indicate that NCX contributes to [Ca2+]i increase and neuronal injury induced by A1254. RT-PCR experiments revealed in SH-SY5Y neuroblastoma cells the expression of NCX1 and NCX3 isoforms. To investigate which isoform was involved in [Ca2+]i increase and neuronal damage induced by A1254, we used specific antisense oligodeoxynucleotides (ODNs) to reduce NCX1 or NCX3 protein expression. The results showed that only NCX1 ODN reduced [Ca2+]i increase and neuronal injury induced by A1254. In conclusion, these results indicate that NCX1 may participate to [Ca2+]i increase and neurotoxicity evoked by A1254 in SH-SY5Y neuroblastoma cells. 相似文献
5.
Inflammation and oxidative stress play important roles in the pathogenesis of neurodegenerative disorders such as stroke, traumatic injury, Parkinson disease, and Alzheimer disease. Paeonol, a natural compound extracted from Moutan cortex, is a potent anti-inflammatory and antioxidative agent. The aim of this study was to investigate the neuroprotective mechanisms of paeonol on lipopolysaccharide (LPS)-induced inflammation in rat primary microglia and 6-hydroxydopamine-induced oxidative damage in cortical neurons. In LPS-treated microglia, paeonol attenuated the overexpression of inducible nitric oxide synthase and cyclooxygenase 2, leading to the decrease in nitric oxide and prostaglandin E2 production, respectively. Paeonol also suppressed LPS-induced phosphorylation of extracellular signal-regulated kinase and Jun N-terminal kinase. In addition, LPS-stimulated NADPH oxidase activation and reactive oxygen species production were attenuated by paeonol. Paeonol-induced upregulation of heme oxygenase 1 was also observed. Moreover, paeonol attenuated LPS-treated microglia culture medium-induced neuron cells death. Posttreatment with paeonol also reduced inflammatory responses in LPS-activated microglia and increased cell viability in LPS-treated microglia culture medium-treated neurons. Furthermore, in 6-hydroxydopamine-treated cortical neurons, paeonol not only decreased reactive oxygen species production but also increased cell viability, superoxide dismutase activity, and the antiapoptotic protein B-cell lymphoma 2 expression. Taken together, the present results suggest that paeonol might be a potential neuroprotective agent via inhibiting microglia-mediated inflammation and oxidative stress-induced neuronal damage. 相似文献
6.
Simian immunodeficiency virus lentivector corrects human X-linked chronic granulomatous disease in the NOD/SCID mouse xenograft 总被引:1,自引:0,他引:1
Naumann N De Ravin SS Choi U Moayeri M Whiting-Theobald N Linton GF Ikeda Y Malech HL 《Gene therapy》2007,14(21):1513-1524
X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the phagocyte nicotinamide dinucleotide phosphate oxidase catalytic subunit gp91(phox). Gene therapy targeting hematopoietic stem cells (HSCs) can correct CGD, but permanent correction remains a challenge. Lentiviral vectors have become attractive tools for gene transfer, and they may have the potential to transduce very primitive HSCs. We used a self-inactivating RD114/TR-pseudotyped simian immunodeficiency virus (SIVmac)-based vector encoding human gp91(phox) for ex vivo transduction of peripheral blood-mobilized stem cells (PBSCs) from patients with X-CGD. In PBSCs from two patients, ex vivo transduction efficiencies of 40.5 and 46% were achieved, and correction of oxidase activity was observed in myeloid cells differentiating in culture. When transduced PBSCs from these patients were transplanted into nonobese diabetic/severe combined immunodeficient mice and compared to normal control, 10.5 and 7.3% of the human myeloid cells in bone marrow developing at 6 weeks from the human xenografts expressed the gp91(phox) transgene. Sustained functional correction of oxidase activity was documented in myeloid cells differentiated from engrafted transduced PBSCs. Transgene marking was polyclonal as assessed by vector integration site analysis. These data suggest that RD114/TR SIVmac-based vectors might be suitable for gene therapy of CGD and other hereditary hematologic diseases. 相似文献
