Downregulation Of Keap1 Research Articles

Salvianolic Acid B Improves Postresuscitation Myocardial and Cerebral Outcomes in a Murine Model of Cardiac Arrest: Involvement of Nrf2 Signaling Pathway.

Survival and outcome of cardiac arrest (CA) are dismal despite improvements in cardiopulmonary resuscitation (CPR). Salvianolic acid B (Sal B), extracted from Salvia miltiorrhiza, has been investigated for its cardioprotective properties in cardiac remodeling and ischemic heart disease, but less is known about its role in CA. The aim of this study was to learn whether Sal B improves cardiac and neurologic outcomes after CA/CPR in mice. Female C57BL/6 mice were subjected to eight minutes of CA induced by an intravenous injection of potassium chloride (KCl), followed by CPR. After 30 seconds of CPR, mice were blindly randomized to receive either Sal B (20 mg/kg) or vehicle (normal saline) intravenously. Hemodynamic variables and indices of left ventricular function were determined before CA and within three hours after CPR, the early postresuscitation period. Sal B administration resulted in a remarkable decrease in the time required for the return of spontaneous circulation (ROSC) in animals that successfully resuscitated compared to the vehicle-treated mice. Myocardial performance, including cardiac output and left ventricular systolic (dp/dtmax) and diastolic (dp/dtmin) function, was clearly ameliorated within three hours of ROSC in the Sal B-treated mice. Moreover, Sal B inhibited CA/CPR-induced cardiomyocyte apoptosis and preserved mitochondrial morphology and function. Mechanistically, Sal B dramatically promoted Nrf2 nuclear translocation through the downregulation of Keap1, which resulted in the expression of antioxidant enzymes, including HO-1 and NQO1, thereby counteracted the oxidative damage in response to CA/CPR. The aforementioned antiapoptotic and antioxidant effects of Sal B were impaired in the setting of gene silencing of Nrf2 with siRNA in vitro model. These improvements were associated with better neurological function and increased survival rate (75% vs. 40%, p < 0.05) up to 72 hours postresuscitation. Our findings suggest that the administration of Sal B improved cardiac function and neurological outcomes in a murine model of CA via activating the Nrf2 antioxidant signaling pathway, which may represent a novel therapeutic strategy for the treatment of CA.

Treatment of dilated cardiomyopathy in a mouse model of Friedreich’s ataxia using N-acetylcysteine and identification of alterations in microRNA expression that could be involved in its pathogenesis

Deficient expression of the mitochondrial protein, frataxin, leads to a deadly cardiomyopathy. Our laboratory reported the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation after frataxin deletion in the heart. This was due, in part, to a pronounced increase in Keap1. To assess if this can be therapeutically targeted, cells were incubated with N-acetylcysteine (NAC), or buthionine sulfoximine (BSO), which increases or decreases glutathione (GSH), respectively, or the NRF2-inducer, sulforaphane (SFN). While SFN significantly (p < 0.05) induced NRF2, KEAP1 and BACH1, NAC attenuated SFN-induced NRF2, KEAP1 and BACH1. The down-regulation of KEAP1 by NAC was of interest, as Keap1 is markedly increased in the MCK conditional frataxin knockout (MCK KO) mouse model and this could lead to the decreased Nrf2 levels. Considering this, MCK KO mice were treated with i.p. NAC (500- or 1500-mg/kg, 5 days/week for 5-weeks) and demonstrated slightly less (p > 0.05) body weight loss versus the vehicle-treated KO. However, NAC did not rescue the cardiomyopathy. To additionally examine the dys-regulation of Nrf2 upon frataxin deletion, studies assessed the role of microRNA (miRNA) in this process. In MCK KO mice, miR-144 was up-regulated, which down-regulates Nrf2. Furthermore, miRNA screening in MCK KO mice demonstrated 23 miRNAs from 756 screened were significantly (p < 0.05) altered in KOs versus WT littermates. Of these, miR-21*, miR-34c*, and miR-200c, demonstrated marked alterations, with functional clustering analysis showing they regulate genes linked to cardiac hypertrophy, cardiomyopathy, and oxidative stress, respectively.

