Heme Oxygenase-1 mRNA Expression Research Articles

Localization of Acrolein-Lysine Adduct in Fibrovascular Tissues of Proliferative Diabetic Retinopathy

ABSTRACTPurpose: To determine the presence of Nε-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), unsaturated aldehyde acrolein-derived lipoxidation end-product, in fibrovascular tissues obtained from patients with proliferative diabetic retinopathy (PDR).Methods: Fibrovascular tissues were collected from 11 eyes of 11 patients with PDR and paraffin-embedded tissue sections were prepared. Tissue localization of FDP-Lys was studied by immunohistochemistry. Signal intensity was quantified by two masked evaluators and graded into three discrete categories. The relationship between FDP-Lys staining and vascular density was analyzed. In addition, subcellular localization of FDP-Lys was studied by immunofluorescent microscopy. The impact of acrolein on cell viability and proliferation was assessed and the expression level of heme oxygenase-1 (HO-1) mRNA was quantified by real-time polymerase chain reaction (PCR) in cultured retinal microvascular endothelial cells.Results: In fibrovascular tissues, FDP-Lys staining was found in vascular components containing CD34-positive cells and alpha smooth muscle actin (α-SMA)-positive cells, and clusters of rabbit anti-glial fibrillary acid protein (GFAP)-positive cells. Immunofluorescent staining depicted subcellular localization of FDP-Lys in the nucleus and cytoplasm of the cells. Morphological analysis revealed that fibrovascular tissues with FDP-Lys staining in vascular components showed high vascular density. Exposure of cultured endothelial cells to high concentration of acrolein resulted in the decrease of cell viability and proliferation, whereas lower concentration of acrolein increased cell viability and proliferation. Sublethal concentration of acrolein upregulated HO-1 mRNA expression in retinal microvascular endothelial cells.Conclusions: The current data demonstrated the presence of FDP-Lys in fibrovascular tissues and indicate its involvement in fibrovascular proliferation in PDR.

Resveratrol-Loaded Liquid-Crystalline System Inhibits UVB-Induced Skin Inflammation and Oxidative Stress in Mice.

Evidence shows beneficial effects of resveratrol (RES) on human health. However, its poor aqueous solubility limits therapeutic effectiveness. Thus, the use of nanostructured delivery systems for RES, such as a liquid-crystalline system (LCS), could be viable. The purpose of this study was to develop, characterize, and determine the in vivo effectiveness of a RES-loaded LCS. We studied an LCS containing silicon glycol copolymer, polyether functional siloxane, and the polymeric dispersion carbomer homopolymer type B (C974) in the ratio 20:55:25 with and without RES. Results obtained using polarized light microscopy, small-angle X-ray scattering, and rheology analysis showed that the RES-loaded LCS system presents a lamellar structure and behaves as a non-Newtonian fluid presenting pseudoplastic (the apparent viscosity decreases as the stress increases) and thixotropic (the apparent viscosity decreases with the duration of stress) behaviors. Cytotoxicity studies showed that the formulation components are noncytotoxic. Topical application of a RES-loaded LCS protected hairless mice from UVB-irradiation-induced skin damage by inhibiting edema, neutrophil recruitment, lipid hydroperoxide and superoxide anion production, gp91phox mRNA expression, and oxidative stress. The RES-loaded LCS maintained 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric reducing abilities, catalase activity, reduced glutathione levels, and mRNA expression of glutathione peroxidase 1 and glutathione reductase. The RES-loaded LCS also up-regulated matrix metalloproteinase-9 activity, IL-10 production, and mRNA expression of transcription factor Nrf2 and heme oxygenase-1. Therefore, a RES-loaded LCS is a promising new therapeutic approach to mitigate skin photodamage.

Overexpression of heme oxygenase-1 induced by constitutively activated NF-κB as a potential therapeutic target for activated B-cell-like diffuse large B-cell lymphoma.

There is an urgent requirement for a new therapeutic target for activated B-cell-like lymphoma (ABC-DLBCL), which is known to have dismal outcome and constitutive activation of NF-κB. Heme oxygenase-1 (HO-1) can inhibit apoptosis and promote proliferation in many cancers. To our knowledge, no studies have been performed on the correlation between HO-1 and DLBCL. In this study, immunohistochemical analysis of 31tumor tissues from DLBCL patients [20 of ABC subtype and 11 of germinal center B-cell-like (GCB) subtype] and 11normal lymph nodes revealed that HO-1 overexpression was characteristic of ABC-DLBCL. In addition, HO-1 mRNA expression levels were consistent with the immunohistochemistry results. High levels of HO-1 expression were significantly correlated with the involvement of more than 1 extranodal site (p=0.025), with a high positivity rate of Ki-67 (p<0.01). Similar to its anti-apoptotic role in other malignancies, HO-1 upregulation suppressed apoptosis of the ABC-DLBCL cell line OCI-ly10, whereas its downregulation sensitized the tumor cells to chemotherapeutic drugs. Further study demonstrated that the HO-1 overexpression was mediated by constitutively activated NF-κB which together played an anti-apoptotic role in ABC-DLBCL. Combination of the NF-κB inhibitor Bay11‑7082 and the lentivirus vector Lenti-siHO-1 significantly decreased HO-1 protein expression and increased apoptosis in OCI-ly10 cells. However, in GCB-DLBCL cells with low levels of NF-κB expression, the TNF-α-mediated activation of NF-κB leading to HO-1 upregulation rescued the cells from apoptosis caused by HO-1 silencing. These results indicated that HO-1 can be a potential target for the treatment of ABC-DLBCL.

