NADPH oxidase 4(NOX4)as a biomarker and therapeutic target in neurodegenerative diseases

Diseases like Alzheimer’s and Parkinson’s diseases are defined by inflammation and the damage neurons undergo due to oxidative stress. A primary reactive oxygen species contributor in the central nervous system, NADPH oxidase 4, is viewed as a potential therapeutic touchstone and indicative marker for these ailments. This in-depth review brings to light distinct features of NADPH oxidase 4, responsible for generating superoxide and hydrogen peroxide, emphasizing its pivotal role in activating glial cells, inciting inflammation, and disturbing neuronal functions. Significantly, malfunctioning astrocytes, forming the majority in the central nervous system, play a part in advancing neurodegenerative diseases, due to their reactive oxygen species and inflammatory factor secretion. Our study reveals that aiming at NADPH oxidase 4 within astrocytes could be a viable treatment pathway to reduce oxidative damage and halt neurodegenerative processes. Adjusting NADPH oxidase 4 activity might influence the neuroinflammatory cytokine levels, including myeloperoxidase and osteopontin, offering better prospects for conditions like Alzheimer’s disease and Parkinson’s disease. This review sheds light on the role of NADPH oxidase 4 in neural degeneration, emphasizing its drug target potential, and paving the path for novel treatment approaches to combat these severe conditions.

Enhancing metformin-induced tumor metabolism destruction by glucose oxidase for triple-combination therapy

Despite decades of laboratory and clinical trials,breast cancer remains the main cause of cancer-related disease burden in women.Considering the metabolism destruction effect of metformin(Met)and cancer cell starvation induced by glucose oxidase(GOx),after their efficient delivery to tumor sites,GOx and Met may consume a large amount of glucose and produce sufficient hydrogen peroxide in situ.Herein,a pH-responsive epigallocatechin gallate(EGCG)-conjugated low-molecular-weight chitosan(LC-EGCG,LE)nanoparticle(Met–GOx/Fe@LE NPs)was constructed.The coordination between iron ions(Fe3+)and EGCG in this nanoplatform can enhance the efficacy of chemodynamic therapy via the Fenton reaction.Met–GOx/Fe@LE NPs allow GOx to retain its enzymatic activity while simultaneously improving its stability.Moreover,this pH-responsive nanoplatform presents controllable drug release behavior.An in vivo biodistribution study showed that the intracranial accumulation of GOx delivered by this nanoplatform was 3.6-fold higher than that of the free drug.The in vivo anticancer results indicated that this metabolism destruction/starvation/chemodynamic triple-combination therapy could induce increased apoptosis/death of tumor cells and reduce their proliferation.This triple-combination therapy approach is promising for efficient and targeted cancer treatment.

Type-B monoamine oxidase inhibitors in neurological diseases:clinical applications based on preclinical findings

Type-B monoamine oxidase inhibitors,encompassing selegiline,rasagiline,and safinamide,are available to treat Parkinson's disease.These drugs ameliorate motor symptoms and improve motor fluctuation in the advanced stages of the disease.There is also evidence suppo rting the benefit of type-B monoamine oxidase inhibitors on non-motor symptoms of Parkinson's disease,such as mood deflection,cognitive impairment,sleep disturbances,and fatigue.Preclinical studies indicate that type-B monoamine oxidase inhibitors hold a strong neuroprotective potential in Parkinson's disease and other neurodegenerative diseases for reducing oxidative stress and stimulating the production and release of neurotrophic factors,particularly glial cell line-derived neurotrophic factor,which suppo rt dopaminergic neurons.Besides,safinamide may interfere with neurodegenerative mechanisms,countera cting excessive glutamate overdrive in basal ganglia motor circuit and reducing death from excitotoxicity.Due to the dual mechanism of action,the new generation of type-B monoamine oxidase inhibitors,including safinamide,is gaining interest in other neurological pathologies,and many supporting preclinical studies are now available.The potential fields of application concern epilepsy,Duchenne muscular dystrophy,multiple scle rosis,and above all,ischemic brain injury.The purpose of this review is to investigate the preclinical and clinical pharmacology of selegiline,rasagiline,and safinamide in Parkinson's disease and beyond,focusing on possible future therapeutic applications.