Sestrin2 overexpression alleviates hydrogen peroxide-induced apoptosis and oxidative stress in retinal ganglion cells by enhancing Nrf2 activation via Keap1 downregulation

Oxidative stress-induced apoptosis of retinal ganglion cells (RGCs) contributes to the development and progression of glaucoma. Sestrin2 (Sesn2), a stress-inducible protein, has a potent antioxidant capacity that can provide cytoprotection against various noxious stimuli. However, whether Sesn2 is involved in protecting RGCs from oxidative stress remains unexplored. The purpose of this study was to evaluate the role of Sesn2 in regulating hydrogen peroxide (H2O2)-induced oxidative stress of RGCs. Here, we showed that Sesn2 expression was induced in RGCs following H2O2 exposure. Sesn2 depletion markedly exacerbated H2O2-induced apoptosis and reactive oxygen species (ROS) generation in RGCs. Notably, upregulation of Sesn2 significantly decreased H2O2-induced apoptosis and ROS generation. Moreover, Sesn2 overexpression increased the nuclear translocation of nuclear factor erythroid-derived 2-like 2 (Nrf2), elevated Nrf2/antioxidant response element (ARE)-mediated transcriptional activity and upregulated the expression of Nrf2 target genes in H2O2-stimulated RGCs. Interestingly, we found that Sesn2 promoted Nrf2/ARE activation through downregulation of kelch-like ECH-associated protein 1 (Keap1). Restoration of Keap1 or inhibition of Nrf2 significantly reversed the Sesn2-mediated protective effect in H2O2-stimulated RGCs. In conclusion, these results elucidated that Sesn2 confers a protective effect in RGCs against H2O2-induced oxidative stress by reinforcing Nrf2/ARE activation via downregulation of Keap1. Our study suggests that the Sesn2/Keap1/Nrf2 axis may play an important role in retinal degeneration in glaucoma.

TRIM16 protects from OGD/R-induced oxidative stress in cultured hippocampal neurons by enhancing Nrf2/ARE antioxidant signaling via downregulation of Keap1

Tripartite motif 16 (TRIM16) has emerged as a novel oxidative stress-responsive protein that confers cytoprotective effects by reinforcing the cellular antioxidant system. However, whether TRIM16 is involved in regulating oxidative stress during cerebral ischemia/reperfusion injury remains unclear. In the present study, we aimed to explore the potential function and molecular mechanism of TRIM16 in regulating oxidative stress in neurons induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Here, we found that OGD/R exposure resulted in a significant induction of TRIM16 expression in neurons. Depletion of TRIM16 by siRNA-mediated gene knockdown markedly upregulated the sensitivity of neurons to OGD/R-induced apoptosis and reactive oxygen species (ROS) generation. Notably, upregulation of TRIM16 expression significantly alleviated OGD/R-induced apoptosis and ROS generation in neurons. Moreover, TRIM16 overexpression markedly increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and enhanced Nrf2/antioxidant response element (ARE) activation associated with downregulation of kelch-like ECH-associated protein 1 (Keap1) expression. Restoration of Keap1 significantly reversed the TRIM16-mediated promotion effect on Nrf2/ARE activation. In addition, knockdown of Nrf2 also markedly abrogated the TRIM16-conferred neuroprotective effect in OGD/R-exposed neurons. Taken together, our results of our study demonstrate that induction of TRIM16 confers a cytoprotective effect in OGD/R-exposed neurons through enhancement of Nrf2/ARE antioxidant signaling via downregulation of Keap1. These findings suggest that TRIM16 may play a critical role in cerebral ischemia/reperfusion injury and serve as a promising target for neuroprotection.

Regulation of Nrf2 by Mitochondrial Reactive Oxygen Species in Physiology and Pathology.

Reactive oxygen species (ROS) are byproducts of aerobic respiration and signaling molecules that control various cellular functions. Nrf2 governs the gene expression of endogenous antioxidant synthesis and ROS-eliminating enzymes in response to various electrophilic compounds that inactivate the negative regulator Keap1. Accumulating evidence has shown that mitochondrial ROS (mtROS) activate Nrf2, often mediated by certain protein kinases, and induce the expression of antioxidant genes and genes involved in mitochondrial quality/quantity control. Mild physiological stress, such as caloric restriction and exercise, elicits beneficial effects through a process known as “mitohormesis”. Exercise induces NOX4 expression in the heart, which activates Nrf2 and increases endurance capacity. Mice transiently depleted of SOD2 or overexpressing skeletal muscle-specific UCP1 exhibit Nrf2-mediated antioxidant gene expression and PGC1α-mediated mitochondrial biogenesis. ATF4 activation may induce a transcriptional program that enhances NADPH synthesis in the mitochondria and might cooperate with the Nrf2 antioxidant system. In response to severe oxidative stress, Nrf2 induces Klf9 expression, which represses mtROS-eliminating enzymes to enhance cell death. Nrf2 is inactivated in certain pathological conditions, such as diabetes, but Keap1 down-regulation or mtROS elimination rescues Nrf2 expression and improves the pathology. These reports aid us in understanding the roles of Nrf2 in pathophysiological alterations involving mtROS.

