mRNA Expression Levels Of Nrf2 Research Articles

Aerobic exercise improves LPS-induced sepsis via regulating the Warburg effect in mice

We investigated the impact of aerobic exercise (AE) on multiple organ dysfunction syndrome (MODS), aortic injury, pathoglycemia, and death during sepsis. ICR mice were randomized into four groups: Control (Con), Lipopolysaccharide (LPS), Exercise (Ex), and Exercise + LPS (Ex + LPS) groups. Mice were trained with low-intensity for 4 weeks. LPS and Ex + LPS mice received 5 mg/kg LPS intraperitoneally for induction of sepsis. Histopathological micrographs showed the organ morphology and damage. This study examined the effects of AE on LPS-induced changes in systemic inflammation, pulmonary inflammation, lung permeability, and bronchoalveolar lavage fluid (BALF) cell count, oxidative stress-related indicators in the lung, blood glucose levels, plasma lactate levels, serum insulin levels, plasma high-mobility group box 1 (HMGB1) levels, glucose transporter 1 (Glut1) and HMGB1, silent information regulator 1 (Sirt-1), and nuclear factor erythroid 2-related factor 2 (Nrf-2) mRNA expression levels in lung tissue. AE improved sepsis-associated multiple organ dysfunction syndrome (MODS), aortic injury, hypoglycemia, and death. AE prominently decreased pulmonary inflammation, pulmonary edema, and modulated redox balance during sepsis. AE prominently decreased neutrophil content in organ. AE prominently downregulated CXCL-1, CXCL-8, IL-6, TNF-α, Glu1, and HMGB1 mRNA expression but activated IL-1RN, IL-10, Sirt-1, and Nrf-2 mRNA expression in the lung during sepsis. AE decreased the serum levels of lactate and HMGB1 but increased blood glucose levels and serum insulin levels during sepsis. A 4-week AE improves sepsis-associated MODS, aortic injury, pathoglycemia, and death. AE impairs LPS-induced lactate and HMGB1 release partly because AE increases serum insulin levels and decreases the levels of Glut1. AE is a novel therapeutic strategy for sepsis targeting aerobic glycolysis.

Paeoniflorin Protects against ANIT-Induced Cholestatic Liver Injury in Rats via the Activation of SIRT1-FXR Signaling Pathway.

Paeoniflorin (PF), a water-soluble monoterpene glycoside, is initially isolated from the dried roots of Paeonia lactiflora Pall., which has effects on ameliorating cholestasis in our previous study. However, comprehensive approaches for understanding the protective effects and mechanisms underlying cholestatic liver injury from the regulating of bile acid metabolism have not been sufficiently elucidated. This study was aimed to explore the effectiveness as well as potential mechanism of PF on alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury. Rats with cholestasis induced by ANIT was used to evaluate the protective effects and mechanism of PF by regulating SIRT1/FXR and NF-κB/NLRP3 signaling pathway. Rats were intragastrically administrated with ANIT to establish cholestatic liver injury model. Serum levels of ALT, AST, TBA, TBIL, ALP, γ-GT and ALB in rats were detected. The histopathology of the liver of rats was analyzed in vivo. The relative mRNA expression and protein expression levels of IL-18, IL-1β, TNF-α, HO-1, Nrf2, TLR4, NLRP3, Caspase-1, ASC, NF-κB, FXR, and SIRT1 in liver of rats were investigated. The results showed that the serum indexes and the liver histopathology were significantly improved by PF. The overexpression of IL-18, IL-1β, TNF-α, NLRP3, ASC, and Caspase-1 in liver was markedly reduced by PF. Furthermore, PF dramatically increased the mRNA and protein expressions of SIRT1, FXR, HO-1, and Nrf2, but decreased NF-κB p65 and TLR4 levels in liver of rats. Taken together, the protective effects of PF on cholestatic liver injury were possibly related to the activation of the SIRT1/FXR and inhibition of NF-κB/NLRP3 inflammasome signaling pathway. These findings might provide a potential protection for cholestatic liver injury.

MAO-A Inhibition by Metaxalone Reverts IL-1β-Induced Inflammatory Phenotype in Microglial Cells.