Amyloid Beta-Mediated Hypomethylation of Heme Oxygenase 1 Correlates with Cognitive Impairment in Alzheimer's Disease.

To identify epigenetically regulated genes involved in the pathogenesis of Alzheimer’s disease (AD) we analyzed global mRNA expression and methylation profiles in amyloid precursor protein (APP)-Swedish mutant-expressing AD model cells, H4-sw and selected heme oxygenase-1 (HMOX1), which is associated with pathological features of AD such as neurofibrillary tangles and senile plaques. We examined the epigenetic regulatory mechanism of HMOX1 and its application as a diagnostic and prognostic biomarker for AD. Our results show that HMOX1 mRNA and protein expression was approximately 12.2-fold and 7.9-fold increased in H4-sw cells, respectively. Increased HMOX1 expression was also detected in the brain, particularly the hippocampus, of AD model transgenic mice. However, the methylation of specific CpG sites within its promoter, particularly at CpG located −374 was significantly decreased in H4-sw cells. Treatment of neuroglioma cells with the demethylating agent 5-aza-2′-deoxycytidine resulted in reduced methylation of HMOX1 promoter accompanied by enhanced HMOX1 expression strongly supporting DNA methylation-dependent transcriptional regulation of HMOX1. Toxic Aβ-induced aberrant hypomethylation of HMOX1 at −374 promoter CpG site was correlated with increased HMOX1expression. In addition to neuroglioma cells, we also found Aβ-induced epigenetic regulation of HMOX1 in human T lymphocyte Jurkat cells. We evaluated DNA methylation status of HMOX1 at −374 promoter CpG site in blood samples from AD patients, patients with mild cognitive impairment (MCI), and control individuals using quantitative methylation-specific polymerase chain reaction. We observed lower methylation of HMOX1 at the −374 promoter CpG site in AD patients compared to MCI and control individuals, and a correlation between Mini-Mental State Examination score and demethylation level. Receiver operating characteristics analysis revealed good discrimination of AD patients from MCI patients and control individuals. Our findings suggest that the methylation status of HMOX1 at a specific promoter CpG site is related to AD progression.

Hyperoside Induces Endogenous Antioxidant System to Alleviate Oxidative Stress.

Background:Hyperoside, a flavonoid which is mainly found in Hypericum perforatum L., has many biological effects. One of the most important effects is to prevent the oxidative stress induced by reactive oxygen species. However, the molecular mechanisms underlying its effect are not fully understood. Oxidative stress is implicated in the occurrence of various physical diseases. A wide array of enzymatic antioxidant defense systems include NADH: quinone oxidoreductase 1, superoxide dismutase, and heme oxygenase-1 (HO-1). In the present study, the protective effects of hyperoside against hydrogen peroxide-induced oxidative stress in human lens epithelial cells, HLE-B3, were investigated in terms of HO-1 induction.Methods:The protein and mRNA expressions of HO-1 were examined by Western blotting and reverse transcriptase-PCR assays, respectively. To evaluate the ability of hyperoside to activate nuclear factor erythroid 2-related factor 2 (Nrf2), Western blotting and electrophoretic mobility shift assay were performed with nuclear extracts prepared from HLE-B3 cells treated with hyperoside. The activation of extracellular signal-regulated kinase (ERK), the upstream kinase of Nrf2 signaling, was monitored by Western blot analysis. The protective effect of hyperoside in HLE-B3 cells against hydrogen peroxide was performed by MTT assay.Results:Hyperoside increased both the mRNA and protein expression of HO-1 in a time- and dose-dependent manner. In addition, hyperoside elevated the level of of Nrf2 and its antioxidant response element-binding activity, which was modulated by upstream of ERK. Moreover, it activated ERK and restored cell viability which was decreased by hydrogen peroxide.Conclusions:Hyperoside is an effective compound to protect cells against oxidative stress via HO-1 induction.