Compound 13 activates AMPK-Nrf2 signaling to protect neuronal cells from oxygen glucose deprivation-reoxygenation.

Oxygen glucose deprivation-reoxygenation (OGD-R) causes the production of reactive oxygen species (ROS) and oxidative injury in neuronal cells. We tested the potential neuroprotective function of compound 13 (C13), a novel AMP-activated protein kinase (AMPK) activator, against OGD-R. We show that C13 pretreatment protected SH-SY5Y neuronal cells and primary hippocampal neurons from OGD-R. C13 activated AMPK signaling in SH-SY5Y cells and primary neurons. It significantly inhibited OGD-R-induced apoptosis activation in neuronal cells. Conversely, AMPKα1 shRNA or knockout reversed C13-mediated neuroprotection against OGD-R. C13 potently inhibited OGD-R-induced ROS production and oxidative stress in SH-SY5Y cells and primary neurons. Furthermore, C13 induced Keap1 downregulation and Nrf2 activation, causing Nrf2 stabilization, nuclear accumulation, and expression of Nrf2-dependent genes. Nrf2 silencing or knockout in SH-SY5Y cells abolished C13-mediated neuroprotection against OGD-R. In conclusion, C13 activates AMPK-Nrf2 signaling to protect neuronal cells from OGD-R.

Sulforaphane Protects the Male Reproductive System of Mice from Obesity-Induced Damage: Involvement of Oxidative Stress and Autophagy.

(1) Background: In recent decades, the prevalence of obesity has grown rapidly worldwide, thus causing many diseases, including male hypogonadism. Sulforaphane (SFN), an isothiocyanate compound, has been reported to protect the reproductive system. This research investigated the protective effect of SFN against obesity-induced impairment in the male reproductive system and explored the potential mechanism involved in mice. (2) Methods: One hundred thirty mice were divided into 5 groups (Control, DIO (diet-induced obesity), DIO + SFN 5 mg/kg, DIO + SFN 10 mg/kg, and DIO + SFN 20 mg/kg). The effects of SFN on the male reproductive system were determined based on the sperm count and motility, relative testes and epididymis weights, hormone levels, and pathological analyses. Oxidative stress was determined by measuring malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione (GSH), H2O2, catalase (CAT), and glutathione peroxidase (GSH-PX) levels. Protein expression of nuclear factor erythroid-2 related factor 2 (Nrf2), Kelch-like ECH-associated protein-1 (Keap1), Microtubule-associated protein light chain 3 (LC3), Beclin1, and P62 were determined by western blotting. (3) Results: High-fat diet (HFD)-induced obesity significantly decreased relative testes and epididymis weights, sperm count and motility, and testosterone levels but increased leptin and estradiol levels. SFN supplementation ameliorated these effects. Additionally, SFN administration inhibited the obesity-induced MDA accumulation and increased the SOD level. Western blot indicated that SFN had an important role in the downregulation of Keap1. Moreover, SFN treatment attenuated obesity-induced autophagy, as detected by LC3 and Beclin1. (4) Conclusions: SFN ameliorated the reproductive toxicity associated with obesity by inhibiting oxidative stress mediated by the nuclear factor erythroid-2 related factor 2/ antioxidant response element (Nrf2/ARE) signaling pathway and recovery of normal autophagy.