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BackgroundMiR-34 family members have been previously shown to play potential functional role in Parkinson’s disease (PD) pathogenesis. However, the regulatory role of miR-34a has not been demonstrated in PD yet. This study aims to clarify the potential neuroprotective effect of Schisandrin B (Sch B) involving miR-34a function in 6-OHDA-induced PD model.MethodsThe expression changes of miR-34a and Nrf2 pathway related genes were detected in 6-OHDA-treated SH-SY5Y cells under Sch B pretreatment. Cell viability and PD feathers of 6-OHDA-induced PD mice were measured for neuroprotection assessment. The regulation of miR-34a on Nrf2 activity and expression was demonstrated through gain-of-function and loss-of-function studies, while the regulatory role of miR-34a in the neuroprotection of Sch B was investigated both in vitro and in vivo.ResultsSch B pretreatment ameliorated 6-OHDA-induced changes in vitro, like upregulated miR-34a expression, inhibited Nrf2 pathways and decreased cell survival, and PD feathers in vivo. Moreover, Nrf2 was negatively regulated by miR-34a, while miR-34a overexpression inhibited the neuroprotection of Sch B in both dopaminergic SH-SY5Y cells and PD mice.ConclusionSch B showed neuroprotective effect in 6-OHDA-induced PD pathogenesis, which could be inhibited by miR-34a, involving the negative regulatory mechanism of miR-34a on Nrf2 pathways. 相似文献
9.
Lekontseva O Chakrabarti S Jiang Y Cheung CC Davidge ST 《The Journal of pharmacology and experimental therapeutics》2011,339(2):367-375
Estrogen has antihypertensive and vasorelaxing properties, partly via activation of endothelial nitric-oxide synthase (eNOS). Recently, neuronal nitric-oxide synthase (nNOS) has been detected in vascular cells, although the significance of this is unclear. Estrogen was found to stimulate nNOS in certain cultured cells. We hypothesized that estrogen regulates vascular tone partly via endothelium-derived nNOS. Human umbilical vein endothelial cells were used to test whether acute (5 min) stimulation with 17β-estradiol (E2) at 1 or 10 nM affected nNOS activity. Small mesenteric arteries from Sprague-Dawley rats were examined for relaxation to E2 (0.001-10 μM) in the absence or presence of selective nNOS inhibitor [N-propyl-L-arginine (L-NPA); 2 μM] or pan-NOS inhibitor [Nω-nitro-L-arginine methyl ester (L-NAME); 100 μM] using a wire myograph. Immunostaining was used to visualize nNOS in rat mesenteric artery cross-sections. Western blotting measured total and phospho-nNOS in endothelial cell lysates and thoracic aorta homogenates. E2 rapidly increased (p < 0.001) activating phosphorylation of nNOS and nitric oxide (NO) production (as measured by 4-amino-5-methylamino-2,7-difluorofluorescein fluorescence) in endothelial cells. Likewise, E2 caused dose-dependent relaxation of arteries from female rats, which was blunted by both l-NPA and l-NAME (p < 0.001). In contrast, E2 response was modest in male animals and unaffected by NOS inhibition. It is noteworthy that there was a greater baseline presence of phospho-nNOS in male relative to female aortas. Although eNOS is believed to be the main source of NO in the vascular endothelium, we confirmed nNOS expression in endothelial cells. Endothelial nNOS mediated E2 relaxation in isolated arteries from female animals. Altogether, these data suggest vascular nNOS as a novel mechanism in E2 signaling. 相似文献
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Sadat MA Pech N Saulnier S Leroy BA Hossle JP Grez M Dinauer MC 《Human gene therapy》2003,14(7):651-666
A murine model of X-linked chronic granulomatous disease (X-CGD), an inherited immune deficiency with absent phagocyte NADPH oxidase activity caused by defects in the gp91(phox) gene, was used to evaluate a bicistronic retroviral vector in which expression of human gp91(phox) and a linked gene for Delta LNGFR, a truncated form of human low-affinity nerve growth factor receptor, are under the control of a spleen focus-forming virus long-terminal repeat (LTR). Four independent cohorts of 11-Gy irradiated X-CGD mice (total, 22 mice) were transplanted with or without preselection of transduced X-CGD bone marrow (BM). Transplanted mice had high-level correction of neutrophil gp91(phox) expression and reconstitution of NADPH oxidase activity. Expression lasted for at least 14 months in primary transplants, and persisted in secondary and tertiary transplants. Both gp91(phox) and Delta LNGFR were detected on circulating granulocytes, lymphocytes, lymphoid, and (for Delta LNGFR) red blood cells. Mice receiving transduced bone marrow [BM] preselected ex vivo for Delta LNGFR expression had high-level (= 80%) reconstitution with transduced cells, with an improved fraction of oxidase-corrected neutrophils posttransplant. Analysis of secondary and tertiary CFU-S showed that silencing of individual provirus integrants can occur even after preselection for Delta LNGFR prior to transplantation, and that persistent provirus expression was associated with multiple integration sites in most cases. No obvious adverse consequences of transgenic protein expression were observed. 相似文献
11.
