Nuclear Factor E2-related Factor-2 Research Articles

Saprirearine protects H9c2 cardiomyocytes against hypoxia/reoxygenation-induced apoptosis by activating Nrf2.

Myocardial infarction is a major cause of mortality and disability worldwide. Ischemia/reperfusion injury is the key factor that results in the increase in infarct size in pathogenesis. To find a novel therapy for myocardial infarction, we have evaluated saprirearine, a natural diterpenoid, using H9c2 cardiomyocytes injured by hypoxia/reoxygenation and explored the possible mechanisms. The results showed that saprirearine improved cell survival by increasing cell viability and blocking the release of lactate dehydrogenase. Meanwhile, saprirearine was found to attenuate mitochondrial dysfunction by inhibiting calcium overload, collapse of the mitochondrial membrane potential, and opening of the mitochondrial permeability transition pore. And oxidative stress resulting from hypoxia/reoxygenation was ameliorated by saprirearine through the reduction of reactive oxygen species and malondialdehyde as well as activation of superoxide dismutase and catalase. Additionally, saprirearine inactivated cysteinyl aspartate-specific proteinase-3, the up-regulated B-cell lymphoma-2 and down-regulated Bcl-2-associated X protein, to inhibit hypoxia/reoxygenation-induced apoptosis. Further research revealed saprirearine-activated nuclear factor E2-related factor-2 in H9c2 cardiomyocytes, which is closely associated with its protective effects. These findings can provide evidence for the discovery of new therapies targeting myocardial infarction and the application of saprirearine in clinical practice.

Psoralidin protects against cerebral hypoxia/reoxygenation injury: Role of GAS6/Axl signaling.

Psoralidin (PSO) is a natural phenolic coumarin extracted from the seeds of Psoralea corylifolia L. Growing preclinical evidence indicates that PSO has anti-inflammatory, anti-vitiligo, anti-bacterial, and anti-viral effects. Growth arrest-specific gene 6 (GAS6) and its receptor, Axl, modulate cellular oxidative stress, apoptosis, survival, proliferation, migration, and mitogenesis. Notably, the neuroprotective role of the GAS6/Axl axis has been identified in previous studies. We hypothesize that PSO ameliorates cerebral hypoxia/reoxygenation (HR) injury via activating the GAS6/Axl signaling. We first confirmed that PSO was not toxic to the cells and upregulated GAS6 and Axl expression after HR injury. Moreover, PSO exerted a marked neuroprotective effect against HR injury, represented by restored cell viability and cell morphology, decreased lactate dehydrogenase (LDH) release, and reactive oxygen species (ROS) generation. Furthermore, PSO pretreatment also elevated the levels of nuclear factor-related factor 2 (Nrf-2), NAD(P)H dehydrogenase quinone-1 (NQO1), heme oxygenase-1 (HO-1), silent information regulator 1 (SIRT1), peroxisome proliferator-activated receptor coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF1), uncoupling protein 2 (UCP2), and B-cell lymphoma 2 (BCl2) both in the condition of baseline and HR injury. However, GAS6 siRNA or Axl siRNA inhibited the neuroprotective effects of PSO. Our findings suggest that PSO pretreatment attenuated HR-induced oxidative stress, apoptosis, and mitochondrial dysfunction in neuroblastoma cells through the activation of GAS6/Axl signaling.

AUF1 protects against ferroptosis to alleviate sepsis-induced acute lung injury by regulating NRF2 and ATF3.

The AU-rich element (ARE)-binding factor 1 (AUF1) acts as a switch for septic shock, although its underlying mechanisms remain largely unknown. In this study, we examined the biological significance and potential molecular mechanism of AUF1 in regulating ferroptosis in sepsis-induced acute lung injury (ALI). Alveolar epithelial cells (AECs) challenged with ferroptosis-inducing compounds and cecum ligation and puncture (CLP)-induced ALI were used as the in vitro and in vivo model, respectively. The stability of AUF1 and its degradation by ubiquitin-proteasome pathway were examined by cycloheximide chase analysis and co-immunoprecipitation assay. The regulation of AUF1 on nuclear factor E2-related factor 2 (NRF2) and activation transcription factor 3 (ATF3) was explored by RNA immunoprecipitation (RIP), RNA pull-down, and mRNA stability assays. Functionally, the effects of altering AUF1, NRF2 or ATF3 on ferroptosis in AECs or ALI mice were evaluated by measuring cell viability, lipid peroxidation, iron accumulation, and total glutathione level. AUF1 was down-regulated in AECs challenged with ferroptosis-inducing compounds, both on mRNA and protein levels. The E3 ubiquitin ligase FBXW7 was responsible for protein degradation of AUF1 during ferroptosis. By up-regulating NRF2 and down-regulating ATF3, AUF1 antagonized ferroptosis in AECs in vitro. In the CLP-induced ALI model, the survival rate of AUF1 knockout mice was significantly reduced and the lung injuries were aggravated, which were related to the enhancement of lung ferroptosis. FBXW7 mediates the ubiquitination and degradation of AUF1 in ferroptosis. AUF1 antagonizes ferroptosis by regulating NRF2 and ATF3 oppositely. Activating AUF1 pathway may be beneficial to the treatment of sepsis-induced ALI.

Galactocerebrosidase deficiency induces an increase in lactosylceramide content: A new hallmark of Krabbe disease?

Galactocerebrosidase (GALC) hydrolyses galactose residues from various substrates, including galactosylceramide, psychosine (galactosylsphingosine), and lactosylceramide. Its severe deficiency has been associated with the accumulation of psychosine, a toxic molecule with detergent-like features, which alters membrane structures and signalling pathways, inducing the death of oligodendrocytes and a sequence of events in the nervous system that explain the appearance of many clinical signs typical of Krabbe disease. Nevertheless, new evidence suggests the existence of other possible links among GALC action, myelination, and myelin stability, apart from psychosine release. In this study, we demonstrated that lactosylceramide metabolism is impaired in fibroblasts isolated from patients with Krabbe disease in the absence of psychosine accumulation. This event is responsible for the aberrant and constitutive activation of the AKT/prolin-rich AKT substrate of 40kDa (PRAS40) signalling axis, inducing B cell lymphoma 2 (BCL2) overexpression and glycogen synthase kinase 3 beta (GSK-3β) inhibition. In addition, nuclear factor E2-related factor 2 (NRF2) showed increased nuclear translocation. Due to the relevance of these molecular alterations in neurodegeneration, lactosylceramide increase should be evaluated as a novel marker of Krabbe disease, and because of its significant connections with signalling pathways.

Fatty acid binding protein 4 regulates pancreatic cancer cell proliferation via activation of nuclear factor E2-related factor 2.

Obesity and diabetes are associated with an increased incidence of pancreatic cancer. Fatty acid binding protein 4 (FABP4), noted to be higher in patients with severe obesity, is linked to the development and progression of several cancers, and its level in the circulation decreases after bariatric surgery. In this paper, we evaluate the role of FABP4 in pancreatic cancer progression. University Hospital and Laboratories, United States. When Panc-1 (human) and Pan02 (mouse) pancreatic cancer cells were treated with FABP4 or the-single-point mutant FABP4 (R126Q, fatty acid binding site mutant), only FABP4 stimulated cellular proliferation. The transcriptional activity of nuclear factor E2-related factor 2 (NRF2) was increased in response to FABP4 but not the R126Q. FABP4 treatment also led to downregulation of reactive oxygen species (ROS) activity. Consistent with induced cell propagation by FABP4, the growth of Pan02 tumor was decreased in FABP4-null animals compared with C57BL/6J controls. These results suggest that FABP4 increases pancreatic cancer proliferation via activation of NRF2 and downregulation of ROS activity.

Nrf2 protects against cerebral ischemia-reperfusion injury by suppressing programmed necrosis and inflammatory signaling pathways.

BackgroundThe NOD-like receptor family pyrin domain-containing 3 (NLRP3) -mediated neuroinflammation is linked to neuronal necroptosis in cerebral ischemia-reperfusion (I/R) injury, especially in cerebral ischemic penumbra. This study was designed to investigate the regulation of nuclear factor E2-related factor-2 (Nrf2) on NLRP3 inflammasome in necroptosis signal pathway induced by I/R injury.MethodsWe investigated the mechanisms of Nrf2-negative regulation in necroptosis signaling pathway by using middle cerebral artery occlusion (MCAO) with Q-VD-OPH injected intraperitoneally. The protein level of the NLRP3 inflammasome was detected by western blot with Nrf2 knockdown and overexpression. NLRP3 inflammasome activation was Reactive oxygen species (ROS) dependent, and the protein level was regulated when N-acetylcysteine (NAC) and adenosine triphosphate (ATP) were selected as tools for regulating ROS.ResultsWe demonstrated the negative regulation of Nrf2 on NLRP3 inflammasome activation in Q-VD-OPH-induced necroptosis in cerebral artery I/R injury through Lentivirus-mediated RNA Interferenc, which mediated knockdown and overexpression of Nrf2. NLRP3 inflammasome activation was sensitive to both ATP-mediated ROS level increases and NAC-mediated ROS inhibition, suggesting that ROS is associated with the activation of NLRP3 inflammasome in necroptosis. In addition, Nrf2-induced NAD(P)H quinone dehydrogenase 1 (NQO1) was involved in the inhibition of NLRP3 inflammasome activation. These results suggest that Nrf2 regulates NQO1 to attenuate ROS, which negatively regulates NLRP3 inflammasome.ConclusionsNrf2/NQO1 was an inhibitor of ROS-induced NLRP3 inflammasome activation in Q-VD-OPH-induced necroptosis following cerebral I/R injury. Therefore, NLRP3 inflammasome could be a potential therapeutic target for cerebral ischemia.

Activation of NRF2/FPN1 pathway attenuates myocardial ischemia\u2013reperfusion injury in diabetic rats by regulating iron homeostasis and ferroptosis

In patients with ischemic heart disease, myocardial ischemia–reperfusion injury (IRI) can aggravate their condition even worse, and diabetes increases their risk of myocardial IRI. Pathological pathways of common diseases and surgical operations like diabetes, obesity, coronary artery angioplasty, and heart transplantation entail disorders of iron metabolism. Ferroportin1 (FPN1) is the only mammalian protein associated with iron release and thus plays a vital role in iron homeostasis, while nuclear factor E2-related factor 2 (NRF2) controls the transcription of FPN1. Since the NRF2/FPN1 pathway may play a favorable role in the therapy of diabetic myocardial IRI, this work investigated the possible mechanism. In this study, we investigated the effects of ferroptosis in STZ-induced diabetic rats following myocardial IRI in vivo, and its alteration in glucose and hypoxia/reoxygenation-induced cardiomyocytes injury in vitro. Rats and H9c2 cardiomyocytes were randomly divided into 6 groups and treated with sulforaphane and erastin besides the establishment of diabetic myocardial IRI and hyperglycemic hypoxia-reoxygenation models. Cardiac functional and structural damage were detected by Evans blue/TTC double staining, echocardiography, HE staining, and serological indices. CCK-8 assay and ROS production were used to measure cardiomyocyte viability and oxidative stress level. Additionally, the changes in cell supernatant levels of Fe2+, SOD, MDA, and mRNA and protein expression of ferroptosis marker proteins confirmed the beneficial effects of the NRF2/FPN1 pathway on diabetic myocardial IRI related to iron metabolism and ferroptosis. Overall, these findings suggest that iron homeostasis-related ferroptosis plays an important role in aggravating myocardial IRI in diabetic rats, and NRF2/FPN1 pathway-mediated iron homeostasis and ferroptosis might be a promising therapeutic target against myocardial IRI in diabetes.

English

Chronic non-communicable diseases are responsible for a large majority of deaths worldwide. These include cardiovascular and respiratory diseases, diabetes and cancer. Inflammation has been reported to be involved in the initiation or progression of these conditions. Populations in developing or low to middle- income countries often rely on traditional medicine using locally available herbs and plants for their medical care. This study examined the anti-inflammatory potential of aqueous extracts of twelve medicinal plants used in Nigeria.  The antioxidant activity was estimated using the total radical-trapping antioxidant parameter (TRAP) and ferric reducing ability (FRAP) assays. The abilities to inhibit nuclear factor kappa light chain enhancer of activated B cells (NfkB), a key regulator of the inflammatory response, and to activate nuclear factorE2 related factor 2 (Nrf2), a transcription factor that regulates cellular antioxidant defense systems, were determined using in vitro cell based assays. Extracts of Erythrina senegalensis (leaves), Sclerocarya birrea (bark), Boswellia dalzielli (leaves and bark), Pseudrocedrila kotschyi (bark), Sterculia setigera (stem bark), and Sarcocephalus esculentus (bark) contained the highest levels of antioxidant activity. Extracts that showed the greatest inhibition of NfkB were S. esculentus (bark), 91.8%; E. senegalensis (leaves), 81.4%; S. birrea (stem bark), 77.5%; and S. setigera (stem bark), 75.5%. B. dalzielli (leaves) and Xylopia aethiopica (leaves) gave 7.4 and 7.7 fold activation of Nrf2, respectively. These were comparable to activation by sulphorophane.   Key words: NfkB inhibition, Nrf2 activation, antioxidant activity, inflammation, medicinal plants, Nigeria.

Protective effect of resveratrol against hydrogen peroxide-induced oxidative stress in bovine skeletal muscle cells

The objective of this study was to investigate the protective effects and the underlying mechanisms of resveratrol (RES) against hydrogen peroxide (H2O2)-induced oxidative stress in bovine skeletal muscle cells (BMCs). Pretreatment of BMCs with RES prior to H2O2 exposure increased cell viability, attenuated reactive oxygen species, and stabilized the redox state. H2O2 exposure activated sirtuin type 1 (SIRT1) and nuclear factor E2-related factor 2 (NRF2)-mediated signaling pathways. Pretreatment with RES did not alter SIRT1-regulated genes but inhibited the upregulation of NRF2, whereas enhanced heme oxygenase 1 (HO-1) expression. Pretreatment with RES prior to H2O2 exposure failed to suppress NRF2 expression when NRF2 was knocked down by RNA interference. However, HO-1 expression still could be induced by RES. These results suggest that RES has benifical effects against oxidative stress. NRF2-mediated pathway play an important role, and HO-1 upregulation is the key process in RES regulation. RES may be used as a therapeutic agent for meat quality improvement in beef cattle.

Bioactive Compounds in Oxidative Stress-Mediated Diseases: Targeting the NRF2/ARE Signaling Pathway and Epigenetic Regulation.

Oxidative stress is a pathological condition occurring due to an imbalance between the oxidants and antioxidant defense systems in the body. Nuclear factor E2-related factor 2 (NRF2), encoded by the gene NFE2L2, is the master regulator of phase II antioxidant enzymes that protect against oxidative stress and inflammation. NRF2/ARE signaling has been considered as a promising target against oxidative stress-mediated diseases like diabetes, fibrosis, neurotoxicity, and cancer. The consumption of dietary phytochemicals acts as an effective modulator of NRF2/ARE in various acute and chronic diseases. In the present review, we discussed the role of NRF2 in diabetes, Alzheimer’s disease (AD), Parkinson’s disease (PD), cancer, and atherosclerosis. Additionally, we discussed the phytochemicals like curcumin, quercetin, resveratrol, epigallocatechin gallate, apigenin, sulforaphane, and ursolic acid that have effectively modified NRF2 signaling and prevented various diseases in both in vitro and in vivo models. Based on the literature, it is clear that dietary phytochemicals can prevent diseases by (1) blocking oxidative stress-inhibiting inflammatory mediators through inhibiting Keap1 or activating Nrf2 expression and its downstream targets in the nucleus, including HO-1, SOD, and CAT; (2) regulating NRF2 signaling by various kinases like GSK3beta, PI3/AKT, and MAPK; and (3) modifying epigenetic modulation, such as methylation, at the NRF2 promoter region; however, further investigation into other upstream signaling molecules like NRF2 and the effect of phytochemicals on them still need to be investigated in the near future.

NRF2 Exerts Anti-Inflammatory Effects in LPS-Induced gEECs by Inhibiting the Activation of the NF-κB.

Nuclear factor E2-related factor 2 (NRF2) plays an anti-inflammatory role in several pathological processes, but its function in lipopolysaccharide- (LPS-) induced goat endometrial epithelial cells (gEECs) is still unknown. We designed a study to investigate the function of NRF2 in LPS-induced gEECs. LPS was found to increase the NRF2 expression and the nuclear abundance of NRF2 in gEECs in a dose-dependent manner. NRF2 knockout (KO) not only increased the expression of LPS-induced proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) but also increased the expression of TLR4, p-IκBα/IκBα, and p-p65/p65 proteins. Immunoprecipitation experiments showed that NRF2 directly binds to p65 in the nucleus and inhibits the binding of p65 to downstream target genes (TNF-α, IL-1β, IL-6, and IL-8). Even though a NF-κB/p65 inhibitor (PDTC) reduced the LPS-induced NRF2 expression and nuclear abundance of NRF2, overexpressing TNF-α reversed the inhibitory effects of PDTC on the NRF2 expression and on its abundance in the nucleus. Similarly, knockdown of the proinflammatory cytokines (TNF-α, IL-1β, IL-6, or IL-8) significantly decreased the LPS-induced NRF2 expression and NRF2 in the nucleus. In conclusion, our data suggest that proinflammatory cytokines induced by LPS through the TLR4/NF-κB pathway promote the NRF2 expression and its translocation into the nucleus. Our work also suggests that NRF2 inhibits the expression of proinflammatory cytokines by directly binding to p65.

Natural Product Erianin Inhibits Bladder Cancer Cell Growth by Inducing Ferroptosis via NRF2 Inactivation.

Erianin, a natural product derived from Dendrobium chrysotoxum Lindl, has been proved to play antitumor activity in various cancers. However, the effects and molecular mechanisms of erianin in bladder cancer cells remain unexplored. In this study, we found that erianin triggered cell death and cell cycle arrest in bladder cancer cells. Then we demonstrated that erianin could promote the accumulation of lethal lipid-based reactive oxygen species (ROS) and the depletion of glutathione (GSH), suggesting the induction of ferroptosis. In the further study, the ferroptosis inhibitor deferoxamine (DFO), N-Acetylcysteine (NAC) and GSH but not necrostatin-1, CQ or Z-VAD-FMK rescued erianin-caused cell death, showing ferroptosis played a major role in erianin-caused cell death. In vivo, we also showed that erianin suppressed the tumor growth by inducing ferroptosis. Mechanistically, we demonstrated that nuclear factor E2-related factor 2 (NRF2) inactivation was a key determinant of ferroptosis caused by erianin. In bladder cancer cells, the compound tert-butylhydro-quinone (TBHQ), an activator of NRF2, suppressed erianin-induced ferroptosis. Whereas, NRF2 inhibition used shRNA augmented the ferroptosis response induced by erianin treatment. In conclusion, our data provide the first evidence that erianin can initiate ferroptosis-like cell death and lipid peroxidation in bladder cancer, which will hopefully become a promising anticancer compound for the treatment of bladder cancer.

Ferulic acid mediates prebiotic responses of cereal-derived arabinoxylans on host health.

