The clinical significance of a myeloperoxidase (MPO) gene polymorphism and inducible nitric oxide synthase (iNOS) expression in cirrhotic patients with hepatopulmonary syndrome (HPS) was explored. Enrolled subjects we...The clinical significance of a myeloperoxidase (MPO) gene polymorphism and inducible nitric oxide synthase (iNOS) expression in cirrhotic patients with hepatopulmonary syndrome (HPS) was explored. Enrolled subjects were divided into three groups according to their disease/health conditions: the HPS group (cirrhotic patients with HPS; n=63), the non-HPS group (cirrhotic patients without HPS; n=182), and the control group (healthy subjects without liver disease; n=35). The distribution of the MPO-463 G/A genotype and its relationship with iNOS expression in a typical cell block from ascitic fluid were detected by immunohistochemistry and polymerase chain reaction-restricted fragment length polymorphism analysis (PCR-RFLP). In the HPS group, the partial pressure of oxygen in blood and ascitic fluid was significantly decreased (8.95±1.58 kPa and 6.81±0.95 kPa, respectively; both P<0.01), while the partial pressure of carbon dioxide significantly increased (4.62±0.20 kPa and 5.92±0.45 kPa, respectively; P<0.01). MPO and iNOS levels were significantly increased in the HPS group as compared with the non-HPS group. These increases were even more remarkable in ascitic fluid (41.36±11.62 and 13.23±4.81 μg/L; 10.27± 3.20 and 4.95±1.12 μg/L) than in blood (16.66±5.24 and 4.87±1.73 μg/L; 5.79±2.31 and 2.35±0.84 μg/L). The distribution of the MPO genotypes GG, GA, and AA were 76.2%, 22.2% and 1.6% in the HPS group, and 57.7%, 37.9% and 4.4% in the non-HPS group (P<0.05). The expression of iNOS was significantly higher in patients with the G alleles (G/G and G/A) (61.54%, 48/78) than in patients with A alleles (G/A and A/A) (38.46%, 30/78) (P<0.01). It was suggested that the expression levels of iNOS and MPO were correlated with HPS-induced hypoxemia. The MPO-463 G/A mutation might be a protective factor that prevents the development of HPS. The MPO might be involved in the regulation of iNOS expression. In humans, MPO pathways, the iNOS/NO system, and their interaction might have an impact on the occurrence and development of HPS.展开更多
BACKGROUND:Previous studies have shown that inducible nitric oxide synthase(iNOS) and cyclooxygenase 2(COX-2) participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury.However...BACKGROUND:Previous studies have shown that inducible nitric oxide synthase(iNOS) and cyclooxygenase 2(COX-2) participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury.However,few reports have addressed time-dependent expression of iNOS and COX-2 following peripheral nerve injury.OBJECTIVE:To investigate spatiotemporal expression of iNOS and COX-2 during early stage sciatic nerve crush injury.DESIGN,TIME AND SETTING:The randomized,controlled,animal experiment was performed at the Laboratory of Applied Anatomy,Department of Human Anatomy and Neurobiology,Central South University,China from September 2006 to September 2007.MATERIALS:Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal antibody(Transduction Laboratory,USA),as well as biotinylated rabbit anti-mouse IgG and biotinylated rabbit anti-goat IgG(Santa Cruz Biotechnology,USA) were used in the present study.METHODS:A total of 48 healthy,adult,Sprague Dawley rats were randomly assigned to three groups.In the model group(n = 32),crush injury to the right sciatic nerve was established using an artery clamp.The model group was further assigned to four subgroups according to survival time(6,12,24,and 72 hours),respectively(n = 8).Sham surgery(n = 8) and normal control(n = 8) groups were also established.MAIN OUTCOME MEASURES:iNOS and COX-2 expression was detected in the L4-6 spinal cord with immunohistochemistry.Gray values of iNOS-and COX-2-postive cells in the anterior horn and posterior horn of spinal cord,as well as quantification of iNOS-and COX-2-positive cells in the anterior horn of spinal cord,were measured.RESULTS:iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury(P < 0.05) and peaked at 72 hours.Simultaneously,the number of iNOS-and COX-2-positive cells similarly increased in the anterior horn of spinal cord on the damaged side(P < 0.05).CONCLUSION:iNOS and COX-2 expression increased in the spinal cord during early stage sciatic nerve crush,which suggested that iNOS and COX-2 participate in occurrence and development of inflammatory immune responses following peripheral nerve injury.展开更多
AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized int...AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized into 7 groups [control(Cont), 3, 6, 12, 24, 48, and 72 h, n=6]. Except Cont group, other groups’ retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of i NOS and tight junction protein zonula occludens(ZO)-1 was detected by Western blotting. Evans blue(EB;3%) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6 h, 12 h groups, the expression of i NOS was localized by immunofluorescence. In order to verify the role of i NOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor(Inh), 6 h and 6 h+Inh, n=9]. After treatment with the i NOSspecific inhibitor 1400 W, the expression of i NOS and ZO-1 and the leakage of BRB were detected again.RESULTS: The immunofluorescence results showed that the expression of i NOS was observed in the Cont group and 6 h group, but not in the 12 h group. i NOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker Neu N but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period(3 h, 6 h) after acute high IOP, the expression of i NOS was upregulated, then the down-regulation of i NOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous downregulation after 6 h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3 h after acute high IOP. At 6 h, the leakage of EB was lower, but at 12 h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of i NOS was down-regulated after 1400 W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6 h group, and significantly down-regulated in the 6 h+Inh group, and the leakage of EB was significantly increased after 1400 W treatment.CONCLUSION: These results suggest that the upregulation of i NOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.展开更多
Pancreatic cancer is a common tumor of the digestive system.At present,the pathogenesis is still unclear,but current research on inducible nitric oxide synthase in relation to the pathogenesis of pancreatic malignant ...Pancreatic cancer is a common tumor of the digestive system.At present,the pathogenesis is still unclear,but current research on inducible nitric oxide synthase in relation to the pathogenesis of pancreatic malignant tumors is particularly extensive.Therefore,this article focuses on the research progress on inducible nitric oxide synthase in the pathogenesis of pancreatic cancer.展开更多
