Association between Nrf2 and CDKN2A expression in patients with end-stage renal disease: a pilot study.

Patients with end-stage renal disease (ESRD) display phenotypic features of premature biological aging, characterized by disproportionately high morbidity and mortality at a younger age. Nuclear factor erythroid 2-related factor 2 (Nrf2) activity, a master regulator of antioxidative responses, declines with age and is implicated in the pathogenesis of age-related disorders; however, little is known about the association between Nrf2 and premature biological aging in ESRD patients. In a cross-sectional pilot cohort of 34 ESRD patients receiving maintenance hemodialysis, we measured the expression of Nrf2 and cyclin-dependent kinase inhibitor 2A (CDKN2A, or p16INK4a, a biomarker of biological aging) genes in whole blood and examined the association of Nrf2 with CDKN2A expression, using Spearman's rank correlation and multivariable linear regression models with adjustment for potential confounders. There was a significant negative correlation between Nrf2 and CDKN2A expression (rho=-0.51, P=0.002); while no significant correlation was found between Nrf2 expression and chronological age (rho=-0.02, P=0.91). After multivariable adjustment, Nrf2 expression remained significantly and negatively associated with CDKN2A expression (ß coefficient=-1.51, P=0.01), independent of chronological age, gender, race, and diabetes status. These findings suggest a potential contribution of Nrf2 dysfunction to the development of premature biological aging and its related morbidities in ESRD patients.

Impact of curcumin supplementation on expression of inflammatory transcription factors in hemodialysis patients: A pilot randomized, double-blind, controlled study.

BACKGROUND & AIMS: Chronic kidney disease (CKD) patients have numerous complications associated with inflammation, which is a potential driver for cardiovascular disease. Curcumin, a compound of the curcuminoid class produced by the Curcuma longa, has been reported to activate nuclear factor erythroid factor 2-related (Nrf2) and inhibit nuclear factor kappa-B (NF-kB). Our aim was to evaluate the effects of curcumin juice on the expression of inflammatory transcription factors in hemodialysis (HD) patients. METHODS AND RESULTS: This double-blind randomized pilot study included 31 HD patients divided into two groups: curcumin group (receiving 100 mL of orange juice with 12 g of carrot and 2.5 g of turmeric after each dialysis session/week for 3 months) and control group (receiving the same juice without curcumin); 14 patients in each arm completed the study. The mRNA expression of Nrf2, NF-kB, NLRP3 inflammasome and IL-1ß in peripheral blood mononuclear cells (PBMC; using real-time quantitative polymerase chain reaction, qPCR) and routine biochemistries, food intake and anthropometrics were analyzed. After three months of supplementation, the curcumin group showed a significant decrease in NF-kB mRNA expression (AU) [from 1.08 (0.77-1.38) to 0.52 (0.32-0.95),p = 0.02] and in plasma high sensitivity C-reactive protein (hsCRP) levels [from 3.8 (2.5-6.8) to 2.0 (1.1-3.8) mg/L, p = 0.04]. There was no change in the other evaluated markers. CONCLUSION: Three months treatment with curcumin in CKD patients undergoing HD resulted in decreased markers of inflammation, NF-kB mRNA expression and hsCRP, suggesting that oral supplementation of curcumin may have an anti-inflammatory effect in this patient group. TRIAL REGISTRATION: Approved by the Ethics Committee of the Faculty of Medicine/UFF, number: 2.346.933. This study was registered within ClinicalTrials.gov under the number NCT03475017.

Mast cell stabilizer modulates Sirt1/Nrf2/TNF pathway and inhibits oxidative stress, inflammation, and apoptosis in rat model of cyclophosphamide hepatotoxicity.