Experimental and clinical studies have suggested that several neurological disorders are associated with the occurrence of central nervous system neuroinflammation. Metaxalone is an FDA-approved muscle relaxant that has been reported to inhibit monoamine oxidase A (MAO-A). The aim of this study was to investigate whether metaxalone might exert antioxidant and anti-inflammatory effects in HMC3 microglial cells. An inflammatory phenotype was induced in HMC3 microglial cells through stimulation with interleukin-1β (IL-1β). Control cells and IL-1β-stimulated cells were subsequently treated with metaxalone (10, 20, and 40 µM) for six hours. IL-1β stimulated the release of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), but reduced the anti-inflammatory cytokine interleukin-13 (IL-13). The upstream signal consisted of an increased priming of nuclear factor-kB (NF-kB), blunted peroxisome proliferator-activated receptor gamma (PPARγ), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression. IL-1β also augmented MAO-A expression/activity and malondialdehyde levels and decreased Nrf2 mRNA expression and protein levels. Metaxalone decreased MAO-A activity and expression, reduced NF-kB, TNF-α, and IL-6, enhanced IL-13, and also increased PPARγ, PGC-1α, and Nrf2 expression. The present experimental study suggests that metaxalone has potential for the treatment of several neurological disorders associated with neuroinflammation.

The effect of Phoenix dactylifera pollen on the expression of NRF2, SOD2, CAT, and GPX4 genes, and sperm parameters of fertile and infertile men: A controlled clinical trial.

BackgroundOxidative stress is caused by the imbalance occurring between the creation and clearance of the reactive oxygen species (ROS), which is responsible for 30–40% of male infertility. The positive impact of phoenix dactylifera pollen (Date palm pollen, DPP) on the improvement of sperm parameters has been well documented in animal models.ObjectiveFor evaluating the effect(s) of DPP on sperm parameters, ROS levels, expression of antioxidant genes, and activity of antioxidant enzymes of infertile men.Materials and Methods In this controlled clinical trial, a total of 60 male case with infertility and 20 normospermic fertile men were recruited. Before and after the treatment with DPP, the case were administered 400 mg/kg of gelatinous capsules daily for 30 consecutive days and semen samples were taken. Quantitative real-time polymerase chain reaction was applied for the evaluation of the mRNA expression levels of Nuclear factor erythroid 2-related factor 2(NRF2), superoxide dismutase (SOD2), glutathione peroxidase 4(GPX4), and catalase (CAT) genes.Results The mRNA expression levels of NRF2, SOD2, GPX4, and CAT (p 0.05 for all) and significantly increased after treatment with DPP. The increased expressions of all antioxidant genes and enzymes significantly correlated with improvement in semen parameters including count (p = 0.01), motility (p = 0.05), and morphology (p = 0.01) of sperm. A significant correlation between the alteration of SOD2 gene expression and SOD activity, GPX4 and GPX, and CAT were also observed (p = 0.05).ConclusionDPP can increase the expressions of NRF2, GPX4, SOD2, and CAT genes and also improve the semen quality in infertile men.

An Investigation of the Antiproliferative Effect of Rhododendron luteum Extract on Cervical Cancer (HeLa) Cells via Nrf2 Signaling Pathway

The aim of the present study was to investigate the role of Rhododendron luteum extract (RLE) in the induction of Nrf2‑related oxidative stress and endoplasmic reticulum (ER) stress in human cervical cancer (HeLa) cells. The antiproliferative effect of RLE on HeLa and fibroblast cells was determined using the MTT assay. The effects of RLE on the cell cycle, apoptosis, and production of reactive oxygen species (ROS) in HeLa cells were evaluated using fluorescent probes. The mRNA expression levels of Nrf2 [and its targets glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD)], and C/EBP homologous protein (CHOP, an ER stress marker were determined using reverse transcription‑quantitative polymerase chain reaction (RT-PCR). The results demonstrated that RLE exhibited a selective cytotoxic effect (2.9-fold) on HeLa cells compared to fibroblast cells. RLE arrested the cell cycle at the S phase, and induced apoptosis, ER stress, and ROS formation. In addition, RLE significantly suppressed the expression levels of Nrf2, GCLC and G6PD (0.65, 0.69, and 0.54-fold, respectively) and increased the expression of CHOP (4.48-fold) in HeLa cells at 72 h of treatment (p < 0.05). These results show that the antiproliferative effect of RLE occurs through the Nrf2 and ER stress pathways, and the results should now be supported by further in vivo studies.

Etomidate attenuates hyperoxia-induced acute lung injury in mice by modulating the Nrf2/HO-1 signaling pathway.