Background:Overexpression of inducible nitric oxide synthase(iNOS)has been reported in diabetic retinopathy(DR).The kinin B1 receptor(B1R)is also overexpressed in DR,and can stimulate iNOS via Gαi/ERK/MAPK pathway.We...Background:Overexpression of inducible nitric oxide synthase(iNOS)has been reported in diabetic retinopathy(DR).The kinin B1 receptor(B1R)is also overexpressed in DR,and can stimulate iNOS via Gαi/ERK/MAPK pathway.We previously showed that the topical administration of a B1R antagonist,LF22-0542,significantly reduces leukocyte infiltration,increased vascular permeability and overexpression of several inflammatory mediators,including iNOS in DR.Thus,the aim of this study was to determine whether the pro-inflammatory effects of B1R are attributed to oxidative stress caused by the activation of iNOS pathway in order to identify new therapeutic targets for the treatment of DR.iNOS and B1R being absent in the normal retina,their inhibition is unlikely to result in undesirable side effects.The approach will be no invasive by eye application of drops.Methods:Diabetes was induced in male Wistar rats(200-230 g)by a single intraperitoneal injection of streptozotocin(STZ,65 mg/kg b.w).One week later,rats were randomly divided into four groups(N=5)and treated for one week as follows:Gr 1:control rats treated with the selective iNOS inhibitor(1,400 W,0.06μM twice a day by eye-drops×7 days),Gr 2,STZ-diabetic rats treated with 1,400 W,Gr 3:control rats received a selective B1R agonist[Sar(D-Phe8)-des-Arg9-BK,100μg twice a week]by intravitreal injections(itrv)and treated with 1,400 W,Gr 4:STZ-diabetic rats+B1R agonist+1,400 W.At the end of treatment and two weeks post-STZ,three series of experiments were carried out to measure vascular permeability(by Evans blue dye method)and the expression of vasoactive and inflammatory mediators,including iNOS,VEGF-A,VEGF-R2,IL-1β,Cox-2,TNF-α,bradykinin 1 and 2 receptors and carboxypeptidase M/kininase 1(by Western Blotting and qRT-PCR).The nitrosative stress(nitrosylation of proteins)was also assessed by Western Blotting.One-way Anova test with Bonferroni post hoc was used for statistical analysis.Results:STZ-diabetic rats showed a significant increase in retinal vascular permeability(22.8μg/g Evans blue dye per g of fresh retinas,P=0.016)compared with control rats and control treated rats(17.2 and 16.8μg/g respectively).The injections of B1R agonist amplified the increase of vascular permeability which was normalized by the 1,400 W.The overexpression of inflammatory markers was also normalized by the 1,400 W in STZ-diabetic rats received or not the B1R agonist.Conclusions:These results support a contribution of iNOS in the deleterious effects of B1R in this model of diabetic retinopathy.Hence,iNOS inhibition by ocular application of 1,400 W may represent a promising and non-invasive therapeutic approach in the treatment of diabetic retinopathy.展开更多
Pancreatic cancer is a common tumor of the digestive system,at present,the pathogenesis is still unclear,but in the current research on the pathogenesis of pancreatic malignant tumors,the research on inducible nitric ...Pancreatic cancer is a common tumor of the digestive system,at present,the pathogenesis is still unclear,but in the current research on the pathogenesis of pancreatic malignant tumors,the research on inducible nitric oxide synthase is particularly extensive.Therefore,this article focuses on the research progress of inducible nitric oxide synthase in the pathogenesis of pancreatic cancer.This is a review.展开更多
The purpose of this study was to investigate the expression of inducible nitric oxide synthase(iNOS)in trophoblasts and deciduas in early medical abortion,and study the relationship of medical abortion through mifepri...The purpose of this study was to investigate the expression of inducible nitric oxide synthase(iNOS)in trophoblasts and deciduas in early medical abortion,and study the relationship of medical abortion through mifepristone and iNOS in early pregnancy.Expression of iNOS in trophoblasts and deciduas was detected by both in situ hybridization and immunohistochemical assay in 40 patients(experimental group);the positive expression of iNOS was represented by number density(N/S)and positive unit(Pu)using computer color magic image analysis system(CMIAS).All results were compared with those obtained from vacuum aspiration.In the experi-mental group,N/S and Pu in trophoblasts were 0.120�0.010 and 15.3�2.6,respectively,while in the control group,they were 0.021�0.003 and 3.1�0.5,respectively,and there were significant differences between the two groups.By immunohistochemical assay,N/S and Pu were 0.090�0.010,10.24�1.55 vs 0.016�0.002,1.26�0.33 in the trophoblasts of the two groups;there were also significant differences between the two groups.There were lower iNOS expression in deciduas by in situ hybridization and immunohistochem-ical assay,and the difference between the two groups was not significant.It was concluded that mifepristone induced medical abortion through the expression of iNOS in trophoblasts but not in deciduas.展开更多
Hepatic ischemia-reperfusion injury(HIRI)is a major clinical cause of morbidity and mortality in liver surgery and transplantation.Many studies have found that nitric oxide(NO)plays an important role in the HIRI and i...Hepatic ischemia-reperfusion injury(HIRI)is a major clinical cause of morbidity and mortality in liver surgery and transplantation.Many studies have found that nitric oxide(NO)plays an important role in the HIRI and its increase or decrease can affect the progression and outcome of HIRI.However,the role of NO in HIRI is controversial and complicated.NO derived by endothelial NO synthase(eNOS)shows a protective role in HIRI,while excessive NO derived by inducible NO synthase(iNOS)accelerates inflammation and increases oxidative stress,further aggravating HIRI.Nevertheless,the overexpression of eNOS may exacerbate HIRI and iNOS-derived NO in some cases reduces HIRI.Here we review the new progress in the understanding of the roles of NO during HIRI:(1)NO possesses different roles in HIRI by increasing NO bioavailability,down-regulating leukotriene C4 synthase,inhibiting the activation of the nuclear factorκB(NFκB)pathway,enhancing cell autophagy,and reducing inflammatory cytokines and reactive oxygen species(ROS).And NO has both protective and deleterious effects by regulating apoptotic factors;(2)eNOS promotes NO production and suppresses its own overexpression,exerting a hepatoprotective effect reversely.Its activation is regulated by the PI3K/Akt and KLF2/AMPK pathways;and(3)iNOS derived NO mainly has deteriorating effects on HIRI,while it may have a protective function under some conditions.Their expression should reach a balance to reduce the adverse side and make NO protective in the treatment of HIRI.Thus,it can be inferred that NO modulating drugs may be a new direction in the treatment of HIRI or may be used as an adjunct to mitigate HIRI for the purpose of protecting the liver.展开更多
