Heme Oxygenase-1 mRNA Expression Research Articles

Reactive oxygen species produced by the corrosion of Fe-based materials induce endothelial dysfunction

Based on their good mechanical properties, bioresorbable Fe-based materials have been proposed during the last decade as candidate alloys for coronary stents. Previous investigations on the biocompatibility of metallic materials often overlooked the complexity of the biodegradation phenomenon. We document, for the first time, the generation of reactive oxygen species (ROS) during Fe-based materials corrosion and their deleterious impacts on endothelial function. ROS generation was evaluated by the terephtalic acid (TA) hydroxylation assay. The endothelium of rat aortic rings was directly exposed (6 h) by inserting an iron wire into the lumen in the presence/absence of catalase. Indirect exposure to iron wire was included to assess the potential role of solubilized iron ions. Nitric oxide (NO) production by rings was measured by EPR spin-trapping, and endothelium-dependent relaxation was assessed with the organ-bath method. Induction of oxidative stress in rings was assessed via the mRNA expression of the oxidative stress response gene heme oxygenase-1 (HO-1). Iron exhibited a strong potential to generate ROS in the TA assay. ROS generation, but not corrosion, was inhibited by catalase. Direct contact with iron significantly impaired endothelium-dependent relaxation of aortic rings. Direct contact with the material in the presence of catalase, or indirect contact did not affect endothelium function. NO production by isolated rat aortic rings was inhibited by ROS generated by the corrosion of iron, as indicated by the protective role of catalase. Expression of HO-1 was increased after direct exposure to iron, not to indirect exposure or in the presence of catalase. The demonstration of ROS production during corrosion, induction of oxidative stress, inhibition of NO production and consequent endothelium dysfunction raise concern about the biocompatibility of biodegradable Fe-based alloys for vascular implants.

Nrf2⁻ARE Signaling Acts as Master Pathway for the Cellular Antioxidant Activity of Fisetin.

Fisetin, a dietary flavonoid, is reported to have cellular antioxidant activity with an unclear mechanism. In this study, we investigated the effect of fisetin on the nuclear factor, erythroid 2-like 2 (Nrf2) signaling pathway in HepG2 cells to explore the cellular antioxidant mechanism. Fisetin upregulated the mRNA expression of heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H quinone oxidoreductase-1 (NQO1), and induced the protein of HO-1 but had no significant effect on the protein of GCLC, GCLM and NQO1. Moreover, nuclear accumulation of Nrf2 was clearly observed by immunofluorescence analysis and western blotting after fisetin treatment, and an enhanced luciferase activity of antioxidant response element (ARE)-regulated transactivation was obtained by dual-luciferase reporter gene assays. In addition, fisetin upregulated the protein level of Nrf2 and downregulated the protein level of Kelch-like ECH-associated protein 1 (Keap1). However, fisetin had no significant effect on Nrf2 mRNA expression. When protein synthesis was inhibited with cycloheximide (CHX), fisetin prolonged the half-life of Nrf2 from 15 min to 45 min. When blocking Nrf2 degradation with proteasome inhibitor MG132, ubiquitinated proteins were enhanced, and fisetin reduced ubiquitination of Nrf2. Taken together, fisetin translocated Nrf2 into the nucleus and upregulated the expression of downstream HO-1 gene by inhibiting the degradation of Nrf2 at the post-transcriptional level. These data provide the molecular mechanism to understand the cellular antioxidant activity of fisetin.

Nrf2 participates in mechanisms for reducing the toxicity and enhancing the antitumour effect of Radix Tripterygium wilfordii to S180-bearing mice by herbal-processing technology