Expression of heme oxygenase-1 in nasal polyps and regulation by glucocorticoid

To evaluate the expression and possible modulation of heme oxygenase-1 (HO-1) in nasal polyps of patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Nasal polyps and uncinate process tissues were collected from 25 CRSwNP patients and 19 healthy controls with nasal septal deviation. HO-1 expression was examined using qRT-PCR, immunohistochemistric staining and Western blot analysis. Moreover, additional uncinate process mucosal samples of 15 healthy controls with nasal septal deviation were harvested for nasal explant culture experiments. HO-1 expression was measured in cultured nasal explant in response to specific inflammatory and glucocorticoid stimulation. SPSS 20.0 software was used to analyze the data. The mRNA and protein expression of HO-1 was significantly increased in polyp tissues, 1.220±0.397 in mRNA and 1.409±0.701 in protein, compared with healthy controls 0.464±0.318 in mRNA and 0.017±0.1147 in protein (U=22.00 in mRNA and U=1.00 in protein, both P< 0.05). The immunohistochemical results showed that HO-1 was mainly distributed in the epithelial layer, submucosal glands and inflammatory cells in nasal tissues. Nasal explant culture experiments demonstrated that HO-1 mRNA was upregulated by IL-17A. The HO-1 mRNA level before the stimulation was 1.000, and 17.264±4.275 after the stimulation of 1 ng/ml IL-17A (U=0, P<0.05), 19.128±4.605 after the stimulation of 10 ng/ml IL-17A (U=0, P<0.05), but was significantly suppressed after stimulation with glucocorticoids (dexamethasone, DEX). The mRNA level after the glucocorticoids stimulation was 0.370±0.101 (U=0, P<0.05) and 0.316±0.167 (U=0, P<0.05) respectively. Furthermore, the HO-1 mRNA was inhibited by TGF-β1, the mRNA level was 0.217±0.322 (U=0, P<0.05), 0.070±0.070 (U=0, P<0.05), respectively. Increased HO-1 expression may play a role in the pathogenesis of CRSwNP, which may be considered as the therapeutic target.

Protective effect of melatonin on myenteric neuron damage in experimental colitis in rats.

Inflammation of the colon in patients with ulcerative colitis (UC) causes pain and altered motility, at least in part through the damage of the myenteric neurons (MNs). Thus, it is important to evaluate new drugs for UC treatment that could also protect myenteric neurons efficiently. As a well-known neural protective and anti-inflammatory agent, melatonin could protect neurons from damage through the activation of the nuclear factor erythroid 2-related factor 2 and antioxidant responsive element (Nrf2-ARE) signaling pathway. Therefore, we investigated the potential protective effect of melatonin against MN damage during colitis induced by 2,4-dinitrobenzene sulfonic acid (DNBS) in rats. Colitis was induced by intracolonic (i.c.) instillation of DNBS and treated with melatonin at a dose of 2.5 mg/kg for 4 days. The damage of MN in the left colon was immunohistochemically evaluated in different groups. Ulcerations and inflammation in the colon were semiquantitatively observed. Myeloperoxidase (MPO), superoxide dismutase (SOD), and malondialdehyde (MDA) levels were detected to evaluate the inflammatory and oxidative stress status. The protein and mRNA expressions of Nrf2 and heme oxygenase-1 (HO-1) in the colon were detected by Western blot and quantitative polymerase chain reaction (qPCR), respectively. Melatonin partially prevented the loss of MN and alleviated the inflammation and oxidative stress induced by DNBS. In addition, melatonin markedly increased the Nrf2 and HO-1 level in the colitis. These results indicate that melatonin protects MN from damage by reducing inflammation and oxidative stress, effects that are partly mediated by the Nrf2-ARE pathway.

β-Caryophyllene Attenuates Focal Cerebral Ischemia-Reperfusion Injury by Nrf2/HO-1 Pathway in Rats.

This study aimed to identify the effect of β-caryophyllene (BCP) pretreatment and elucidate the Nrf2/HO-1 signaling mechanism after focal cerebral ischemia-reperfusion (I-R) injury in rats. Adult male Sprague-Dawley rats were randomly assigned to the sham-operated group, I-R group and BCP pretreated I-R group. At 24h after reperfusion, neurological deficits and infarct volume were evaluated. Pathological changes of neuron in hippocampuses were observed by Nissil staining and transmission electron microscopy (TEM). Oxidative stress was assessed by malondialdehyde (MDA) level, lipid peroxidation (LPO),nitric oxide (NO), superoxide dismutase (SOD) and Catalase (CAT)activity. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were analysed by Western blotting and real-timequantitative polymerase chain reaction (Q-PCR). The protein expression of Bcl-2 and Bax was determined by immunohistochemistry. Apoptotic cells were detected using TUNEL staining. In I-R group, neurological deficit scores, cerebral infarct volume, MDA levels, LPO content, NO level, expression of Bax and TUNEL-positive cells were found to be increased at 24h after I-R injury, while SOD activity, CAT activity and expression of Bcl-2 were decreased. However, results in the BCP pretreatment groups were reversed. And the protein and mRNA expressions of Nrf2 and HO-1 were significantly up-regulated in the BCP pretreated I-R group. Results of Nissil staining and TEM scan manifested that BCP remarkablely improved neuronal injury after I-R in rats. All the above suggested that BCP pretreatment played a neuroprotective role in cerebral I-R injury, which might be exerted by upregulating the expression of Nrf2 and HO-1 to ameliorate oxidative damage and neuronal apoptosis.

Involvement of Heme Oxygenase-1 in particulate matter-induced impairment of NO-dependent relaxation in rat intralobar pulmonary arteries

Particulate air pollution exerts deleterious effects on cardiovascular system. We previously described that exposure to urban particulate matter (SRM1648) impairs nitric oxide (NO, a major vasculoprotective factor) responsiveness in intrapulmonary arteries. As Heme Oxygenase-1 (HO-1) is induced by urban particles in some cell types and is known to alter NO-dependent signaling pathway, the objective was to characterize HO-1 involvement in SRM1648-induced impairment of NO-dependent relaxation in intrapulmonary arteries.Rat intrapulmonary artery rings were exposed or not to Co (III) Protoporphyrin IX Chloride (HO-1 inducer) or SRM1648 in the absence or presence of Cr (III) Mesoporphyrin IX Chloride (HO-1 activity inhibitor). NO-dependent relaxation was assessed with DEA-NOnoate (DEA-NO) on pre-contracted arteries. HO-1 and soluble guanylyl-cyclase (sGC) mRNA and protein expressions were assessed by qRT-PCR and Western blotting, respectively.SRM1648 or Co (III) Protoporphyrin IX Chloride exposure (24) impaired DEA-NO-dependent relaxation. The SRM-induced alteration of DEA-NO responsiveness was partially prevented by Cr (III) Mesoporphyrin IX Chloride. Co (III) Protoporphyrin IX Chloride induced HO-1 mRNA and protein expressions, whereas SRM1648 only induced HO-1 protein expression without affecting its mRNA level. Exposure to either SRM1648 or to Co (III) Protoporphyrin IX Chloride did not affect the expression levels of sGC.In conclusion, this study provides some evidence that impairment of NO signaling pathway in intrapulmonary arteries involves HO-1. Therefore it highlights the role of HO-1 in particulate matter-induced detrimental effects in pulmonary circulation.

Topical Formulation Containing Naringenin: Efficacy against Ultraviolet B Irradiation-Induced Skin Inflammation and Oxidative Stress in Mice.

Naringenin (NGN) exhibits anti-inflammatory and antioxidant activities, but it remains undetermined its topical actions against ultraviolet B (UVB)-induced inflammation and oxidative stress in vivo. The purpose of this study was to evaluate the physicochemical and functional antioxidant stability of NGN containing formulations, and the effects of selected NGN containing formulation on UVB irradiation-induced skin inflammation and oxidative damage in hairless mice. NGN presented ferric reducing power, ability to scavenge 2,2′-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) and hydroxyl radical, and inhibited iron-independent and dependent lipid peroxidation. Among the three formulations containing NGN, only the F3 kept its physicochemical and functional stability over 180 days. Topical application of F3 in mice protected from UVB-induced skin damage by inhibiting edema and cytokine production (TNF-α, IL-1β, IL-6, and IL-10). Furthermore, F3 inhibited superoxide anion and lipid hydroperoxides production and maintained ferric reducing and ABTS scavenging abilities, catalase activity, and reduced glutathione levels. In addition, F3 maintained mRNA expression of cellular antioxidants glutathione peroxidase 1, glutathione reductase and transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), and induced mRNA expression of heme oxygenase-1. In conclusion, a formulation containing NGN may be a promising approach to protecting the skin from the deleterious effects of UVB irradiation.

HIV Protease Inhibitors Apoptotic Effect in SH-SY5Y Neuronal Cell Line

Background: Prophylactic treatment regimens to prevent mother-to-child HIV transmission include protease inhibitors Lopinavir and Ritonavir. Lopinavir and Ritonavir have been reported to be able to induce intracellular oxidative stress in diverse cellular models, however scarce informations are available about protease inhibitor effects of in the central nervous system (CNS). In our study we evaluated the impact of protease inhibitors on a cell neuronal model. Methods: We treated a neuroblastoma cell line (SH-SY5Y) with increasing doses of Lopinavir and Ritonavir (0.1-1-10-25-50 µM), used alone or in combination, evaluating the impact of these drugs in terms of mitochondrial activity, with MTT cell proliferation assay; mRNA expression of heme oxygenase (HemeOH) and reactive oxygen species (ROS) levels with 2',7'-dichlorofluorescin diacetate (H2DCFDA) in order to assess oxidative stress; apoptotic cell death with flow cytometry. Results: We observed that Lopinavir and Ritonavir treatment, at 25 and/or 50 µM concentrations, induced mitochondrial damage, increase of heme oxygenase RNA expression levels and ROS generation, followed by apoptosis in SH-SY5Y. Conclusions: Our in vitro model demonstrates a damaging effect of HIV protease inhibitors on the neuroblastoma cell line, thus partially mimicking the impact of these drugs on the CNS of children born to HIV positive mothers undergone to antiretroviral treatment.

Changes in expression and production of heme oxygenase-1 in rats with acute liver injury induced by lipopolysaccharide.