AIM:To study the effects of preconditioning on inducible nitric oxide synthase(iNOS)and interleukin 1(IL-1)receptor transcription in rat liver ischemia/reperfusion injury(IRI).METHODS:Seventy-two male rats were random...AIM:To study the effects of preconditioning on inducible nitric oxide synthase(iNOS)and interleukin 1(IL-1)receptor transcription in rat liver ischemia/reperfusion injury(IRI).METHODS:Seventy-two male rats were randomized into 3 groups:the one-hour segmental ischemia(IRI,n=24)group,the ischemic preconditioning(IPC,n=24)group or the remote ischemic preconditioning(RIPC,n=24)group.The IPC and R-IPC were performed as 10 min of ischemia and 10 min of reperfusion.The iNOS and the IL-1 receptor mRNA in the liver tissue was analyzed with real time PCR.The total Nitrite and Nitrate(NOx)in continuously sampled microdialysate(MD)from the liver was analyzed.In addition,the NOx levels in the serum were analyzed.RESULTS:After 4 h of reperfusion,the iNOS mRNA was significantly higher in the R-IPC(ΔCt:3.44±0.57)group than in the IPC(ΔCt:5.86±0.82)group(P=0.025).The IL-1 receptor transcription activity was reduced in the IPC group(ΔCt:1.88±0.53 to 4.81±0.21),but not in the R-IPC group,during reperfusion(P=0.027).In the MD,a significant drop in the NOx levels was noted in the R-IPC group(12.3±2.2 to 4.7±1.2μmol/L)at the end of ischemia compared with the levels in early ischemia(P=0.008).A similar trend was observed in the IPC group(11.8±2.1 to 6.4±1.5μmol/L),although this difference was not statistically significant.The levels of NOx rose quickly during reperfusion in both groups.CONCLUSION:IPC,but not R-IPC,reduces iNOS and IL-1 receptor transcription during early reperfusion,indicating a lower inflammatory reaction.NOx is consumed in the ischemic liver lobe.展开更多
Progesterone has nongenomic effects on inducible nitric oxide synthase(iNOS),which is mediated by mitogen activated protein kinase(MAPK)pathways.This effect is supposed to have some potential association with asymptom...Progesterone has nongenomic effects on inducible nitric oxide synthase(iNOS),which is mediated by mitogen activated protein kinase(MAPK)pathways.This effect is supposed to have some potential association with asymptomatic gonococcal infections in women by immunological depression.In this study,polymorphonuclear leukocytes(PMNs)challenged by gonococci were used to study the nongenomic effects of progesterone.The activation of iNOS was assessed by measuring [3H] L-arginine converses to [3H] L-citrulline,and the activity of MAPK was detected by Western blot.It was found that the activity of iNOS and the yields of NO were enhanced significantly in gonococci-challenged PMNs compared with the controls(P<0.01).Progesterone could repress the activation of iNOS through P38MAPK pathway within PMNs(P<0.05),which could be blocked by SB203580(P<0.01),but not by actinomycin D(P>0.05).It was also found subsequently that in the serum specimens collected from gonococci-infected but asymptomatic women,the progesterone level was higher than that in women with severe symptoms(P<0.01).Moreover,the yield of NO had an inverse correlation with progesterone.With these results it suggested that the rapid nongenomic effects of progesterone may inhibit iNOS activation and NO yields mediated by P38MAPK pathways,which were supposed to be concerned with asymptomatic women infected with gonococci.展开更多
Glioblastoma multiforme is a highly aggressive primary brain malignancy that resists most conventional chemoand radiotherapeutic interventions.Nitric oxide(NO),a short lived free radical molecule produced by inducible...Glioblastoma multiforme is a highly aggressive primary brain malignancy that resists most conventional chemoand radiotherapeutic interventions.Nitric oxide(NO),a short lived free radical molecule produced by inducible NO synthase(iNOS)in glioblastomas and other tumors,is known to play a key role in tumor persistence,progression,and chemo/radiotherapy resistance.Site-specific and minimally invasive photodynamic therapy(PDT),based on oxidative damage resulting from non-ionizing photoactivation of a sensitizing agent,is highly effective against glioblastoma,but resistance also exists in this case.Studies in the authors’laboratory have shown that much of the latter is mediated by iNOS/NO.For example,when glioblastoma U87 or U251 cells sensitized in mitochondria with 5-aminolevulinic acid-induced protoporphyrin IX were exposed to a moderate dose of visible light,the observed apoptosis was strongly enhanced by an iNOS activity inhibitor or NO scavenger,indicating that iNOS/NO had increased cell resistance to photokilling.Moreover,cells that survived the photochallenge proliferated,migrated,and invaded more aggressively than controls,and these responses were also driven predominantly by iNOS/NO.Photostress-upregulated iNOS rather than basal enzyme was found to be responsible for all the negative effects described.Recognition of NO-mediated hyper-resistance/hyper-aggression in PDT-stressed glioblastoma has stimulated interest in how these responses can be prevented or at least minimized by pharmacologic adjuvants such as inhibitors of iNOS activity or transcription.Recent developments along these lines and their clinical potential for improving anti-glioblastoma PDT are discussed.展开更多
Glycyrrhizae Radix et Rhizoma,a traditional Chinese medicine also known as Gan Cao(GC),is frequently included in clinical prescriptions for the treatment of pneumonia.However,the pharmacological components of GC for p...Glycyrrhizae Radix et Rhizoma,a traditional Chinese medicine also known as Gan Cao(GC),is frequently included in clinical prescriptions for the treatment of pneumonia.However,the pharmacological components of GC for pneumonia treatment are rarely explored.Gan An He Ji oral liquid(GAHJ)has a simple composition and contains GC liquid extracts and paregoric,and has been used clinically for many years.Therefore,GAHJ was selected as a compound preparation for the study of GC in the treatment of pneumonia.We conducted an in vivo study of patients with pneumonia undergoing GAHJ treatments for three days.Using the intelligent mass spectrometry data-processing technologies to analyze the metabolism of GC in vivo,we obtained 168 related components of GC in humans,consisting of 24 prototype components and 144 metabolites,with 135 compounds screened in plasma and 82 in urine.After analysis of the metabolic transformation relationship and relative exposure,six components(liquiritin,liquiritigenin,glycyrrhizin,glycyrrhetinic acid,daidzin,and formononetin)were selected as potential effective components.The experimental results based on two animal pneumonia models and the inflammatory cell model showed that the mixture of these six components was effective in the treatment of pneumonia and lung injury and could effectively downregulate the level of inducible nitric oxide synthase(iNOS).Interestingly,glycyrrhetinic acid exhibited the strongest inhibition on iNOS and the highest exposure in vivo.The following molecular dynamic simulations indicated a strong bond between glycyrrhetinic acid and iNOS.Thus,the current study provides a pharmaceutical basis for GC and reveals the possible corresponding mechanisms in pneumonia treatment.展开更多