Context: Radix Tripterygium wilfordii Hook. f. (Celastraceae) (LGT) has outstanding curative efficacy; however, side effects include high toxicity, particularly hepatotoxicity and nephrotoxicity.Objective: To investigate detoxification mechanisms of LGT through processing separately with each of these medicinal herbs including Flower Lonicera japonica Thunb. (Caprifoliaceae) (JYH), Radix Paeonia lactiflora Pall. (Ranunculaceae) (BS), Herba Lysimachia christinae Hance (Primulaceae) (JQC), Radix et Rhizoma Glycyrrhiza uralensis Fisch. (Fabaceae) (GC) and Seed Phaseolus radiatus L. (Fabaceae) (LD) in S180-bearing mice by involving nuclear factor (erythroid-derived 2)-like 2 (Nrf2).Materials and methods: LGT raw and processed products were orally administered at 60 mg/kg to KM male mice inoculated with S180 tumour cells for 14 consecutive days, and blood, tumour, liver and kidney were taken to observe the detoxifying effects and biological mechanisms.Results: Herbal-processing technology significantly weakened hepatotoxicity and nephrotoxicity evoked by LGT with ED50 of the converted triptolide in each processed-herb product for serum alanine transaminase, aspartate transaminase, creatinine and urea nitrogen of 9.3, 16.6, 2.5 and 4.2 μg/kg, for liver glutathione, glutathione S-transferase, catalase, tumour necrosis factor-α and interleukin-10 of 114.9, 67.8, 134.1, 7.7, 4171.6 μg/kg, and for kidney 21.9, 20.5, 145.0, 529.7, 19.4 μg/kg, respectively. Moreover, herbal-processing technology promoted the accumulation of Nrf2 into the nucleus, and upregulated mRNA expression of Nrf2 and heme oxygenase-1. Additionally, herbal-processing technology enhanced the tumour inhibition rate with ED50 12.2 μg/kg.Discussion and conclusions: Herbal-processing technology improves the safety and effectiveness of LGT in cancer treatment, and future research may be focused on the Nrf2-related molecules.

Resveratrol reduces brain injury after subarachnoid hemorrhage by inhibiting oxidative stress and endoplasmic reticulum stress.

Previous studies have shown that resveratrol, a bioactive substance found in many plants, can reduce early brain injury after subarachnoid hemorrhage, but how it acts is still unclear. This study explored the mechanism using the experimental subarachnoid hemorrhage rat model established by injecting autologous blood into the cerebellomedullary cistern. Rat models were treated with an intraperitoneal injection of 60 mg/kg resveratrol 2, 6, 24 and 46 hours after injury. At 48 hours after injury, their neurological function was assessed using a modified Garcia score. Brain edema was measured by the wet-dry method. Neuronal apoptosis in the prefrontal cortex was detected by terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick-end labeling assay. Levels of reactive oxygen species and malondialdehyde in the prefrontal cortex were determined by colorimetry. CHOP, glucose-regulated protein 78, nuclear factor-erythroid 2-related factor 2 and heme oxygenase-1 mRNA expression levels in the prefrontal cortex were measured by reverse transcription polymerase chain reaction. Tumor necrosis factor-alpha content in the prefrontal cortex was detected by enzyme linked immunosorbent assay. Immunohistochemical staining was used to detect the number of positive cells of nuclear factor-erythroid 2-related factor 2, heme oxygenase 1, glucose-regulated protein 78, CHOP and glial fibrillary acidic protein. Western blot assay was utilized to analyze the expression levels of nuclear factor-erythroid 2-related factor 2, heme oxygenase 1, glucose-regulated protein 78 and CHOP protein expression levels in the prefrontal cortex. The results showed that resveratrol treatment markedly alleviated neurological deficits and brain edema in experimental subarachnoid hemorrhage rats, and reduced neuronal apoptosis in the prefrontal cortex. Resveratrol reduced the levels of reactive oxygen species and malondialdehyde, and increased the expression of nuclear factor-erythroid 2-related factor 2, heme oxygenase-1 mRNA and protein in the prefrontal cortex. Resveratrol decreased glucose-regulated protein 78, CHOP mRNA and protein expression and tumor necrosis factor-alpha level. It also activated astrocytes. The results suggest that resveratrol exerted neuroprotective effect on subarachnoid hemorrhage by reducing oxidative damage, endoplasmic reticulum stress and neuroinflammation. The study was approved by the Animals Ethics Committee of Shandong University, China on February 22, 2016 (approval No. LL-201602022).

Diverse associations between oxidative stress and thromboxane A2 in hypertensive glomerular injury

We examined the potential contributions of oxidative stress and thromboxane A2 (TXA2) to the development of regional heterogeneity in hypertensive glomerular injury using stroke-prone spontaneously hypertensive rats (SHRSP), an animal model of human essential hypertension. We also examined the effect of antioxidant treatment on the regional expression of thromboxane synthase (TXAS) mRNA using a microdissection method. Increases in the glomerular expression of TXAS mRNA were observed in the SHRSP at 15 weeks of age compared with those in the age-matched normotensive control Wistar–Kyoto (WKY) rats: 2.4-fold and 3.1-fold in the superficial and juxtamedullary glomeruli, respectively (P < 0.05). The heme oxygenase-1 mRNA expression was markedly increased (greater than eightfold, P < 0.05) in both the superficial and juxtamedullary glomeruli in the SHRSP compared with the expression in the WKY rats. In contrast to our expectations, the treatment of SHRSP with tempol (a superoxide dismutase mimetic) significantly (P < 0.05) increased the TXAS mRNA expression in the superficial glomeruli and did not improve the histological injury or albuminuria, which were both aggravated. Moreover, ozagrel (a TXAS inhibitor) had a suppressive effect on the TXAS mRNA expression and significantly (P < 0.05) improved the histological injury. These results indicated that although TXA2 and oxidative stress are linked to each other, TXA2 rather than oxidative stress may be a better therapeutic target to improve hypertensive glomerular injury.