Dietary fiber is named as “the 7th nutrient” for humans, which is beneficial to improve intestinal health and prevent metabolic disease of the host. Mechanisms of dietary fiber administration on improved host health are mediated by short chain fatty acids (SCFA), which are reported to activate G protein-coupled receptors (GPR) and suppress activity of histone deacetylase (HDAC) to down-regulate expression of nuclear factor-к-gene binding (NF-кB) signaling. Arabinoxylan is fermented by gut microbiota to produce SCFA and improved microbial community composition, intestinal barrier functions and host health. Interestingly, the latest publications have observed that ferulic acid combined with the arabinose in arabinoxylans from various cereal grains can be released through gut microbial fermentation. Ferulic acid can improve antioxidase activity and decrease reactive oxygen species (ROS) concentration by activating the signaling pathway of Kelch-like ECH-associated protein-1 and nuclear factor E2-related factor-2 (Keap1-Nrf2). However, the role of ferulic acid in cooperation with SCFA produced from microbial fermentation of cereal-derived arabinoxylan to regulate the intestinal health and host metabolisms, has been widely unclear. This review summarizes the potential mechanisms of ferulic acid from microbial fermentation of cereal-derived arabinoxylans on immunological functions and physiological metabolisms of the host. The evidence presented in the review indicates that dietary supplementation with cereal-derived arabinoxylans improves antioxidant capacity of intestinal epithelial cells due to the production of ferulic acid and SCFA from microbial fermentation. Ferulic acid can cooperate with SCFA to regulate intestinal integrity and immunological functions of the host. Peroxisome proliferator activated-receptor γ (PPARγ) may play an important role in integrating ferulic acid and SCFA to regulate host health and metabolism.

Investigation of the effect of watercress via nuclear factor-E2-related factor-2 pathway on 7,12-dimethylbenz[a]anthracene-induced oxidative damage in rat liver

Aim: Nuclear factor E2-related factor-2 (Nrf2) regulates many cytoprotective antioxidants, and detoxifying enzymes, has been a hopeful approach for chemoprevention of cancer. We researched the chemopreventive impact of watercress on the DMBA-induced oxidative damage in the rat liver via the Nrf2/ARE signalling pathway in the present study. 
 Material and Method: Female Wistar albino rats have categorized into three groups. The control was the first group, rats in the second and third groups were administered 7,12-dimethylbenz[a]anthracene (DMBA) (20 mg/kg b.w., i.p.) and rats in the third group were given daily doses of watercress (250 mg/kg b.w. Nasturtium officinale R.Br. for 4 weeks by oral gavage). Antioxidant and phase II detoxification enzyme activities, Nrf2 transcription factor level in liver, and serum ALT were determined. Also, histopathological analysis of the liver was performed.
 Results: We observed that watercress induces DNA-binding of Nrf2. It was related to increased enzyme activities of phase II detoxifying and the antioxidant. Our results also demonstrated that watercress ameliorated liver injury.
 Conclusion: Our data ensured considerable evidence that the dietary watercress ameliorates DMBA induced liver toxicity via regulating the activation of the Nrf2/ARE pathway which increases the expression of cytoprotective enzymes.

Erratum.

Sorafenib, a multityrosine kinase inhibitor, is a standard treatment for advanced hepatocellular carcinoma (HCC), but the clinical response to sorafenib is seriously limited by drug resistance. Programmed death ligand-1 (PD-L1) is one of the most important inhibitory molecules involved in tumor immune evasion. Recently, it has been reported that PD-L1 could play crucial roles in drug resistance of many kinds of cancers. However, the expression, function, and regulation of PD-L1 in sorafenib-resistant hepatoma cells remain unclear. In this study, we reported that PD-L1 was overexpressed in sorafenib-resistant hepatoma cells, and shRNA-mediated PD-L1 depletion attenuated drug resistance and suppressed the migration, invasion, colony formation, and tumorigenesis in sorafenib-resistant hepatoma cells in vitro and in vivo. Mechanistic investigations indicated that loss of microRNA-1 (miR-1), a tumor-suppressive microRNA, contributed to the PD-L1 upregulation in sorafenib-resistant hepatoma cells, and PD-L1 was a direct regulatory target of miR-1. Further study revealed that an oncogenic transcriptional factor, nuclear factor E2-related factor 2 (NRF-2), was induced in sorafenib-resistant hepatoma cells and inhibited expression of miR-1 in vitro. From molecular mechanism insight back to the functional verification, we eventually demonstrated that miR-1 executed its tumor-suppressive effects on drug resistance and other malignant properties in sorafenib-resistant hepatoma cells partially by PD-L1 inhibition in vitro and in vivo. In conclusion, our data suggested that a NRF-2/miR-1/PD-L1 regulatory axis contributed to the development and maintenance of drug resistance and other tumorigenic properties in sorafenib-resistant hepatoma cells and provided a potential therapeutic target for overcoming sorafenib resistance in HCC.

Ginsenoside Rg1 prevents bone marrow mesenchymal stem cell senescence via NRF2 and PI3K/Akt signaling

Senescence limits the characteristics and functionality of mesenchymal stem cells (MSCs), thereby severely restricting their application in tissue engineering. Here, we investigated ways to prevent MSCs from entering a state of senescence. We found that Rg1, an extract of natural ginseng, can reduce the expression of senescence markers in cultured cells in vitro and in various tissues in vivo. Simultaneously, ginsenoside Rg1 improved the antioxidant capacity of cells, and the senescence-inhibiting and antioxidant effect of Rg1 were associated with the activation of the nuclear factor E2-related factor 2 (NRF2) signaling pathway. Furthermore, Rg1 may activate the NRF2 pathway by increasing the interaction between P62 and KEAP1through P62 upregulation and AKT activation. Taken together, our findings indicate that Rg1 prevents cell senescence via NRF2 and AKT, and activation of AKT or NRF2 may thus represent therapeutic targets for preventing cell senescence.

Manganese Mitigates Heat Stress–Induced Apoptosis by Alleviating Endoplasmic Reticulum Stress and Activating the NRF2/SOD2 Pathway in Primary Chick Embryonic Myocardial Cells

Heat stress leads to oxidative stress and induces apoptosis in various cells. Endoplasmic reticulum (ER) stress is an important apoptosis pathway. Manganese (Mn) has been shown to enhance the activity of manganese superoxide dismutase (MnSOD). To explore the potential effect of Mn on ER stress and apoptosis induced by heat stress, we examined crucial factors associated with heat stress, ER stress, and apoptosis in cultured primary chick embryonic myocardial cells that had been pretreated with 20μM Mn for 24h and then subjected to 4h of heat stress. The results showed that Mn decreased (P < 0.05) heat stress-induced reactive oxygen species (ROS) production and exerted antiapoptotic effects by increasing MnSOD enzymatic activity. The heat stress-induced accumulation of intracellular calcium was dramatically reduced (P < 0.05). Mn treatment significantly decreased (P < 0.05) the expression levels of the apoptosis-related gene Bax and ER stress markers glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in primary chick embryonic myocardial cells. Additionally, Mn reduced oxidative stress by activating the nuclear factor E2-related factor 2 (NRF2)/SOD2 signaling pathway. Taken together, our findings indicate that Mn attenuates heat stress-induced apoptosis by inhibiting ROS generation, intracellular calcium accumulation, and the ER stress pathway and activating the NRF2/SOD2 signaling pathway to protect myocardial cells from oxidative stress during chick embryonic development.

Farnesoid X Receptor as Target for Therapies to Treat Cholestasis-Induced Liver Injury.

Recent studies on liver disease burden worldwide estimated that cirrhosis is the 11th most common cause of death globally, and there is a great need for new therapies to limit the progression of liver injuries in the early stages. Cholestasis is caused by accumulation of hydrophobic bile acids (BA) in the liver due to dysfunctional BA efflux or bile flow into the gall bladder. Therefore, strategies to increase detoxification of hydrophobic BA and downregulate genes involved in BA production are largely investigated. Farnesoid X receptor (FXR) has a central role in BA homeostasis and recent publications revealed that changes in autophagy due to BA-induced reactive oxygen species and increased anti-oxidant response via nuclear factor E2-related factor 2 (NRF2), result in dysregulation of FXR signaling. Several mechanistic studies have identified new dysfunctions of the cholestatic liver at cellular and molecular level, opening new venues for developing more performant therapies.

Astaxanthin Protects Human Granulosa Cells against Oxidative Stress through Activation of NRF2/ARE Pathway and Its Downstream Phase II Enzymes.

Objective Astaxanthin (AST) has been introduced as a radical scavenger and an anti-apoptotic factor that acts via regulating the nuclear factor-E2-related factor 2 (NRF2) and related factors. Here, we intended to examine the effect of AST on granulosa cells (GCs) against oxidative stress by examining NRF2 and downstream phase II antioxidant enzymes. Materials and Methods In this experimental study, we used cultured human primary GCs for the study. First, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test to evaluate cells viability after treatment with hydrogen peroxide (H2O2) and AST. The apoptosis rate and ROS levels were measured by flow cytometry. To determine NRF2 and phase II enzymes expression, we performed real-time polymerase chain reaction (PCR). Finally, we used western blot to measure the protein levels of NRF2 and Kelch-like ECsH-associated protein 1 (KEAP1). Enzyme activity analysis was also performed to detect NRF2 activity. Results This study showed that AST suppressed ROS generation (P<0.01) and cell death (P<0.05) in GCs induced by oxidative stress. AST also elevated gene and protein expression and nuclear localization of NRF2 and had an inhibitory effect on the protein levels of KEAP1 (P<0.05). Furthermore, when we used trigonelline (Trig) as a known inhibitor of NRF2, it attenuated the protective effects of AST by decreasing NRF2 activity and gene expression of phase II enzymes (P<0.05). ConclusionOur results presented the protective role of AST against oxidative stress in GCs which was mediated through up-regulating the phase II enzymes as a result of NRF2 activation. Our study may help in improving in vitro fertilization (IVF) outcomes and treatment of infertility.

Oxidative stress in corneal stromal cells contributes to the development of keratoconus in a rabbit model.

To investigate the role of oxidative stress in keratocytes in the pathogenesis of keratoconus (KC) using the rabbit cornea as a model. Immerse the rabbit cornea in collagenase type II solution at room temperature for 30 min in the KC group. The central cornea thickness (CCT), and mean keratometry (Km) were examined before and after the procedure. Reactive oxygen species (ROS), the nuclear translocation of nuclear factor E2-related factor 2 (NRF-2), the expression of heme oxygenase-1 (HO-1) protein, and nicotinamide adenine dinucleotide phosphate (NADPH) Oxidase (NOX) family members NOX-2 and NOX-4 protein levels were examined by immunohistochemistry analysis and Western Blot. The expression levels of HO-1, NOX-2, NOX-4, and NRF-2 mRNA were quantitatively detected by Real-time PCR. A significant increase in Km and a significant decrease in CCT were observed in the KC group compared with the control group after the surgery (both p < 0.001). Immunofluorescence staining showed the rabbit KC model induced a significant increase in ROS production (p < 0.001). The expression of HO-1, NOX-2, NOX-4, and NRF-2 proteins in the KC group were significantly higher than those in the control group (all p < 0.001). RT-PCR results showed the levels of HO-1, NOX-2, NOX-4, and NRF-2 mRNA in KC groups were all significantly increased compared with control groups (all p < 0.05). Oxidative stress and compensatory activation of antioxidant proteins suggest oxidative stress injury in corneal stromal cells plays an important role in the development of KC in a rabbit model.