Objectives: Cyclophosphamide (CYC) is the most common cytotoxic alkylating agent which considered as chemotherapy but its clinical usefulness is challenged with different forms of organ damage including hepatotoxicity. Hepatic mast cells (MC) have an important role in the pathophysiology of liver toxicity. We aimed to evaluate the possible protective effect of mast cell stabilizer, ketotifen in CYC induced-hepatotoxicity.Materials and methods: Twenty-four adult male albino Wistar rats were divided into four groups: control group, ketotifen group (received ketotifen 10 mg/kg/day, p.o.) for 14 days, CYC group (received CYC 200 mg/kg i.p.) as a single dose at the ninth day and ketotifen plus CYC group (received ketotifen and CYC). We measured serum enzyme biomarkers [alanine transaminase (ALT) and aspartate transaminase (AST)], total antioxidant capacity (TAC), interluken-1ß (IL-1ß), tissue malondialdehyde (MDA), nitric oxide (NOx), reduced glutathione (GSH), P-glycoprotein (P-gp), Sirtuin type 1 (Sirt1) and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Furthermore; histological changes, tumor necrosis factor (TNF) and caspase-3 immuno-expressions were evaluated.Results: CYC group showed hepatotoxic effect in the form of a significant increase in ALT, AST, MDA, NOx, IL-1ß levels; TNF and caspase-3 immuno-expression. Moreover; it showed toxic histological changes of marked liver injury meanwhile, there is a significant decrease in TAC, GSH, P-gp, Sirt1, and Nrf2 levels. Ketotifen showed a significant improvement in all parameters.Conclusion: Mast cell stabilizer, ketotifen possesses potent ameliorative effects against the hepatotoxic effect of CYC by reducing oxidative stress, inflammatory process, and apoptosis through regulation of Sirt1/Nrf2/TNF pathway.

Effects of Low Protein Diet on Nuclear Factor Erythroid 2-Related Factor 2 Gene Expression in Nondialysis Chronic Kidney Disease Patients.

OBJECTIVE(S): Low protein diets (LPD; 0.6 g/kg/day), prescribed for nondialysis chronic kidney disease (CKD) patients, have demonstrated numerous benefits. LPDs may modulate inflammation and oxidative stress through the nuclear factor erythroid 2-related factor 2 (Nrf2), which encodes antioxidant and phase II detoxifying enzymes. LPDs also inhibit or antagonize nuclear factor kB (NF-kB) activity, which orchestrates inflammatory and oxidative stress responses. The objective of this study was to evaluate the effects of LPD on Nfr2 and NF-κB messenger RNA (mRNA) expression in nondialysis CKD patients. METHODS: In this longitudinal study, a LPD was prescribed for 30 nondialysis CKD patients for 6 months. Peripheral blood mononuclear cells were isolated, and quantitative real-time polymerase chain reaction analysis was performed to evaluate Nrf2, NF-κB, and NADPH quinine oxidoreductase-1 mRNA expression. Thiobarbituric acid-reactive substance (TBARS) levels, a marker of lipid peroxidation, were also evaluated. RESULTS: (Age 55.5 ± 14.0 years; body mass index 29.1 ± 5.9 kg/m2; glomerular filtration rate 35.6 ± 12.2 mL/minute). After 6 months of nutritional intervention, Nrf2 mRNA expression increased from 0.85 (0.47-1.56) to 1.28 (0.63-2.63) nmol/mL (P = .03), and TBARS levels were significantly decreased from 1.78 (1.31-2.38) to 1.30 (1.07-2.22) nmol/mL (P = .04). NF-κB mRNA expression showed no significant difference after 6 months, but the Nrf2/NF-κB ratio was increased. CONCLUSION(S): In this study, a LPD appeared to modulate Nrf2 expression and decrease the levels of TBARS in nondialysis CKD patients. However, more studies are needed to confirm the effectiveness of LPD on the modulation of transcription factors involved with oxidative stress and inflammation in nondialysis CKD patients.

Crosstalk of Nrf2 with the Trace Elements Selenium, Iron, Zinc, and Copper.

Trace elements, like Cu, Zn, Fe, or Se, are important for the proper functioning of antioxidant enzymes. However, in excessive amounts, they can also act as pro-oxidants. Accordingly, trace elements influence redox-modulated signaling pathways, such as the Nrf2 pathway. Vice versa, Nrf2 target genes belong to the group of transport and metal binding proteins. In order to investigate whether Nrf2 directly regulates the systemic trace element status, we used mice to study the effect of a constitutive, whole-body Nrf2 knockout on the systemic status of Cu, Zn, Fe, and Se. As the loss of selenoproteins under Se-deprived conditions has been described to further enhance Nrf2 activity, we additionally analyzed the combination of Nrf2 knockout with feeding diets that provide either suboptimal, adequate, or supplemented amounts of Se. Experiments revealed that the Nrf2 knockout partially affected the trace element concentrations of Cu, Zn, Fe, or Se in the intestine, liver, and/or plasma. However, aside from Fe, the other three trace elements were only marginally modulated in an Nrf2-dependent manner. Selenium deficiency mainly resulted in increased plasma Zn levels. One putative mediator could be the metal regulatory transcription factor 1, which was up-regulated with an increasing Se supply and downregulated in Se-supplemented Nrf2 knockout mice.

Cellular adaptation mediated through Nrf2-induced glutamate cysteine ligase up-regulation against oxidative stress caused by iron overload in ß-thalassemia/HbE patients.

Oxidative stress caused as a result of iron overload is implicated in clinical manifestation of beta-thalassemia/haemoglobin E (ß-Thal/HbE). In this study, we investigated the cellular adaptation against oxidative stress in ß-Thal/HbE patients. Twenty-four paediatric ß-Thal/HbE patients and 22 healthy controls were recruited in the study. Blood samples from patients exhibited iron overload, elevation of lipid peroxidation, and marked diminution in the reduced glutathione (GSH) level. However, expression of glutamate-cysteine ligase catalytic (GCLC) subunit, a key enzyme in GSH biosynthesis, was up-regulated when compared with that in controls. GCLC protein levels were correlated with serum iron. There was an enhanced binding activity of the oligonucleotide probe for Nrf2-driven antioxidant response element (ARE) to nuclear protein from blood mononuclear cells of thalassemia subjects. In conclusion, ß-Thal/HbE patients exhibit elevated plasma levels of GCLC expression and Nrf2-ARE binding activity, which may account for their adaptive survival response to oxidative stress.

Modulation of inflammatory and hormonal parameters in response to testosterone therapy: Effects on the ventral prostate of adult rats.

Testosterone is often recommended in the treatment of several aging-related conditions. However, there are still questions about the consequences of this therapy in terms of hormonal and inflammatory parameters that are crucial for prostate homeostasis. Thus, we investigate if the testosterone therapy (TT) modulates the hormone receptors and inflammatory cytokines in the ventral prostate of adult rats. Wistar rats aging 150 days were divided into two experimental groups (n = 10/group): T: received subcutaneous injections of testosterone cypionate (5 mg/kg body weight) diluted in corn oil every other day for 4 weeks; and C: received corn oil as vehicle. Animals were euthanized at 180 days old by decapitation. Blood was collected to obtain hormone and cytokines concentrations. The ventral prostate was dissected and processed for light microscope and molecular analyses. Relative ventral prostate weight and epithelial compartment were increased after TT. The number of intact and degranulated mast cells was reduced in the T group. Plasma testosterone, DHT and intraprostatic testosterone concentrations were higher in the T group. TT leads to an increase in cell proliferation and up-regulation of AR, ERß, PAR-4, and NRF2. Importantly, plasma concentration and tissue expression of IL-10 and TNF-α were higher after TT. In summary, these results indicate that TT can regulate inflammatory response, with impacts in cytokines and mast cell population, and modulates steroids receptors, important parameters for prostatic homeostasis.

Effects of grape consumption on biomarkers of inflammation, endothelial function, and PBMC gene expression in obese subjects.

This study investigated effects of grape consumption on biomarkers of cardiovascular health in obese participants in both postprandial and chronic settings. Twenty obese adults participated in this randomized, placebo controlled, double-blinded crossover trial. Participants were randomized to consume 60 g freeze-dried polyphenol-rich whole grape powder (GP) or placebo (PBO) followed by high fat high carbohydrate (HFHC) meal challenge. Following acute challenge, participants consumed their respective treatment daily for 4 weeks to determine effects of chronic consumption. Consumption of GP with HFHC meal significantly increased nuclear factor (erythroid-derived 2)-like 2 (NRF2) expression in peripheral blood mononuclear cells (PBMC) at 3 h (p < 0.05) and decreased plasma endothelin-1 (ET-1) concentration at 5 h (p < 0.05) after meal challenge compared with PBO. Following 4 weeks of daily GP consumption, soluble vascular cell adhesion molecule 1 (sVCAM-1) plasma concentration increased compared with PBO (p < 0.05), however baseline values differed between treatments. In conclusion, GP consumption resulted in decreased vasoconstrictor ET-1 concentration and increased gene expression related to oxidative stress defense following HFHC meal. Except for increase in sVCAM-1 concentration, 4 weeks of chronic GP consumption had little effect on cardiovascular biomarkers measured in this study. This trial was registered: clinicaltrials.gov NCT01674231.

Nrf2 expression is increased in peripheral blood mononuclear cells derived from mild-moderate ex-smoker COPD patients with persistent oxidative stress.

Inadequacy of antioxidant nuclear factor-E2-related factor 2 (Nrf2) and endoplasmic reticulum stress-mediated unfolded protein response has been implicated in severe chronic obstructive pulmonary disease (COPD) and cigarette smoking-induced emphysema. As evidence suggests that the ability to upregulate Nrf2 expression may influence the progression of COPD and no data exist up to now in ex-smokers with mild-moderate COPD, this study was first aimed to evaluate Nrf2 and unfolded protein response expression in peripheral blood mononuclear cells (PBMC) of mild-moderate ex-smokers with COPD compared to smoking habit-matched non-COPD subjects. Then, we tested whether oxidative stress persists after cigarette smoking cessation and whether the concentrations of oxidized phospholipids (oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine [oxPAPC]) in the PBMC of the same subjects may have a causative role in determining the upregulation of Nrf2. The expression (mRNA and protein) of Nrf2 and of its related gene heme oxygenase-1 was significantly increased in COPD group without differences in the unfolded protein response. Plasma malondialdehyde, the circulating marker of oxidative stress, and oxPAPC in PBMC were significantly higher in COPD than in non-COPD subjects. The fact that the expression of p47phox, a subunit of NADPH oxidase, was increased in PBMC of COPD patients and that it was directly correlated with oxPAPC may indicate that oxPAPC may be one of the determinants of oxidative stress-induced Nrf2 upregulation. Finally, we also demonstrated that lung function inversely correlated with plasma malondialdehyde and with Nrf2 and heme oxygenase-1 mRNA expression in all subjects. Our results indicate that mild-moderate ex-smokers with COPD may be able to counteract oxidative stress by increasing the expression of Nrf2/antioxidant-response elements. Because Nrf2 failure significantly contributes to the development of COPD, our findings suggest that the possibility to prevent Nrf2 reduction may open a new scenario in helping to prevent the oxidative stress-associated lung function decline.

Effects of Silicon vs. Hydroxytyrosol-Enriched Restructured Pork on Liver Oxidation Status of Aged Rats Fed High-Saturated/High-Cholesterol Diets.

BACKGROUND: Pork is an essential component of the diet that has been linked with major degenerative diseases and development of non-alcoholic steatohepatitis (NASH). Previous studies have. Previous studies have demonstrated the in vitro antioxidant activity of silicon (Si). Furthermore, when Si is added to restructured pork (RP) strongly counterbalances the negative effect of high-cholesterol-ingestion, acting as an active hypocholesterolemic and hypolipemic dietary ingredient in aged rats. OBJECTIVE: This study was designed to evaluate the effects of Si vs hydroxytyrosol (HxT) RP on liver antioxidant defense in aged rats fed cholesterol-enriched high saturated/high cholesterol diets as a NASH model. METHODS: Four diets were prepared: Control RP diet (C) with non-added cholesterol; Cholesterol-enriched high-saturated/high-cholesterol control RP diet (CHOL-C) with added cholesterol and cholic acid; Si- or HxT-RP cholesterol-enriched high-saturated/high-cholesterol diets (CHOL-Si and CHOL-HxT). Groups of six male Wistar rats (1-yr old) were fed these modified diets for eight weeks. Total cholesterol, hepatosomatic index, liver Nrf2 and antioxidant (CAT, SOD, GSH, GSSG, GR, GPx) markers were determined. RESULTS: Both CHOL-Si and CHOL-HxT diets enhanced the liver antioxidant status, reduced hepatosomatic index and increased SOD actvity. Hydrogen peroxide removal seemed to be involved, explaining that the value of redox index was even lower than C without changing the CAT activity. CHOL-Si results were quite better than CHOL-HxT in most measured parameters. CONCLUSIONS: Our study suggests that Si incorporated into RP matrix was able to counterbalance, more efficiently than HxT, the deleterious effect of consuming a high-saturated/high-cholesterol diet, by improving the liver antioxidant defenses in the context of NASH.

[Study on anti-oxidase in smoking-related laryngeal squamous cell carcinoma].

Objective:To investigate the role of oxidative stress in smoking-related laryngeal squamous carcinoma through detecting the expression of antioxidant enzymes in smoking patients. Method:A total of 138 cases with laryngeal squamous cell carcinoma enrolled in the first hospital affiliated the northern he bei college from 2012 to 2015 and forty five volunteers were conducted. All participants were divided into three groups according to smoking index: group A(heavy smoking, 88 cases of laryngeal cancer patients) and group B(no smoking 50 cases of laryngeal cancer patients) and C group(45 heavy smoking volunteers).Catalase(CAT), glutathione peroxidase(GSH-px) and malondialdehyde(MDA) and the expression of NRF2 in serum, tissue adjacent to carcinoma, and carcinoma tissues from each groups were measured, respectively. Result:①the expression of the CAT and GSH-px in group A were significantly lower than that of group B(P <0.05), but higher than that of group C(P <0.05); ②the MDA level of group A is significantly higher than group B(P <0.05) and C group(P <0.01);③NRF2 was highly expressed in carcinoma tissues, and the expression level was negatively correlated with degree of carcinoma differentiation (P <0.05). Conclusion:Compared with nonsmoking patients, heavy smoking patients with laryngeal cancer were under more severe oxidative stress. NRF2 expression level in patients with laryngeal squamous cell carcinomas was associated with pathological stage.

Amelioration by catalpol of atherosclerotic lesions in hypercholesterolemic rabbits.

The aim of the present study was to evaluate the effects of catalpol administration on atherosclerosis. Atherogenesis was induced by a high-cholesterol chow in male New Zealand White rabbits that were randomly assigned to receive atorvastatin (5 mg/kg/day), catalpol (5 mg/kg/day), or vehicle by oral gavage for 12 weeks. The rabbits were sacrificed after 12 weeks, and the thoracic aorta and serum were collected for further pathological and molecular biological analysis. Catalpol administration resulted in significantly attenuated atherosclerotic lesions. Total cholesterol, triglycerides, and low-density lipoprotein cholesterol were remarkably reduced, and high-density lipid cholesterol was elevated in the catalpol-treated group. Catalpol reduced the levels of tumor necrosis factor-α, interleukin-6, monocyte chemoattractant protein-1, soluble vascular cell adhesion molecule-1, and soluble intercellular adhesion molecule-1 in the serum, as well as vascular cell adhesion molecule-1, monocyte chemoattractant protein-1, tumor necrosis factor-α protein, inducible nitric oxide synthase, matrix metalloproteinases-9, and nuclear factor-κB protein65 in the aortic arch. In addition, catalpol treatment reduced the lipid peroxidation levels, while elevating antioxidant capacity. Catolpol pretreatment inhibited the nuclear translocation and DNA binding activity of nuclear factor-κB protein in oxygenized low-density lipoprotein-stimulated EA.hy926 cells. Furthermore, catolpol pretreatment activated nuclear factor erythroid 2-related factor 2 and upregulated the expression of its downstream antioxidant enzyme heme oxygenase. In summary, catalpol attenuated atherosclerotic lesions by the inhibition of inflammatory cytokines and oxidative stress status in a rabbit atherosclerotic model and enhanced the antioxidant capacity in oxygenized low-density lipoprotein-stimulated EA.hy926 cells. These results suggest that catalpol may be used to prevent and attenuate atherosclerosis.

Effects of sevelamer carbonate on advanced glycation end products and antioxidant/pro-oxidant status in patients with diabetic kidney disease.

BACKGROUND AND OBJECTIVES: The primary goals were to re-examine whether sevelamer carbonate (SC) reduces advanced glycation end products (AGEs) (methylglyoxal and carboxymethyllysine [CML]), increases antioxidant defenses, reduces pro-oxidants, and improves hemoglobin A1c (HbA1c) in patients with type 2 diabetes mellitus (T2DM) and diabetic kidney disease (DKD). Secondary goals examined albuminuria, age, race, sex, and metformin prescription. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This two-center, randomized, intention-to-treat, open-label study evaluated 117 patients with T2DM (HbA1c >6.5%) and stages 2-4 DKD (urinary albumin/creatinine ratio ≥200 mg/g) treated with SC (1600 mg) or calcium carbonate (1200 mg), three times a day, without changing medications or diet. Statistical analyses used linear mixed models adjusted for randomization levels. Preselected subgroup analyses of sex, race, age, and metformin were conducted. RESULTS: SC lowered serum methylglyoxal (95% confidence interval [CI], -0.72 to -0.29; P<0.001), serum CML (95% CI, -5.08 to -1.35; P≤0.001), and intracellular CML (95% CI, -1.63 to -0.28; P=0.01). SC increased anti-inflammatory defenses, including nuclear factor like-2 (95% CI, 0.58 to 1.29; P=0.001), AGE receptor 1 (95% CI, 0.23 to 0.96; P=0.001), NAD-dependent deacetylase sirtuin-1 (95% CI, 0.20 to 0.86; P=0.002), and estrogen receptor α (95% CI, 1.38 to 2.73; P ≤0.001). SC also decreased proinflammatory factors such as TNF receptor 1 (95% CI, -1.56 to -0.72; P≤0.001) and the receptor for AGEs (95% CI, -0.58 to 1.53; P≤0.001). There were no differences in HbA1c, GFR, or albuminuria in the overall group. Subanalyses showed that SC lowered HbA1c in women (95% CI, -1.71 to -0.27; P=0.01, interaction P=0.002), and reduced albuminuria in those aged <65 years (95% CI, -1.15 to -0.07; P=0.03, interaction P=0.02) and non-Caucasians (95% CI, -1.11 to -0.22; P=0.003, interaction P≤0.001), whereas albuminuria increased after SC and calcium carbonate in Caucasians. CONCLUSIONS: SC reduced circulating and cellular AGEs, increased antioxidants, and decreased pro-oxidants, but did not change HbA1c or the albumin/creatinine ratio overall in patients with T2DM and DKD. Because subanalyses revealed that SC may reduce HbA1c and albuminuria in some patients with T2DM with DKD, further studies may be warranted.

5-lipoxygenase deficiency reduces acetaminophen-induced hepatotoxicity and lethality.

5-Lipoxygenase (5-LO) converts arachidonic acid into leukotrienes (LTs) and is involved in inflammation. At present, the participation of 5-LO in acetaminophen (APAP)-induced hepatotoxicity and liver damage has not been addressed. 5-LO deficient (5-LO⁻/⁻) mice and background wild type mice were challenged with APAP (0.3-6 g/kg) or saline. The lethality, liver damage, neutrophil and macrophage recruitment, LTB4, cytokine production, and oxidative stress were assessed. APAP induced a dose-dependent mortality, and the dose of 3 g/kg was selected for next experiments. APAP induced LTB4 production in the liver, the primary target organ in APAP toxicity. Histopathological analysis revealed that 5-LO⁻/⁻ mice presented reduced APAP-induced liver necrosis and inflammation compared with WT mice. APAP-induced lethality, increase of plasma levels of aspartate aminotransferase and alanine aminotransferase, liver cytokine (IL-1ß, TNF-α , IFN- γ, and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-ß-D-glucosaminidase activity, Nrf2 and gp91(phox) mRNA expression, and decrease of reduced glutathione and antioxidant capacity measured by 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) assay were prevented in 5-LO⁻/⁻ mice compared to WT mice. Therefore, 5-LO deficiency resulted in reduced mortality due to reduced liver inflammatory and oxidative damage, suggesting 5-LO is a promising target to reduce APAP-induced lethality and liver inflammatory/oxidative damage.

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 (. -) by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 (. -) production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis.

Relation of signal in mononuclear cell with endotoxin response and clinical outcome after trauma.

BACKGROUND: We investigated the correlation of proinflammatory transcript nuclear factor κB (NF-κB) and antioxidative gene transcript nuclear factor-erythroid 2-related factor 2 (Nrf2) expressions in peripheral blood mononuclear cells (PBMCs) with the tumor necrosis factor α (TNF-α) response after endotoxin stimulation and the clinical outcome of severely injured patients. METHODS: Thirty-two severe blunt trauma patients (injury severity score>16) with systemic inflammatory response syndrome were enrolled. Age- and sex-matched healthy persons were the controls. Patients' blood samples were obtained at 24 and 72 hours after injury. Peripheral blood mononuclear cells were isolated, and measurements for NF-κB p65 translocation, Nrf2 and phosphorylated inhibitory κB-α expressions, and TNF-α levels were assayed after endotoxin stimulation. RESULTS: In the trauma patients, TNF-α hyporesponse, depressed NF-κB p65 translocation, and phosphorylated inhibitory κB-α expression in PBMCs were found at 24 and 72 hours after injury; the Nrf2 expressions in PBMCs were not significantly different between patients and controls. The TNF-α levels had significant correlation with the NF-κB translocation and the trend of negative correlation with Nrf2 expression. Fifteen patients had critical injury (injury severity score≥25). Patients with critical injury had a lower NF-κB signal and a lower TNF-α response than did the counter group. Twelve patients developed organ failure; their Nrf2 expressions were significantly lower than those of patients without organ failure. CONCLUSIONS: The endotoxin hyporesponse associated with NF-κB and Nrf2 signal alternations in PBMCs of injured patients develops early after injury. The hyporesponse of PBMCs with a lower TNF-α level correlates with a lower NF-κB signal and is associated with critical injury, whereas a depressed Nrf2 expression in PBMCs is associated with later organ failure in trauma patients.