To investigate the changes of heme oxygenase-1 (HO-1) expression and production in rats with acute liver injury induced by lipopolysaccharide (LPS), and explore the role of HO-1 in the pathogenesis of liver injury. Liver injury was assessed histologically and the serum level of alanine transaminase (ALT) and aspartate transaminase (AST) were examined. The activity of super oxide dismutase (SOD) and the content of malondialdehyde (MDA) and carbon monoxide (CO) in liver tissues were also examined at the same time. HO-1 mRNA expression was examined at different time points following LPS treatment and the expression of HO-1 protein was determined by immunohistochemical staining. Administration of LPS caused severe hepatic damage, characterized by significant elevation of serum ALT and AST levels and hepatic MDA content as well as a remarkable reduction of liver SOD activity at 24 hr as compared with those in the control group. HO-1 activity was elevated significantly after modeling, showing a time-dependent manner from 6 to 24 hr, while expression of HO-1 protein was increased remarkably from 6 to 24 hr. Endogenous CO concentration in the liver of control rats remained very low but was elevated significantly after LPS treatment (6, 12, 24 hr), which was in accordance with the changes of HO-1. HO-1 activity and protein are increased significantly in rats with acute liver injury induced by LPS, suggesting that HO-1 plays an important role in the pathogenesis of acute hepatic damage.

Comparison of viability and antioxidant capacity between canine adipose-derived mesenchymal stem cells and heme oxygenase-1-overexpressed cells after freeze-thawing.

Allogenic adipose-derived mesenchymal stem cells (Ad-MSCs) are an alternative source for cytotherapy owing to their antioxidant and anti-inflammatory effects. Frozen-thawed allogenic Ad-MSCs can be used instantly for this purpose. However, the viability and function of frozen-thawed Ad-MSCs have not been clearly evaluated. The purpose of this study was to compare the viability and function of Ad-MSCs and heme oxygenase-1 (HO-1)-overexpressed Ad-MSCs in vitro after freeze-thawing. The viability, proliferation, antioxidant capacity and mRNA gene expression of growth factors were evaluated. Frozen-thawed cells showed significantly lower viability than fresh cells (77% for Ad-MSCs and 71% for HO-1 Ad-MSCs, P<0.01). However, the proliferation rate of frozen-thawed Ad-MSCs increased and did not differ from that of fresh Ad-MSCs after 3 days of culture. In contrast, the proliferation rate of HO-1-overexpressed Ad-MSCs was lower than that of Ad-MSCs. The mRNA expression levels of TGF-β, HGF and VEGF did not differ between fresh and frozen-thawed Ad-MSCs, but COX-2 and IL-6 had significantly higher mRNA expression in frozen cells than fresh cells (P<0.05). Fresh Ad-MSCs exhibited higher HO-1 mRNA expression than frozen-thawed Ad-MSCs, and fresh HO-1-overexpressed Ad-MSCs exhibited higher than fresh Ad-MSCs (P<0.05). However, there was no significant difference between fresh and frozen HO-1-overexpressed Ad-MSCs. The antioxidant capacity of HO-1-overexpressed Ad-MSCs was significantly higher than that of Ad-MSCs. Cryopreservation of Ad-MSCs negatively affects viability and antioxidant capacity, and HO-1-overexpressed Ad-MSCs might be useful to maximize the effect of Ad-MSCs for cytotherapy.

Moderate Exercise Training Attenuates Inflammation in Transgenic Sickle Cell Mice