Objective:To investigate the biochemical capacity,and in vitro inhibitory effects of hairy roots from two cultivars of Ficus carica L.(Sabz and Siah)on Leishmania major promastigotes and amastigotes.Methods:In the hai...Objective:To investigate the biochemical capacity,and in vitro inhibitory effects of hairy roots from two cultivars of Ficus carica L.(Sabz and Siah)on Leishmania major promastigotes and amastigotes.Methods:In the hairy roots,the activity of antioxidant enzymes compared to normal leaves and roots,and the presence of some phenolic compounds in comparison with fruits were investigated.The IC_(50) values of hairy roots in promastigotes was determined by tetrazolium-dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays.By calculating the infectivity index of peripheral blood mononuclear cells(PBMCs),the leishmanicidal activity(IC_(50) values)of hairy roots for amastigotes was estimated.The effects of hairy roots(IC_(50) values)treatment on the levels of IFN-γ and iNOS expression,intracellular reactive oxygen species,and iNOS protein expression in infected-PBMCs were determined.Results:Based on antioxidant enzyme assays and high performance liquid chromatography analysis,hairy roots exhibited high antioxidant capacity and contained high levels of phenolic compounds.According to the results of tetrazolium-dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays,the hairy root extracts of both cultivars showed considerable dose-dependent inhibitory effects against Leishmania major promastigotes.Depending on the concentration and exposure time,treatment of infected-PBMCs with hairy root extracts caused the generation of a significant reactive oxygen species,up-regulation of IFN-γ and iNOS genes expression,and high value of iNOS protein compared to controls.Conclusions:The findings of this study suggest that the hairy roots of Ficus carica can be considered as a promising natural source of antileishmanial agents.展开更多
Neocryptotanshinone(NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide(LPS)-stimulated mouse...Neocryptotanshinone(NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide(LPS)-stimulated mouse macrophage(RAW264.7) cells. MTT results showed that NCTS partly reversed LPS-induced cytotoxicity. Real-time PCR results showed that NCTS suppressed LPS-induced mRNA expression of inflammatory cytokines, including tumor necrosis factor α(TNFα), interleukin-6(IL-6) and interleukin-1β(IL-1β). Moreover, NCTS could decrease LPS-induced nitric oxide(NO)production. Western blotting results showed that NCTS could down-regulate LPS-induced expression of inducible nitric oxide synthase(iNOS), p-IκBα, p-IKKβ and p-NF-κB p65 without affecting cyclooxygenase-2(COX-2). In addition, NCTS inhibited LPS-induced p-NF-κB p65 nuclear translocation.In conclusion, these data demonstrated that NCTS showed anti-inflammatory effect by suppression of NF-κB and iNOS signaling pathways.展开更多
Although previous studies have demonstrated the involvement of nitrated a-synuclein in neurodegenerative disorders(synucleinopathies),the effects of nitrated α-synuclein and the molecular mechanisms underlying its t...Although previous studies have demonstrated the involvement of nitrated a-synuclein in neurodegenerative disorders(synucleinopathies),the effects of nitrated α-synuclein and the molecular mechanisms underlying its toxicity are still unclear.In the present study,nitrated α-synuclein with four 3-nitrotyrosines(Tyr^(39),Tyr^(125),Tyr^(133),and Tyr^(136))was obtained non-enzymatically by incubation with nitrite.The nitrated protein existed as a mixture of monomers,dimers,and polymers in solution.The nitrated a-synuclein could induce cell death in a time-and concentration-dependent manner when SH-SY5Y cells(a human neuroblastoma cell line)were incubated with the dimers and polymers.Treatment with anti-integrin α5β1 antibody partially rescued the SH-SY5Y cells from the cell death.Dot blotting and immunoprecipitation revealed that the nitrated protein bound to integrin on the cell membranes.Level of nitric oxide(NO)and calcium-independent inducible NO synthase(iNOS)activity increased during the initial stages of the treatment.The expression of phosphorylated focal adhesion kinase(FAK)decreased in the cells.Subsequently,an increase in caspase 3 activity was observed in SH-SY5Y cells.Our results demonstrate that activation of iNOS and inhibition of FAK may both be responsible for the cell death induced by nitrated α-synuclein.These data suggest that the cytotoxicity of nitrated a-synuclein is mediated via an integrin-iNOS/-FAK signaling pathway,and that the nitration of α-synuclein plays a role in neuronal degeneration.展开更多
The present study provides supportive evid-ence for the effective prevention and treatment of lipopo-lysaccharide(LPS)-induced hepatocyte injury in neonatal mice by N-acetylcysteine(NAC).Hepatocytes of neonatal mice w...The present study provides supportive evid-ence for the effective prevention and treatment of lipopo-lysaccharide(LPS)-induced hepatocyte injury in neonatal mice by N-acetylcysteine(NAC).Hepatocytes of neonatal mice were obtained through collagenase digestion of the liver.The hepatocytes were treated either with LPS(10 mg/mL)alone or with NAC(5 mmol/L)for 1 h before the addition of LPS(10 mg/mL).After LPS treatment,12 wells of the cultured hepatocytes and supernatants were harvested at 0,6,and 12 h,respectively.Levels of alanine aminotransferase(ALT)and nitric oxide(NO)in the supernatant were biochemically quantified and reverse transcription-polymerase chain reaction(RT-PCR)was performed to detect the expression of inducible nitric oxide synthase(iNOS)mRNA after different treatments.At 0 h following the treatment of primary cultured hepa-tocytes with LPS,the levels of ALT,NO and iNOS mRNA in the supernatant were(21.1±4.78)u/L,(1.6±0.31)mmol/L and 0.17±0.023,respectively;at 6 h,(59.8±8.59)u/L,(6.6±0.81)mmol/L,and 0.71±0.091;and at 12 h,(89.6±15.30)u/L,(7.8±1.01)mmol/L,and 0.71±0.097.The levels of ALT,NO and iNOS mRNA at 6 and 12 h increased significantly,compared to those at 0 h(P<0.01).In contrast to LPS treatment alone,pre-treatment with NAC before LPS addition significantly reduced the levels of ALT,NO and iNOS mRNA in the supernatant at 6 h to(40.8±7.30)u/L,(3.2±0.71)mmol/L,and 0.41±0.060;and at 12 h to(55.4±5.48)u/L,(4.0±0.71)mmol/L,and 0.40¡0.067,respectively(P<0.01).However,the levels of ALT,NO and iNOS mRNA at 0 h did not change significantly with both treat-ment approaches.NAC has protective effects in hepato-cytes of neonatal mice against LPS-induced injury as shown by the reduced levels of ALT,NO and iNOS mRNA when primary hepatocytes were treated with NAC prior to LPS stimulation.We postulate that NAC exhibits its protective function by inhibiting LPS-induced transcription of iNOS,resulting in decreased levels of NO.展开更多