Neutrophils from p40phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing
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Ellson CD Davidson K Ferguson GJ O'Connor R Stephens LR Hawkins PT 《The Journal of experimental medicine》2006,203(8):1927-1937
The generation of reactive oxygen species (ROS) by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex plays a critical role in the antimicrobial functions of the phagocytic cells of the immune system. The catalytic core of this oxidase consists of a complex between gp91(phox), p22(phox), p47(phox), p67(phox), p40(phox), and rac-2. Mutations in each of the phox components, except p40(phox), have been described in cases of chronic granulomatous disease (CGD), defining their essential role in oxidase function. We sought to establish the role of p40(phox) by investigating the NADPH oxidase responses of neutrophils isolated from p40(phox-/-) mice. In the absence of p40(phox), the expression of p67(phox) is reduced by approximately 55% and oxidase responses to tumor necrosis factor alpha/fibrinogen, immunoglobulin G latex beads, Staphylococcus aureus, formyl-methionyl-leucyl-phenylalanine, and zymosan were reduced by approximately 97, 85, 84, 75, and 30%, respectively. The defect in ROS production by p40(phox-/-) neutrophils in response to S. aureus translated into a severe, CGD-like defect in the killing of this organism both in vitro and in vivo, defining p40(phox) as an essential component in bacterial killing. 相似文献
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Moreno-Carranza B Gentsch M Stein S Schambach A Santilli G Rudolf E Ryser MF Haria S Thrasher AJ Baum C Brenner S Grez M 《Gene therapy》2009,16(1):111-118
Gene therapy has proven to be of potential value for the correction of inherited hematopoietic disorders. However, the occurrence of severe side effects in some of the clinical trials has questioned the safety of this approach and has hampered the use of long terminal repeat-driven vectors for the treatment of a large number of patients. The development of self-inactivating (SIN) vectors with reduced genotoxicity provides an alternative to the currently used vectors. Our initial attempts to use SIN vectors for the correction of a myeloid disorder, chronic granulomatous disease, failed due to low vector titers and poor transgene expression. The optimization of the transgene cDNA (gp91(phox)) resulted in substantially increased titers and transgene expression. Most notably, transgene optimization significantly improved expression of a second cistron located downstream of gp91(phox). Thus, optimization of the transgene sequence results in higher expression levels and increased therapeutic index allowing the use of low vector copy numbers per transduced cell and weaker internal promoters. 相似文献
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Pithon-Curi TC Levada AC Lopes LR Doi SQ Curi R 《Clinical science (London, England : 1979)》2002,103(4):403-408
The effect of glutamine on the activity of the NADPH oxidase complex from rat neutrophils was investigated. Superoxide anion (O(2)(-)) production was assessed: (1) by scintillation counting by using lucigenin, and (2) by reduction of cytochrome c over 10 min. The effects of glutamine and PMA on the expression of the NADPH oxidase components p22( phox ), gp91( phox ) and p47( phox ) were also determined. Glutamine at 1 and 2 mM increased O(2)(-) generation in the presence of PMA by 100% and 74% respectively, in neutrophils maintained previously for 3 h in medium deprived of this amino acid. DON (6-diazo-5-oxo-L-norleucine), an inhibitor of phosphate-dependent glutaminase and thus of glutamine metabolism, caused a significant decrease in O(2)(-) production by neutrophils stimulated with PMA both in the absence (44%) and in the presence (66%) of glutamine. PMA markedly increased the expression of gp91( phox ), p22( phox ) and p47( phox ) mRNAs. Glutamine (2 mM) increased the expression of these three proteins both in the absence and in the presence of PMA. We postulate that glutamine leads to O(2)(-) production in neutrophils, probably via the generation of ATP and regulation of the expression of components of NADPH oxidase. 相似文献
14.