Sickle cell disease (SCD) is a monogenic disorder characterized by severe systemic and vascular inflammation. There is strong evidence that moderate exercise training reduces inflammation in healthy people as well as in multiple diseases conditions. The aim of this study was to characterize the effects of moderate exercise training in a humanized transgenic mouse model of SCD (Townes) in mice expressing exclusively human sickle hemoglobin (SS). The SS mice display many of the features of human SCD including systemic inflammation and chronic anemia. We designed a treadmill training protocol of 1hr/day, 5 days a week for 8 weeks. Prior to the training, the maximal oxygen uptake (V̇O2max) was assessed by an exhaustive incremental exercise. SS mice (n=22) had lower V̇O2max (mean ± SEM: 7,068 ml/kg/hr ± 133 vs. 8,407 ml/kg/hr ± 224.5, p<0.0001) and lower maximal speed (14.3 m/min ± 0.7 vs. 29.3 m/min ± 1.7, p<0.0001) than control AA mice expressing exclusively normal human hemoglobin (n=12). Together these results indicate SS mice have an inherently reduced aerobic exercise capacity; nonetheless, they tolerated a training regimen at 60% of V̇O2max. After eight weeks of training, SS mice had a lower hematocrit (20.3% ± 0.8% vs. 24.4% ± 1.8%, p=0.04; n=8) than untrained SS littermates that were exposed to all other experimental conditions except training. There were only moderate changes in red blood cell count (Trained: 4.8.106/µL ± 0.2, Untrained: 5.7.106/µL ± 0.5, p>0.05) or total hemoglobin (Trained: 5.6 g/dL ± 0.2 vs 6.3 g/dL ± 0.4, p>0.05) between the two groups suggesting the low hematocrit in the trained mice was probably due to increased plasma volume, which is a normal physiological response to exercise training. The spleen/body mass ratio was significantly lower in the trained SS mice compared to the untrained littermates (5.7 mg/g body weight ± 0.3 vs. 7.0 mg/g body weight ± 0.9, p=0.04). Importantly, the spleen was less congested in the trained SS mice, and the magnitude of congestion in all animals (trained and untrained) correlated very strongly with the relative spleen mass (r=0.7, p=0.009). Exercise training significantly increased the percent of oxyhemoglobin in the venous blood of SS mice (Trained: 39% ± 4.3, Untrained: 22.3 ± 4.1, p=0.016). And, there was a corresponding decrease in the percent carboxyhemoglobin (Trained: 2.3% ± 0.7, Untrained 6.2% ± 0.5, p=0.001). These data suggest moderate exercise training may reduce the proportion HbS that is deoxygenated in the venous circulation. In addition, exercise training significantly reduced the peripheral blood white cell count in the SS mice (Training: 14.7.103/µL ± 0.9, Untrained: 21.6.103/µL ± 3.1, p=0.03); this change was reflected by a corresponding lower lymphocyte count in the trained mice (12.103/µL ± 0.8 vs. 17.3.103/µL ± 2.3, p=0.028). Finally, running decreased the liver expression of heme oxygenase-1 (HO-1) mRNA (p=0.04) and the plasma concentration of macrophage inflammatory protein-1β (MIP-1β, p=0.03). Interestingly, HO-1 is implicated in metabolic inflammation and MIP-1β promotes endothelial adhesion of CD8+ T cell through a VCAM-1 dependent pathway. Together with the reductions in leukocyte counts, we suggest that moderate exercise training may reduce sequestration of sickle erythrocytes/congestion, resting blood deoxygenation and inflammation in SCD. DisclosuresNo relevant conflicts of interest to declare.

Constitutive Effects of Lead on Aryl Hydrocarbon Receptor Gene Battery and Protection by β-carotene and Ascorbic Acid in Human HepG2 Cells.

Lead (Pb) is an environmental pollutant that can get entry into human body through contaminated foods, drinks, and inhaled air leading to severe biological consequences, and has been responsible for many deaths worldwide. The objectives of this study were 1st to investigate the modulatory effects of environmentally relevant concentrations of Pb on AhR gene battery, which is controlling xenobiotics metabolism. 2nd, trials to reduce Pb-induced adverse effects were done using some phytochemicals like β-carotene or ascorbic acid. Human hepatoma (HepG2) cell lines were exposed to a wide range of Pb concentrations varying from physiological to toxic levels (0 to 10 mg/L) for 24 h. High Pb concentrations (1 to 10 mg/L) significantly reduced phase I (CYP1A1 and 1A2) and phase II (UGT1A6 and NQO1) xenobiotic metabolizing enzyme mRNA expression in a mechanistic manner through the AhR regulation pathway. Additionally, these Pb concentrations induced oxidative stress in HepG2 cells in terms of production of reactive oxygen species (ROS) and induced heme oxygenase-1 mRNA expression in a concentration-dependent phenomenon. Coexposure of HepG2 cells to physiological concentrations of some micronutrients, like β-carotene (10 μM) or ascorbic acid (0.1 mM), along with Pb (1 mg/L) for 24 h significantly reduced the levels of ROS production and recovered AhR mRNA expression into the normal levels. Thus, consumption of foods rich in these micronutrients may help to reduce the adverse effects of lead in areas with high levels of pollution.

The influence of tert-butyl hydroquinone on retinal nuclear factor E2-related factor 2 and heme oxygenase-1 in type 2 diabetic rats