基金supported by a grant from Heilongjiang Provincial Science and Technology Breakthrough Project Foundation (No. GB07C32506)
文摘The clinical significance of a myeloperoxidase (MPO) gene polymorphism and inducible nitric oxide synthase (iNOS) expression in cirrhotic patients with hepatopulmonary syndrome (HPS) was explored. Enrolled subjects were divided into three groups according to their disease/health conditions: the HPS group (cirrhotic patients with HPS; n=63), the non-HPS group (cirrhotic patients without HPS; n=182), and the control group (healthy subjects without liver disease; n=35). The distribution of the MPO-463 G/A genotype and its relationship with iNOS expression in a typical cell block from ascitic fluid were detected by immunohistochemistry and polymerase chain reaction-restricted fragment length polymorphism analysis (PCR-RFLP). In the HPS group, the partial pressure of oxygen in blood and ascitic fluid was significantly decreased (8.95±1.58 kPa and 6.81±0.95 kPa, respectively; both P<0.01), while the partial pressure of carbon dioxide significantly increased (4.62±0.20 kPa and 5.92±0.45 kPa, respectively; P<0.01). MPO and iNOS levels were significantly increased in the HPS group as compared with the non-HPS group. These increases were even more remarkable in ascitic fluid (41.36±11.62 and 13.23±4.81 μg/L; 10.27± 3.20 and 4.95±1.12 μg/L) than in blood (16.66±5.24 and 4.87±1.73 μg/L; 5.79±2.31 and 2.35±0.84 μg/L). The distribution of the MPO genotypes GG, GA, and AA were 76.2%, 22.2% and 1.6% in the HPS group, and 57.7%, 37.9% and 4.4% in the non-HPS group (P<0.05). The expression of iNOS was significantly higher in patients with the G alleles (G/G and G/A) (61.54%, 48/78) than in patients with A alleles (G/A and A/A) (38.46%, 30/78) (P<0.01). It was suggested that the expression levels of iNOS and MPO were correlated with HPS-induced hypoxemia. The MPO-463 G/A mutation might be a protective factor that prevents the development of HPS. The MPO might be involved in the regulation of iNOS expression. In humans, MPO pathways, the iNOS/NO system, and their interaction might have an impact on the occurrence and development of HPS.
基金the National Natural Science Foundation of China,No. 30860290the Scientific Research Foundation of Department of Health of Hunan Province of China,No. C2007038+2 种基金the Scientific Research Program of Department of Education of Hunan Province,No. 08C816the Nanometer Specific Foundation of Shanghai of China,No. 1052nm05800the Doctor Innovation Foundation of Medical College of Shanghai Jiao Tong University of China,No. BXJ201043
文摘BACKGROUND:Previous studies have shown that inducible nitric oxide synthase(iNOS) and cyclooxygenase 2(COX-2) participate in inflammatory immune responses and neuropathic pain following peripheral nerve injury.However,few reports have addressed time-dependent expression of iNOS and COX-2 following peripheral nerve injury.OBJECTIVE:To investigate spatiotemporal expression of iNOS and COX-2 during early stage sciatic nerve crush injury.DESIGN,TIME AND SETTING:The randomized,controlled,animal experiment was performed at the Laboratory of Applied Anatomy,Department of Human Anatomy and Neurobiology,Central South University,China from September 2006 to September 2007.MATERIALS:Mouse anti-rat iNOS monoclonal antibody and goat anti-rat COX-2 monoclonal antibody(Transduction Laboratory,USA),as well as biotinylated rabbit anti-mouse IgG and biotinylated rabbit anti-goat IgG(Santa Cruz Biotechnology,USA) were used in the present study.METHODS:A total of 48 healthy,adult,Sprague Dawley rats were randomly assigned to three groups.In the model group(n = 32),crush injury to the right sciatic nerve was established using an artery clamp.The model group was further assigned to four subgroups according to survival time(6,12,24,and 72 hours),respectively(n = 8).Sham surgery(n = 8) and normal control(n = 8) groups were also established.MAIN OUTCOME MEASURES:iNOS and COX-2 expression was detected in the L4-6 spinal cord with immunohistochemistry.Gray values of iNOS-and COX-2-postive cells in the anterior horn and posterior horn of spinal cord,as well as quantification of iNOS-and COX-2-positive cells in the anterior horn of spinal cord,were measured.RESULTS:iNOS and COX-2 expression gradually increased in the anterior horn and posterior horn of the spinal cord on the damaged side over time from 6 hours following sciatic nerve injury(P < 0.05) and peaked at 72 hours.Simultaneously,the number of iNOS-and COX-2-positive cells similarly increased in the anterior horn of spinal cord on the damaged side(P < 0.05).CONCLUSION:iNOS and COX-2 expression increased in the spinal cord during early stage sciatic nerve crush,which suggested that iNOS and COX-2 participate in occurrence and development of inflammatory immune responses following peripheral nerve injury.
基金Supported by the National Natural Science(No.81660217)National College Students Innovation and Entrepreneurship Training Program Project (No.201911810004)。
文摘AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized into 7 groups [control(Cont), 3, 6, 12, 24, 48, and 72 h, n=6]. Except Cont group, other groups’ retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of i NOS and tight junction protein zonula occludens(ZO)-1 was detected by Western blotting. Evans blue(EB;3%) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6 h, 12 h groups, the expression of i NOS was localized by immunofluorescence. In order to verify the role of i NOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor(Inh), 6 h and 6 h+Inh, n=9]. After treatment with the i NOSspecific inhibitor 1400 W, the expression of i NOS and ZO-1 and the leakage of BRB were detected again.RESULTS: The immunofluorescence results showed that the expression of i NOS was observed in the Cont group and 6 h group, but not in the 12 h group. i NOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker Neu N but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period(3 h, 6 h) after acute high IOP, the expression of i NOS was upregulated, then the down-regulation of i NOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous downregulation after 6 h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3 h after acute high IOP. At 6 h, the leakage of EB was lower, but at 12 h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of i NOS was down-regulated after 1400 W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6 h group, and significantly down-regulated in the 6 h+Inh group, and the leakage of EB was significantly increased after 1400 W treatment.CONCLUSION: These results suggest that the upregulation of i NOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.
