mRNA Expression Levels Of Nrf2 Research Articles

Impairment of PGC-1α-mediated mitochondrial biogenesis precedes mitochondrial dysfunction and Alzheimer's pathology in the 3xTg mouse model of Alzheimer's disease.

Impairment of mitochondrial biogenesis and mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD). However, the extent to which the impairment of mitochondrial biogenesis influences mitochondrial dysfunction at the onset and during progression of AD is still unclear. Our study demonstrated that the protein expression pattern of the transcription factor pCREB/CREB, together with the protein expression of PGC-1α, NRF1 and TFAM are all significantly reduced in early ages of 3xTg-AD mice. We also found reduced mRNA expression levels of PKAC-α, CREB, PGC-1α, NRF1, NRF2 and TFAM as early as 1month-of-age, an age at which there was no significant Aβ oligomer deposition, suggesting that mitochondrial biogenesis is likely impaired in ages preceding the development of the AD pathology. In addition, there was a decrease in VDAC2 expression, which is related to mitochondrial content and mitochondrial function, as demonstrated by protein expression of complex IV, as well as complex II+III, and complex IV activities, at later ages in 3xTg-AD mice. These results suggest that the impairment in mitochondrial biogenesis signaling mediated by PGC-1α at early ages of the AD mice model likely resulted in mitochondrial dysfunction and manifestation of the AD pathology at later ages. Taken together, enhancing mitochondrial biogenesis may represent a potential pharmacological approach for the treatment of AD.

Nrf2-ME-1 axis is associated with 5-FU resistance in gastric cancer cell line

The aim of this study was to investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) and key downstream targeting molecule, malic enzyme-1 (ME-1), in the induction of 5-fluorouracil (5-FU) resistance. Resistant MKN-45 (MKN-45/DR) cell line was generated by employing IC50 concentration of the agent followed by a resting cycles. Cell viability and apoptosis were evaluated by MTT assay, DAPI staining and flow cytometry. mRNA expression levels of Nrf2 and ME-1 were measured by qRT-PCR technique. Morphology analysis and combination index calculation were assessed by ImagJ and compuSyn softwares, respectively. The value for IC50 in 5-FU resistant cells increased from 8.81 ± 0.209–142.4 ± 0.060 μM with a considerable morphological changes from round to elongated shape (p = 0.016). Nrf2 and ME-1 expression levels were decreased in resistant cells with a marked increase in MDR1 mRNA level compared to sensitive cells. Combination of Nrf2 inhibitors, luteolin and brusatol had synergistic effects on 5-FU induced cytotoxicity. Moreover, combined incubation of MKN-45/DR cells with these inhibitors, exponentially increased Nrf2 and ME-1 mRNA levels (p < 0.001). Our findings suggest that the combined application of Nrf2 inhibitors, can be considered as a novel strategy to overcome 5-FU resistance in GC patients.

Upregulation of nuclear factor (erythroid-derived 2)-like 2 protein level in the human colorectal adenocarcinoma cell line DLD-1 by a heterocyclic organobismuth(III) compound: Effect of organobismuth(III) compound on NRF2 signaling

An increasing number of metal-based compounds, including arsenic trioxide, auranofin, and cisplatin, have been reported to have antitumor activity. Their beneficial effects are controlled by a transcription factor, nuclear factor (erythroid-derived 2)-like 2 (NRF2). In response to oxidative stress, NRF2 induces the expression of cytoprotective genes. NRF2 protein levels are regulated by Kelch-like ECH-associated protein 1 (KEAP1) via ubiquitination. Bi-chlorodibenzo[c,f][1,5]thiabismocine (compound 3), a bismuth compound, is known for its potent anti-proliferative activity against various cancer cell lines. In the present study, we investigated the effect of compound 3 on NRF2 signaling in the human colorectal adenocarcinoma cell line DLD-1 in terms of cell viability as well as mRNA and protein expression levels of NRF2. Compound 3 upregulated NRF2 protein levels in a time- and concentration-dependent manner, accompanied by a marked increase in heme-oxygenase-1 (HO-1) mRNA and protein levels. We observed that brusatol, an NRF2 inhibitor, as well as small interfering RNA (siRNA)-mediated knockdown of NRF2 in DLD-1 cells suppressed compound 3-induced HO-1 expression. The anticancer activity of compound 3 was enhanced by compounds that downregulate NRF2. These results suggest that compound 3 upregulates HO-1 via NRF2 activation and that the NRF2-HO-1 pathway is the cellular response to compound 3. We also discovered that compound 3 slightly downregulated KEAP1; thus, NRF2 activation may be associated with KEAP1 modification. Collectively, our results indicate that compound 3 simultaneously activates an anti-oxidative stress pathway, such as NRF2 and HO-1, and a pro-cell death signal in DLD-1 cells. Our findings may provide useful information for the development of a potent anticancer organobismuth(III) compound.