Objective To observe the effect of tert-butyl hydroquinone (tBHQ) on type 2 diabetic rats retinal nuclear factor E2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Methods 60 Sprague Dawley rats were randomly divided into normal control group ( NC group, n=20) and model group (n=40). The rats in model group were intraperitoneal injected with streptozotocin (30 mg/kg) to establishing type 2 diabetic mellitus (DM). There were 35 rats successfully established and they were randomly divided into diabetic group (DM group, 17 rats) and tBHQ group (18 rats). The rats in tBHQ group were fed with high fat and sugar diet with 1% tBHQ. After 4 weeks and 12 weeks of tBHQ intervention, hematoxylin eosin staining of retinal sections, immunohistochemical staining and quantitative polymerase chain reaction (PCR) of Nrf2 and HO-1 were performed. Results In tBHQ control, the retina of rats was normal and individual cells showed slightly edema at 4 weeks; the retinal structure of rats was clear and part of cells showed edema at 12 weeks. At 4 and 12 weeks, the expression of Nrf2 (t=3.115, 3.781) and HO-1 (t=3.485, 3.785) protein in DM group were higher than that in NC group (P<0.05); the expression of Nrf2 (t=2.473, 2.576) and HO-1 (t=2.785, 2.879) protein in tBHQ group were higher than that in DM group (P<0.05). In DM group, the expression of Nrf2 protein at 12 weeks was higher than that at 4 weeks (t=0.276, P<0.05). In tBHQ group, the expression of Nrf2 (t=2.516) and HO-1 (t=2.776) protein at 12 weeks were higher than that at 4 weeks (P<0.05). 4 and 12 weeks, the expression of Nrf2 (t=4.758, 4.285) and HO-1 (t=5.114, 4.514) mRNA in DM group were higher than that in NC group (P<0.05); the expression of Nrf2 (t=5.133, 4.976) and HO-1 (t=4.758, 4.251) mRNA in tBHQ group were higher than that in DM group (P<0.05). In DM gruop, the expression of Nrf2 protein at 12 weeks was higher than that at 4 weeks (t=5.114, P<0.05). In tBHQ group, the expression of Nrf2 (t=4.292) and HO-1 (t=4.974) protein at 12 weeks were higher than that at 4 weeks (P<0.05). Conclusion tBHQ intervention can increased the expression of Nrf2, HO-1 significantly in the retina of type 2 diabetic rats. Key words: Diabetic retinopathy/physiopathology; NF-E2-related factor 2; Heme oxygenase (decyclizing); Animal experimentation

Heme oxygenase-1-mediated anti-inflammatory effect of tussilagonone in RAW 264.7 macrophages

The dried flower buds of Tussilago farfara have been used in traditional medicine, mainly as antitussive in treatment of coughs and other respiratory problems. In the present study, we investigated the anti-inflammatory signaling pathway by up-regulation of heme oxygenase-1 (HO-1) expression in response to tussilagonone (TGN), a sesquiterpene compound isolated from Tussilago farfara. Treatment of RAW 264.7 cells with TGN induced HO-1 protein and mRNA expression. TGN increased protein but not mRNA level of nuclear factor-E2-related factor 2 (Nrf2), suggesting that TGN regulated Nrf2 at the translational level. Nuclear translocation of Nrf2 by TGN is also increased in a time- and dose-dependent manner indicating that TGN induced HO-1 via Nrf2 pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, TGN suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and also reduced the mRNA expression of pro-inflammatory cytokines, including iNOS, COX-2, TNF-α and IL-6, as well as nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Furthermore, the LPS-induced SEAP expression in NF-κB-SEAP-NPT construct-transfected RAW 264.7 macrophages was inhibited by TGN. Moreover, TGN inhibited the phosphorylation and degradation of inhibitory κB (IκB)-α and the nuclear translocation of NF- κB. However, a specific inhibitor of HO-1, SnPP, reversed TGN-mediated suppression of NO production and knockdown of HO-1 by small interfering RNA abrogated inhibitory effects of TGN on iNOS and COX-2 protein expression and NF-κB nuclear translocation in LPS-stimulated RAW 264.7 cells. Taken together, these findings suggest an important role of TGN-induced HO-1 activation in regulating inflammatory responses and TGN is a potent therapeutic candidate targeting the crosstalk between Nrf2/HO-1 and NF-κB signaling pathway for preventing or treating inflammation-associated diseases.

Molecular mechanism of lutein protecting Müller cells against oxidative stress induced by H2O2

Background Oxidative stress is a main cause of age-related macular degeneration (AMD). Lutein has a preventive role for AMD, but its antioxidant mechanism remains unclear. Objective Present study was to investigate the effect of lutein on oxidative stress of Muller cells and its signaling pathway. Methods Human Muller cells (human Muller cell strain) were cultured, and the cells at logarithmimic growth phase were incubated in 96 well plate overnightly.Oxidative stress cell models were established by adding 160 μmol/L H2O2, a median lethal dose for Muller cells.The models were divided into the model control group and 12.5, 25.0, 50.0 mg/L lutein groups, and the different concentrations of lutein were used to culture the cells for 24 hours, respectively.The routine cultured cells served as the blank control group.Growth of the cells was assayed by MTT method (absorbancy); the reactive oxygen species (ROS) content in the cells was assayed by flow cytometry; the mRNA and protein levels of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the cells were detected by quantitative real-time PCR and Western blot, respectively. Results The inhibitory effects on the cells were gradually enhanced with the increase of H2O2 concentrations, showing a significant difference among the groups (F=43.890, P<0.01). A significant difference was found in apoptotic rate of the cells among the blank control group, model control group and 12.5, 25.0, 50.0 mg/L lutein groups (F=346.770, P=0.000), and the apoptosis rate was significant elevated with the increase of lutein dose (all at P<0.05). The ROS contents in the cells were 1.92±0.18, 64.89±2.86, 52.70±2.80, 32.61±4.20 and 5.68±1.35 in the blank control group, model control group and 12.5, 25.0, 50.0 mg/L group, respectively, with significant difference among the groups (F=324.900, P=0.000), and the ROS content was gradually reduced as the increase of lutein dose (all at P<0.05). The relative mRNA and protein expressions of Nrf2 and HO-1 were remarkedly higher in the 12.5, 25.0, 50.0 mg/L lutein groups than those in the model control group (F=236.960, 242.620, 186.830, 263.120, all at P=0.000), and no significant difference was seen in the relative expression level of nuclear Nrf2 protein among the groups (F=1.790, P=0.210). Conclusions Lutein can induce the expression of antioxidant enzymes by inducing the expression of nuclear translocation of Nrf2 and consequently inhibit the oxidative stress status. Key words: Oxidative stress/physiology; Lutein/pharmocology; Muller cells; Macular degeneration, age-related; Nuclear factor-E2-related factor 2/metabolism; Heme oxygenase-1/metabolism