Quercetin suppressed NADPH oxidase-derived oxidative stress via heme oxygenase-1 induction in macrophages

NADPH oxidase-derived superoxide (O2.-) generation and oxidative stress is usually considered as an important factor to the pathogenesis of inflammatory diseases. Quercetin, widely known for their anti-oxidant and anti-inflammatory properties in vitro and in vivo, is recently identified to induce expression of antioxidant enzyme heme oxygenase-1 (HO-1). Previous studies suggest that HO-1 induction and/or subsequent HO-1 end product generation in vitro and in vivo may suppress NADPH oxidase-derived oxidative stress. In this study, we tested whether quercetin might modulate NADPH oxidase activity in macrophages via induction of HO-1. In RAW264.7 macrophages, quercetin significantly attenuated NADPH oxidase-derived O2.- generation via a HO-1-dependent mechanism. Mechanistically, the protective effects of quercetin were (1) linked to increased expression of HO-1 in the presence or absence of lipopolysaccharide (LPS), (2) similar to that observed with the NADPH oxidase inhibitor apocynin, and (3) could be abolished by the specific small-interfering RNA against HO-1 expression or HO-1 activity inhibitor tin protoporphyrin. The induction of HO-1 by quercetin was associated with the nuclear accumulation of Nrf2 and downregulation of Keap1, a negative regulator of Nrf2. In addition, this flavonoid also inhibited the overproduction of nitric oxide and inflammatory cytokines in LPS-stimulated macrophages via simultaneous induction of HO-1 expression. In agreement with the observations in macrophages, pretreatment with quercetin significantly alleviated LPS-induced inflammation in mice which was concomitant with decreased NADPH oxidase activity and increased HO-1 expression. Our results suggested that quercein could modulate NADPH oxidase-derived O2.- production in macrophages at least partly through HO-1 induction. Suppression of NADPH oxidase-dependent oxidative stress may represent a novel mechanism underlying the anti-oxidant and anti-inflammatory properties of quercetin/HO-1 pathway.

Overexpression of CKIP-1 alleviates hypoxia-induced cardiomyocyte injury by up-regulating Nrf2 antioxidant signaling via Keap1 inhibition

Acute myocardial infarction causes irreversible myocardial damage and is a leading cause of death and disability worldwide. Casein kinase 2 interacting protein-1 (CKIP-1) has been suggested to confer cytoprotection against various pathologic injuries. However, it remains unclear whether CKIP-1 regulates myocardial infarction-induced cardiomyocyte injury. This study aimed to explore the potential role of CKIP-1 in regulating hypoxia-induced cardiomyocyte injury and reveal the underlying mechanism. The results demonstrated that hypoxia-exposed cardiomyocytes showed lower CKIP-1 expression. CKIP-1 restoration by transfecting a CKIP-1 expression vector significantly improved viability and reduced apoptosis in hypoxia-treated cardiomyocytes. Moreover, CKIP-1 overexpression suppressed hypoxia-induced oxidative stress in cardiomyocytes. Mechanism research revealed that CKIP-1 overexpression reduced the expression of kelch-like ECH-associated protein 1 (Keap1) and increased the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), actions which resulted in an increase in the transcription of Nrf2 target genes. However, Keap1 overexpression partially reversed CKIP-1-mediated Nrf2 promotion and cardioprotection. Notably, the blockade of Nrf2 signaling also significantly abolished CKIP-1-mediated cardioprotection. Overall, our findings demonstrate that CKIP-1 alleviates hypoxia-induced cardiomyocyte injury through the up-regulation of Nrf2 antioxidant signaling via the down-regulation of Keap1, suggesting a potential role for CKIP-1 in myocardial infarction.

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.

Downregulation of Keap1 contributes to poor prognosis and Axitinib resistance of renal cell carcinoma via upregulation of Nrf2 expression.

Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling has a protective effect on normal cells. A number of previous studies demonstrated that Keap1/Nrf2 signaling is associated with drug resistance in numerous tumors. The aim of the present study was to investigate the roles of Keap1 in renal cell carcinoma (RCC) and its effect on sensitivity to chemotherapy. Reverse transcription-quantitative polymerase chain reaction was used to detect the mRNA expression of Keap1 in 45 cases of RCC tumors and adjacent normal tissues. A total of five randomly selected patients with RCC, five RCC cell lines and normal renal tubular cells were examined to detect the protein and mRNA expressions of Keap1. The 5-year survival rate was analyzed by Kaplan-Meier analysis. The cell viability was assessed by a Cell Counting kit-8 assay. The cell apoptosis and reactive oxygen species (ROS) were determined by flow cytometry. The expressions of associated proteins were determined by western blot analysis. It was identified that in RCC tissues and RCC cell lines, the expression of Keap1 was downregulated, which was considered to be associated with poor prognosis. In total, 1 µM Axitinib significantly decreased cell viability, promoted ROS release and induced cell apoptosis in ACHN cells. Silencing Keap1 was able to reverse the inhibitory effect of Axitinib and enhance the protein expressions of Nrf2, NAD(P)H dehydrogenase [quinone] 1 and heme oxygenase 1. However, silencing Nrf2 increased the cell sensitivity to Axitinib. Under Axitinib condition, overexpressing Nrf2 was able to increase cell viability; however, overexpressing Keap1 resulted in an opposite effect. Keap1 serves as a tumor suppressor; its low expression was associated with poor prognosis and a decreased sensitivity of RCC cells to Axitinib. A possible mechanism underlying Axitinib resistance may involve Nrf2 overexpression.