The present study aimed to investigate the protective effects of etomidate on hyperoxia-induced acute lung injury in mice, particularly on the nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway. Fifty specific pathogen-free mice were randomly divided into the blank control, model, high oxygen exposure + low etomidate dose (0.3 mg·kg-1), a high oxygen exposure + moderate etomidate dose (3 mg·kg-1), and a high oxygen exposure + high etomidate dose (10 mg·kg-1) groups, with ten mice allotted per group. After 72 h, the mice were sacrificed and the lung tissues were harvested, and the wet-to-dry (W/D) ratio of the tissues was calculated. Hematoxylin-eosin staining was performed to observe the pathological changes in the lung tissues, and the lung injury score (LIS) was calculated. The mRNA and protein expression levels of Nrf2 and HO-1 were measured. The malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD) and catalase (CAT) levels were also measured, and interleukin (IL)-1β, IL-6, tumor necrosis factor alpha (TNF-α) and IL-10 concentrations in the bronchoalveolar lavage fluid were determined. At low and moderate doses, etomidate decreased pathological damage in the lung tissue, decreased the LIS and W/D ratio, upregulated Nrf2 and HO-1 mRNA and protein expression, decreased IL-1β, IL-6, and TNF-α concentrations, increased MPO activity and IL-10 levels, suppressed the production of the oxidation product MDA, and enhanced the activities of the antioxidant enzymes CAT and SOD. Within a certain dose range, etomidate enhanced antioxidant and anti-inflammatory effects in mice, thereby decreasing lung injury induced by the chronic inhalation of oxygen at high concentrations. Furthermore, the underlying mechanism may be associate with the upregulation of the Nrf2/HO-1 signaling pathway.

Hydrogen sulfide attenuates renal I/R‑induced activation of the inflammatory response and apoptosis via regulating Nrf2‑mediated NLRP3 signaling pathway inhibition.

Renal ischemia/reperfusion (I/R) injury can lead to acute renal failure, delayed graft function and graft rejection. Nucleotide-binding oligomerization domain NOD-like receptor containing pyrin domain 3 (NLRP3)-mediated inflammation participates in the development of renal injury. Nrf2 accelerates NLRP3 signaling pathway activation and further regulates the inflammatory response. In addition, hydrogen sulfide serves a protective role in renal injury; however, the detailed underlying mechanism remains poorly understood. The present study investigated whether Nrf2 and NLRP3 pathway participate in hydrogen sulfide-regulated renal I/R-induced activation of the inflammatory response and apoptosis. Wild-type and Nrf2-knockout (KO) mice underwent surgery to induce renal I/R via clamping of the bilateral renal pedicles. A total of 20 mg/kg MCC950 (an NLRP3 inhibitor) was injected intraperitoneally daily for 14 days prior to surgery. Renal tissue and blood were collected from the I/R model mice to analyze NLRP3 and Nrf2 mRNA expression levels, NLRP3, PYD and CARD domain containing, caspase-1, IL-1β, Nrf2 and heme oxygenase 1 protein expression levels, cell apoptosis, the secretion of tumor necrosis factor-α, IL-1β and IL-6 cytokines and renal histopathology and function. Renal I/R activated the NLRP3 and Nrf2 signaling pathways. Conversely, MCC950 treatment inhibited activation of the NLRP3 signaling pathway, and prevented I/R-induced renal injury, release of cytokines and apoptosis in renal I/R model mice. Sodium hydrosulfide (NaHS) not only alleviated upregulation of NLRP3 protein expression levels, but also relieved renal injury, release of cytokines and cell apoptosis induced by renal I/R in wild-type mice, but not in Nrf2-KO mice. NaHS alleviated NLRP3 inflammasome activation, renal injury, the inflammatory response and cell apoptosis via the Nrf2 signaling pathway in renal I/R model mice.

Co-treatment of vitamin D supplementation with enriched environment improves synaptic plasticity and spatial learning and memory in aged rats.

Environmental enrichment (EE) has been shown in old rats to improve learning and memory. Vitamin D (VitD) has also been shown to modulate age-related, cognitive dysfunction. As both EE and VitD could work to improve cognition via enhancement of neurotrophic factors, their effects might occlude one another. Therefore, a clinically relevant question is whether noted cognition-promoting effects of EE and VitD can co-occur. Aged rats were housed for 6weeks in one of three housing conditions: environmentally enriched (EE), socially enriched (SE), or standard condition (SC). Further, a 4th group was co-treated with VitDsupplementation (400IUkg-1 daily, 6weeks)under EE conditions (EE + VitD). Treatment with VitD and EE housingwere associated with higher score on measures of learning and memory and exhibited lower anxiety scores compared to EE alone, SE or SC as assayed in the elevated plus maze, Morris water maze, passive avoidance, and open field tasks. Additionally, inthe EE + VitD group, mRNA expression levels of NGF, TrkA, BDNF, Nrf2, and IGF-1 were significantly higher compared to expression seen in the EE group. Furthermore, field potential recordings showed that EE + VitD resulted in a greater enhancement of hippocampal LTP and neuronal excitability when compared to EE alone. These findings demonstrate that in aged rats exposure to EE and VitD results in effects on hippocampal cognitive dysfunction and molecular mechanisms which are greater than effects of EE alone, suggesting potential for synergistic therapeutic effects for management of age-related cognitive decline.