Knockdown of NADPH oxidase 4 reduces mitochondrial oxidative stress and neuronal pyroptosis following intracerebral hemorrhage

Intracerebral hemorrhage is often accompanied by oxidative stress induced by reactive oxygen species,which causes abnormal mitochondrial function and secondary reactive oxygen species generation.This creates a vicious cycle leading to reactive oxygen species accumulation,resulting in progression of the pathological process.Therefore,breaking the cycle to inhibit reactive oxygen species accumulation is critical for reducing neuronal death after intracerebral hemorrhage.Our previous study found that increased expression of nicotinamide adenine dinucleotide phosphate oxidase 4(NADPH oxidase 4,NOX4)led to neuronal apoptosis and damage to the blood-brain barrier after intracerebral hemorrhage.The purpose of this study was to investigate the role of NOX4 in the circle involving the neuronal tolerance to oxidative stress,mitochondrial reactive oxygen species and modes of neuronal death other than apoptosis after intracerebral hemorrhage.We found that NOX4 knockdown by adeno-associated virus(AAV-NOX4)in rats enhanced neuronal tolerance to oxidative stress,enabling them to better resist the oxidative stress caused by intracerebral hemorrhage.Knockdown of NOX4 also reduced the production of reactive oxygen species in the mitochondria,relieved mitochondrial damage,prevented secondary reactive oxygen species accumulation,reduced neuronal pyroptosis and contributed to relieving secondary brain injury after intracerebral hemorrhage in rats.Finally,we used a mitochondria-targeted superoxide dismutase mimetic to explore the relationship between reactive oxygen species and NOX4.The mitochondria-targeted superoxide dismutase mimetic inhibited the expression of NOX4 and neuronal pyroptosis,which is similar to the effect of AAV-NOX4.This indicates that NOX4 is likely to be an important target for inhibiting mitochondrial reactive oxygen species production,and NOX4 inhibitors can be used to alleviate oxidative stress response induced by intracerebral hemorrhage.

Value of combining the serum D-lactate, diamine oxidase, and endotoxin levels to predict gut-derived infections in cancer patients

Objective:This is a retrospective observational cohort study.The objective of this retrospective observational cohort study was to evaluate the value of the combined serum D-lactic acid,diamine oxidase(DAO),and endotoxin levels to predict intestinal barrier impairment and gut-derived infection(GDI)in cancer patients.Methods:Cancer patients receiving chemotherapy or palliative care treatment at our hospital were enrolled in the study.The serum concentrations of DAO,D-lactic acid,and endotoxin were determined using the intestinal barrier function biochemical index analysis system.The patients'infection information came from the hospital's Medicom Prescription Automatic Screening System and themedical records.Three hundred fifty-three cancer patients were included in the study(53.8%female,73.7%cancer stage IV,27.8%had bowel obstruction).Results:The total incidence of GDI was 33.4%(118/353).The median length of hospital stay was 16 days for patients with GDI,compared with 7 days for patients without GDI(P<0.001).The media hospitalization costs were¥27,362.35 for patients with GDI compared with¥11,614.08 for patients without GDI(P<0.001).The serum concentrations of DAO,D-lactic acid,and endotoxin were significantly higher in patients with GDI.As malignant bowel obstruction(MBO)worsened,the concentrations of DAO,D-lactic acid,and endotoxin increased.Multivariate logistic regression models revealed that the DAO,endotoxin,IL-6,and C-reactive protein levels were significantly associated with an increased risk of GDI.In addition,we also found a fivefold increased risk of infection in patients withMBO compared with those without bowel obstruction(OR=6.210,P<0.001).All of the areas under the receiver operating characteristic curve(AUCs)for DAO,D-lactate,and endotoxin to predict GDI were<0.7(AUC=0.648,P<0.001;AUC=0.624,P<0.01;AUC=0.620,P<0.01,respectively).However,when the parameters were combined(DAO+D-lactate+endotoxin),the predictive power increased significantly(AUC=0.797,P<0.001).Moreover,combining these intestinal barrier indicators and the presence of MBO had better power to predict GDI than either alone(AUC=0.837,P<0.001).Conclusions:Combining the serum DAO,D-lactic acid,and endotoxin levels was a better predictor of GDI than any of the indicators alone,and combining these with the diagnosis of MBO could further improve the efficacy for predicting GDI.

