Nuclear Factor Erythroid 2-related Factor 2/antioxidant Response Element Research Articles

Chitosan oligosaccharides prevent doxorubicin-induced oxidative stress and cardiac apoptosis through activating p38 and JNK MAPK mediated Nrf2/ARE pathway

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, the incidence of cardiotoxicity compromises its therapeutic index. Oxidative stress and apoptosis are believed to be involved in DOX-induced cardiotoxicity. Chitosan oligosaccharides (COS), the enzymatic hydrolysates of chitosan, have been reported to possess diverse biological activities including antioxidant and anti-apoptotic properties. The objective of the present study was to investigate the potential role of COS against DOX-induced cardiotoxicity, and the effects of COS on apoptosis and oxidative stress in rats and H9C2 cells. Furthermore, we also shed light on the involved pathways during the whole process. For this purpose, first, we demonstrated that COS exhibited a significant protective effect on cardiac tissue by not only inducing a decrease in body and heart growth but also ameliorated oxidative damage and ECG alterations in DOX-treated rats. Second, we found that COS reversed the decrease of cell viability induced by DOX, reduced the intracellular reactive oxygen species (ROS), increased the mitochondrial membrane potential (MMP) and Bcl-2/Bax ratio. COS treatment also results in reduced caspase-3 and caspase-9 expressions, and an increase in the phosphorylation of MAPKs (mitogen-activated protein kinases) in DOX-exposed H9C2 cells. Additionally, cellular homeostasis was re-established via stabilization of MAPK mediated nuclear factor erythroid 2-related factor 2/antioxidant-response element (Nrf2/ARE) signaling and transcription of downstream cytoprotective genes. In summary, these findings suggest that COS could be a potential candidate for the prevention and treatment of DOX-induced cardiotoxicity.

Effect of the Nrf2-ARE signaling pathway on biological characteristics and sensitivity to sunitinib in renal cell carcinoma.

The aim of the present study was to examine the effects of the nuclear factor erythroid-2 related factor 2-antioxidant-responsive element (Nrf2-ARE) signaling pathway on the biological characteristics and sensitivity to targeted therapy in human renal cell carcinoma (RCC) cells. RCC tissues and adjacent tissues were collected and assessed by immunohistochemistry to determine the expression of Nrf2, NAD(P)H dehydrogenase [quinone] 1 (NQO1) and heme oxygenase-1 (HO-1) to analyze the clinicopathological features of RCC. A series of in vitro experiments were conducted to analyze the biological characteristics of Nrf2-ARE signaling in RCC. The renal cancer cell line, 786-0 was used, and cells was divided into a mock group, negative control group and small hairpin (sh)RNA-Nrf2 group. A Cell Counting Kit-8 assay was performed alongside flow cytometry to detect cell viability, cell cycle stage and apoptosis following treatment with sunitinib. The results demonstrated that Nrf2, NQO1 and HO-1 were significantly upregulated in RCC tissues compared with adjacent tissues and were associated with tumor node metastasis stage, Fuhrman classification and lymph node metastasis. Following shRNA-Nrf2 transfection, the 786-0 cells demonstrated a significant decrease in viability, cell invasion and scratch healing rate, and the mRNA and protein expression levels of Nrf2, NQO1, HO-1 and glutathione transferase were significantly decreased, which enhanced the sensitivity to sunitinib, arrested cells in the G0/G1 phase and increased apoptosis. In conclusion, Nrf2-ARE signaling is important for RCC progression, and its inhibition may increase sensitivity to targeted drugs to provide novel developments for RCC treatment.

Effects of penehyclidine hydrochloride on Nrf2/ARE signaling pathway during endotoxin-induced acute lung injury in neonate rats

Objective: To evaluate the effects of penehyclidine hydrochloride on the nuclear factor erythroid 2-related factor 2/antioxidant responsive element (Nrf2/ARE) signaling pathway during endotoxin-induced acute lung injury(ALI) in neonate rats. Methods: Forty 7-day-old Wistar rats weighing 12-18 g were randomly divided into 4 groups (n=10) using a random number table: normal saline group(NS group), acute lung injury(ALI group), penehyclidine hydrochloride group(PHC group) and penehyclidine hydrochloride+ Nrf2 siRNA plasmid group(PNS group). The ALI model was induced with intraperitoneal endotoxin (5.0 mg/kg) in groups ALI, PHC and PNS. In groups PHC and PNS, penehyclidine hydrochloride (2.0 mg/kg) was injected intraperitoneally at 1 h before ALI respectively, while the equal volume of normal saline was administered in groups NS and ALI. The animal of PNS group were inhaled adenovirus packaging of Nrf2-siRNA three times (one time a day) before modeling. At 4 h after endotoxin injection, the rats were sacrificed. The lungs were collected to determine the wet/dry(W/D) lung weight ratio. The expression of Nrf2 and heme oxygen and enzyme 1(HO-1) were determined by Western blotting, contents of tumor necrosis factor-alpha(TNF-α),interleukin10 (IL-10)were determined by enzyme-linked immunosorbent assay(ELISA). The cell apoptosis were determined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL),and the apoptotic index was calculated. Results: The W/D ratio in NS, ALI, PHC and PNS groups were (4.2±0.1), (9.6±0.7), (6.5±0.6), (8.3±1.3) respectively. The apoptotic index were (3.7±0.5)%, (31.5±3.2)%, (17.6±4.2)%, (28.1±3.5)%respectively.The contents of TNF-α were (10.3±1.6), (98.5±8.5), (68.5±6.7), (89.9±8.5) pg/ml respectively. The contents of IL-10 were (7.9±0.6), (102.8±9.3), (72.5±5.8), (97.7±9.1) pg/ml respectively.The expression of Nrf2 were (23.2±7.6), (79.8±13.0), (155.5±16.7), (12.0±3.3) respectively. The expression of HO-1 were (31.7±8.6), (90.8±10.3), (147.6±22.5), (61.4±9.7) respectively. There were statistically significant differences among different groups (F=86.013, 154.897, 328.810, 374.198, 333.965, 125.274, all P<0.05). Compared with group NS, the W/D ratio, apoptotic index and the contents of TNF-α, IL-10 increased, the expression of Nrf2 and HO-1 up-regulated in group ALI and group PHC (all P<0.05). Compared with group ALI, the W/D ratio, apoptotic index and the contents of TNF-α, IL-10 decreased, the expression of Nrf2 and HO-1 up-regulated in group PHC (all P<0.05). Compared with group ALI, no significant differences were found in the W/D ratio, apoptotic index and the contents of TNF-α, IL-10 in group PNS(all P>0.05), while the expression of Nrf2 and HO-1 down-regulated in group PNS (all P<0.05). Compared with group PHC, the W/D ratio,apoptotic index and the contents of TNF-α,IL-10 increased, the expression of Nrf2 and HO-1 down-regulated in group PNS (all P<0.05). Conclusion: Nrf2/ARE signaling pathway is involved in the reduction of ALI by penehyclidine hydrochloride in neonate rats.

Berberine attenuates hepatic oxidative stress in rats with non-alcoholic fatty liver disease via the Nrf2/ARE signalling pathway.

The present study investigated the effects of berberine (BBR) on hepatic oxidative stress and the nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signalling pathway in rats in which non-alcoholic fatty liver disease (NAFLD) was induced by a high-fat diet. Rats were randomly divided into three groups: The normal control (NC), high-fat diet (HFD) and BBR groups. The NC group received a normal diet, while the other two groups were fed a high-fat diet. The rats in the BBR group were also fed BBR (100 mg/kg body weight) daily. A total of 8 weeks later, serum and liver lipid levels were measured. Hepatic histopathological changes were observed with haematoxylin and eosin and Oil Red O staining. Transmission electron microscopy was performed to observe the ultrastructural changes of the liver. The levels of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) in the liver were measured. Quantitative polymerase chain reaction and western blotting were performed to investigate the expression of genes in the Nrf2/ARE signalling pathway in the liver. Histopathological results demonstrated that rats fed a high-fat diet for 8 weeks developed NAFLD, characterized by hepatic steatosis. BBR significantly decreased the body weight and liver weight. BBR markedly reduced hepatic steatosis, and the serum and liver lipid levels. Hepatic SOD and GSH levels were increased, while MDA levels were decreased by BBR co-administered with a high-fat diet. Additionally, the Nrf2/ARE signalling pathway was revealed to be involved in the protective effect of BBR on rats fed a high-fat diet. In conclusion, BBR may alleviate hepatic oxidative stress in rats with NAFLD, which may be partly attributed to the activation of the Nrf2/ARE signalling pathway.

Amelioratory Effects of Testosterone Propionate on Age-related Renal Fibrosis via Suppression of TGF-\u03b21/Smad Signaling and Activation of Nrf2-ARE Signaling

Androgen plays a pivotal role in the progression of renal fibrosis. However, whether exogenous androgen treatment to aged male rats can improve the age-related renal fibrosis was not explored. In our study, the changes of morphological structure, renal fibrosis, ultrastructure and renal function, the expressions of extracellular matrix (ECM), matrix metalloproteinases (MMPs) and its tissue inhibitors of metalloproteinases (TIMPs), the expressions of tumor growth factor β1 (TGF-β1)/Smad signaling and oxidative stress parameters as well as nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) signaling were tested in kidney of aged male Wistar rats after subcutaneous testosterone propionate (TP, 2 mg/kg/d, 84-day) injection. Aged rats showed significantly renal histopathological changes, increased renal fibrosis, increased thickening of the glomerular basement membrane and the Bowman’s capsule basement membrane, declined renal functional, increased ECM, lower expressions of MMP-2 and MMP-9 and higher expressions of TIMP-1 and TIMP-2 in renal tissues and higher expressions of TGF-β1/Smad signaling, as well as lower expressions of Nrf2-ARE signaling compared to young rats. TP treatment significantly improved age-related above indexes. These results suggested that TP supplement may alleviate age-related renal fibrosis via suppression of TGF-β1/Smad signaling and activation of Nrf2-ARE signaling in aged rats.

Sirt1 activation prevents anti-Thy 1.1 mesangial proliferative glomerulonephritis in the rat through the Nrf2/ARE pathway

Mesangial proliferative glomerulonephritis (MsPGN) is characterized by glomerular mesangial cells proliferation and extracellular matrix deposition in mesangial area, which develop into glomerulosclerosis. Both silent information regulator 2-related protein 1 (Sirt1) and nuclear factor erythroid 2-related factor 2/anti-oxidant response element (Nrf2/ARE) pathway had remarkable renoprotective effects. However, whether Sirt1 and Nrf2/ARE pathway can regulate the pathological process of MsPGN remains unknown. Here, we found that Sirt1 activation by SRT1720 decreased mesangial hypercellularity and mesangial matrix areas, reduced renal Col4 and α-SMA expressions, lowered 24 h proteinuria, and eventually reduced FN and TGF-β1 expressions in rats received anti-Thy 1.1 IgG. Further study showed that SRT1720 markedly enhanced the activity of Nrf2/ARE pathway including promoting the nuclear content and ARE-binding ability of Nrf2, elevating the protein levels of HO-1 and SOD1, two target genes of Nrf2, which eventually increased total SOD activity and decreased malondialdehyde level in the kidney tissues of experimental anti-Thy 1.1 MsPGN rats. Taken together, Sirt1 prevented the pathological process of experimental anti-Thy 1.1 MsPGN through promoting the activation of Nrf2/ARE pathway, which warrants further elucidation. Sirt1 might be a potential therapeutic target for treating MsPGN.

Docosahexaenoic Acid (DHA) Provides Neuroprotection in Traumatic Brain Injury Models via Activating Nrf2-ARE Signaling.

In this study, we explored the neuroprotective effects of docosahexaenoic acid (DHA) in traumatic brain injury (TBI) models. In this study, we first confirmed that DHA was neuroprotective against TBI via the NSS test and Morris water maze experiment. Western blot was conducted to identify the expression of Bax, caspase-3, and Bcl-2. And the cell apoptosis of the TBI models was validated by TUNEL staining. Relationships between nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway-related genes and DHA were explored by RT-PCR and Western blot. Rats of the DHA group performed remarkably better than those of the TBI group in both NSS test and water maze experiment. DHA conspicuously promoted the expression of Bcl-2 and diminished that of cleaved caspase-3 and Bax, indicating the anti-apoptotic role of DHA. Superoxide dismutase (SOD) activity and cortical malondialdehyde content, glutathione peroxidase (GPx) activity were renovated in rats receiving DHA treatment, implying that the neuroprotective influence of DHA was derived from lightening the oxidative stress caused by TBI. Moreover, immunofluorescence and Western blot experiments revealed that DHA facilitated the translocation of Nrf2 to the nucleus. DHA administration also notably increased the expression of the downstream factors NAD(P)H:quinone oxidoreductase (NQO-1) and heme oxygenase 1(HO-1). DHA exerted neuroprotective influence on the TBI models, potentially through activating the Nrf2- ARE pathway.

Plumbagin ameliorates memory dysfunction in streptozotocin induced Alzheimer’s disease via activation of Nrf2/ARE pathway and inhibition of β-secretase

Although plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) protects against cerebral ischemia and spinal cord injury-induced oxidative stress and inflammation by activating the nuclear factor-erythroid 2-related factor-2 /antioxidant response element (Nrf2/ARE) pathway, its role in the amelioration of neurodegenerative diseases remains unexplored. In the present study, we investigated the effect of plumbagin on Alzheimer’s disease (AD)-like condition in mice. The animals were treated intracerebroventricularly with streptozotocin (STZ; 3 mg/kg) twice, on day 1 and 3, to induce AD-like condition, and the symptoms were evaluated after 14 days. While the loss of learning and memory performance was evident in the mice subjected to Morris water maze (MWM), there was a striking increase in the population of astrocytes labelled with glial fibrillary acidic protein (GFAP) in the hippocampus. Daily intraperitoneal (i.p.) treatment with plumbagin (0.5 and 1 mg/kg), starting from 1 h prior to first dose of STZ, significantly prevented the cognitive deficits in MWM. On the other hand, administration of Nrf2/ARE pathway inhibitor, trigonelline (10 and 15 mg/kg, i.p.) enhanced the effects of STZ. Pre-treatment with subeffective dose of trigonelline (5 mg/kg) significantly attenuated the effects of plumbagin in MWM. While plumbagin prevented the STZ induced GFAP expression, this effect of plumbagin was attenuated by trigonelline. Moreover, the in silico docking study revealed potent inhibitory effect of plumbagin on β-secretase enzyme. The results of the present study suggest that plumbagin improves cognitive function in STZ induced mouse model of AD possibly via Nrf2/ARE mediated suppression of astrogliosis and inhibition of β-secretase enzyme.

Borago officinalis L. attenuates UVB-induced skin photodamage via regulation of AP-1 and Nrf2/ARE pathway in normal human dermal fibroblasts and promotion of collagen synthesis in hairless mice

Ultraviolet B (UVB) irradiation is regarded as the main cause of skin photodamage. After exposure to UVB irradiation, collagen degradation is accelerated by upregulation of matrix metalloproteinases (MMPs), and collagen synthesis is decreased via downregulation of transforming growth factor (TGF)-β1 signaling. Borago officinalis L. (BO) is an annual herb with medicinal and culinary applications. Although BO has been demonstrated to have antioxidant and anti-inflammatory activities, its potential anti-photoaging effects have not been examined. In this study, we examined the protective effects of BO against skin photodamage in UVB-exposed normal human dermal fibroblasts (NHDFs) in vitro and hairless mice in vivo. BO downregulated the expression of MMP-1, MMP-3, and IL-6, and enhanced TGF-β1 by modulating activator protein (AP-1) and nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signaling in UVB-irradiated NHDFs. We also found that dietary BO reduced wrinkle formation, epidermal thickness, and erythema in UVB-exposed skin. Moreover, skin hydration and collagen synthesis were improved by dietary BO treatment. Our results demonstrate that BO can be used in functional foods, cosmetic products, and medicines for prevention and treatment of UVB-induced skin photodamage.

Therapeutic relevance of ozone therapy in degenerative diseases: Focus on diabetes and spinal pain.

Ozone, one of the most important air pollutants, is a triatomic molecule containing three atoms of oxygen that results in an unstable form due to its mesomeric structure. It has been well-known that ozone has potent ability to oxidize organic compounds and can induce respiratory irritation. Although ozone has deleterious effects, many therapeutic effects have also been suggested. Since last few decades, the therapeutic potential of ozone has gained much attention through its strong capacity to induce controlled and moderated oxidative stress when administered in precise therapeutic doses. A plethora of scientific evidence showed that the activation of hypoxia inducible factor-1α (HIF-1a), nuclear factor of activated T-cells (NFAT), nuclear factor-erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE), and activated protein-1 (AP-1) pathways are the main molecular mechanisms underlying the therapeutic effects of ozone therapy. Activation of these molecular pathways leads to up-regulation of endogenous antioxidant systems, activation of immune functions as well as suppression of inflammatory processes, which is important for correcting oxidative stress in diabetes and spinal pain. The present study intended to review critically the available scientific evidence concerning the beneficial properties of ozone therapy for treatment of diabetic complications and spinal pain. It finds benefit for integrating the therapy with ozone into pharmacological procedures, instead of a substitutive or additional option to therapy.

Retracted: Activation of the Nrf2‐ARE Signaling Pathway Prevents Hyperphosphatemia‐Induced Vascular Calcification by Inducing Autophagy in Renal Vascular Smooth Muscle Cells

This study investigates the effect of nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) signaling pathway in vascular calcification (VC) via inducing Autophagy in renal vascular smooth muscle cells (VSMCs). VSMCs were assigned into six experimental groups: the normal control, high phosphorus, si-negative control (si-NC), Nrf2-siRNA, over-expressed Nrf2, and negative control (NC) groups. RT-PCR was applied to detect the mRNA expressions of the desired Nrf2-ARE signaling pathway-related genes (Nrf2, NQO-1, HO-1, γ-GCS). The protein products of these genes: apoptosis-related genes (LC3I and LC3II), osteogenic marker proetins (Runt-related transcription factor 2) Runx2 and BMP2 were all detected by Western blotting. Autophagosomes in VSMCs were observed under a transmission electron microscope. We discovered an increased calcium ion concentration and upregulated Runx2, BMP2, Nrf2, HO-1, γ-GCS, NQO-1, and LC3II/LC3I expressions in the high phosphorous, si-NC and Nrf2-siRNA, and NC groups, compared with the normal control group. Compared to the high phosphorus and si-NC groups, higher levels of Runx2 and BMP2 but decreased Nrf2, HO-1, γ-GCS, NQO-1, and LC3II/LC3I expressions were detected in the Nrf2-siRNA group. The high phosphorus, si-NC and over-expressed Nrf2 experimental groups all had increased Nrf2, NQO-1, HO-1, γ-GCS, and LC3II/LC3I expressions as well as high numbers of autophagosomes compared with the normal control group. Finally, we detected a lower amount of autophagosomes presence and Nrf2, NQO-1, HO-1 γ-GCS, and LC3II/LC3 protein expression of Nrf2-siRNA group than that of the high phosphorus and si-NC groups. Activation of Nrf2-ARE signaling pathway may prevent hyperphosphatemia-induced VC by inducing autophagy in VSMCs. J. Cell. Biochem. 118: 4708-4715, 2017. © 2017 Wiley Periodicals, Inc.

Airborne nitro-PAHs induce Nrf2/ARE defense system against oxidative stress and promote inflammatory process by activating PI3K/Akt pathway in A549 cells

Ambient particulate matter (PM) is a worldwide health issue of concern. However, limited information is available regarding the toxic contributions of the nitro-derivatives of polycyclic aromatic hydrocarbons (nitro-PAHs). This study intend to examine whether 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) could activate the nuclear factor-erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) antioxidant defense system, and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway participates in regulating pro-inflammatory responses in A549 cells. Firstly, 1-NP and 3-NF concentration-dependently induced cellular apoptosis, reactive oxygen species (ROS) generation, DNA damage, S phase cell cycle arrest and differential expression of related cytokine genes. Secondly, 1-NP and 3-NF activated the Nrf2/ARE defense system, as evidenced by increased protein expression levels and nuclear translocation of transcription factor Nrf2, elevated Nrf2/ARE binding activity, up-regulated expression of the target gene heme oxygenase-1 (HO-1). Significantly increased protein expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and phosphorylation level of Akt indicated that the PI3K/Akt pathway was activated during pro-inflammatory process. Further, both PI3K inhibitor (LY294002) and Akt inhibitor (MK-2206) reversed the elevated TNF-α expression to control level. Our results suggested that Nrf2/ARE pathway activation might cause an initiation step in cellular protection against oxidative stress caused by nitro-PAHs, and the PI3K/Akt pathway participated in regulating inflammatory responses.

Pinocembrin Attenuates Mitochondrial Dysfunction in Human Neuroblastoma SH-SY5Y Cells Exposed to Methylglyoxal: Role for the Erk1/2-Nrf2 Signaling Pathway.

Pinocembrin (PB; 5,7-dihydroxyflavanone) is found in propolis and exhibits antioxidant activity in several experimental models. The antioxidant capacity of PB is associated with the activation of the nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signaling pathway. The Nrf2/ARE axis mediates the expression of antioxidant and detoxifying enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), heme oxygenase-1 (HO-1), and the catalytic (GCLC) and regulatory (GCLM) subunits of the rate-limiting enzyme in the synthesis of glutathione (GSH), γ-glutamate-cysteine ligase (γ-GCL). Nonetheless, it is not clear how PB exerts mitochondrial protection in mammalian cells. Human neuroblastoma SH-SY5Y cells were pretreated (4h) with PB (0-25µM) and then exposed to methylglyoxal (MG; 500µM) for further 24h. Mitochondria were isolated by differential centrifugation. PB (25µM) provided mitochondrial protection (decreased lipid peroxidation, protein carbonylation, and protein nitration in mitochondrial membranes; decreased mitochondrial free radical production; enhanced the content of GSH in mitochondria; rescued mitochondrial membrane potential-MMP) and blocked MG-triggered cell death by a mechanism dependent on the activation of the extracellular-related kinase (Erk1/2) and consequent upregulation of Nrf2. PB increased the levels of GPx, GR, HO-1, and mitochondrial GSH. The PB-induced effects were suppressed by silencing of Nrf2 with siRNA. Therefore, PB activated the Erk1/2-Nrf2 signaling pathway resulting in mitochondrial protection in SH-SY5Y cells exposed to MG. Our work shows that PB is a strong candidate to figure among mitochondria-focusing agents with pharmacological potential.

Protecting effects and mechanism of tert-butyl hydroquinone on retinal cells in type 2 diabetic rats

Background Apoptosis is a primary clinical pathological mechanism of diabetic retinopathy (DR). Oxidative stress and high glucose can activate cell apoptosis pathway and thus leads to cellular damage.It is confirmed that tert-butyl hydroquinone (tBHQ) plays an antioxidation effect, however, whether it has a protective role on retinal cells in DR is still unelucidated. Objective This study was to investigate the effect of tBHQ on vascular endothelial growth factor (VEGF) and bcl-2 expressions in retina of type 2 diabetic rats and its possible mechanism via nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signal pathway. Methods Fifty clean healthy male SD rats were included in this experimental study.Ten rats were fed with normal diet as the normal control group, and other rats were fed with high fatty and high sugar food for 4 weeks.After 12 hours of fasting, streptozotoin (STZ) (30 mg/kg) was intraperitoneally injected to induce the type 2 diabetic models.The model rats were randomly divided into the diabetic control group and tBHQ group and 1% tBHQ was added into the high fatty and sugar food 1 week after modeling in the tBHQ group.Fasting plasma glucose (FPG) level, blood total cholesterol (TC) level, blood triglyceride (TG) level, high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and fasting serum insulin (FINs) were detected 4 and 12 weeks after modeling, respectively, and radio immunoassay was used to detect the FIN levels of the rats.The relative expression of VEGF and bcl-2 in retinas of the rats were assayed by immunohistochemistry and fluorescence real-time quantitative PCR (qRT-PCR). The use of the animals complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by State and Technology Commission. Results Type 2 diabetic models were successfully established in 35 rats with successful rate 92.1%.The FIN levels were significantly different among different groups and time points (Fgroup=22.480, P=0.000; Ftime=7.636, P=0.008). The FPG, TC, TG and LDL-C levels were significantly different among the groups (FPG: Fgroup=78.531, P=0.000; TC: Fgroup=28.049, P=0.000; TG: Fgroup=13.108, P=0.000; LDL-C: Fgroup=6.804, P<0.05). Immunohistochemistry showed that VEGF and bcl-2 were mainly expressed in retinal ganglion cell layer, inner plexiform layer and outer plexiform layer.The expressions of VEGF and bcl-2 proteins were significantly different among different groups (VEGF: Fgroup=11.805, P=0.000; bcl-2: Fgroup=22.943, P=0.000); the expression level of bcl-2 protein was higher in 12 weeks after modeling than that in 4 weeks after modeling in the tBHQ group (P<0.05). The expressions of VEGF and Bcl-2 mRNA in rat retinas were significantly different among different groups and time points (VEGF: Fgroup=79.220, P=0.000; Ftime=6.090, P<0.05; Bcl-2: Fgroup=105.000, P=0.000; Ftime=13.170, P=0.001). Four and eight weeks after modeling, the expressions of VEGF and Bcl-2 mRNA in the diabetic control group and tBHQ group were significantly higher than that in the normal control group, and the expressions of Bcl-2 mRNA in the tBHQ group were significantly higher than that in the model control group (all at P<0.05); the expression of Bcl-2 mRNA was higher at 12 weeks after modeling than that at 4 weeks in the tBHQ group (P<0.05). Conclusions tBHQ produces anti-oxidative-damage and anti-apoptosis effects on retinal cells by up-regulating VEGF expression and down-regulating bcl-2 expression in DR rats.In addition, tBHQ may have effects on lowering high blood sugar, regulating insulin and blood lipid levels. Key words: Diabetes mellitus/complication; Retinopathy; Vascular endothelial growth factor; Bcl-2/metabolism; Disease models, animal; SD rats; Tert-butyl hydroquinone

Eriodictyol Attenuates β-Amyloid 25–35 Peptide-Induced Oxidative Cell Death in Primary Cultured Neurons by Activation of Nrf2

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). Eriodictyol, a flavonoid isolated from the Chinese herb Dracocephalum rupestre, has long been established as an antioxidant. The present study was designed to investigate the effect of eriodictyol on β-amyloid 25-35 peptide (Aβ25-35)-induced oxidative cell death in primary neurons and to explore the role of the nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway in this process. For this purpose, primary cultures of cortical neurons were exposed to 15μM Aβ25-35 in the absence or presence of eriodictyol (20, 40 and 80μM). The results revealed that Aβ25-35-induced cytotoxicity and apoptotic characteristics such as activation of JNK/p38 apoptotic signaling pathway were effectively attenuated by eriodictyol pretreatment. Eriodictyol treatment also resulted in an increase in Nrf2 protein levels and subsequent activation of ARE pathway genes in primary cultured neurons. The protective effects of eriodictyol were attenuated by RNA interference-mediated knockdown of Nrf2 expression. Taken together, these results clearly demonstrate that eriodictyol protects neurons against Aβ25-35-induced cell death partially through Nrf2/ARE signaling pathway, which further supports that eriodictyol might be a promising novel therapeutic agent for AD.

Role of Nrf2/ARE signaling pathway in reduction of myocardial ischemia-reperfusion injury by propofol in rats

Objective To evaluate the role of nuclear factor erythroid 2-related factor 2/antioxidant responsive element (Nrt2/ARE) signaling pathway inthe reduction of myocardial ischemia-reperfusion (I/R) injury by propofol in rats.Methods Sixty adult male Sprague-Dawley rats,weighing 200-240 g,were randomly divided into 5 groups (n =12 each) using a random number table:sham operation group (S group),I/R group,propofol group (P group),propofol + Nrf2 vehicle-plasmid group (PNV group) and propofol + Nrf2 siRNA plasmid group (PNS group).The animals were anesthetized with inhalation of 2％ isoflurane,tracheally intubated and mechanically ventilated.Myocardial I/R was produced by 5 min occlusion of left anterior descending branch of coronary artery followed by 60 min reperfusion.In P,PNV and PNS groups,isoflurane inhalation was stopped after successful intubation and propofol was infused via the caudal vein at 6 mg· kg-1 · h-1 until 30 of reperfusion.At 30 min of propofol infusion,Nrf2 vehicle-plasmid 10 μg (100 μl) was injected intramyocardially before myocardial ischemia in group PNV,and Nrf2 siRNA 10 μg (100 μl) was injected intramyocardially before myocardial ischemia in group PNS.The animals were sacrificed at 60 min of reperfusion and myocardial specimens were taken for determination of the infarct size,apoptosis index,and the expression of Nrf2 and heme oxygenase-1 (HO-1).Results Compared with group S,the infarct size and apoptosis index were significantly increased,and the expression of Nrf2 and HO-1 was up-regulated in I/R and P groups.Compared with group I/R,the infarct size and apoptosis index were significantly decreased,and the expression of Nrf2 and HO-1 was up-regulated in group P.Compared with group P,no significant changes were found in the infarct size,apoptosis index and expression of Nrf2 and HO-1 in group PNV,and the infarct size and apoptosis index were significantly increased,and the expression of Nrf2 and HO-1 was down-regulated in group PNS.Conclusion Nrf2/ARE signaling pathway is involved in the reduction of myocardial I/R injury by propofol in rats. Key words: Propofol ; Myocardial reperfusion injury ; NF-E2-related factor 2 ; Response elements

Natural product-derived pharmacological modulators of Nrf2/ARE pathway for chronic diseases

Covering: 2000 to 2013. Oxidative stress is the central component of chronic diseases. The nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway is vital in the up-regulation of cytoprotective genes and enzymes in response to oxidative stress and treatment with certain dietary phytochemicals. Herein, we classify bioactive compounds derived from natural products that are Nrf2/ARE pathway activators and recapitulate the molecular mechanisms for inducing Nrf2 to provide favorable effects in experimental models of chronic diseases. Moreover, pharmacological inhibition of Nrf2 signalling has emerged as promising strategy against multi-drug resistance thereby improving the treatment efficacy. We have also enlisted natural product-derived inhibitors of Nrf2/ARE pathway.

Eriodictyol-7-O-glucoside activates Nrf2 and protects against cerebral ischemic injury

Stroke is a complex disease that may involve oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and thus has been considered a potential target for neuroprotection in stroke. The aim of the present study was to determine whether eriodictyol-7-O-glucoside (E7G), a novel Nrf2 activator, can protect against cerebral ischemic injury and to understand the role of the Nrf2/ARE pathway in neuroprotection. In primary cultured astrocytes, E7G increased the nuclear localization of Nrf2 and induced the expression of the Nrf2/ARE-dependent genes. Exposure of astrocytes to E7G provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. The protective effect of E7G was abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo administration of E7G in a rat model of focal cerebral ischemia significantly reduced the amount of brain damage and ameliorated neurological deficits. These data demonstrate that activation of Nrf2/ARE signaling by E7G is directly associated with its neuroprotection against oxidative stress-induced ischemic injury and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in stroke.

PI3K/Akt Pathway Mediates Nrf2/ARE Activation in Human L02 Hepatocytes Exposed to Low-Concentration HBCDs

We investigated the effects of hexabromocyclododecanes (HBCDs) at environmentally relevant concentrations on human L02 hepatocytes and explored possible underlying molecular mechanism(s), focusing on functional interactions between the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and nuclear factor-erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathways. The results showed that low concentrations of HBCDs could stimulate cell proliferation in a "DNA-dependent protein kinase catalytic subunit" (DNA-PKcs)-dependent manner, increase protein levels and nuclear translocation of transcription factor Nrf2, and upregulate expression of its target gene heme oxygenase-1 (HO-1). Electrophoretic mobility-shift assays (EMSAs) showed that ARE was a prominent element for HO-1 induction after low-concentration HBCDs exposure. The relationship between PI3K/Akt pathway and Nrf2/HO-1 axis was demonstrated by the finding that pretreatment with PI3K inhibitors (wortmannin, LY294002) attenuated the upregulation of Nrf2 expression induced by HBCDs exposure. Furthermore, knock-down of DNA-PKcs through small interfering RNA blocked Nrf2/HO-1 axis activation in L02 cells exposed to low-concentration HBCDs. Moreover, DNA-PKcs and phosphorylated Akt at Ser(473) proved to be crucial in regulating the Nrf2-ARE pathway. Thus, the PI3K/Akt pathway is essential in regulating Nrf2-ARE pathway activation in L02 cells induced by low-concentration HBCDs.

Sulforaphane enhances the activity of the Nrf2–ARE pathway and attenuates inflammation in OxyHb-induced rat vascular smooth muscle cells

A growing body of evidence indicates that the nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway plays a protective role in many physiological stress processes such as inflammatory damage, oxidative stress, and the accumulation of toxic metabolites, which are all involved in the cerebral vasospasm following subarachnoid hemorrhage (SAH). We hypothesized that the Nrf2-ARE pathway might have a protective role in cerebral vasospasm following SAH. In our study, we investigate whether the oxyhemoglobin (OxyHb) can induce the activation of the Nrf2-ARE pathway in vascular smooth muscle cells (VSMCs), and evaluate the modulatory effects of sulforaphane (SUL) on OxyHb-induced inflammation in VSMCs. As a result, both the protein level and the mRNA level of the nuclear Nrf2 were significantly increased, while the mRNA levels of two Nrf2-regulated gene products, both heme oxygenase-1 and NAD(P)H: quinone oxidoreductase-1, were also up-regulated in VSMCs induced with OxyHb. A marked increase of inflammatory cytokines such as IL-1β, IL-6 and TNF-α release was observed at 48h after cells were treated with OxyHb. SUL enhanced the activity of the Nrf2-ARE pathway and suppressed cytokine release. Our results indicate that the Nrf2-ARE pathway was activated in OxyHb-induced VSMCs. SUL suppressed cytokine release via the activation of the Nrf2-ARE pathway in OxyHb-induced VSMCs.