Factors contributing to the resistance of the thyrocyte to hydrogen peroxide

We studied the mechanism that may explain the relative resistance of thyrocytes to H2O2 compared to other cell types. Ability to degrade H2O2, glutathione peroxidase (GPx) activity, heme oxygenase-1 (HO-1) expression, cell survival and capacity to repair DNA damage after H2O2 exposure or irradiation were measured in human thyrocytes in primary culture and compared to the values obtained in human T-cells and different cell lines. Compared to other cell types, thyrocytes presented a low mortality rate after H2O2 exposure, rapidly degraded extracellular H2O2 and presented a high basal seleno-dependent GPx activity. Only in thyrocytes, H2O2 up-regulated GPx activity and expression of HO-1 mRNA. These effects were not reproduced by irradiation. DNA damage caused by H2O2 was more slowly repaired than that caused by irradiation and not repaired at all in T-cells. Our study demonstrates that the thyrocyte has specific protective mechanisms against H2O2 and its mutagenic effects.

METH-Induced Neurotoxicity Is Alleviated by Lactulose Pretreatment Through Suppressing Oxidative Stress and Neuroinflammation in Rat Striatum.

Abuse of methamphetamine (METH) results in neurological and psychiatric abnormalities. Lactulose is a poorly absorbed derivative of lactose and can effectively alleviate METH-induced neurotoxicity in rats. The present study was designed to investigate the effects of lactulose on METH-induced neurotoxicity. Rats received METH (15 mg/kg, 8 intraperitoneal injections, 12-h interval) or saline and received lactulose (5.3 g/kg, oral gavage, 12-h interval) or vehicle 2 days prior to the METH administration. Reactive oxygen species (ROS) and malondialdehyde (MDA) were measured. Protein levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6), nuclear factor κB (NFκB), interleukin (IL)-1β, IL-6, TNF-α, cleaved caspase 3, and poly(ADP-ribose) polymerase-1 (PARP-1) were determined by western blotting. mRNA expressions of nuclear factor erythroid 2-relatted factor-2 (Nrf2), p62, and heme oxygenase-1 (HO-1) were assessed by RT-qPCR. The lactulose pretreatment decreased METH-induced cytoplasmic damage in rat livers according to histopathological observation. Compared to the control group, overproduction of ROS and MDA were observed in rat striatums in the METH alone-treated group, while the lactulose pretreatment significantly attenuated the METH-induced up-regulation of oxidative stress. The lactulose pretreatment significantly repressed over-expressions of proteins of TLR4, MyD88, TRAF6, NFκB, IL-1β, IL-6, TNF-α, cleaved caspase 3, PARP-1. The lactulose pretreatment increased mRNA expressions of Nrf2, p62, and HO-1. These findings suggest that lactulose pretreatment can alleviate METH-induced neurotoxicity through suppressing neuroinflammation and oxidative stress, which might be attributed to the activation of the Nrf2/HO-1 axis.

Protocatechuic acid-mediated DJ-1/PARK7 activation followed by PI3K/mTOR signaling pathway activation as a novel mechanism for protection against ketoprofen-induced oxidative damage in the gastrointestinal mucosa

Oxidative stress contributes to the progression of non-steroidal anti-inflammatory drug (NSAID)-induced gastrointestinal (GI) cell apoptosis. In our previous study, we reported that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a protective role against ketoprofen-induced GI mucosal oxidative injury. Recent reports suggest that Nrf2 could exhibit antioxidative and antiapoptosis responses through up-regulation of DJ-1 (PARK7). In the current study, we proposed that induction of DJ-1 expression by protocatechuic acid (PCA) might provide a potential therapeutic approach for treating oxidative stress-associated GI ulcer diseases. The results indicated that PCA increased mRNA expression of glutathione peroxidase and heme oxygenase-1 through up-regulation of DJ-1 followed by Nrf2 translocation. Furthermore, PCA protected Int-407 cells against ketoprofen-induced oxidative stress by regulating the DJ-1, PI3K, and mTOR pathways. Pretreatment with PCA inhibited mitochondrial ROS generation, up-regulated the mitochondrial membrane potential, and down-regulated pro-apoptotic Bax as well as downstream caspase-8, caspase-9, and caspase-3 activity, and reversed impaired DJ-1 and anti-apoptotic Bcl-2 protein expression in Int-407 cells induced by ketoprofen. Similar to the in vitro results, SD rats treated with PCA before administration of ketoprofen exhibited decreased caspase-3 protein expression as well as oxidative damage, and impairment of the antioxidant system and DJ-1 protein expression in the GI mucosa were reversed. The administration of lansoprazole, a type of proton pump inhibitor (PPI), strongly inhibited ketoprofen-induced GI mucosal injuries via up-regulation of DJ-1, indicating that DJ-1 is essential for the dietary antioxidant- and PPI drug-mediated mechanism of ulcer therapy. These results suggest that DJ-1 could be a novel target for protection against ketoprofen-induced GI ulcers due to its antioxidant and anti-apoptosis characteristics.

Rapamycin induces the expression of heme oxygenase-1 and peroxyredoxin-1 in normal hepatocytes but not in tumorigenic liver cells

Rapamycin (sirolimus) is employed as an immunosuppressant following liver transplant, to inhibit the re-growth of cancer cells following liver resection for hepatocellular carcinoma (HCC), and for the treatment of advanced HCC. Rapamycin also induces the expression of antioxidant enzymes in the liver, suggesting that pretreatment with the drug could provide a potential strategy to reduce ischemia reperfusion injury following liver surgery. The aim of this study was to further investigate the actions of rapamycin in inducing expression of the antioxidant enzymes heme oxygenase-1 (HO-1) and peroxiredoxin-1 (Prx-1) in normal liver and in tumorigenic liver cells. A rat model of segmental hepatic ischemia and reperfusion, cultured freshly-isolated rat hepatocytes, and tumorigenic H4IIE rat liver cells in culture were employed. Expression of HO-1 and Prx-1 was measured using quantitative PCR and western blot. Rapamycin pre-treatment of normal liver in vivo or normal hepatocytes in vitro led to a substantial induction of mRNA encoding HO-1 and Prx-1. The dose-response curve for the action of rapamycin on mRNA expression was biphasic, showing an increase in expression at 0 - 0.1 μM rapamycin but a decrease from maximum at concentrations greater than 0.1 μM. By contrast, in H4IIE cells, rapamycin inhibited the expression of HO-1 and Prx-1 mRNA. Oltipraz, an established activator of transcription factor Nrf2, caused a large induction of HO-1 and Prx-1 mRNA. The dose response curve for the inhibition by rapamycin of HO-1 and Prx-4 mRNA expression, determined in the presence of oltipraz, was monophasic with half maximal inhibition at about 0.01 μM. It is concluded that, at concentrations comparable to those used clinically, pre-treatment of the liver with rapamycin induces the expression of HO-1 and Prx-1. However, the actions of rapamycin on the expression of these two antioxidant enzymes in normal hepatocytes are complex and, in tumorigenic liver cells, differ from those in normal hepatocytes. Further studies are warranted to evaluate preconditioning the livers of patients subject to liver resection or liver transplant with rapamycin as a viable strategy to reduce IR injury following liver surgery.

Dysfunction of Nrf2-ARE Signaling Pathway: Potential Pathogenesis in the Development of Neurocognitive Impairment in Patients with Moderate to Severe Obstructive Sleep Apnea-Hypopnea Syndrome.