Protective Effects of Silymarin Against D-Gal/LPS-Induced Organ Damage and Inflammation in Mice.

AimSilymarin contains various flavonoids and exhibits antioxidative, anti-inflammatory, and anticancer effects, in addition to other pharmacological properties. This study explored the alleviating effect of silymarin on multiple-organ damage induced by D-galactose/lipopolysaccharide in Kunming mice.MethodsKunming mice were injected intraperitoneally with D-galactose (30 mg/kg·BW)/LPS (3 μg/kg·BW) and then treated using silymarin with different doses (75 mg/kg·bw and 150 mg/kg·bw) via intragastric administration. Changes in organ indexes, pathological changes, liver-function index, biochemical indexes, molecular biological indexes, and genes related to the oxidation and inflammation of main organs were evaluated.ResultsAfter the mice were treated with silymarin, their body weight showed no significant change, and the liver, kidney, and lung indexes of the treated mice were higher than those of the model group; meanwhile, the corresponding histopathological formation was reduced. Compared with the model group, the silymarin-treated group showed reductions in ALT, AST, and liver function indexes in the mouse serum. Silymarin treatment also increased the SOD, CAT, GSH, GSH-Px, T-AOC, IL-10, and IL-12 levels, as well as reduced the MDA, NO, IL-6, IL-1β, TNF-α, IFN-γ levels in the mouse serum and liver tissues. In addition, quantitative polymerase chain reaction analysis indicated that the mRNA expression levels of SOD1, SOD2, CAT, GSH-Px, IL-10, Nrf2, HO-1, NQO1, Trx, and IκB-α were higher in the liver tissue of the silymarin-treated mice than in those of the model group; meanwhile, the mRNA expression levels of IL-6, IL-1β, TNF-α, IFN-γ, NF-κB, NLRP3, COX2, and p38 were lower than those in the model group.ConclusionSilymarin, which exhibits antioxidative and anti-inflammatory effects, can alleviate the liver, lung, and kidney damage induced by D-galactose/lipopolysaccharide. High-dose (150 mg/kg·bw) silymarin can more effectively inhibit organ damage, compared with low-dose silymarin (75 mg/kg·bw) in Kunming mice.

Fructus Lycii and Salvia miltiorrhiza Bunge extract alleviate retinitis pigmentosa through Nrf2/HO-1 signaling pathway

Ethnopharmacological relevanceFructus Lycii and Salvia miltiorrhiza Bunge (FS) are popular Chinese herbs for the treatment of retinitis pigmentosa (RP). Aim of the studyThis study was to evaluate protective effects of FS extract on RP and to explore whether FS extract exerts its protective effects via oxidative stress by regulating Nrf2/HO-1 signaling pathway. Material and methodsFS extract were identified by UPLC chromatographic analysis. Rd10 mice as the model of RP, followed by a 4-week FS extract treatment by intragastric administration. After the animal sacrifice, histopathological examination and Scotopic electroretinography (ERG) analysis were assessed. The oxidative stress markers were determined and the expression levels of Nrf2 and HO-1 mRNA were evaluated by qRT-PCR. The expression and distribution of Nrf2 and HO-1 protein were determined by Western blot and immunohistochemistry. ResultsThe morphological changes of Outer nuclear layer (ONL) thickness and number of the ONL were observed with a significant increased, and the functional changes of a-amplitude and b-wave amplitude were measured with a markedly increased. Treatment with FS extract remarkably increased levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and decreased level of malondialdehyde (MDA). Moreover, FS extract up-regulated mRNA and protein expression of Nrf2 and HO-1. ConclusionsThis study indicated that FS extract can improve retinal morphology and function, which may have occurred through the regulation of the Nrf2/HO-1 pathway to inhibit the oxidative reaction.

Nephroprotective activity of Aframomum melegueta seeds extract against diclofenac-induced acute kidney injury: A mechanistic study

Ethnopharmacological relevanceIn Africa, Aframomum species have been traditionally used to treat illnesses such as inflammation, hypertension, diarrhea, stomachache and fever. Moreover, Aframomum melegueta seed extracts (AMSE) are used in traditional medicine to relieve stomachaches and inflammatory diseases. AimChronic administration of diclofenac (DIC) has been reported to cause acute kidney injury (AKI), which is a serious health condition. The nephroprotective effect of AMSE is yet to be elucidated. Accordingly, this study aims to investigate the phytoconstituents of standardized AMSE, evaluate its nephroprotective effects against DIC-induced AKI in rats, and elaborate its underlying molecular mechanisms. Materials and methodsThe quantitative estimation of major AMSE constituents and profiling of its secondary metabolites were conducted via RP-HPLC and LC-ESI/Triple TOF/MS, respectively. Next, DIC (50 mg/kg)-induced AKI was achieved in Sprague-Dawley rats and DIC-challenged rats were administered AMSE (100 and 200 mg/kg) orally. All treatments were administered for five consecutive days. Blood samples were collected and the sera were used for estimating creatinine, urea and, kidney injury molecule (KIM)-1 levels. Kidney specimens were histopathologically assessed and immunohistochemically examined for c-Myc expression. A portion of the kidney tissue was homogenized and examined for levels of oxidative stress markers (MDA and GSH). Heme oxygenase (HO)-1, TNF-α, IL-6, Bax, Bcl2 and caspase-3 renal levels were quantified by ELISA. Moreover, the protein expression levels of NF-ҡB p65 was quantified using Western blot analysis, whereas mRNA expression levels of AMPK, SIRT-1, nuclear factor erythroid-2-related factor (Nrf2) and STAT3 were detected using qRT-PCR in the remaining kidney tissues. ResultsStandardized AMSE was shown to primarily contain 6-gingerol, 6-shogaol and 6-paradol among the 73 compounds that were detected via LC-ESI/Triple TOF/MS including phenolic acids, hydroxyphenylalkanes, diarylheptanoids and fatty acids. Relative to DIC-intoxicated rats, AMSE modulated serum creatinine, urea, KIM-1, renal MDA, TNF-α, IL-6, Bax, and caspase-3 levels. AMSE has also improved renal tissue architecture, enhanced GSH and HO-1 levels, and upregulated renal Nrf2, AMPK, and SIRT-1 mRNA expression levels. Furthermore, AMSE suppressed NF–ҡB p65 protein and STAT3 mRNA expression, and further reduced c-Myc immunohistochemical expression in renal tissues. Overall, our findings revealed that AMSE counteracted DIC-induced AKI via its antioxidant, anti-inflammatory, and antiapoptotic activities. Moreover, AMSE activated Nrf2/HO1 and AMPK/SIRT1, and inhibited NF-ҡB/STAT3 signaling pathways. Therefore, AMSE is a promising agent for inhibiting DIC-induced nephrotoxicity.

Effects of Long-Term Exposure to Copper on the Keap1/Nrf2 Signaling Pathway and Msr-Related Redox Status in the Kidneys of Rats.

The objective of the present study was to examine the effects of long-term exposure on oxidative damage, Keap1/Nrf2 signaling pathway, and Msr-related redox status in the kidneys of rats. Therefore, in this experimental study, a total of 32 CD-1 rats were randomized into 4 groups and treated with 30-, 60-, and 120-mg/kg Cu for 24 weeks. Different serum biomarkers suggestive of renal functions, pathological changes, and oxidative stress were analyzed in kidney tissues. Moreover, the levels of the Keap1/Nrf2 signaling pathway and redox status-related gene mRNA and proteins were also detected. The results indicated that Cu exposure dramatically increased the contents of creatinine and carbamide. Furthermore, histopathological alterations and mitochondrial damage in kidneys of rats of different Cu-treated groups were obviously observed. In addition, Cu exposure markedly changed the levels of glutathione, catalase, and total antioxidant capacity, and upregulated the contents of protein carbonyl, nitric oxide, and malondialdehyde. Moreover, higher levels of Cu treatments significantly increased the expression of Keap1/Nrf2 signaling pathway and redox status-related genes (NQO1, SOD-1, TRX, MsrA, MsrB1, MsrB2, MsrB3). Simultaneously, the mRNA expression levels of Nrf2, HO-1, and CAT were upregulated in rats exposed to 30- and 60-mg/kg Cu, but downregulated in the 120-mg/kg Cu group compared with the control group. Moreover, the Keap1/Nrf2 signaling pathway and redox status-related protein expression levels (HO-1, SOD-1, TRX, MsrA, MsrB1, MsrB2) were significantly increased in treated rats. In summary, it is suggested that the Keap1/Nrf2 signaling pathway and activation of Msr prevent Cu-induced nephrotoxicity and attenuate oxidative damage.