Sebastian Brenner Martin F Ryser Uimook Choi Narda Whiting-Theobald Eberhard Kuhlisch Gilda Linton Elizabeth Kang Romy Lehmann Angela Rosen-Wolff Andrew G Rudikoff Ann M Farese Thomas J Macvittie Joachim Roesler Mitchell E Horwitz Harry L Malech 《Molecular therapy》2006,14(2):202-211
We have recently reported that the RD114-pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the NADPH-oxidase gp91phox (approved gene symbol CYBB) defect in CD34(+) cells from patients with X-linked chronic granulomatous disease in the NOD/SCID mouse model. Considering clinical use of this vector, we transplanted autologous mobilized peripheral blood CD34(+) progenitor cells, transduced with the RD114-MFGS-gp91phox vector, into two healthy rhesus macaques following nonmyeloablative conditioning. The moderately high levels of in vivo marking seen in the first months following transduction decreased and stabilized at about 8 months posttransplant. Marking for both healthy animals after 15 months was 0.3 to 1.3 vector copies per 100 cells in lymphocytes, neutrophils, and monocytes. Vector insertion analyses performed by linear amplification-mediated PCR and sequencing identified 32 and 45 separate insertion sites in the animals. Identical insertion sites were found in myeloid cells and lymphocytes, demonstrating the successful transduction of lymphomyeloid progenitors. Some inserts landed in the vicinity of genes controlling cell cycle and proliferation. Statistical analyses of insertion sites 1 year posttransplant suggest a high diversity of insertion sites despite low marking. 相似文献
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目的探讨扇贝裙边糖胺聚糖(SS-GAG)对6-羟基多巴胺(6-OHDA)诱导的SH-SY5Y细胞丙二醛(MDA)、乳酸脱氢酶(LDH)含量及凋亡相关基因Bax、Bcl-2、Caspase-9表达的影响。方法体外培养SH-SY5Y细胞,随机分为对照组、6-OHDA损伤组和SS-GAG(200、400和800mg/L)处理组,测定各组细胞中MDA含量、LDH活性,实时荧光(real-time)PCR检测细胞Bcl-2、Bax mRNA表达的变化,流式细胞技术(FCM)检测活化Caspase-9蛋白表达。结果与对照组相比较,6-OHDA损伤组的MDA含量、LDH活性增加,Bcl-2mRNA表达降低,Bax mRNA表达升高,表达活化Caspase-9蛋白的细胞数增加,差异均有统计学意义(F=27.699-280.567,P〈0.01)。与6-OHDA损伤组比较,各浓度SS-GAG处理组SH-SY5Y细胞中MDA含量、LDH活性降低,Bcl-2mRNA表达升高,Bax mRNA表达降低,活化的Caspase-9蛋白表达量减少,并且呈剂量依赖性,差异均有显著意义(F=27.699-280.567,P〈0.01)。结论 SS-GAG能降低6-OHDA诱导的氧化应激水平,保护生物膜的完整性。SS-GAG通过上调Bcl-2基因的表达和下调Bax基因的表达,抑制Caspase-9蛋白的活化,从而抑制6-OHDA诱导的SH-SY5Y细胞凋亡。 相似文献
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Tsou PS Addanki V Fung HL 《The Journal of pharmacology and experimental therapeutics》2008,327(1):97-104
We hypothesize that superoxide (O(2)(*-)) accumulation is not a crucial causative factor in inducing nitroglycerin (NTG) tolerance. In LLC-PK1 cells, pre-exposure to NTG resulted in increased O(2)(*-) accumulation and reduced cGMP response to NTG versus vehicle control. O(2)(*-) stimulated by NTG was reduced by oxypurinol (100 microM), a xanthine oxidase inhibitor. Exposure to angiotensin II (Ang II) increased O(2)(*-) but did not reduce cGMP response. The O(2)(*-) scavenger tiron reduced Ang II-induced O(2)(*-) production but did not increase NTG-stimulated cGMP production. Using p47(phox-/-) and gp91(phox-/-) mice versus their respective wild-type controls (WT), we showed that aorta from mice null of these critical NADPH oxidase subunits exhibited similar vascular tolerance after NTG dosing (20 mg/kg s.c., t.i.d. for 3 days), as indicated by their ex vivo pEC(50) and cGMP accumulation upon NTG challenge. In vitro aorta O(2)(*-) production was enhanced by NTG incubation in both p47(phox) null and WT mice. Pre-exposure of isolated mice aorta to 100 microM NTG for 1 h resulted in vascular tolerance toward NTG and increased O(2)(*-) accumulation. Oxypurinol (1 mM) reduced O(2)(*-) but did not attenuate vascular tolerance. These results suggest that O(2)(*-) does not initiate either in vitro and in vivo NTG tolerance, and that the p47(phox) and gp91(phox) subunits of NADPH oxidase are not critically required. Increased O(2)(*-) accumulation may be an effect, rather than an initiating cause, of NTG tolerance. 相似文献
19.
Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts
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Nakahira K Kim HP Geng XH Nakao A Wang X Murase N Drain PF Wang X Sasidhar M Nabel EG Takahashi T Lukacs NW Ryter SW Morita K Choi AM 《The Journal of experimental medicine》2006,203(10):2377-2389
Carbon monoxide (CO), a byproduct of heme catabolism by heme oxygenase (HO), confers potent antiinflammatory effects. Here we demonstrate that CO derived from HO-1 inhibited Toll-like receptor (TLR) 2, 4, 5, and 9 signaling, but not TLR3-dependent signaling, in macrophages. Ligand-mediated receptor trafficking to lipid rafts represents an early event in signal initiation of immune cells. Trafficking of TLR4 to lipid rafts in response to LPS was reactive oxygen species (ROS) dependent because it was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and in gp91(phox)-deficient macrophages. CO selectively inhibited ligand-induced recruitment of TLR4 to lipid rafts, which was also associated with the inhibition of ligand-induced ROS production in macrophages. TLR3 did not translocate to lipid rafts by polyinosine-polycytidylic acid (poly(I:C)). CO had no effect on poly(I:C)-induced ROS production and TLR3 signaling. The inhibitory effect of CO on TLR-induced cytokine production was abolished in gp91(phox)-deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91(phox). Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase-dependent ROS generation. 相似文献
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Cañas N Valero T Villarroya M Montell E Vergés J García AG López MG 《The Journal of pharmacology and experimental therapeutics》2007,323(3):946-953
We investigated the mechanism of the neuroprotective properties of chondroitin sulfate (CS), an endogenous perineuronal net glycosaminoglycan, in human neuroblastoma SH-SY5Y cells subjected to oxidative stress. Preincubation with CS for 24 h afforded concentration-dependent protection against H2O2-induced toxicity (50 microM for 24 h) measured as lactic dehydrogenase released to the incubation media; cell death was prevented at the concentrations of 600 and 1000 microM. Cell death caused by a combination of 10 microM rotenone plus 1 microM oligomycin-A (Rot/oligo) was also reduced by CS at concentrations ranging from 0.3 to 100 microM; in this toxicity model, maximum protection was achieved at 3 microM (48%). No significant protection was observed in a cell death model of Ca2+ overload (70 mM K+, for 24 h). H2O2 and Rot/oligo generated reactive oxygen species (ROS) measured as an increase in the fluorescence of dichlorofluorescein diacetate-loaded cells. CS drastically reduced ROS generation induced by both H2O2 (extracellular ROS) and Rot/oligo (intracellular ROS). CS also increased the expression of phosphorylated Akt and heme oxygenase-1 by 2-fold. The protective effects of CS were prevented by chelerythrine, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), cycloheximide, and Sn(IV)-protoporphyrin IX. Taken together, these results show that CS can protect SH-SY5Y cells under oxidative stress conditions by activating protein kinase C, which phosphorylates Akt that, via the phosphatidylinositol 3-kinase/Akt pathway, induces the synthesis of the antioxidant protein heme oxygenase-1. 相似文献