The present study investigated the nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling pathway in patients with moderate to severe obstructive sleep apnea-hypopnea syndrome (OSAHS). Their correlation with neurocognitive impairment metrics was investigated to explore potential pathogenesis in OSAHS. Forty-eight patients with OSAHS and 28 controls underwent testing with the Epworth Sleep Scale (ESS), MATRICS Consensus Cognitive Battery (MCCB), Stroop Color and Word Test, polysomnography (PSG), and measurements of the concentration of plasma superoxide dismutase (SOD) and thioredoxin (Trx). Further, 20 pairs of matched patients with OSAHS and controls were selected for measurement of the expression (protein and mRNA) of Nrf2 and of its downstream antioxidase, heme oxygenase-1 (HO-1), in peripheral mononuclear cells (PBMCs). Finally, correlations between neurocognitive impairment and the above metrics were analyzed. Expression of Nrf2 and HO-1 mRNA and protein in the PBMCs, as well as plasma SOD and Trx levels, were significantly reduced in patients with OSAHS. After adjusting for education, sex, age, and smoking index, the expression of Nrf2-ARE signaling pathway proteins (or mRNA) was closely correlated with sleep respiratory parameters. An inverse relationship was demonstrated between the expression of nuclear Nrf2 in PBMCs, concentration of plasma SOD and Trx, and apnea-hypopnea index (AHI) in patients with OSAHS. Trx, nuclear Nrf2 protein, and HO-1 protein were also negatively correlated with the percent of time that SaO2 was less than 90% (TSat90). Total Nrf2 protein level was positively correlated with AHI and TSat90 and negatively correlated with minimum SaO2 (LSaO2), while nuclear Nrf2 protein and HO-1 protein were positively correlated with LSaO2. Moreover, significant positive correlations were found between maze scores and expression of nuclear Nrf2 protein, HO-1 protein, and SOD and Trx levels. Furthermore, inverse relationships between total Nrf2 protein in PBMCs and HVLT-R and maze scores were found. Multiple linear regression showed plasma Trx concentration as a potential predictor of maze and BVMT-R scores. In conclusion, the expression of Nrf2-ARE molecules and related antioxidases is significantly decreased in patients with OSAHS and is correlated with neurocognitive dysfunction. The Nrf2-ARE signaling pathway may play a crucial role in neurocognitive impairment in patients with moderate to severe OSAHS. Further studies are needed to explore the exact mechanisms and potential treatment interventions.

Microsatellite polymorphism in the Heme oxygenase-1 gene promoter is associated with dermal collagen density in Japanese obese male subjects.

We previously reported elevated oxidative stress-related mechanical vulnerabilities of the skin as sparse distributions of hyperechoic areas. Although this helped establish a personalized skin care system to prevent skin disorders related to mechanical stress, obesity-related skin vulnerability involves individual differences. Here, we hypothesized that individual differences are caused by polymorphisms of GT repetitive sequences in the heme oxygenase1 (HMOX1) promoter region, which encodes an antioxidant enzyme. This cross-sectional study enrolled healthy male volunteers in a walking classroom aimed at weight control. Subjects with a body mass index <25 kg/m2 were classified as non-obese and those with body mass index ≥25 kg/m2 were classified as obese. Subject skin was categorized into sparse dermis or normal groups according to the distribution of hyperechoic areas by high-resolution skin ultrasonography (20 MHz). Genomic DNA and mRNA extracted from three body hairs with attached follicle cells were used to analyze GT repetitive sequences of the HMOX1 promoter, HMOX1 mRNA expression levels, and oxidative stress levels (8-hydroxy-2’-deoxyguanosine). Classifications of GT repetitive sequence of HMOX1 promoter were Short (<27 times) and Long (≥27 times). Higher numbers of subjects with sparse dermis were in the obese group compared with the non-obese group. In obese subjects, the number of subjects that had the Long allele of the HMOX1 promoter with sparse dermis was significantly higher compared with the normal group, whereas no association was observed between the polymorphism and ultrasonographic features in non-obese subjects. Thus, HMOX1 polymorphisms detected a risk of low collagen density in Japanese obese male subjects.

Expression of the oxygen-sensitive transcription factor subunit HIF-1α in patients suffering from secondary Raynaud syndrome.

Anti-ischemic therapy remains a challenge due to the complexity of hypoxia response pathways. Hypoxia-inducible factor (HIF)-1 is a heterodimer transcription factor consisting of 2 subunits, HIF-1α and HIF-1β. Hypoxia-dependent activation of HIF-1α regulates cellular O2 homeostasis. Raynaud syndrome (RS), as a comorbidity of the autoimmune disease systemic sclerosis (SS), is characterized by vasospasms that limit blood flow to the limbs, resulting in hypoxia. A single-center randomized study was conducted to compare prostaglandin E1 (PgE1) therapy with a treatment combining PgE1 and an endothelin-1 blocker, bosentan. A total of 30 patients suffering from SS with RS were enrolled. We examined the regulation of HIF-1α, its target heme oxygenase-1 (HMOX-1), and the serum levels of the HIF-1α protein in a subset of patients as well as in ten healthy individuals. The expression of HIF-1α and HMOX-1 in monocytes was measured using absolute plasmid-based quantitative real-time PCR, whereas serum HIF-1α levels were measured with ELISA. Samples were taken at the time of randomization and after 24 weeks. We found that HIF-1α and HMOX-1 mRNA expression in monocytes and serum HIF-1α protein levels were significantly higher in the SS/RS patients compared to the healthy control group. Single-drug therapy significantly increased HIF-1α and HMOX-1 mRNA expression in monocytes and serum HIF-1α protein levels in the SS/RS patients compared to those at the time of randomization, whereas combining PgE1 with an endothelin-1 blocker prevented the further increases in HIF-1α and HMOX-1 expression. We propose HIF-1α and HMOX-1 as novel markers for anti-ischemic therapy in RS.