高等植物赤霉素生物合成关键组分GA20-oxidase氧化酶基因的研究进展

GA-20氧化酶(GA-20 oxidase)是重要的GA生物合成和调控酶,直接催化生成有生物活性的GAs,是一种多功能酶,最显著的特点就是负反馈调节。GA20-氧化酶在植物发育和生理过程中起着重要的调控作用。综述了高等植物体内GA20氧化酶基因的克隆及表达调控研究及其对株高、纤维、开花、产量性状等影响,重点阐述了GA20氧化酶基因与激素、光周期、抗性等之间的相互作用,以便更好地揭示GA-20氧化酶信号网络系统及其作用机制。

赤霉素诱导甘蔗GA20-Oxidase基因实时荧光定量PCR分析

本研究以喷清水甘蔗幼茎作为对照(即未处理),经过赤霉素处理后6h、12h、24h和48h的甘蔗幼茎为处理,分别进行取样并提取RNA,反转录获得cDNA作模板,利用SYBR GreenⅠ成功构建了目的基因(GA20-oxidase)和内参基因(GAPDH)的标准品和标准曲线。结果表明:内参基因与目的基因的起始模板浓度与Ct之间呈良好线性关系,决定系数分别为r2=0.998、r2=0.995;溶解曲线均显单峰型,证明扩增的特异性比较好,可对基因表达进行相对定量。定量结果表明:目的基因未处理的表达量最大,赤霉素处理后表达量持续下降,在6h达到最低值,12h后逐渐上升,但到48h又明显下降,且所有处理的表达量均低于未处理,6h、12h、24h和48h比对照的表达量分别下降了24.75%、13.18%、10.23%和20.34%。本实验研究GA20-oxidase基因在赤霉素处理后的表达情况,为进一步克隆甘蔗GA20氧化酶基因全长序列、研究该基因在甘蔗节间伸长过程中的表达调控及甘蔗茎间伸长中的作用机理提供理论依据。

Semicarbazide-Sensitive Amine Oxidase对血糖调节作用研究

Semicarbazide-Sensitive Amine Oxidase(SSAO)是一类在人体内广泛分布但生理功能尚不清楚的酶。本研究中我们首次在动物模型上发现了SSAO活性对大鼠血糖的影响。通过使用SSAO的高选择性抑制剂MDL-72974A以及它的内源性底物甲胺等对动物进行处理,研究血糖与SSAO活性的关系。SSAO活性用同位素标记法以14C-苯甲胺为底物进行测定,血糖则采用己糖激酶法测定。此外我们还测定了尿甲醛排泄以考察SSAO催化脱氨反应是否发生。实验结果显示,抑制SSAO活性可以导致动物血糖升高,并且SSAO底物甲胺可以降低糖尿病鼠血糖。这些结果表明SSAO可能在血糖调节方面有某种作用,进一步研究将对发展糖尿病治疗策略有重要意义。

Changes of plasma D(-) -lactate, diamine oxidase and endotoxin in patients with liver cirrhosis

BACKGROUND: Plasma D(-)-lactate and diamine oxidase (DAO) can reflect patients' intestinal mucosal condition. We evaluated the changes of plasma D (-)-lactate, DAO and endotoxin activities and their significance in patients with liver cirrhosis. METHODS: Fifty liver cirrhosis patients were enrolled into experimental group and 30 healthy people into control group. The plasma levels of D(-)-lactate, DAO and endo- toxin were detected spectrophotographically. RESULTS: The level of D(-)-lactate was significantly high- er in the experimental group than that in the control group (P<0.01). Significant differences of D (-)-lactate levels were observed in Child-Pugh subgroups of the experimen- tal group (P <0. 01). The level of DAO was significantly higher in the experimental group than that in the control group (P <0.01), but the level of DAO in Child-Pugh sub- group C was significantly lower than that in Child-Pugh subgroup B (P<0.01). The level of endotoxin was signifi- cantly increased in the experimental group except Child Pugh subgroup A (P<0.01). The plasma levels of D(-) lactate, DAO and endotoxin were positively correlated with each other (P<0.01). CONCLUSIONS: The data suggest that both plasma D(-) lactate and DAO activity are sensitive markers for early diagnosis of gut failure and endotoxemia in patients with liver cirrhosis. The impairment of intestinal barrier func- tion may be one of the critical reasons for deterioration of liver cirrhosis.

Inhibition of Xanthine Oxidase Activity by Gnaphalium Affine Extract

Objective To evaluate the inhibitory effect of Gnaphalium affine extracts on xanthine oxidase(XO) activity in vitro and to analyze the mechanism of this effect. Methods In this in vitro study, Kinetic measurements were performed in 4 different inhibitor concentrations and 5 different xanthine concentrations(60, 100, 200, 300, 400 μmol/L). Dixon and Lineweaver-Burk plot analysis were used to determine Ki values and the inhibition mode for the compounds isolated from Gnaphalium affine extract. Results Four potent xanthine oxidase inhibitors were found in 95% ethanolic(v/v) Gnaphalium affine extract. Among them, the f lavone Eupatilin exhibited the strongest inhibitory effect on XO with a inhibition constant(Ki) of 0.37 μmol/L, lower than the Ki of allopurinol(4.56 mol/L), a known synthetic XO inhibitor. Apigenin(Ki of 0.56 μmol/L, a proportion of 0.0053‰ in Gnaphalium affine), luteolin(Ki of 2.63 μmol/L, 0.0032‰ in Gnaphalium affine) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone(Ki of 3.15 μmol/L, 0.0043‰ in Gnaphalium affine) also contributed to the inhibitory effect of Gnaphalium affine extract on XO activity. Conclusions These results suggest that the use of Gnaphalium affine in the treatment of gout could be attributed to its inhibitory effect on XO. This study provides a rational basis for the traditional use of Gnaphalium affine against gout.

Inhibitory activities of microalgal fucoxanthin againstα-amylase,α-glucosidase, and glucose oxidase in 3T3-L1cells linked to type 2 diabetes

Postprandial hyperglycemia is an early indication of type 2 diabetes and the target of many anti-diabetic and anti-obesity studies.α-Glucosidase and α-amylase are the crucial factors in regulating starch digestion and glucose absorption,making them key targets for many studies to treat postprandial hyperglycemia.We studied the inhibitory activities of microalgal fucoxanthin against rat-intestinalα-glucosidase and pancreaticα-amylase along with the antidiabetic eff ect to induce diff erentiation in 3T3-L1 pre-adipocytes using Oil Red-O staining.Fucoxanthin displayed strong hindrance activities towardα-amylase in a concentration-dependent manner,with an IC50 value of 0.68mmol/L,whereas weak inhibitory activity against α-glucosidase,with an IC 50 value of 4.75 mmol/L.Fucoxanthin also considerably elevated glucose oxidase activity in 3T3-L1 cells by 31.3%at 5μmol/L.During adipocyte differentiation,fucoxanthin showed lipid accumulation in 3T3-L1 cells with no cytotoxicity up to 20μmol/L.However,fucoxanthin had no inhibitory activity on glucose-6-phosphate dehydrogenase.These results suggest that fucoxanthin might be useful for the prevention of obesity or diabetes by inhibiting carbohydrate-hydrolyzing enzymes and lipid accumulation and be utilized as an ingredient for a functional food or dietary supplement.

Polyphenol Oxidase, Peroxidase and PhenylalanineAmmonium Lyase Induced in Postharvest Peach Fruitsby Inoculation with Pichia membranefaciensor Rhizopus stolonifer

Rhizopus rot of peach fruits could be significantly suppressed by Pichia membranefaciens. Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonium-lyase (PAL) activities induced by inoculation with P. membrane faciens or R. stolonifer were studied in postharvest peach fruits. The activities of PPO and PAL in peaches increased significantly after being inoculated with P. membrane faciens + R. stolonifer by 24 h, the activities maintained at a high level throughout the experiment. Under the condition of infected with R. stolonifer alone, activity of PPO and PAL could also increased, but the levels were lower than those treated with P. membrane faciens+ R. stolonifer. However, fruits inoculaed with P. membrane-faciens + R. stolonifer or R. stolonifer alone did not stimulated POD activity. The results suggest that the activation of these defense enzymes is involved in the action of P. membrane faciens against R. stolonifer.

Nicotinamide adenine dinucleotide phosphate oxidase activation and neuronal death after ischemic stroke

Nicotinamide adenine dinucleotide phosphate oxidase(NOX) is a multisubunit enzyme complex that utilizes nicotinamide adenine dinucleotide phosphate to produce superoxide anions and other reactive oxygen species. Under normal circumstances, reactive oxygen species mediate a number of important cellular functions, including the facilitation of adaptive immunity. In pathogenic circumstances, however,excess reactive oxygen species generated by NOX promotes apoptotic cell death. In ischemic stroke, in particular, it has been shown that both NOX activation and derangements in glucose metabolism result in increased apoptosis. Moreover, recent studies have established that glucose, as a NOX substrate, plays a vital role in the pathogenesis of reperfusion injury. Thus, NOX inhibition has the potential to mitigate the deleterious impact of hyperglycemia on stroke. In this paper, we provide an overview of this research,coupled with a discussion of its implications for the development of NOX inhibition as a strategy for the treatment of ischemic stroke. Both inhibition using apocynin, as well as the prospect of developing more specific inhibitors based on what is now understood of the biology of NOX assembly and activation, will be highlighted in the course of our discussion.

Xanthine oxidase inhibitors from Archidendron clypearia(Jack.) I.C. Nielsen: Results from systematic screening of Vietnamese medicinal plants

Objective: To screen Vietnamese medicinal plants for xanthine oxidase(XO) inhibitory activity and to isolate XO inhibitor(s) from the most active plant. Methods: The plants materials were extracted by methanol. The active plant materials were fractionated using different organic solvents, including n-hexane, ethyl acetate, and n-butanol. Bioassay-guided fractionation and column chromatography were used to isolate compounds. The compounds structures were elucidated by analysis of spectroscopic data, including IR, MS, and NMR. Results: Three hundreds and eleven methanol extracts(CME) belonging to 301 Vietnamese herbs were screened for XO inhibitory activity. Among these plants, 57 extracts displayed XO inhibitory activity at 100 μg/m L with inhibition rates of over 50%. The extracts of Archidendron clypearia, Smilax poilanei, Linociera ramiflora and Passiflora foetida exhibited the greatest potency with IC_(50) values below 30 μg/m L. Chemical study performed on the extract of Archidendron clypearia resulted in the isolation of six compounds, including 1-octacosanol, docosenoic acid, daucosterol, methyl gallate, quercitrin and(-)-7-O-galloyltricetiflavan. The compound(-)-7-O-galloyltricetiflavan showed the most potent XO inhibitory activity with an IC_(50) value of 25.5 μmol/L. Conclusions: From this investigation, four Vietnamese medicinal plants were identified to have XO inhibitory effects with IC_(50) values of the methanol extracts below 30 μg/m L. Compound(-)-7-O-galloyltricetiflavan was identified as an XO inhibitor from Archidendron clypearia with IC_(50) value of 25.5 μmol/L.

Regulatory Function of Polyamine Oxidase-Generated Hydrogen Peroxide in Ethylene-Induced Stomatal Closure in Arabidopsis thaliana

Hydrogen peroxide （H2O2） is an important signaling molecule in ethylene-induced stomatal closure in Arabidopsis thaliana. Early studies on the sources of H2O2 mainly focused on NADPH oxidases and cell-wall peroxidases. Here, we report the involvement of polyamine oxidases （PAOs） in ethylene-induced H2O2 production in guard cells. In Arabidopsis epidermal peels, application of PAO inhibitors caused the failure of ethylene to induce H2O2 production and stomatal closure. Results of quantitative RT-PCR analysis and pharmacological experiments showed that AtPAO2 and AtPAO4 transcripts and activities of PAOs were both induced by ethylene. In transgenic Arabidopsis plants over-expressing AtPAO2 and AtPAO4, stomatal movement was more sensitive to ethylene treatment and H2O2 production was also significantly induced. The increased H2O2 production in the transgenic lines compared to the wild-type plants suggests that AtPAO2 and AtPAO4 probably are involved in ethylene-induced H2O2 production. Several factors which induce stomatal closure such as dehydration and high salinity all enhanced the expression of AtPAO2 and AtPAO4 to different degrees. Moreover, GFP- AtPAOs fusion protein localized in the nucleus, cytoplasm, and cell wall of the guard cells. Therefore, our results strongly indicated that PAO is a source of H2O2 generation in Arabidopsis guard cells and plays crucial roles in stomatal movement.

Inhibitory effect of tartary buckwheat seedling extracts and associated flavonoid compounds on the polyphenol oxidase activity in potatoes(Solanum tuberosum L.)

To improve the processing quality of potatoes,phosphate buffer extract(PBE),50%ethanol(E50),and aqueous extract(AE)of tartary buckwheat seedlings were evaluated for their ability to inhibit the enzymatic browning of potatoes.The results suggest that all extracts of tartary buckwheat seedlings exert significant inhibitory effects on the polyphenol oxidase(PPO)activity in potatoes.The relative concentrations required for a 50%reduction in the PPO activity(IC50)were 0.21,0.28 and 0.41 mg mL^-1,for PBE,E50 and AE,respectively.The strongest inhibitory activity was observed for PBE,followed by E50 and AE.Four flavone compounds in the PBE of tartary buckwheat seedlings(i.e.,rutin,kaempferol-3-O-rutinoside,quercetin,and kaempferol)were identified by high-performance liquid chromatography.These compounds were subsequently evaluated for their roles in the inhibition of PPO from potatoes using a model system.The results indicated that rutin exhibited the highest inhibition rate on the PPO of potato.A synergistic inhibitory effect was observed by mixing rutin,kaempferol-3-Orutinoside,quercetin,and proteins.The inhibitory patterns of rutin,kaempferol-3-O-rutinoside,and quercetin on the enzyme were noncompetitive and reversible,with inhibitory constants of 0.12,0.31,and 0.40 mg mL^-1,respectively.Flavonoids from tartary buckwheat seedlings may exhibit a common mechanism with phenolic compounds,involving the blockage of the reaction of oxygen with PPO leading to the inhibition of the enzymes involved in browning.Based on these results,extracts of tartary buckwheat seedlings can be used as potent natural inhibitors.

Purification and Characterization of a Low-temperature Hydroxylamine Oxidase from Heterotrophic Nitrifier Acinetobacter sp.Y16

Objective To purify a low-temperature hydroxylamine oxidase （HAO） from a heterotrophic nitrifying bacterium Acinetobacter sp. Y26 and investigate the enzyme property. Methods A HAO was purified by an anion-exchange and gel-filtration chromatography from strain Y16. The purity and molecular mass were determined by RP-HPLC and SDS-PAGE. The HAO activity was detected by monitoring the reduction of potassium ferricyanide using hydroxylamine as substrate and ferricyanide as electron acceptor. The partial amino acid sequence was determined by mass spectrometry. Results The low-temperature HAO with a molecular mass of 61 kDa was purified from strain Y26 by an anion-exchange and gel-filtration chromatography. The enzyme exhibited an ability to oxidize hydroxylamine in wide temperature range （4-40 ℃） in vitro using hydroxylamine as substrate and ferricyanide as electron acceptor. It was stable in the temperature range of 4 to 25 ℃ and pH range of 6.0 to 8.5 with less than 30% change in its activity. The optimal temperature and pH were 15 ℃ and 7.5, respectively. Three peptides were determined by mass spectrometry which were shown to be not identical to other reported HAOs. Conclusion This is the first study to purify a low-temperature HAO from a heterotrophic nitrifier Acinetobecter sp. It differs from other reported HAOs in molecular mass and enzyme properties. The findings of the present study have suggested that the strain Y26 passes through a hydroxylamine-oxidizing process catalyzed by a low-temperature HAO for ammonium removal.

Lysyl oxidase and hypoxia-inducible factor 1α: biomarkers of gastric cancer

BACKGROUND Gastric cancer(GC) is one of the main causes of cancer mortality worldwide.Recent studies on tumor microenvironments have shown that tumor metabolism exerts a vital role in cancer progression.AIM To investigate whether lysyl oxidase(LOX) and hypoxia-inducible factor 1α(HIF1α) are prognostic and predictive biomarkers in GC.METHODS A total of 80 tissue and blood samples were collected from 140 patients admitted to our hospital between August 2008 and March 2012. Immunohistochemical staining was performed to measure the expression of LOX and HIF1α in tumor and adjacent tissues collected from patients with GC. Real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR) analysis was used to detect the mRNA expression levels of LOX and HIF1α in patients with GC. In addition, single-factor analysis was applied to analyze the relationship between LOX, HIF1α and prognosis of GC.RESULTS Immunohistochemical staining suggested that the expression levels of LOX and HIF1α increased in tumor tissues from patients with GC. QRT-PCR analysis indicated that mRNA expression of LOX and HIF1α was also upregulated in tumor tissues, which was in accordance with the above results. We also detected expression of these two genes in blood samples. The expression level of LOX and HIF1α was higher in patients with GC than in healthy controls. Additional analysis showed that the expression level of LOX and HIF1α was related to the clinicopathological characteristics of GC. Expression of LOX and HIF1α increased with the number of lymph node metastases, deeper infiltration depth and later tumor–node–metastasis stages. Single-factor analysis showed that high expression of LOX and HIF1α led to poor prognosis of patients with GC.CONCLUSION LOX and HIF1α can be used as prognostic and predictive biomarkers for GC.

Cancer and age related colonic crypt deficiencies in cytochrome c oxidase Ⅰ

AIM: To investigate whether defi ciency of expressionof cytochrome c oxidase I (CcOI) in colonic crypts is associated with colon cancer.METHODS: The pattern and level of expression of CcOI in non-neoplastic colonic crypts,and in dysplastic tissues,was assessed using standard immunohis-tochemical methods.Biopsies were obtained from individuals undergoing colonoscopies for screening purposes or for a medically indicated reason.Tissue samples were also obtained from surgical colonic resections.Samples from resections were taken from colonic mucosa 1 and 10 cm from tumors and from the tumors themselves.Samples were evaluated for frequency of crypts with reduced or absent expression of CcOI.In most crypts the loss was apparent throughout the entire crypt,while in a small minority the loss was segmental.The strong immunoreactivity using this monoclonal antibody makes the scoring unambiguous.The percent of crypts with reduced or absent expression of CcOI or (infrequent) segmented loss of expression was then calculated.Data analyses were performed using SPSS statistical package 17.0.RESULTS: The average frequency of CcOI deficient crypts (CcOI-DC) is low in individuals between 20 and 39 years of age,with 0.48% ± 0.40% CcOI-DC for women and 1.80% ± 0.35% for men.CcOI-DC increases after age 40 years,so that between the ages of 40 and 44 years the average frequency of CcOI- DC goes up to 5.89% ± 0.84% in women and 2.15% ± 1.27% in men.By 80-84 years of age,the average frequency of CcOI-DC goes up in women to 15.77% ± 0.97% and in men to 22.6% ± 0.65%.The increases in CcOI-DC from ages 40-44 years compared to 80-84 years in women and men are significantly different with P < 0.01.For women over age 60 years,deficiency of CcOI expression is greater in those women who have had a cancer in their colon.The frequency of CcOI-DC,measured in men,increased in tissues adjacent to colon cancer,being 4.03% ± 0.27% in individuals free of neoplasia in the age range 55-64 yearsand 14.13% ± 0.35% in resected histologically normal tissue of men with cancer in the same age range,P < 0.001.Similar signifi cant differences were noted in older age ranges.The frequency of CcOI-DC crypts in the cecum and sigmoid colon of an individual are signifi cantly correlated,with an R2 = 0.414 for women and R2 = 0.528 for men,P < 0.001.This suggests that the factors determining the level of CcOI deficiency act throughout the colon.Most defective crypts are in clusters of two or more,a likely consequence of crypt fission.In the non-neoplastic margins of cancers,crypts are frequently defi cient for CcOI,and such crypts may appear in large clusters,some containing more than 100 defi cient crypts.CcOI defi ciency is also apparent in colon cancers and sometimes involves a large section of the tumor.Overall,CcOI deficient cells can be visualized in segments of crypts,in whole crypts that increase in frequency with age,in crypts undergoing f ission,in clusters of crypts where the clusters increase in size with age,in increased frequency near tumors,in large clusters in the intimate margins of tumors,and in the tumors themselves.There is no clear dividing line between early stages that can be considered aspects of aging and later stages that can be considered aspects of the progression to cancer.This ambiguity may re ect a rather general situation leading to adult cancer where the early stages of cellular change appear to be relatively innocuous features of the aging process but over decades may evolve into malignancy.CONCLUSION: CcOI defi cient crypts increase in frequency with age,and clusters of defi cient crypts are associated with,and may give rise to,colon cancer.

Urotensin Ⅱ-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in Hep G2 cells

AIM: To investigated the effects of urotensin Ⅱ(UII) on hepatic insulin resistance in Hep G2 cells and the potential mechanisms involved.METHODS: Human hepatoma Hep G2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucoseoxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species(ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase(JNK), insulin signal essential molecules such as insulin receptor substrate-1(IRS-1), protein kinase B(Akt), glycogen synthase kinase-3β(GSK-3β), and glucose transporter-2(Glut 2), and NADPH oxidase subunits such as gp91 phox, p67 phox, p47 phox, p40 phox, and p22 phox were evaluated by Western blot.RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption(P < 0.05)and glycogen content(P < 0.01) in Hep G2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression(P < 0.01) and phosphorylation of IRS-1(P < 0.05), associated with down-regulation of Akt(P < 0.05) and GSK-3β(P < 0.05) phosphorylation levels, and the expression of Glut 2(P < 0.001), indicating an insulin-resistance state in Hep G2 cells. Furthermore, UII enhanced the phosphorylation of JNK(P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1(P < 0.001), phosphorylation of IRS-1(P < 0.001) and GSK-3β(P < 0.05), and glycogen synthesis(P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation(P < 0.05) and NADPH oxidase subunit expression(P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production(P < 0.05), JNK phosphorylation(P < 0.05), and insulin resistance(P < 0.05) in HepG 2 cells. CONCLUSION: UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG 2 cells.