The Effect of N-Acetyl-Cysteine on NRF2 Antioxidant Gene Expression in Asthenoteratozoospermia Men: A Clinical Trial Study.

BackgroundOne of the important factor associated with male infertility is high production of reactive oxygen species (ROS). The main function of Nuclear factor erythroid 2-related factor 2 (NRF2) is to activate the cellular anti- oxidant response by inducing the transcription of a wide array of genes that can combat the harmful effects of factors such as oxidative stress. The purpose of this study was to evaluate the effect of N-acetyl-L-cysteine (NAC), as an antioxidant drug, on NRF2 Gene Expression in Asthenoteratozoospermia Men.Materials and Methods In this randomized, blinded clinical trial study, included 50 infertile men with asthenoteratozoo- spermia, who received NAC (600 mg, three times daily). Sperm parameters analyzed according to the world health organiza- tion (WHO; 2010). Sperm DNA fragmentation, relative NRF2 expression, and seminal plasma level of antioxidant enzymes were measured by TUNEL assay, reverse transcription polymerase chain reaction (RT-PCR) and ELISA test, respectively. ResultsAfter NAC treatment, findings showed a significant increase in sperm concentration and motility compared to pre-treatment status, whereas the percentage of abnormal morphology and DNA fragmentation was significantly decreased (P<0.05). A significant improvement in expression of NRF2 gene and antioxidant enzyme levels were ob- served compared to pre-treatment by NAC (P<0.05). Significant correlations were observed between NRF2 mRNA expression level, specific sperm parameters and level of antioxidant enzymes (P<0.05).ConclusionThe results demonstrated that NAC oral supplementation protected against oxidative stress by enhancing NRF2 expression. This could improve semen parameters quality parameters in asthenoteratozoospermia men (Regis- tration number: IRCT20170830035998N4).

Cypermethrin induces cell injury in primary cortical neurons of C57BL/6 mice by inhibiting Nrf2/ARE signaling pathway

To study the role of Nrf2/ARE signaling pathway in cypermethrin-induced oxidative stress and apoptosis of cerebral cortex neurons in C57BL/6 mice. The cortical neurons of C57BL/6 mice were cultured and identified, and a cypermethrin-induced cell injury model was established by treating the cells with 0, 25, 50 and 100 μmol/L of cypermethrin for 48 h. CCK-8 assay was used to analyze the effects of cypermethrin on the cell viability, and the fluorescence probe DCFH-DA was used for detecting intracellular reactive oxygen species (ROS); flow cytometry was performed for determining the apoptosis rate of the cells. The mRNA and protein expression levels of Nrf2 and its downstream genes HO-1 and NQO1 were detected using qPCR and Western blotting. Exposure to cypermethrin at different doses inhibited the viability of the cultured cortical neurons. With the increase of cypermethrin dose, the viability of the neurons decreased progressively, the intracellular ROS and the cell apoptosis rate increased, and the neuronal injury worsened. At the dose of 50 and 100 μmol/L, cypermethrin significantly down-regulated the expressions of HO-1, NQO1 and Nrf2 at both the mRNA and protein levels in the cells (P < 0.01). Cypermethrin exposure shows a dose-dependent neurotoxicity by inhibiting Nrf2/ARE signaling pathway, down-regulating the expression of Nrf2 and its downstream genes HO-1, NQO1 mRNA and protein, and inducing oxidative damage and apoptosis in primary mouse cortical neurons, .