Canagliflozin Inhibits Human Endothelial Cell Inflammation via the Induction of Heme Oxygenase-1

Vascular disease is the major cause of mortality in diabetes. Although many factors contribute to diabetes-associated vascular disease, inflammation of endothelial cells (ECs) plays a critical role. Sodium-glucose transporter 2 (SGLT2) inhibitors are recently approved anti-hyperglycemic drugs that cause a significant reduction in cardiovascular events in type 2 diabetic patients, but the mechanisms underlying this protection are not fully known. In the present study, we investigated the effect of SGLT2 inhibitors on the expression of the vasoprotective protein heme oxygenase-1 (HO-1) in human umbilical vein ECs. Treatment of ECs with canagliflozin, dapagliflozin, or empagliflozin stimulated the expression of HO-1 mRNA and protein; however, only clinically achievable concentrations of canagliflozin (3-10µM) increased HO-1 levels. The induction of HO-1 by canagliflozin was associated with an increase in Nrf2 activity and reactive oxygen species (ROS) formation. Canagliflozin also stimulated HO-1 promoter activity, and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the canagliflozin-mediated induction of HO-1 was abolished by N-acetyl-L-cysteine or rotenone. Significantly, prior treatment of ECs with canagliflozin inhibited the induction of the adhesion molecules ICAM-1 and VCAM-1 in response to high concentrations of glucose (25mM) and oxidized low-density lipoprotein (50mg/L) that mimic the diabetic milieu. Significantly, the anti-inflammatory action of canagliflozin was abrogated by blocking HO-1 activity or expression. In conclusion, this study demonstrates that canagliflozin stimulates HO-1 gene expression in the vascular endothelium via the ROS-Nrf2 pathway, and that canagliflozin mitigates inflammation in human ECs exposed to a diabetic environment via the induction of HO-1. Thus, canagliflozin may exert some of it cardiovascular benefit in diabetes by targeting HO-1. Disclosure K.J. Peyton: None. G. Behnammanesh: None. W. Durante: None.

Effect of penehychdine hydrochloride pretreatment on Nrf2/ARE signaling pathway during myocardial ischemia-reperfusion in rats

Objective To evaluate the effect of penehychdine hydrochloride pretreatment on nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway during myocardial ischemia-reperfusion(I/R)in rats. Methods Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 2-3 months, weighing 220-240 g, were divided into 3 groups (n=12 each) using a random number table method: sham operation group (group S), myocardial I/R group and penehyelidine hydrochloride pretreatment group (group PHC). Myocardial I/R was induced by occlusion of the anterior descending branch of left coronary artery for 30 min followed by 120 min reperfusion.At 30 min before ischemia, penehyelidine hydrochloride 2 mg/kg was injected intraperitoneally in group PHC, and the anterior descending branch of left coronary artery was only exposed but not ligated in group S. Rats were sacrificed at the end of reperfusion, and hearts were removed for measurement of the myocardial infarct size(by 2, 3, 5-triphenyltetrazolium chloride staining), cell apoptosis (by TUNEL) and expression of Nrf2, heme oxygenase-1(HO-1), NQO1 and γ-glutamylcysteine synthetase(γ-GCS) protein and mRNA (by using Western blot or real-time fluorescence quantitative polymerase chain reaction). The percentage of myocardial infarct size and apoptosis index were calculated. Results Compared with group S, the percentage of myocardial infarct size and apoptosis index were significantly increased, and the expression of Nrf2, HO-1, NQO1 and γ-GCS protein and mRNA was down-regulated in I/R and PHC groups (P<0.05). Compared with group I/R, the percentage of myocardial infarct size and apoptosis index were significantly decreased, and the expression of Nrf2, HO-1, NQO1 and γ-GCS protein and mRNA was up-regulated in group PHC (P<0.05). Conclusion Penehyclidine hydrochloride pretreatment attenuates myocardial I/R injury through activating Nrf2-ARE signaling pathway in rats. Key words: Cholinergic antagonists; Myocardial reperfusion injury; Nuclear factor-E2 related factor 2; Response elements; Signal transduction

Neuroprotection against 6-OHDA toxicity in PC12 cells and mice through the Nrf2 pathway by a sesquiterpenoid from Tussilago farfara