Porphyromonas gingivalis infection alters Nrf2-phase II enzymes and nitric oxide in primary human aortic endothelial cells.

Periodontal disease (PD) is known to be associated with endothelial dysfunction in patients with coronary artery and/or cardiovascular disease. In our study, we sought to explore the virulence of P. gingivalis (Pg) affecting glycogen synthase kinase 3 beta (GSK-3β)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/tetrahydrobiopterin (BH4 )/ nitric oxide synthase (NOS) expression in primary human aortic endothelial cells (pHAECs). pHAECs were infected for 48 hours with Pg in vitro using the Human oxygen-Bacteria anaerobic coculture technique. Cell viability was determined, and target gene expression changes were evaluated by quantitative real-time polymerase chain reaction at the end of each incubation period. Pg impaired pHAEC viability 24 hours post-infection. Pg infection reduced mRNA expression levels of endothelial NOS (eNOS), Nrf2, and Phase II enzymes (heme oxygenase-1, catalase, superoxide dismutase-1) in a time-dependent manner. Significant (P<0.05) increase in the inflammatory markers (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α) were observed in the medium as well as in the infected cells. Interestingly, inducible NOS mRNA levels showed a significant (P<0.05) increase at 12 hours and 24 hours and were reduced at later time points. BH4 (cofactor of eNOS) biosynthesis enzyme dihydrofolate reductase (DHFR, salvage pathway) mRNA levels showed a significant (P<0.05) decrease, while mRNA levels of GSK-3β were elevated. These results suggest that periodontal bacterial infection may cause significant changes in the endothelial GSK-3β/BH4 /eNOS/Nrf2 pathways, which may lead to impaired vascular relaxation. Greater understanding of the factors that adversely affect endothelial cell function could contribute to the development of new therapeutic compounds to treat PD-induced vascular diseases.

Improvement of cerebral ischemia–reperfusion injury by l-3-n-butylphthalide through promoting angiogenesis

Cerebral ischemia/reperfusion (I/R) injury may lead to a poor prognosis for ischemic stroke patients after reperfusion therapy, and currently, lacks effective therapeutic intervention. This study aimed to investigate the effects of L-3-n-butylphthalide (L-NBP) on cerebral I/R injury in rats. Rat models of cerebral I/R injury were established using the middle cerebral artery occlusion/refusion (MACO/R) surgery and were administrated intragastrically with L-NBP or vehicle. We found that L-NBP attenuated the histological damages and reduced the brain hematoma in MACO/R rats. L-NBP also significantly improved the neurological function, alleviated the brain edema, and reduced the permeability of blood-brain barrier of MACO/R rats. Moreover, we detected that L-NBP considerably facilitated microvessel formation in the lesion area of brain in MACO/R rats. Finally, we found that L-NBP significantly increased the protein and mRNA expression levels of Nrf2, HIF-1α, and VEGF in the brain of MACO/R rats. In conclusion, our results demonstrated that L-NBP exerted significant beneficial effects on cerebral I/R injury in rats through promoting angiogenesis, which may be associated with the activation of Nrf2/HIF-1α/VEGF signaling pathway. Our results suggested that L-NBP could be a potential therapeutic drug for cerebral I/R injury.

6-shogaol suppresses oxidative damage in L6 muscle cells

Ginger (Zingiber Officinale Roscoe) has been known reduce muscle pain after exercise, and 6-shogaol {(E)-1-(4-Hydroxy-3-methoxyphenyl)dec-4-en-3-one)} is the major essential oil contained in ginger. In this study, the protective effect of 6-shogaol on L6 muscle cells against oxidative damage was measured. 6-shagol inhibited the damage of L6 cell induced by H2O2, and allowed the increase in mRNA and protein expression levels of intracellular HO-1 and NRF2. 6-shogaol also reduced the production of intracellular ROS. These results suggested that 6-shagol effectively inhibits oxidative damage of skeletal muscle cell.

Chemoprevention of Prostate Cancer Cells by Vitamin C plus Quercetin: role of Nrf2 in Inducing Oxidative Stress

To assess the effect of sequential treatment with Vitamin C (VC) and Quercetin (Q) on Nrf2-related oxidative stress in PC3 and DU145 cells, viability was measured by MTT assay. Intracellular ROS levels were determined, using 2'-7'-dichlorodihydrofluorescein diacetate fluorescent as a probe. Nrf2 gene expression was investigated by quantitative reverse transcription polymerase chain reaction, and Nrf2 protein levels were defined by western blot analysis. The activity of glutathione peroxidase (GPx), glutathione reductase (GR), nicotinamide adenine dinucleotide phosphate dehydrogenase quinone 1 (NQO1) and hemeoxygenase 1 (HO-1) enzymes were measured. The IC50 values for VC + Q were 263.03–372.1 µM and 144.2–194.1 µM respectively and 200 µM VC + 50 µM Q (dose no.1) and 100 µM VC + 75 µM Q (dose no.2) were selected. Sequential treatment of PC3 cells led to a significant reduction of Nrf2 mRNA expression and protein levels in addition to a significant reduction of GPx, GR and NQO1 enzymatic activity. Although the data was slightly different for DU145 cells after the treatments, in terms of Nrf2 gene expression, we obtained the same results. Our study revealed the significant effects of sequential treatment with VC + Q on Nrf2 suppression in prostate cancer cells.

Paeoniflorin Improves Acute Lung Injury in Sepsis by Activating Nrf2/Keap1 Signaling Pathway

To investigate the effect of paeoniflorin (PF) on sepsis-induced acute lung injury and its relationship with nuclear factor erythyroid 2-related factor 2 (Nrf2)/Kelch-like ECH2 associated protein 1 (Keap1) signaling pathway. Cecal ligation and puncture (CLP) was used to induce the sepsis rat model. Rats were randomly divided into 5 groups (n=10): sham group (Sham), model group (Model), low dose PF group (L-PF group, 50 mg/kg PF), high dose PF group (H-PF group, 150 mg/kg PF) and high dose PF+Nrf2 inhibitor group (H-PF+ML385 group, 150 mg/kg PF+30 mg/kg ML385). The severity of sepsis in rats was scored according to the improved severity scale of sepsis; the pathological changes of lung tissue were observed by HE staining; the activity of superoxide dismutase (SOD) in lung tissue was determined by xanthine oxidase method; the content of malondialdehyde (MDA) in lung tissue was measured by thiobarbituric acid method; and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in lung tissues were detected by ELISA. Western blot was used to detect the expression of Nrf2, Keap1 and HO-1 proteins in lung tissues. Compared with Sham group, the sepsis symptoms in Model group were more serious, severe inflammatory infiltration in lung tissue, macrophages and interstitial enlargement, and the contents of pro-inflammatory factors IL-1β, TNF-α, IL-6 and oxidative index MDA in lung tissues were significantly increased (P<0.05), while the activity of antioxidant index SOD were significantly decreased (P<0.05). The protein and mRNA expression levels of Nrf2 and HO-1 were significantly decreased (P<0.05), while the protein and mRNA expression levels of Keap1 were markedly increased (P<0.05). Compared with the Model group, the severity of sepsis in PF groups was decreased, the lung tissue injury was improved, the contents of IL-1β, TNF-α, IL-6 and MDA in lung tissue were significantly decreased (P<0.05), the activity of SOD was markedly increased (P<0.05), the protein and mRNA expression levels of Nrf2 and HO-1 were significantly increased (P<0.05), and the expression levels of Keap1 protein and mRNA were significantly decreased (P<0.05). Compared with the H-PF group, Nrf2 inhibitor ML385 significantly inhibited the improvement of PF on lung injury in sepsis rats. PF can reduce oxidative stress and inflammation by activating Nrf2/Keap1 signaling pathway, and improve sepsis-induced acute lung injury.

Aerobic exercise combined with huwentoxin-I upregulates phase-Ⅱ detoxification enzymes to alleviate obstructive jaundice-induced central nervous system injury in mice

To explore the effects of aerobic exercise combined with huwentoxin-I (HWTX-I)-mediated Keap1-Nrf2-ARE pathway on phase II detoxification enzymes HO-1 and NQO1 and their protective effects against obstructive jaundice (OJ)-induced central nervous system injury in mice. 50 male KM mice were randomly divided into blank group (GO), model group (M), aerobic exercise group (T), HWTX-I group (H), and aerobic exercise combined with HWTX-I group (TH). Mouse models of OJ were established with surgical suture for 72 h in the mice in all the groups except for the blank control group. The mice received interventions by aerobic exercise and tail vein injection of HWTX-I (0.05 μg/g) and were assessed by behavioral observation, Clark's neurological function scores, enzyme-linked immunosorbent assay (ELISA), brain tissue Nissl staining, hippocampal tissue Western blotting, and liver tissue mRNA expression profiling and sequencing. The mice in group M had obvious jaundice symptoms after the operation with significantly increased Clark's neurological score (P &lt; 0.01). Compared with those in group M, the mice in group T, group H, and group TH showed significantly decreased serum levels of ALT, AST, TBIL, and TBA (P &lt; 0.01) with increased contents of 5-HT and BDNF and decreased contents of S100B and NSE in the hippocampus (P &lt; 0.01). Synergistic effects between aerobic exercise and HWTX-I were noted on the above parameters except for the liver function indicators. Interventions with aerobic exercise and HWTX-I, alone or in combination, obviously lessened pathologies in the brain tissue induced by OJ, and the combined treatment produced the strongest effect. The treatment also increased the expression levels of Nrf2, HO-1, and NQO1 mRNA and protein in brain tissues (P &lt; 0.01 or 0.05) with a synergistic effect between aerobic exercise and HWTX-I. Illumina high-throughput sequencing showed that the differentially expressed factors participated mainly in such neural regulatory pathways as neuroactive ligand-receptor interaction, GABAergic synapses, dopaminergic synapses, synaptic vesicle circulation, and axon guidance, involving tissue cell neuronal signal transduction, apoptosis inhibition, immune response, and toxicity. Aerobic exercise and HWTX-I synergistically increased the accumulation of the signal pathways related with neuron damage repair and proliferation. Aerobic exercise combined with HWTX-I can up-regulate the expression of phase Ⅱ detoxification enzymes HO-1 and NQO1 through the Keap1-Nrf2-ARE pathway to protect the central nervous system against OJ-induced damage in mice.

Polysaccharides from Hemp Seed Protect against Cyclophosphamide-Induced Intestinal Oxidative Damage via Nrf2-Keap1 Signaling Pathway in Mice.

Hemp seed has been used as a traditional oriental medicine and health food in China for centuries. Polysaccharides from hemp seed (HSP) exhibit important properties of intestinal protection, but there are limited data on the specific underlying mechanism. The primary objective of this study was to investigate the protective effect of HSP on intestinal oxidative damage induced by cyclophosphamide (Cy) in mice. The results showed that pretreatment with HSP significantly increased the average daily gain, thymus index, spleen index, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity in serum and ileal homogenate and significantly reduced malondialdehyde (MDA) content in ileal homogenate. In addition, the expression levels of SOD, GSH-Px, Nrf2, heme oxidase-1 (HO-1), and quinoneoxidoreductase-1 (NQO1) mRNA in ileal homogenate were significantly increased. Western blot results showed that HSP significantly upregulated the expression of Nrf2 protein and downregulated the expression of Keap1 protein in the ileum. Collectively, our findings indicated that HSP had protective effects on intestinal oxidative damage induced by Cy in mice, and its mechanism might be related to the activation of Nrf2-Keap1 signaling pathway.

Rosemary extract reverses oxidative stress through activation of Nrf2 signaling pathway in hamsters fed on high fat diet and HepG2 cells

• Diet rosemary extract (RE) alleviated high-fat induced oxidative damage in vivo and in vitro. • Diet RE enhanced the antioxidant enzyme activity, gene and protein expression level. • The protective effect of diet RE was achieved by regulating Nrf2 pathway. Rosemary extract (RE), a useful component isolated from Rosmarinus officinalis L. , was recently reported to protect cells against the harmful effects of ROS. However, the underlying mechanisms of this action remained unknown. We investigated the defensive effect of RE on the oxidative stress injury resulted from high fat diet in hamsters and oleic acid (OA) in HepG2 cells respectively, and its underlying mechanisms. Treatment with RE alleviated liver steatosis and reduced plasma and liver content of TG and TC, as well as plasma content of AST and ALT. RE apparently increased the activity of SOD, GSH-Px, CAT and decreased MDA content. RE notably increased CuZn-SOD, Mn-SOD, CAT, GSH-Px, GCLC, GCLM and Nrf2 mRNA and protein expression level. Nrf2 shRNA knockdown in HepG2 cells increased OA-induced ROS generation, and effectively altered OA-induced cytotoxicity, suggesting that Nrf2 is a crucial mediator against high-fat induced oxidative damage.