The impact of Keap1/Nrf2, P38MAPK/NF-κB and Bax/Bcl2/caspase-3 signaling pathways in the protective effects of berberine against methotrexate-induced nephrotoxicity

Berberine (BBR) is a natural compound of plant origin belonging to isoquinoline type of alkaloid. Methotrexate (MTX) is an anti-metabolite used widely for a variety of tumors and autoimmune conditions. Clinical uses of MTX were severely limited by its concomitant renal intoxication. The current study was designed to investigate the efficacy of BBR against MTX-induced nephrotoxicity and for exploring the underlying molecular mechanisms through examining the Keap1/Nrf2, NF-κB/P38MAPK and Bax/Bcl2/caspase-3 pathways. Adults male rats were assigned to 4 groups: control, BBR, MTX and MTX + BBR. As compared to MTX-treated group, BBR effectively reduced the serum levels of creatinine, urea, uric acid and kidney/body weight ratio with a remarkable increase in serum level of albumin and the final body weight. Moreover, down-regulation of Keap1, P38MAPK and NF-κB genes along with marked up-regulation of Nrf2 gene were observed. In addition, BBR negatively regulated both Bax and caspase-3 proteins expression along with increased expression of the Bcl2 protein. Also, BBR restored GSH content and SOD activity while it decreased both TBARS and NO2− contents. Biochemical findings confirmed and markedly supported by alleviation of histopathological changes in kidney tissues. Furthermore, MTX cytotoxic activity was markedly enhanced by BBR in vitro using some human cancer cell lines. In conclusion, the current findings indicated that co-administration of BBR with MTX may be a reasonable therapeutic strategy for attenuation of MTX -induced renal damage.

Inhibition of SETD7 protects cardiomyocytes against hypoxia/reoxygenation-induced injury through regulating Keap1/Nrf2 signaling

The protein SET domain-containing lysine methyltransferase 7 (SETD7) has recently been shown to regulate apoptosis in various cells. However, the role of SETD7 on cardiomyocyte apoptosis during myocardial ischemia/reperfusion injury remains unclear. This study aimed to investigate the potential role of SETD7 in hypoxia/reoxygenation (H/R)-induced apoptosis of rat cardiomyocytes and reveal the underlying mechanism. Our results demonstrated that SETD7 expression was significantly up-regulated in cardiomyocytes in response to H/R injury. The inhibition of SETD7 by siRNA-mediated gene silencing significantly suppressed H/R-induced apoptosis and decreased the production of reactive oxygen species (ROS). The overexpression of SETD7 markedly enhanced H/R-induced apoptosis and ROS production. Moreover, the knockdown of SETD7 reduced the expression of Keap1 and promoted the expression of Nrf2. In addition, the knockdown of SETD7 increased the activity of antioxidant response element and promoted the expression of heme oxygenase-1 and NADPH-quinone oxidoreductase 1. However, the knockdown of Nrf2 partially abrogated the SETD7 inhibition-mediated protective effect against H/R injury. Taken together, these results indicate that the inhibition of SETD7 attenuates H/R-induced injury of cardiomyocytes via the down-regulation of Keap1 and promotion of the Nrf2-mediated anti-oxidation signaling pathway.

Caffeic acid inhibits HCV replication via induction of IFNα antiviral response through p62-mediated Keap1/Nrf2 signaling pathway