Oxidative stress plays a key role in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Therefore, the nuclear factor-E2-related factor 2 (Nrf2), a key regulator of the antioxidative response, is considered to be important as a therapeutic target for neurodegenerative diseases. We investigated the underlying mechanism of Nrf2-mediated neuroprotective effects against oxidative stress in the PC12 cell line by 7β-(3-ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), one of the sesquiterpenoids in Farfarae Flos. Pretreatment of PC12 cells with ECN had a protective effect against hydrogen peroxide (H2O2)- or 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. ECN upregulated the ARE-luciferase activity and induced the mRNA expression of Nrf2 and antioxidant enzyme heme oxygenase-1 (HO-1). Knockdown of Nrf2 by small, interfering RNA (siRNA) abrogated the upregulation of HO-1, indicating that ECN had induced HO-1 via the Nrf2 pathway. Pretreatment with the thiol reducing agents, N-acetylcysteine (NAC) or dithiothreitol (DTT), attenuated Nrf2 activation and HO-1 expression. However, the non-thiol reducing antioxidant, Trolox, failed to inhibit HO-1 induction by ECN. These results suggest that ECN may directly interact with Kelch-like ECH-associated protein 1 (Keap1) and modify critical cysteine thiols present in the proteins responsible for Nrf2-mediated upregulation of HO-1. In a 6-OHDA-induced mouse model of PD, administration of ECN ameliorated motor impairments and dopaminergic neuronal damage. Taken together, ECN exerts neuroprotective effects by activating the Nrf2/HO-1 signaling pathway in both PC12 cells and mice. Thus, ECN, as an Nrf2 activator, could be an attractive therapeutic candidate for the neuroprotection or treatment of neurodegenerative diseases.

Heme oxygenase-1 exerts pro-apoptotic effects on hepatic stellate cells in vitro through regulation of nuclear factor-κB.

Heme oxygenase-1 (HO-1) is an antioxidant and cytoprotective protein, which has been proven to alleviate the proliferation of hepatic stellate cells (HSCs) and the development of liver fibrosis. However, the role of HO-1 in HSC apoptosis remains unclear. The aim of the present study was to investigate the effect of HO-1 on HSC apoptosis and its possible underlying mechanisms. HSCs-T6 were incubated with different concentrations of hemin (HO-1 chemical inducer) and Znpp-IX (HO-1 chemical inhibitor) for 12, 24 and 48 h. Cell viability was determined using an MTT assay. HSCs were classified into 4 groups as follows: Control, hemin, Znpp-IX and hemin+Znpp-IX co-treatment groups. Apoptosis was quantitatively measured by Annexin V/propidium iodide double staining and a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The mRNA and protein expression of HO-1, α-smooth muscle actin, B-cell lymphoma (Bcl)-2, caspase-3 and nuclear factor (NF)-κB p65 were measured using quantitative polymerase chain reaction and western blotting. The levels of tumor growth factor (TGF)-β and interleukin (IL)-6 in HSC supernatants were examined by ELISA. The results demonstrated that HO-1 exerted antiproliferative effects on HSCs in a time- and concentration-dependent manner. Increasing HO-1 expression induced HSC apoptosis in vitro as demonstrated by a significant decrease in Bcl-2 and an increase in caspase-3 expression. Additionally, the expression of NF-κB p65 and its downstream inflammatory factors TGF-β and IL-6 in the HO-1 overexpression group was significantly decreased compared with the control group. Therefore, the present study provided evidence that HO-1 serves an anti-fibrosis role in the liver by enhancing HSC apoptosis, which was partially associated with the regulation of NF-κB and its downstream effectors.

Prophylactic Effects of Polymethoxyflavone-Rich Orange Peel Oil on Nω-Nitro-L-Arginine-Induced Hypertensive Rats

The prophylactic effects of the polymethoxyflavones (PMFs) in long-leaf orange peel oil (OPO) were determined using an Nω-nitro-L-arginine-induced hypertensive rat model. The OPO contained eight PMF components, namely sinensetin, hexamethoxyflavone, tetramethyl-O-isoscutellarein, nobiletin, tetramethyl-O-scutellarein, heptamethoxyflavone, 5-demethylnobiletin and tangeretin. After treatment with OPO, the SP (systolic pressure) and DP (diastolic pressure) in hypertensive rats were reduced. The NO (nitric oxide) contents in serum, heart, liver and kidney of OPO-treated Nω-nitro-L-arginine (L-NNA)-induced hypertensive rats were higher than those in untreated hypertensive rats, but the MDA (malondialdehyde) contents in OPO-treated rats were lower than those of the control rats (untreated hypertensive rats). ET-1 (endothelin-1), VEGF (vascular endothelial growth factor) and E-selectin serum levels in hypertensive rats could be reduced, but the CGRP (calcium gene-related peptide) level could be increased by OPO treatment. The results of the qPCR assay showed that OPO upregulated HO-1 (heme oxygenase-1), nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) mRNA expression and downregulated ADM (adrenomedullin), RAMP2 (receptor activity modifying protein 2) and iNOS (inducible nitric oxide synthase) expression in hypertensive rats. The Western blot results also proved that OPO upregulated nNOS and eNOS protein expression and downregulated iNOS expression in hypertensive rats. Based on this study, we could conclude that OPO showed good antihypertensive effects, and the effect was concentration dependent.

Expression of Cyclooxygenase-2, Nitric Oxide Synthase 2 and Heme Oxygenase-1 mRNA Induced by Bis-Eugenol in RAW264.7 Cells and their Antioxidant Activity Determined Using the Induction Period Method

To clarify the mechanisms responsible for the anti-inflammatory/proinflammatory activities of eugenol-related compounds, we investigated the cytotoxicity and up-regulatory/down-refgulatory effects of the biphenols curcumin, bis-eugenol, magnolol and honokiol, and the monophenols eugenol and isoeugenol, on major regulators of cyclooxygenase-2 (Cox-2), nitric oxide synthase 2 (Nos2) and heme oxygenase-1 (HO-1) mRNA in RAW264.7 cells. mRNA expression was investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and the theoretical parameters were calculated using the DFT/B3LYP/6-31* method. Also, the antioxidant activity of eugenol-related compounds in combination with 2-mercapto-1-methylimidazole (MMI, as a model for glutathione (GSH)) was investigated using the induction period method for polymerization of methyl methacrylate initiated by benzoyl peroxide (BPO). The cytotoxicity of eugenol-related compounds showed a linear relationship with their softness (σ) and electrophilicity (ω). At a concentration of 50 μM, biphenols except for bis-eugenol elicited the expression of mRNA for both Cox-2 and Nos2, but monophenols did not. In contrast, bis-eugenol elicited Cox-2 gene expression, but down-regulated Nos2 gene expression. bis-Eugenol alone induced the expression of HO-1 mRNA, and when combined with MMI it showed a potent antagonistic effect on BPO-induced antioxidant activity. The ability of methoxyphenols to inhibit LPS-stimulated Cox-2 gene expression declined in the order curcumin >> isoeugenol > bis-eugenol >> eugenol, and the rank of ability was related to their ω value. Most eugenol-related compounds had proinflammatory activity at high concentrations. However, they had also anti-inflammatory activity at lower concentrations. Eugenol-related compounds may exert antioxidant and anti-inflammatory activity in LPS-stimulated RAW264.7 cells possibly by inhibiting the activation of nuclear factor-kappa B (Nf-ĸB), whereas bis-eugenol requires induction of HO-1 expression. bis-Eugenol as well as curcumin, may have anti-inflammatory and anticancer therapeutic applications.

Mangosteen peel extract exhibits cellular antioxidant activity by induction of catalase and heme oxygenase-1 mRNA expression

Mangosteen peel extract exhibits cellular antioxidant activity by induction of catalase and heme oxygenase-1 mRNA expression

Astaxanthin Induces the Nrf2/HO-1 Antioxidant Pathway in Human Umbilical Vein Endothelial Cells by Generating Trace Amounts of ROS.

Astaxanthin is a powerful antioxidant that possesses potent protective effects against various human diseases and physiological disorders. However, the mechanisms underlying its antioxidant functions in cells are not fully understood. In the present study, the effects of astaxanthin on reactive oxygen species (ROS) production and antioxidant enzyme activity, as well as mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K)/Akt, and the nuclear factor erythroid 2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) pathways in human umbilical vein endothelial cells (HUVECs), were examined. It was shown that astaxanthin (0.1, 1, and 10 μM) induced ROS production by 9.35%, 14.8%, and 18.06% compared to control, respectively, in HUVECs. In addition, astaxanthin increased the mRNA levels of phase II enzymes HO-1 and also promoted GSH-Px enzyme activity. Furthermore, we observed ERK phosphorylation, nuclear translocation of Nrf-2, and activation of antioxidant response element-driven luciferase activity upon astaxanthin treatment. Knockdown of Nrf-2 by small interfering RNA inhibited HO-1 mRNA expression by 60%, indicating that the Nrf-2/ARE signaling pathway is activated by astaxanthin. Our results suggest that astaxanthin activates the Nrf-2/HO-1 antioxidant pathway by generating small amounts of ROS.