文摘Pancreatic cancer is a common tumor of the digestive system.At present,the pathogenesis is still unclear,but current research on inducible nitric oxide synthase in relation to the pathogenesis of pancreatic malignant tumors is particularly extensive.Therefore,this article focuses on the research progress on inducible nitric oxide synthase in the pathogenesis of pancreatic cancer.
文摘Background:Overexpression of inducible nitric oxide synthase(iNOS)has been reported in diabetic retinopathy(DR).The kinin B1 receptor(B1R)is also overexpressed in DR,and can stimulate iNOS via Gαi/ERK/MAPK pathway.We previously showed that the topical administration of a B1R antagonist,LF22-0542,significantly reduces leukocyte infiltration,increased vascular permeability and overexpression of several inflammatory mediators,including iNOS in DR.Thus,the aim of this study was to determine whether the pro-inflammatory effects of B1R are attributed to oxidative stress caused by the activation of iNOS pathway in order to identify new therapeutic targets for the treatment of DR.iNOS and B1R being absent in the normal retina,their inhibition is unlikely to result in undesirable side effects.The approach will be no invasive by eye application of drops.Methods:Diabetes was induced in male Wistar rats(200-230 g)by a single intraperitoneal injection of streptozotocin(STZ,65 mg/kg b.w).One week later,rats were randomly divided into four groups(N=5)and treated for one week as follows:Gr 1:control rats treated with the selective iNOS inhibitor(1,400 W,0.06μM twice a day by eye-drops×7 days),Gr 2,STZ-diabetic rats treated with 1,400 W,Gr 3:control rats received a selective B1R agonist[Sar(D-Phe8)-des-Arg9-BK,100μg twice a week]by intravitreal injections(itrv)and treated with 1,400 W,Gr 4:STZ-diabetic rats+B1R agonist+1,400 W.At the end of treatment and two weeks post-STZ,three series of experiments were carried out to measure vascular permeability(by Evans blue dye method)and the expression of vasoactive and inflammatory mediators,including iNOS,VEGF-A,VEGF-R2,IL-1β,Cox-2,TNF-α,bradykinin 1 and 2 receptors and carboxypeptidase M/kininase 1(by Western Blotting and qRT-PCR).The nitrosative stress(nitrosylation of proteins)was also assessed by Western Blotting.One-way Anova test with Bonferroni post hoc was used for statistical analysis.Results:STZ-diabetic rats showed a significant increase in retinal vascular permeability(22.8μg/g Evans blue dye per g of fresh retinas,P=0.016)compared with control rats and control treated rats(17.2 and 16.8μg/g respectively).The injections of B1R agonist amplified the increase of vascular permeability which was normalized by the 1,400 W.The overexpression of inflammatory markers was also normalized by the 1,400 W in STZ-diabetic rats received or not the B1R agonist.Conclusions:These results support a contribution of iNOS in the deleterious effects of B1R in this model of diabetic retinopathy.Hence,iNOS inhibition by ocular application of 1,400 W may represent a promising and non-invasive therapeutic approach in the treatment of diabetic retinopathy.
文摘Pancreatic cancer is a common tumor of the digestive system,at present,the pathogenesis is still unclear,but in the current research on the pathogenesis of pancreatic malignant tumors,the research on inducible nitric oxide synthase is particularly extensive.Therefore,this article focuses on the research progress of inducible nitric oxide synthase in the pathogenesis of pancreatic cancer.This is a review.
文摘The purpose of this study was to investigate the expression of inducible nitric oxide synthase(iNOS)in trophoblasts and deciduas in early medical abortion,and study the relationship of medical abortion through mifepristone and iNOS in early pregnancy.Expression of iNOS in trophoblasts and deciduas was detected by both in situ hybridization and immunohistochemical assay in 40 patients(experimental group);the positive expression of iNOS was represented by number density(N/S)and positive unit(Pu)using computer color magic image analysis system(CMIAS).All results were compared with those obtained from vacuum aspiration.In the experi-mental group,N/S and Pu in trophoblasts were 0.120�0.010 and 15.3�2.6,respectively,while in the control group,they were 0.021�0.003 and 3.1�0.5,respectively,and there were significant differences between the two groups.By immunohistochemical assay,N/S and Pu were 0.090�0.010,10.24�1.55 vs 0.016�0.002,1.26�0.33 in the trophoblasts of the two groups;there were also significant differences between the two groups.There were lower iNOS expression in deciduas by in situ hybridization and immunohistochem-ical assay,and the difference between the two groups was not significant.It was concluded that mifepristone induced medical abortion through the expression of iNOS in trophoblasts but not in deciduas.
文摘Hepatic ischemia-reperfusion injury(HIRI)is a major clinical cause of morbidity and mortality in liver surgery and transplantation.Many studies have found that nitric oxide(NO)plays an important role in the HIRI and its increase or decrease can affect the progression and outcome of HIRI.However,the role of NO in HIRI is controversial and complicated.NO derived by endothelial NO synthase(eNOS)shows a protective role in HIRI,while excessive NO derived by inducible NO synthase(iNOS)accelerates inflammation and increases oxidative stress,further aggravating HIRI.Nevertheless,the overexpression of eNOS may exacerbate HIRI and iNOS-derived NO in some cases reduces HIRI.Here we review the new progress in the understanding of the roles of NO during HIRI:(1)NO possesses different roles in HIRI by increasing NO bioavailability,down-regulating leukotriene C4 synthase,inhibiting the activation of the nuclear factorκB(NFκB)pathway,enhancing cell autophagy,and reducing inflammatory cytokines and reactive oxygen species(ROS).And NO has both protective and deleterious effects by regulating apoptotic factors;(2)eNOS promotes NO production and suppresses its own overexpression,exerting a hepatoprotective effect reversely.Its activation is regulated by the PI3K/Akt and KLF2/AMPK pathways;and(3)iNOS derived NO mainly has deteriorating effects on HIRI,while it may have a protective function under some conditions.Their expression should reach a balance to reduce the adverse side and make NO protective in the treatment of HIRI.Thus,it can be inferred that NO modulating drugs may be a new direction in the treatment of HIRI or may be used as an adjunct to mitigate HIRI for the purpose of protecting the liver.
文摘AIM:To study the effects of preconditioning on inducible nitric oxide synthase(iNOS)and interleukin 1(IL-1)receptor transcription in rat liver ischemia/reperfusion injury(IRI).METHODS:Seventy-two male rats were randomized into 3 groups:the one-hour segmental ischemia(IRI,n=24)group,the ischemic preconditioning(IPC,n=24)group or the remote ischemic preconditioning(RIPC,n=24)group.The IPC and R-IPC were performed as 10 min of ischemia and 10 min of reperfusion.The iNOS and the IL-1 receptor mRNA in the liver tissue was analyzed with real time PCR.The total Nitrite and Nitrate(NOx)in continuously sampled microdialysate(MD)from the liver was analyzed.In addition,the NOx levels in the serum were analyzed.RESULTS:After 4 h of reperfusion,the iNOS mRNA was significantly higher in the R-IPC(ΔCt:3.44±0.57)group than in the IPC(ΔCt:5.86±0.82)group(P=0.025).The IL-1 receptor transcription activity was reduced in the IPC group(ΔCt:1.88±0.53 to 4.81±0.21),but not in the R-IPC group,during reperfusion(P=0.027).In the MD,a significant drop in the NOx levels was noted in the R-IPC group(12.3±2.2 to 4.7±1.2μmol/L)at the end of ischemia compared with the levels in early ischemia(P=0.008).A similar trend was observed in the IPC group(11.8±2.1 to 6.4±1.5μmol/L),although this difference was not statistically significant.The levels of NOx rose quickly during reperfusion in both groups.CONCLUSION:IPC,but not R-IPC,reduces iNOS and IL-1 receptor transcription during early reperfusion,indicating a lower inflammatory reaction.NOx is consumed in the ischemic liver lobe.
基金supported by the Young Scientists Fund Program of National Natural Science Fund Program from National Natural Sciences Foundation of China(No.30371293)Medical Research Foundation of Guangdong Province of China(No.B2009193)
文摘Progesterone has nongenomic effects on inducible nitric oxide synthase(iNOS),which is mediated by mitogen activated protein kinase(MAPK)pathways.This effect is supposed to have some potential association with asymptomatic gonococcal infections in women by immunological depression.In this study,polymorphonuclear leukocytes(PMNs)challenged by gonococci were used to study the nongenomic effects of progesterone.The activation of iNOS was assessed by measuring [3H] L-arginine converses to [3H] L-citrulline,and the activity of MAPK was detected by Western blot.It was found that the activity of iNOS and the yields of NO were enhanced significantly in gonococci-challenged PMNs compared with the controls(P<0.01).Progesterone could repress the activation of iNOS through P38MAPK pathway within PMNs(P<0.05),which could be blocked by SB203580(P<0.01),but not by actinomycin D(P>0.05).It was also found subsequently that in the serum specimens collected from gonococci-infected but asymptomatic women,the progesterone level was higher than that in women with severe symptoms(P<0.01).Moreover,the yield of NO had an inverse correlation with progesterone.With these results it suggested that the rapid nongenomic effects of progesterone may inhibit iNOS activation and NO yields mediated by P38MAPK pathways,which were supposed to be concerned with asymptomatic women infected with gonococci.
基金USPHS Grant(CA70823)the National Cancer Institute,BSC Grant(FP12605)+2 种基金Rock River Grant(FP14869)the MCW Cancer Center(to Girotti AW)NCN Grant 2017/27/B/NZ5/02620(to Korytowski W).
文摘Glioblastoma multiforme is a highly aggressive primary brain malignancy that resists most conventional chemoand radiotherapeutic interventions.Nitric oxide(NO),a short lived free radical molecule produced by inducible NO synthase(iNOS)in glioblastomas and other tumors,is known to play a key role in tumor persistence,progression,and chemo/radiotherapy resistance.Site-specific and minimally invasive photodynamic therapy(PDT),based on oxidative damage resulting from non-ionizing photoactivation of a sensitizing agent,is highly effective against glioblastoma,but resistance also exists in this case.Studies in the authors’laboratory have shown that much of the latter is mediated by iNOS/NO.For example,when glioblastoma U87 or U251 cells sensitized in mitochondria with 5-aminolevulinic acid-induced protoporphyrin IX were exposed to a moderate dose of visible light,the observed apoptosis was strongly enhanced by an iNOS activity inhibitor or NO scavenger,indicating that iNOS/NO had increased cell resistance to photokilling.Moreover,cells that survived the photochallenge proliferated,migrated,and invaded more aggressively than controls,and these responses were also driven predominantly by iNOS/NO.Photostress-upregulated iNOS rather than basal enzyme was found to be responsible for all the negative effects described.Recognition of NO-mediated hyper-resistance/hyper-aggression in PDT-stressed glioblastoma has stimulated interest in how these responses can be prevented or at least minimized by pharmacologic adjuvants such as inhibitors of iNOS activity or transcription.Recent developments along these lines and their clinical potential for improving anti-glioblastoma PDT are discussed.
基金funded by the National Natural Science Foundation of China(Grant Nos.:82141215,82173694,82173779,82222068,and U1903119)Fujian Province Science and Technology Project(Grant Nos.:2021J011340 and 2020Y0013)Xiamen Municipal Bureau of Science and Technology Planning Project(Grant No.:3502Z2021YJ11).
文摘Glycyrrhizae Radix et Rhizoma,a traditional Chinese medicine also known as Gan Cao(GC),is frequently included in clinical prescriptions for the treatment of pneumonia.However,the pharmacological components of GC for pneumonia treatment are rarely explored.Gan An He Ji oral liquid(GAHJ)has a simple composition and contains GC liquid extracts and paregoric,and has been used clinically for many years.Therefore,GAHJ was selected as a compound preparation for the study of GC in the treatment of pneumonia.We conducted an in vivo study of patients with pneumonia undergoing GAHJ treatments for three days.Using the intelligent mass spectrometry data-processing technologies to analyze the metabolism of GC in vivo,we obtained 168 related components of GC in humans,consisting of 24 prototype components and 144 metabolites,with 135 compounds screened in plasma and 82 in urine.After analysis of the metabolic transformation relationship and relative exposure,six components(liquiritin,liquiritigenin,glycyrrhizin,glycyrrhetinic acid,daidzin,and formononetin)were selected as potential effective components.The experimental results based on two animal pneumonia models and the inflammatory cell model showed that the mixture of these six components was effective in the treatment of pneumonia and lung injury and could effectively downregulate the level of inducible nitric oxide synthase(iNOS).Interestingly,glycyrrhetinic acid exhibited the strongest inhibition on iNOS and the highest exposure in vivo.The following molecular dynamic simulations indicated a strong bond between glycyrrhetinic acid and iNOS.Thus,the current study provides a pharmaceutical basis for GC and reveals the possible corresponding mechanisms in pneumonia treatment.
文摘Objective:To investigate the biochemical capacity,and in vitro inhibitory effects of hairy roots from two cultivars of Ficus carica L.(Sabz and Siah)on Leishmania major promastigotes and amastigotes.Methods:In the hairy roots,the activity of antioxidant enzymes compared to normal leaves and roots,and the presence of some phenolic compounds in comparison with fruits were investigated.The IC_(50) values of hairy roots in promastigotes was determined by tetrazolium-dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays.By calculating the infectivity index of peripheral blood mononuclear cells(PBMCs),the leishmanicidal activity(IC_(50) values)of hairy roots for amastigotes was estimated.The effects of hairy roots(IC_(50) values)treatment on the levels of IFN-γ and iNOS expression,intracellular reactive oxygen species,and iNOS protein expression in infected-PBMCs were determined.Results:Based on antioxidant enzyme assays and high performance liquid chromatography analysis,hairy roots exhibited high antioxidant capacity and contained high levels of phenolic compounds.According to the results of tetrazolium-dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays,the hairy root extracts of both cultivars showed considerable dose-dependent inhibitory effects against Leishmania major promastigotes.Depending on the concentration and exposure time,treatment of infected-PBMCs with hairy root extracts caused the generation of a significant reactive oxygen species,up-regulation of IFN-γ and iNOS genes expression,and high value of iNOS protein compared to controls.Conclusions:The findings of this study suggest that the hairy roots of Ficus carica can be considered as a promising natural source of antileishmanial agents.
基金supported by the Science and Technology Development Fund of Macao Special Administrative Region (No. 021/2012/A1)the Research Fund of University of Macao (No. MRG007/CXP/2013/ICMS)
文摘Neocryptotanshinone(NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide(LPS)-stimulated mouse macrophage(RAW264.7) cells. MTT results showed that NCTS partly reversed LPS-induced cytotoxicity. Real-time PCR results showed that NCTS suppressed LPS-induced mRNA expression of inflammatory cytokines, including tumor necrosis factor α(TNFα), interleukin-6(IL-6) and interleukin-1β(IL-1β). Moreover, NCTS could decrease LPS-induced nitric oxide(NO)production. Western blotting results showed that NCTS could down-regulate LPS-induced expression of inducible nitric oxide synthase(iNOS), p-IκBα, p-IKKβ and p-NF-κB p65 without affecting cyclooxygenase-2(COX-2). In addition, NCTS inhibited LPS-induced p-NF-κB p65 nuclear translocation.In conclusion, these data demonstrated that NCTS showed anti-inflammatory effect by suppression of NF-κB and iNOS signaling pathways.
基金supported by the grants from the China Postdoctoral Science Foundation(20070410153,200801120)the National Science Foundation of China(30970695)+1 种基金the Major State Basic Research Development Program of China(973 Program)(2010CB912303)the Chinese Academy of Sciences(KSCX2-YW-R-119,KSCX2-YW-R-256).
文摘Although previous studies have demonstrated the involvement of nitrated a-synuclein in neurodegenerative disorders(synucleinopathies),the effects of nitrated α-synuclein and the molecular mechanisms underlying its toxicity are still unclear.In the present study,nitrated α-synuclein with four 3-nitrotyrosines(Tyr^(39),Tyr^(125),Tyr^(133),and Tyr^(136))was obtained non-enzymatically by incubation with nitrite.The nitrated protein existed as a mixture of monomers,dimers,and polymers in solution.The nitrated a-synuclein could induce cell death in a time-and concentration-dependent manner when SH-SY5Y cells(a human neuroblastoma cell line)were incubated with the dimers and polymers.Treatment with anti-integrin α5β1 antibody partially rescued the SH-SY5Y cells from the cell death.Dot blotting and immunoprecipitation revealed that the nitrated protein bound to integrin on the cell membranes.Level of nitric oxide(NO)and calcium-independent inducible NO synthase(iNOS)activity increased during the initial stages of the treatment.The expression of phosphorylated focal adhesion kinase(FAK)decreased in the cells.Subsequently,an increase in caspase 3 activity was observed in SH-SY5Y cells.Our results demonstrate that activation of iNOS and inhibition of FAK may both be responsible for the cell death induced by nitrated α-synuclein.These data suggest that the cytotoxicity of nitrated a-synuclein is mediated via an integrin-iNOS/-FAK signaling pathway,and that the nitration of α-synuclein plays a role in neuronal degeneration.
文摘The present study provides supportive evid-ence for the effective prevention and treatment of lipopo-lysaccharide(LPS)-induced hepatocyte injury in neonatal mice by N-acetylcysteine(NAC).Hepatocytes of neonatal mice were obtained through collagenase digestion of the liver.The hepatocytes were treated either with LPS(10 mg/mL)alone or with NAC(5 mmol/L)for 1 h before the addition of LPS(10 mg/mL).After LPS treatment,12 wells of the cultured hepatocytes and supernatants were harvested at 0,6,and 12 h,respectively.Levels of alanine aminotransferase(ALT)and nitric oxide(NO)in the supernatant were biochemically quantified and reverse transcription-polymerase chain reaction(RT-PCR)was performed to detect the expression of inducible nitric oxide synthase(iNOS)mRNA after different treatments.At 0 h following the treatment of primary cultured hepa-tocytes with LPS,the levels of ALT,NO and iNOS mRNA in the supernatant were(21.1±4.78)u/L,(1.6±0.31)mmol/L and 0.17±0.023,respectively;at 6 h,(59.8±8.59)u/L,(6.6±0.81)mmol/L,and 0.71±0.091;and at 12 h,(89.6±15.30)u/L,(7.8±1.01)mmol/L,and 0.71±0.097.The levels of ALT,NO and iNOS mRNA at 6 and 12 h increased significantly,compared to those at 0 h(P<0.01).In contrast to LPS treatment alone,pre-treatment with NAC before LPS addition significantly reduced the levels of ALT,NO and iNOS mRNA in the supernatant at 6 h to(40.8±7.30)u/L,(3.2±0.71)mmol/L,and 0.41±0.060;and at 12 h to(55.4±5.48)u/L,(4.0±0.71)mmol/L,and 0.40¡0.067,respectively(P<0.01).However,the levels of ALT,NO and iNOS mRNA at 0 h did not change significantly with both treat-ment approaches.NAC has protective effects in hepato-cytes of neonatal mice against LPS-induced injury as shown by the reduced levels of ALT,NO and iNOS mRNA when primary hepatocytes were treated with NAC prior to LPS stimulation.We postulate that NAC exhibits its protective function by inhibiting LPS-induced transcription of iNOS,resulting in decreased levels of NO.