Hepatitis C virus (HCV) infection and its related liver disease have constituted a heavy burden worldwide. It had been reported that Drinking coffee could decrease mortality risk of HCV infected patients. Caffeic Acid (CA), the Coffee-related organic acid could inhibit HCV replication, however, the detailed mechanism of CA against HCV is unclear. In this study, we showed that CA could notably inhibit HCV replication. Mechanism study demonstrated that CA could induce HO-1 expression, which would trigger the IFNα antiviral response, and the antiviral effect of CA was attenuated when HO-1 activity was inhibited by SnPP (an HO-1 inhibitor). CA could also increase erythroid 2-related factor 2 (Nrf2) expression. When Nrf2 was knocked down by specific siRNA, HO-1 expression was concomitantly decreased while HCV expression was restored. Further study indicated that kelch-like ECH-associated protein 1 (keap1) expression was decreased by CA through p62/Sequestosome1 (p62)-mediated autophagy, which would lead to the stabilization and accumulation of Nrf2. The decrease of keap1 was restored when p62 was silenced by specific p62 siRNA and when autophagy was inhibited, suggesting p62-mediated autophagy was required for CA-mediated keap1 downregulation. Taken together, the results demonstrated that CA could modulate Keap1/Nrf2 interaction via increasing p62 expression, leading to stabilization of Nrf2 and HO-1 induction, and elicit IFNα antiviral response to suppress HCV replication.

Renoprotective effects of umbelliferone on methotrexate-induced renal injury through regulation of Nrf-2/Keap-1, P38MAPK/NF-κB, and apoptosis signaling pathways

Nephrotoxicity is the major dose-limiting adverse effect of methotrexate (MTX). Umbelliferone (UMB) is a known coumarin derivative. The current study aimed to investigate possible protective effects of UMB against MTX-induced nephrotoxicity. Adult male albino rats were divided into: control group, UMB group (30 mg/kg, p.o), MTX group (single i.p. injection of 20 mg/kg) and MTX + UMB group. Serum urea and creatinine were evaluated. The renoprotective effects of UMB were evaluated by estimation of renal Nrf-2/Keap-1 and P38MAPK/NF-κB, GSH, MDA, NO2− contents and SOD activity. Moreover, expression of Bcl-2, Bax and caspase-3 were determined. The results demonstrated that UMB significantly reduced serum creatinine and urea levels with alleviations of histopathological abrasions induced by MTX. It limited oxidative stress via lowering both renal MDA and NO2− contents and restoring renal content of reduced GSH and SOD activity with downregulation of Keap-1 and upregulation of Nrf-2. UMB downregulated P38MAPK and NF-κB expression levels. In addition, UMB increased Bcl-2 protein expression while decreasing both Bax and caspase-3 expression levels. Importantly, UMB enhanced the cytotoxic activity of MTX human cancer cell lines. In conclusion, UMB possess marked renoprotective effects against MTX-induced renal damage through modulating oxidative stress, inflammation and apoptosis with enhancement of its cytotoxic activity.

Activation of Nrf2 by Ginsenoside Rh3 protects retinal pigment epithelium cells and retinal ganglion cells from UV.

Excessive Ultra-violet (UV) radiation shall induce damages to resident retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs). Here we tested the potential activity of Ginsenoside Rh3 ("Rh3") against the process. In cultured human RPEs and RGCs, pretreatment with Rh3 inhibited UV-induced reactive oxygen species (ROS) production and following apoptotic/non-apoptotic cell death. Rh3 treatment in retinal cells induced nuclear-factor-E2-related factor 2 (Nrf2) activation, which was evidenced by Nrf2 protein stabilization and its nuclear translocation, along with transcription of antioxidant responsive element (ARE)-dependent genes (HO1, NOQ1 and GCLC). Nrf2 knockdown by targeted-shRNA almost abolished Rh3-induced retinal cell protection against UV. Further studies found that Rh3 induced microRNA-141 ("miR-141") expression, causing downregulation of its targeted gene Keap1 in RPEs and RGCs. On the other hand, Rh3-induced Nrf2 activation and retinal cell protection were largely attenuated by the miR-141's inhibitor, antagomiR-141. In vivo, intravitreal injection of Rh3 inhibited retinal dysfunction by light damage in mice. Rh3 intravitreal injection also induced miR-141 expression, Keap1 downregulation and Nrf2 activation in mouse retinas. We conclude that Rh3 protects retinal cells from UV via activating Nrf2 signaling.