Glutamine improves heat stress–induced oxidative damage in the broiler thigh muscle by activating the nuclear factor erythroid 2–related 2/Kelch-like ECH-associated protein 1 signaling pathway

The aim of the present study was to evaluate the effect of glutamine (Gln) on modulating heat stress–induced oxidative damage in the broiler thigh muscle through nuclear factor erythroid 2–related 2/Kelch-like ECH-associated protein 1 (Nrf2-Keap1) pathway. Three-hundred 22-day-old Arbor Acres broilers were reallocated into 5 groups: a control group (24 °C) fed with basal diet and 4 heat stress (HS) groups (34 °C for 8 h/D) fed with basal diet containing 0, 0.5, 1.0, and 1.5% Gln. This experiment lasted 21 D. Heat stress decreased (P < 0.05) pH, redness, and Gln levels, and increased (P < 0.05) luminance, water loss rate, and cooking loss (CL) values of the thigh meat. Compared with the HS group, supplementation with 1.5% Gln increased (P < 0.05) pH, redness, and Gln levels, but decreased (P < 0.05) luminance and CL values in the thigh meat. There were significant decreases (P < 0.05) in glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and Nrf2 levels, but significant increases (P < 0.05) in the malondialdehyde (MDA) and Keap1 levels of the thigh muscle after HS treatment. Compared with the HS group, supplementation with 1.0, and 1.5% Gln decreased (P < 0.05) MDA and Keap1 levels; supplementation with 1.5% Gln increased (P < 0.05) GSH, GSH-Px, T-AOC, CAT, SOD, and Nrf2 levels in the thigh muscle of heat-stressed broilers. Furthermore, HS decreased (P < 0.05) Nrf2, SOD, CAT, and GSH-Px mRNA expression levels, but increased (P < 0.05) Keap1 mRNA level in the thigh muscle of broiler. Dietary supplementation with 1.5% Gln increased (P < 0.05) Nrf2, GSH-Px, CAT, and SOD mRNA expression levels, but decreased (P < 0.05) Keap1 mRNA level in the thigh muscle of heat-stressed broilers. In conclusion, dietary Gln improved the resistance of heat-stressed broiler muscles to oxidative damage possibly through reversing the muscle Gln level and inducing the expression of the Nrf2-Keap1 pathway.

Imatinib inhibits oxidative stress response in spinal cord injury rats by activating Nrf2/HO-1 signaling pathway.

Effect of imatinib on rats with spinal cord injury (SCI) was investigated through the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Forty-eight Sprague-Dawley rats were randomly divided into sham operation group (n=12), model group (n=12), imatinib group (n=12) and inhibitor group (n=12). The results of immunohistochemistry showed that in comparison with sham operation group, the other three groups had overtly increased positive expression level of Bax and evidently reduced positive expression level of Bcl-2 (P<0.05). Compared with those in model group and inhibitor group, the positive expression level of Bax was distinctly lower, while that of Bcl-2 was notably increased in imatinib group (P<0.05). According to western blot analysis, the protein expression levels of Nrf2 and HO-1 were obviously higher in the other three groups than those in sham operation group (P<0.05), and they were remarkably higher in imatinib group than those in model group and inhibitor group (P<0.05). The results of qPCR assay revealed that the Nrf2 and HO-1 mRNA expression levels were markedly elevated in the other three groups compared with those in sham operation group (P<0.05). Based on ELISA, the other three groups exhibited notably raised content of IL-6, TNF-α, ROS and SOD compared with sham operation group (P<0.05), and imatinib group displayed remarkably decreased content of IL-6, TNF-α and ROS and markedly elevated SOD content in comparison with model group and inhibitor group (P<0.05). The results of TUNEL assay demonstrated that the rate of apoptosis was significantly raised in the other three groups compared with that in sham operation group (P<0.05), and it declined obviously in imatinib group compared with that in model group and inhibitor group (P<0.05). Imatinib inhibits oxidative stress response in SCI rats by activating the Nrf2/HO-1 signaling pathway, thus repressing apoptosis and inflammation.

Nrf2 mediates the antinociceptive activity of dexmedetomidine in an acute inflammatory visceral pain rat model by activating the NF-κB sensor.

Dexmedetomidine (DEX) is a highly selective α2 adrenoceptor agonist. In this study, we evaluated the antalgic effect of DEX on acetic acid-induced acute inflammatory visceral pain (AIVP) in rats. Additionally, we evaluated the role of Nrf2 signalling in antinociception. We administered acetic acid to male Sprague Dawley rats that were treated with DEX or saline. Twenty rats were randomly classified into the following groups: normal, model, vehicle, or DEX group. Both q-RT PCR and enzyme-linked immunosorbent assay data suggested that interleukin 1β (IL-1β), tumour necrosis factor α, and IL-6 were upregulated in the spinal cord. Western blotting and q-RT PCR analyses were performed to detect the protein and mRNA expression levels of Nrf2, Keap1, and HO-1 in the spinal cord. The DEX group exhibited a significant downregulation in Nrf2/Keap1/HO-1 signal activation compared with the model group. Furthermore, we used the Nrf2-/- knockout AIVP rat model to determine the role of Nrf2 in the antinociceptive effect of DEX. We observed that the Nrf2 knockout blocked the Keap1/Nrf2/HO-1 signal transduction and partially abated the antinociceptive and the anti-inflammatory effects of DEX. Moreover, our data also indicated that DEX treatment decreased the activation and expression of nuclear factor (NF)-κB. However, Nrf2 silencing restored the expression of NF-κB and its phosphorylated form to physiological levels. In summary, our results suggested that Nrf2 signalling plays an important role in the antinociceptive effect of DEX in the AIVP rat model and that Nrf2 exerts its function by enhancing the activation of the NF-κB sensor. SIGNIFICANCE OF THE STUDY: Currently, using the behavioural parameters is not adequate for the diagnosis of AIVP, and there are no studies that have investigated the role and the mechanism of DEX in ameliorating visceral pain. In this study, we demonstrated that acetic acid stimulation in rats induces AIVP. Additionally, the administration of DEX inhibited the acute inflammation response and decreased the visceromotor reaction (behavioural) to algesia. Further, DEX inhibited the Keap1/Nrf2 pathway, which was activated by acetic acid treatment. We suggest that suppressing the inflammatory response could partially regulate the antinociceptive effect of DEX through Nrf2-mediated NF-κB activation associated with AIVP.

Inhibition of the Nrf2-TrxR Axis Sensitizes the Drug-Resistant Chronic Myelogenous Leukemia Cell Line K562/G01 to Imatinib Treatments.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in tumor drug resistance, but its role in imatinib resistance of chronic myeloid leukemia (CML) remains elusive. We aimed to investigate the effects of Nrf2 on drug sensitivity, thioredoxin reductase (TrxR) expression, reactive oxygen species (ROS) production, and apoptosis induction in imatinib-resistant CML K562/G01 cells and explored their potential mechanisms. Stable K562/G01 cells with knockdown of Nrf2 were established by infection of siRNA-expressing lentivirus. The mRNA and protein expression levels of Nrf2 and TrxR were determined by real-time quantitative polymerase chain reaction and western blot, respectively. ROS generation and apoptosis were assayed by flow cytometry, while drug sensitivity was measured by the Cell Counting Kit-8 assay. Imatinib-resistant K562/G01 cells had higher levels of Nrf2 expression than the parental K562 cells at both mRNA and protein levels. Expression levels of Nrf2 and TrxR were positively correlated in K562/G01 cells. Knockdown of Nrf2 in K562/G01 cells enhanced the intracellular ROS level, suppressed cell proliferation, and increased apoptosis in response to imatinib treatments. Nrf2 expression contributes to the imatinib resistance of K562/G01 cells and is positively correlated with TrxR expression. Targeted inhibition of the Nrf2-TrxR axis represents a potential therapeutic approach for imatinib-resistant CML.

Pioglitazone Attenuates Lipopolysaccharide-Induced Oxidative Stress, Dopaminergic Neuronal Loss and Neurobehavioral Impairment by Activating Nrf2/ARE/HO-1.

The aim of the present study was to examine the neuroprotective potential of pioglitazone via activation of Nrf2/ARE-dependent HO-1 signaling pathway in chronic neuroinflammation and progressive neurodegeneration mouse model induced by lipopolysaccharide (LPS). After assessing spatial memory, anxiety and motor-coordination, TH+ neurons in substantia nigra (SN) were counted. The oxidative stress marker carbonyl protein levels and HO-1 enzyme activity were also evaluated. RT-qPCR was conducted to detect HO-1, Nrf2 and NF-κp65 mRNA expression levels and Nrf2 transcriptional activation of antioxidant response element (ARE) of HO-1 was investigated. Pioglitazone ameliorated LPS-induced dopaminergic neuronal loss, as well as mitigated neurobehavioral impairments. It enhanced Nrf2 mRNA expression, and augmented Nrf2/ARE-dependent HO-1 pathway activation by amplifying HO-1 mRNA expression. Moreover, it induced a significant decrease in NF-κB p65 mRNA expression, while reducing carbonyl protein levels and restoring the HO-1 enzyme activity. Interestingly, LPS induced Nrf2/antioxidant response element (ARE) of HO-1 activation, ultimately resulting in slight enhanced HO-1 mRNA expression. However, LPS elicited decrease in HO-1 enzyme activity. Zinc protoporphyrin-IX (ZnPPIX) administrated with pioglitazone abolished its effects in the LPS mouse model. The study results demonstrate that coordinated activation of Nrf2/ARE-dependent HO-1 pathway defense mechanism by the PPARγ agonist pioglitazone mediated its neuroprotective effects.

Icariin Alleviates IL-1β-Induced Matrix Degradation By Activating The Nrf2/ARE Pathway In Human Chondrocytes.

ObjectiveOsteoarthritis (OA) is characterized by progressive matrix destruction of articular cartilage. This study aimed to investigate the potential antioxidative and chondroprotective effects and underlying mechanism of Icariin (ICA) in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage.MethodsHuman chondrocyte cell line HC-A was treated with different doses of ICA, and then MTT assay and PI staining were used to estimate ICA-induced chondrocyte apoptosis. Intracellular ROS and superoxide dismutase (SOD) and glutathione peroxidase (GPX) were measured after treatment by IL-1β with or without ICA. The mRNA and protein expression levels of redox transcription factor Nrf2 and the downstream effector SOD-1, SOD-2, NQO-1 and HO-1 were assayed to explore the detailed mechanism by which ICA alleviates ECM degradation. Finally, to expound the role of Nrf2 in ICA-mediated chondroprotection, we specifically depleted Nrf2 in human chondrocytes and then pretreated them with ICA followed by IL-1β.ResultsICA had no cytotoxic effects on human chondrocytes and 10−9 M was selected as the optimum concentration. ROS induced by IL-1β could drastically activate matrix-degrading proteases and ICA could significantly rescue the matrix degradation and excess ROS generation caused by IL-1β. We observed that ICA activated the Nrf2/ARE pathway, consequently upregulating the generation of GPX and SOD. Ablation of Nrf2 abrogated the chondroprotective and antioxidative effects of ICA in IL-1β-treated chondrocytes.ConclusionICA alleviates IL-1β-induced matrix degradation and eliminates ROS by activating the Nrf2/ARE pathway.

Toxic effects of glyphosate on intestinal morphology, antioxidant capacity and barrier function in weaned piglets

At present, the public is paying more attention to the adverse effects of pesticides on human and animal health and the environment. Glyphosate is a broad-spectrum pesticide that is widely used in agricultural production. In this manuscript, the effects of diets containing glyphosate on intestinal morphology, intestinal immune factors, intestinal antioxidant capacity and the mRNA expression associated with the Nrf2 signaling pathway were investigated in weaned piglets. Twenty-eight healthy female hybrid weaned piglets (Duroc × Landrace × Yorkshire) were randomly selected with an average weight of 12.24 ± 0.61 kg. Weaned piglets were randomly assigned into 4 treatment groups and fed a basal diet supplemented with 0, 10, 20, and 40 mg/kg glyphosate for a 35-day feeding trial. We found that glyphosate had no effect on intestinal morphology. In the duodenum, glyphosate increased the activities of CAT and SOD (linear, P < 0.05) and increased the levels of MDA (linear and quadratic, P < 0.05). In the duodenum, glyphosate remarkably increased the relative mRNA expression levels of Nrf2 (linear and quadratic, P < 0.05) and NQO1 (linear and quadratic, P < 0.05) and reduced the relative mRNA expression levels of GPx1, HO-1 and GCLM (linear and quadratic, P < 0.05). In the jejunum, glyphosate remarkably increased the relative mRNA expression levels of Nrf2 (linear and quadratic, P < 0.05) and decreased the relative mRNA expression levels of GCLM (linear and quadratic, P < 0.05). Glyphosate increased the mRNA expression levels of IL-6 in the duodenum (linear and quadratic, P < 0.05) and the mRNA expression levels of IL-6 in the jejunum (linear, P < 0.05). Glyphosate increased the mRNA expression of NF-κB in the jejunum (linear, P = 0.05). Additionally, the results demonstrated that glyphosate linearly decreased the ZO-1 mRNA expression levels in the jejunum and the mRNA expression of claudin-1 in the duodenum (P < 0.05). In the duodenum, glyphosate increased the protein expression levels of Nrf2 (linear, P = 0.025). Overall, glyphosate exposure may result in oxidative stress in the intestines of piglets, which can be alleviated by enhancing the activities of antioxidant enzymes and self-detoxification.

Impact of Gene Expression Associated with Glucocorticoid-Induced Transcript 1 (GLCCI1) on Severe Asthma and Future Exacerbation.

Genetic variations in glucocorticoid-induced transcript 1 (GLCCI1) have been associated with the response to corticosteroid treatment. However, the associations of GLCCI1 polymorphisms or gene expression with the prognosis of asthma and pathophysiological factors related to steroid insensitivity remain unclear. We sought to investigate the associations of GLCCI1, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and histone deacetylase 2 (HDAC2) mRNA expression levels and the GLCCI1 rs37973 polymorphism with asthma severity and future exacerbation in patients with asthma. Subjects included 25 patients with severe asthma and 127 patients with nonsevere asthma. mRNA expression levels in peripheral blood mononuclear cells were measured and evaluated as predictors of severe asthma using receiver operating characteristic (ROC) analysis. The hazard ratios of the mRNA expression levels for time to first exacerbation in the 1-year follow-up period were calculated. GLCCI1, Nrf2, and HDAC2 mRNA expression levels were significantly lower in patients with severe asthma than in patients with nonsevere asthma and could predict severe asthma with an area under the ROC curve of 0.68, 0.71, and 0.65, respectively. In contrast, no relationship was found between the GLCCI1 rs37973 polymorphism and severe asthma. The hazard ratios for asthma exacerbation in patients with low GLCCI1, Nrf2, and HDAC2 mRNA expression levels were 3.24 (95% confidence interval, 1.42-7.40), 3.13 (1.37-7.16), and 2.98 (1.22-7.25), respectively. Patients with severe asthma could be distinguished by lower GLCCI1, Nrf2, and HDAC2 mRNA levels in peripheral blood cells, and all of these gene signatures could predict future asthma exacerbations.

Puerarin prevents cataract development and progression in diabetic rats through Nrf2/HO‑1 signaling.

Puerarin is the major bioactive ingredient isolated from the dry root of Pueraria lobata, a plant used in traditional Chinese medicine. Puerarin has been used to treat diabetes and cataracts in China; however, its underlying mechanism of action remains unclear. The aim of the present study was to investigate the effectiveness and mechanism of puerarin in preventing cataracts in diabetic rats. Diabetes was induced by streptozocin (STZ) administration and rats were intraperitoneally injected with puerarin (25, 50 and 100 mg/kg). Blood glucose levels and cataract development were examined in the different experimental groups. In addition, the expression levels of markers associated with oxidative stress, including nuclear factor erythroid 2 like 2 (Nrf2) and heme oxygenase-1 (HO-1), were analyzed. The present results suggested that treatment with puerarin at 25, 50 and 100 mg/kg significantly reduced blood glucose levels and the incidence of cataract in STZ-induced diabetic rats. Additionally, puerarin treatment reduced oxidative stress, restoring the levels of malondialdehyde and glutathione, and the activity of glutathione peroxidase. Furthermore, puerarin administration decreased the expression levels of retinal vascular endothelial growth factor and interleukin-1β and increased the mRNA expression levels of Nrf2 and HO-1, thus inhibiting oxidative stress. The present findings suggested that puerarin had hypoglycemic effects and that it prevented cataract development and progression in diabetic rats by reducing oxidative stress through the Nrf2/HO-1 signaling pathway.

Expression of the receptor of advanced glycation end-products (RAGE) and membranal location in peripheral blood mononuclear cells (PBMC) in obesity and insulin resistance.

Objective(s):The present study aimed to evaluate the receptor of advanced glycation end-products (RAGE), NF-kB, NRF2 gene expression, and RAGE cell distribution in peripheral blood mononuclear cells (PBMC) in subjects with obesity and IR compared with healthy subjects.Materials and Methods:The mRNA expression levels of RAGE, NF-kB, NRF2, and GAPDH were determined in PBMC by qPCR in 20 obese (OB), 17 obese with insulin resistance (OB-IR), and 20 healthy subjects (HS), matched by age and sex. RAGE protein expression and its localization were determined by Western Blot and immunocytochemistry (ICC) analysis, total soluble RAGE (sRAGE) and MCP-1 plasma levels by ELISA. Results: :RAGE, NF-kB, and NRF2 genes mRNA expression in PBMCs did not show variation between groups. RAGE protein was lower in OB and OB-IR groups; RAGE was located predominantly on the cell-surface in the OB-IR group compared to the HS group (22% vs 9.5%, P<0.001). OB-IR group showed lower sRAGE plasma levels, and correlated negatively with HOMA-IR, ALT parameters (r= -0.374, r= -0.429, respectively), and positively with NFE2L2 mRNA (r= 0.540) P<0.05. Conclusion:In this study, OB-IR subjects did not reflect significant differences in gene expression; however, correlations detected between sRAGE, biochemical parameters, and NRF2, besides the predominant RAGE distribution on the cell membrane in PBMC could be evidence of the early phase of the inflammatory cascade and the subsequent damage in specific tissues in subjects with OB-IR.

Protective properties of glycogen synthase kinase-3 inhibition against doxorubicin-induced oxidative damage to mouse ovarian reserve

Chemotherapy induces ovarian failure in female children and young female cancer survivors. It has been shown that doxorubicin (DOX), an antitumor drug of the anthracycline group, causes gonadotoxicity via the stimulation of oxidative stress. The inhibition of glycogen synthase kinase-3 (GSK-3) was reported to be able to regulate oxidative stress. The present study assessed whether GSK-3 inhibition confers protection to the ovary against DOX-induced oxidative stress damage. An intraperitoneal injection of DOX was used to induce oxidative damage in the mouse ovary, while the inhibition of GSK-3 was achieved by the administration of SB216763, a small and potent GSK-3 inhibitor. DOX increased the mRNA expression levels of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the antioxidant genes heme-oxygenase-1 (HO-1) and catalase (CAT), while reducing the levels of glutathione peroxidase (GSH-Px) and superoxide dismutase-1 (SOD-1) compared with those of the control. When the GSK-3 inhibitor was combined with DOX increased more expression levels of Nrf2 mRNA and restored levels of GSH-Px and SOD-1 mRNA similar to those in the control group. DOX remarkably decreased the Nrf2 protein expression compared to that in the control, which was significantly associated with increased MDA levels. Interestingly, the SB216763 and DOX coadministration restored the Nrf2 protein expression and MDA levels to levels that were similar to those in the control. DOX significantly decreased the number of primordial, primary, preantral and antral follicles while increasing the number of atretic follicles compared to those in the control. SB216763 caused the drastic recovery of these follicles from the DOX effects. SB216763 and DOX coadministration significantly reduced the apoptotic index compared with that with DOX treatment. DOX decreased the serum AMH and E2 levels and increased the FSH levels compared to those in the controls. However, SB216763 and DOX coadministration restored AMH and E2 while decreasing the FSH levels compared to those in the DOX-treated group. In addition, SB216763 and DOX coadministration reduced the mature oocyte abnormalities that resulted from DOX-induced ovarian damage. Given these results, we suggest that GSK-3/Nrf2 is a promising protective pathway against doxorubicin-induced oxidative damage to the ovaries of females at reproductive ages. Thus, GSK-3 could be an attractive target for the research and development of new drugs for preserving ovarian function during chemotherapy.

829 Folic acid protects melanocytes from oxidative damage by Keap1-Nrf2 pathway

This study aims to investigate whether folic acid could protect human melanocyte against oxidative damage and to elucidate the underlying pharmacological mechanism. And we demonstrate that folic acid could protect melanocytes form oxidative damage. Folic acid is of great therapeutic potential in the treatment of vitiligo. Vitiligo is a common skin disease characterized by the loss of functional melanocytes. Previous studies have indicated that oxidative stress plays a pivotal role in the onset of vitiligo. Thus, antioxidant therapy is a promising therapeutic strategy to prevent or even reverse the progression of depigmentation. Folic acid is a conventional antioxidant, but whether folic acid can be used to treat vitiligo is unclear. PIG1 cells that are an immortalized human epidermal melanocyte cell line were cultured in Medium 254. H2O2 at 800 μM was chosen to induce oxidative stress. CCK8 assay was used to estimate the cell survival rate. Flow cytometry was used to measure the intracellular ROS level and apoptosis rate. The nuclear translocation of Nrf2 was tested by immunofluorescence. Western blot and qRT-PCR were used to analyze the mRNA and protein expression levels of Keap1, Nrf2, HO-1, SOD2 and NQO1. We initially found that folic acid was able to ameliorate H2O2-induced oxidative damage in human melanocytes. In parallel, folic acid lessened the accumulation of intracellular ROS by potentiating the activity of antioxidant enzymes. Furthermore, folic acid protected melanocyte against oxidative stress by activating Nrf2 and its downstream genes HO-1, SOD2 and NQO1, and interfering with Nrf2 diminished the protection of folic acid against H2O2-induced apoptosis. At last, the down-regulation of Keap1 contributed to the activation of Nrf2 induced by folic acid.

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.

Nrf2 deficiency exacerbates frailty and sarcopenia by impairing skeletal muscle mitochondrial biogenesis and dynamics in an age-dependent manner.

Mitochondrial dysfunction during aging is a key factor that contributes to sarcopenia. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been increasingly recognized to regulate mitochondrial function. The present study aimed to investigate the role of Nrf2 in the development of frailty and sarcopenia during aging, and to demonstrate whether Nrf2 contributes to the maintenance of muscle mass and function by regulation of mitochondrial biogenesis and dynamics during the aging process. Young (5-6 months), middle-aged (11-13 months), old (20-24 months) Nrf2-/- (knockout, KO) mice and age-matched wild-type (WT) C57/BL6 mice were used in this study. Physical function of the mice in the 6 groups was assessed by grip strength test, four paw inverted hanging test, rotarod analysis, open field analysis, and treadmill endurance test. Muscle mass was measured by cross-sectional area (CSA) of tibialis anterior muscles and gastrocnemius muscle weight. The frailty status of the 25 old WT mice and 23 old KO mice were assessed based on the mouse frailty phenotype assessment. Expression levels of genes involved in mitochondrial biogenesis (nuclear respiratory factor 1 (Nrf1), peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (PGC-1α), mitochondrial transcription factor A (TFAM)) and mitochondrial dynamics (optic atrophy protein 1 (Opa1), mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and dynamin-related protein 1 (Drp1)) were measured in the skeletal muscle. SDH staining was performed and mitochondrial DNA (mtDNA) copy number was measured. Transmission electron microscopy was used to measure the mitochondria number and morphology. Physical function and muscle mass decreased during aging. The mRNA expression levels of Nrf2 decreased with increasing frailty phenotype scores in the old WT mice. There were minimal differences in the physical function and muscle mass between the WT and KO mice in the young groups, whereas Nrf2 deficiency caused a declined physical function and muscle mass in the middle-aged and old mice, and exacerbated frailty in the old mice. The decreases of the physical function and muscle mass were accompanied by the reduced expression levels of genes involved in mitochondrial biogenesis and dynamics, as well as a reduction of mitochondrial number, mitochondrial content, mtDNA copy number, and an impaired mitochondria morphology in the skeletal muscle. Nrf2 deficiency exacerbated frailty and sarcopenia during aging, at least partially by impairing skeletal muscle mitochondrial biogenesis and dynamics in an age-dependent manner.

Proanthocyanidins Protect against β-Hydroxybutyrate-Induced Oxidative Damage in Bovine Endometrial Cells.

Metabolic diseases, such as ketosis, are closely associated with decreased reproductive performance (such as delayed estrus and decreased pregnancy rate) in dairy cows. The change of β-hydroxybutyrate (BHBA) concentration in dairy cattle is an important mechanism leading to ketosis, and its blood concentration in ketotic cows is always significantly higher than in nonketotic cows. Many studies indicated that BHBA can induce oxidative damage in liver and other organs. Proanthocyanidins (PCs) have gained substantial attention in the last decade as strong antioxidative substances. This study aimed to demonstrate a protective effect of PCs against BHBA-induced oxidative stress damage in bovine endometrial (BEND) cells by activating the nuclear erythroid2-related factor2 (Nrf2) signaling pathway. Our research show that PCs could significantly increase activities of catalase (CAT) and glutathione peroxidase (GSH-PX), glutathione (GSH) content, and antioxidant capacity (T-AOC), while significantly decreasing malondialdehyde (MDA) content in BEND cells. Both mRNA and protein expression levels of Nrf2 were significantly increased in BEND cells, and glutamate–cysteine ligase catalytic subunit (GCLC), heme oxygenase 1 (HO-1), manganese superoxide dismutase (Mn-SOD), and NAD(P)H quinone dehydrogenase 1 (NQO-1) were also significantly increased. These results indicate that PCs can antagonize BHBA-induced oxidative damage by activating the Nrf2 signaling pathway to exert an antioxidant effect.