HO-1 inhibits IL-13-induced goblet cell hyperplasia associated with CLCA1 suppression in normal human bronchial epithelial cells

Mucus hypersecretion and goblet cell hyperplasia are common features that characterize asthma. IL-13 increases mucin (MUC) 5AC, the major component of airway mucus, in airway epithelial cells. According to the literature, IL-13 receptor activation leads to STAT6 activation and consequent induction of chloride channel accessory 1 (CLCA1) gene expression, associated with the induction of MUC5AC. Heme oxygenase-1 (HO-1) is an enzyme that catalyzes oxidation of heme to biliverdin, and has anti-inflammatory and anti-oxidant properties. We examined the effects of HO-1 on mucin production and goblet cell hyperplasia induced by IL-13. Moreover, we assessed the cell signaling intermediates that appear to be responsible for mucin production. Normal human bronchial epithelial (NHBE) cells were grown at air liquid interface (ALI) in the presence or absence of IL-13 and hemin, a HO-1 inducer, for 14days. Protein concentration was analyzed using ELISA, and mRNA expression was examined by real-time PCR. Histochemical analysis was performed using HE staining, and Western blotting was performed to evaluate signaling transduction pathway. Hemin (4μM) significantly increased HO-1 protein expression (p<0.01) and HO-1 mRNA expression (p<0.001). IL-13 significantly increased goblet cells, MUC5AC protein secretion (p<0.01) and MUC5AC mRNA (p<0.001), and these were decreased by hemin by way of HO-1. Tin protoporphyrin (SnPP)-IX, a HO-1 inhibitor, blocked the effect of hemin restoring MUC5AC protein secretion (p<0.05) and goblet cell hyperplasia. Hemin decreased the expression of CLCA1 mRNA (p<0.05) and it was reversed by SnPP-IX, but could not suppress IL-13-induced phosphorylation of STAT6 or SAM pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box A2 (FOXA2) mRNA expression.In summary, HO-1 overexpression suppressed IL-13-induced goblet cell hyperplasia and MUC5AC production, and involvement of CLCA1 in the mechanism was suggested.

Role of PI3K/Akt/Nrf2 signaling pathway in endotoxic shock-induced acute lung injury in rabbits

Objective To evaluate the role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B(Akt)/nuclear factor E2-related factor 2 (Nrf2) signaling pathway in endotoxic shock-induced acute lung injury (ALI) in rabbits. Methods Thirty healthy male New Zealand white rabbits, aged 2 months, weighing 1.5–2.0 kg, were randomly divided into 3 groups (n = 10 each) using a random number table: control group (group C), ALI group, and wortmannin group (group W). In group W, wortmannin 0.6 mg/kg (in 0.08 ml/kg dimethyl sulfoxide) was injected via the auricular vein, while the equal volume of normal saline was given in C and ALI groups.And 30 min later, lipopolysaccharide (LPS) 5 mg/kg (in 2 ml of normal saline) was injected via the auricular vein in ALI and W groups, while the equal volume of normal saline was given in group C. The rabbits were sacrificed at 6 h after LPS or normal saline injection.The lung was immediately removed for microscopic examination and for determination of wet/dry lung weight ratio (W/D ratio), expression of phosphorylated Akt (p-Akt), Nrf2 and heme oxygenase-1 (HO-1) (by Western blot), and expression of Nrf2 and HO-1 mRNA (using fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction). The pathological changes of the lung were scored. Results Compared with group C, the lung injury scores and W/D ratio were significantly increased, and the expression of p-Akt, Nrf2 protein and mRNA, and HO-1 protein and mRNA was up-regulated in ALI and W groups (P<0.05). Compared with group ALI, the lung injury scores and W/D ratio were significantly increased, and the expression of p-Akt, Nrf2 protein and mRNA, and HO-1 protein and mRNA was up-regulated in group W (P<0.05). Conclusion Activation of PI3K/Akt/Nrf2 pathway is the regulatory mechanism of the body adapting to the development of endotoxic shock-induced ALI in rabbits. Key words: 1-phosphatidylinositol 3-kinase; Protein-serine-threonine kinases; NF-E2-related factor 2; Endotoxemia; Respiratory distress syndrome, adult