The long non-coding RNA MALAT1 activates Nrf2 signaling to protect human umbilical vein endothelial cells from hydrogen peroxide

The potential effect of the long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) against hydrogen peroxide (H2O2)-induced oxidative injury in endothelial cells was tested. We show that forced-expression of MALAT1 using a lentiviral vector (“LV-MALAT1”) significantly attenuated H2O2-induced death and apoptosis of human umbilical vein endothelial cells (HUVECs). Conversely, knocking down of MALAT1 by targeted siRNA exacerbated H2O2-induced HUVEC injury. For the mechanism study, we show that LV-MALAT1 induced Keap1 downregulation, leading to nuclear-factor-E2-related factor 2 (Nrf2) stabilization and activation. Critically, Nrf2 shRNA almost completely abolished LV-MALAT1-mediated HUVEC protection against H2O2. Significantly, H2O2-induced oxidative stress, lipid peroxidation and DNA damages in HUVECs were attenuated by LV-MALAT1, but were intensified with MALAT1 siRNA. In summary, we identified a novel signaling axis involving MALAT1, Keap1 and Nrf2, which in turn protects HUVECs from oxidative injury.

Keap1 knockdown in melanocytes induces cell proliferation and survival via HO-1-associated β-catenin signaling

BackgroundNrf2-Keap1 signaling pathway protects cells against photo-oxidative stress. Yet in recent works, its role in melanogenesis together with cell protection functions against oxidative stress has been gaining interest. However, its effect on melanogenesis still has contradictory results from different studies. ObjectiveThe aims of our study were to investigate the effect of Keap1 silencing in melanocyte on melanogenesis and its associated mechanism. MethodsPrimary human epidermal melanocytes and melan-a cell line were used for this experiment. RNA sequencing was done to identify genes involved in melanocyte biology using Keap1 knockdown through siRNA techniques. And melanogenesis and the expression of melanogenesis-associated molecules were evaluated in Keap1 silenced melanocyte to examine the effects of Keap1 on melanogenesis, melanocyte growth, and related pathways. ResultsRNA-sequencing data revealed that Keap1 knockdown in primary human epidermal melanocytes (PHEMs) induced cell survival-related gene expression. Additionally, siRNA-mediated inhibition of Keap1 led to upregulation of MITF and melanogenesis-associated molecules along with Nrf2 activation in PHEMs. HO-1, a major gene that is upregulated in RNA-sequencing using Keap1-silenced PHEMs, protected melanocytes against H2O2-induced cell death and upregulated MITF and β-catenin expression. Further, increased expression of melanogenesis-associated molecules after Keap1 silencing was validated to occur through HO-1-associated β-catenin activation in a Keap1 and HO-1 double knockdown experiment. ConclusionThis work suggests that Keap1 silencing in melanocytes induced melanogenesis and the expression of melanogenesis-associated molecules through HO-1-associated β-catenin activation. Keap1 downregulation in melanocytes is important for cell proliferation and survival.

Nuclear lamins and progerin are dispensable for antioxidant Nrf2 response to arsenic and cadmium

Lamins are important constituents of the nuclear inner membrane and provide a platform for transcription factors and chromatin. Progerin, a C-terminal truncated lamin A mutant, causes premature aging termed Hutchinson-Gilford Progeria Syndrome (HGPS). Oxidative stress appears to be involved in the pathogenesis of HGPS, although the mechanistic role of progerin remains elusive. Here we examined whether nuclear lamins are important for a cellular antioxidant mechanism, and whether progerin compromises it. We investigated the activation of nuclear factor-E2-related factor 2 (Nrf2) which regulates various antioxidant genes including heme oxygenase-1 (HMOX1), following exposure to sodium arsenite or cadmium chloride in lamin knockdown human cell lines and primary HGPS human fibroblasts. Knocking down lamin A/C, or B, or all nuclear lamins simultaneously in three human cell lines (HaCaT, SW480, and K562) did not impair arsenite- or cadmium-induced activation of Nrf2. Progerin-expressing human primary HGPS fibroblasts showed lower basal levels of HMOX1 and NQO1 expression; however, in response to arsenic stress both normal and HGPS primary fibroblasts showed Nrf2 nuclear accumulation along with upregulation and phosphorylation of p62/SQSTM1 at Ser351, downregulation of Keap1, and comparable expression of an array of downstream Nrf2-regulated antioxidant genes. We also observed new forms of cleaved lamin A, B1 and B2 induced by cadmium stress although their roles in the Nrf2 antioxidant system need further investigation. These results suggest that the nuclear lamins and progerin have marginal roles in the activation of the antioxidant Nrf2 response to arsenic and cadmium.

RNAi Screen for NRF2 Inducers Identifies Targets That Rescue Primary Lung Epithelial Cells from Cigarette Smoke Induced Radical Stress.

Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the ‘druggable’ genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism.