Impaired Nrf2 Activation Research Articles

Puerarin alleviates acrolein-induced atherosclerosis by activating the MYH9-mediated SIRT1/Nrf2 cascade to inhibit the activation of inflammasome.

Puerarin (Pue) has significant antioxidant and anti-inflammatory properties. This work was designed to clarify and investigate the potential mechanisms of Pue in atherosclerosis (AS) progression. In vivo, acrolein (Acr) was inhaled through drinking water to construct AS model. In vitro, CCK-8 assay and lactate dehydrogenase (LDH) assay kit were used to detect cell viability. Apoptosis was detected by flow cytometry. The content of malondialdehyde (MDA) was determined by commercial kit, the level of inflammatory factors was detected by ELISA, and proteins were determined by western blot. Pue administration could effectively reduce blood lipid level in Acr-fed mice. Pue suppressed oxidative stress, the formation of atherosclerotic plaques, and the process of aortic histological changes. Pue pretreatment decreased MDA in HUVECs and maintained the activity of antioxidant enzymes. Pue upregulated SIRT1/Nrf2 cascade in HUVECs. Pue increased MYH9 and inhibited NLRP3 inflammasome-related proteins, and the inhibition of MYH9 significantly impaired Pue-induced Nrf2 activation. Moreover, HUVEC cytotoxicity and apoptosis are alleviated by Pue, in addition to NLRP3-mediated pyroptosis in HUVECs induced by Acr. MYH9 inhibitors effectively suppressed the pyroptosis induced by Acr and prevented injury to HUVECs. In addition, Pue promoted SIRT1/Nrf2 cascade activation in HUVECs. Pue may alleviate Acr-induced AS by activating the MYH9-mediated SIRT1/Nrf2 cascade to inhibit inflammasome activation.

Quantitative Proteomics Identifies Reduced NRF2 Activity and Mitochondrial Dysfunction in Atopic Dermatitis

Atopic dermatitis is the most common inflammatory skin disease and is characterized by a deficient epidermal barrier and cutaneous inflammation. Genetic studies suggest a key role of keratinocytes in atopic dermatitis pathogenesis, but the alterations in the proteome that occur in the full epidermis have not been defined. Using a pressure-cycling technology and data-independent acquisition approach, we performed quantitative proteomics of epidermis from healthy volunteers and lesional and nonlesional patient skin. Results were validated by targeted proteomics using parallel reaction monitoring mass spectrometry and immunofluorescence staining. Proteins that were differentially abundant in the epidermis of patients with atopic dermatitis versus in healthy control reflect the strong inflammation in lesional skin and the defect in keratinocyte differentiation and epidermal stratification that already characterizes nonlesional skin. Most importantly, they reveal impaired activation of the NRF2-antioxidant pathway and reduced abundance of mitochondrial proteins involved in key metabolic pathways in the affected epidermis. Analysis of primary human keratinocytes with small interfering RNA‒mediated NRF2 knockdown revealed that the impaired NRF2 activation and mitochondrial abnormalities are partially interlinked. These results provide insight into the molecular alterations in the epidermis of patients with atopic dermatitis and identify potential targets for pharmaceutical intervention.

Axonal pathology in hPSC-based models of Parkinson’s disease results from loss of Nrf2 transcriptional activity at the Map1b gene locus

While mutations in the SNCA gene (α-synuclein [α-syn]) are causal in rare familial forms of Parkinson's disease (PD), the prevalence of α-syn aggregates in the cortices of sporadic disease cases emphasizes the need to understand the link between α-syn accumulation and disease pathogenesis. By employing a combination of human pluripotent stem cells (hPSCs) that harbor the SNCA-A53T mutation contrasted against isogenic controls, we evaluated the consequences of α-syn accumulation in human A9-type dopaminergic (DA) neurons (hNs). We show that the early accumulation of α-syn in SNCA-A53T hNs results in changes in gene expression consistent with the expression profile of the substantia nigra (SN) from PD patients, analyzed post mortem. Differentially expressed genes from both PD patient SN and SNCA-A53T hNs were associated with regulatory motifs transcriptionally activated by the antioxidant response pathway, particularly Nrf2 gene targets. Differentially expressed gene targets were also enriched for gene ontologies related to microtubule binding processes. We thus assessed the relationship between Nrf2-mediated gene expression and neuritic pathology in SNCA-A53T hNs. We show that SNCA-mutant hNs have deficits in neuritic length and complexity relative to isogenic controls as well as contorted axons with Tau-positive varicosities. Furthermore, we show that mutant α-syn fails to complex with protein kinase C (PKC), which, in turn, results in impaired activation of Nrf2. These neuritic defects result from impaired Nrf2 activity on antioxidant response elements (AREs) localized to a microtubule-associated protein (Map1b) gene enhancer and are rescued by forced expression of Map1b as well as by both Nrf2 overexpression and pharmaceutical activation in PD neurons.

Xin-Ji-Er-Kang ameliorates kidney injury following myocardial infarction by inhibiting oxidative stress via Nrf2/HO-1 pathway in rats

AimCardiovascular diseases, such as coronary heart disease and myocardial infarction (MI) are currently considered as the leading causes of death and disability. The aim of the present study is to investigate the effects of Xin-Ji-Er-Kang (XJEK) on kidney injury and renal oxidative stress. In addition, the associated mechanism involved in these processes was examined in an MI model, and particularly focused on the nuclear factor erythroid 2‐related factor (NRF2)/heme oxygenase‐1 (HO‐1) pathway. Materials and methodsA total of 138 Sprague-Dawley rats were used in the present study. The control group was designated as 0 wk (n = 8). A total of 3 phases (2, 4, 6 wk) of administration were used in the sham-operated groups (sham, n = 10), MI groups (MI, n = 10), MI + XJEK groups (XJEK, n = 10) and MI + fosinopril groups (fosinopril, n = 10). Additional 10 rats were used to evaluate the infarct area. At 2, 4 or 6 wk post-MI, the hemodynamic parameters were monitored, the rats were sacrificed, then blood, heart and renal tissues were collected for furtherly analysis. ResultsThe results indicated that XJEK administration continuously ameliorated renal hypertrophy index, blood urea nitrogen and cystatin C concentrations. XJEK further improved post-MI cardiac function by limiting scar formation and caused a downregulation in the hemodynamic parameters at the end of 2 and 4 wk. The hemodynamic parameters were upregulated after 6 wk treatment with XJEKcompared with those noted in the MI groups. Similarly, XJEK treatment for 2 wk potentiated Nrf2 nuclear translocation and HO-1 expression and inhibited the deficiency of nuclear Nrf2 and HO-1 at 6 wk post-MI compared with that of the MI groups, indicating the attenuation of the renal oxidative stress condition. The levels of malondialdehyde and angiotensin II (Ang II) in plasma and renal tissues, as well as the levels of aldosterone, 8-hydroxydeoxyguanosine, angiotensin II type 1 receptor and NADPH Oxidase-4 in the kidney tissue significantly decreased following XJEK treatment for 6 wk. In addition, the XJEK treatment groups revealed a significant upregulation in the activity of superoxide dismutase and in the total antioxidant capacity activity compared with those noted in the corresponding MI groups. ConclusionThese results demonstrated that progressive nephropathy in MI rats was associated with intrarenal activation of the renin-angiotensin-aldosterone system. Concomitantly, this process was associated with oxidative stress and impaired Nrf2 activation. The improvement in the severity of nephropathy by XJEK in this model may be associated with the reversal of these abnormalities.

Inflammatory and Senescent Phenotype of Pancreatic Stellate Cells Induced by Sqstm1 Downregulation Facilitates Pancreatic Cancer Progression

Pancreatic ductal adenocarcinoma (PDAC) has unique microenvironment with extensive infiltration of fibroblasts, which are mainly derived from the resident pancreatic stellate cells (PaSCs). As activated PaSCs constitute a major contributor to pancreatic cancer progression, the mechanisms underlying their activation have been being intensively studied. Previous studies showed that Sequestosome-1 (sqstm1) can modulate the functional status of fibroblasts in cancer. Here, we further delineated the role of sqstm1 in PaSCs. The analysis of PDAC patient samples revealed reduction of sqstm1 expression in activated PaSCs in both mRNA and protein level. Downregulated sqstm1 via shRNA in PaSCs led to an inflammatory and senescent phenotype with increased IL8, CXCL1, and CXCL2 expression. Further analysis demonstrated that increased intracellular reactive oxygen species level contributed to the senescence in sqstm1-downregulated PaSCs. This was mediated via impaired NRF2 activity since reduced sqstm1 resulted in accumulation of KEAP1. Meanwhile, we found that sqstm1 degradation caused by enhanced autophagy was not associated with transformation of senescent phenotype. At last, the data revealed that sqstm1-downregulated PaSCs promoted pancreatic tumor cell growth, invasion, and macrophage phenotype transformation. Collectively, the current study indicated that sqstm1 controlled transformation of senescent phenotype of PaSCs, which in turn is pro-tumorigenic.

Design and development of Nrf2 modulators for cancer chemoprevention and therapy: a review.

A major cell defense mechanism against oxidative and xenobiotic stress is mediated by the Nrf2/Keap1 signaling pathway. The Nrf2/Keap1 pathway regulates gene expression of many cytoprotective and detoxifying enzymes, thus playing a pivotal role in maintaining redox cellular homeostasis. Many diseases including cancer have been closely related to impaired Nrf2 activity. Targeting Nrf2 and modulating its activity represents a novel modern strategy for cancer chemoprevention and therapy. In this review, different design strategies used for the development of Nrf2 modulators are described in detail. Moreover, the main focus is on important and recently developed Nrf2 activators and inhibitors, their in vitro and in vivo studies, and their potential use as chemopreventive agents and/or cancer therapeutics.

Increased Nrf2 expression by renal cell carcinoma is associated with postoperative chronic kidney disease and an unfavorable prognosis.

Chronic kidney disease (CKD) is a worldwide health problem, and prevention of CKD is important for preservation of renal function after kidney surgery. There is evidence that transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) has a vital antioxidant and detoxifying role in protecting the kidneys against various diseases. Impaired activation of Nrf2 is associated with oxidative stress related to CKD, and Nrf2 is also a key player in the development of cancer. However, the clinical impact of Nrf2 has not been investigated in patients with renal cell carcinoma (RCC). A retrospective study was performed in 89 patients undergoing nephrectomy for RCC. The estimated glomerular filtration rate (eGFR) and serum uric acid (SUA) were investigated over time after surgery. We investigated Nrf2 protein expression in all tumors and single nucleotide polymorphisms (SNPs) of the Nrf2 gene in 7 tumors. In patients whose tumors showed higher Nrf2 expression, there was a more rapid decrease of eGFR and increase of SUA after nephrectomy. Multivariate analysis confirmed that increased Nrf2 expression was an independent poor prognostic factor related to shorter overall survival. Among the 7 tumor samples, an SNP on exon 5 of the Nrf2 gene in one tumor and three genotypes (C/C, C/A, and A/A) of rs6721961 at the promoter region of the Nrf2 gene were observed. Although the mechanisms underlying the influence of Nrf2 are still unclear, our findings suggested that elevated tumor expression of Nrf2 was associated with postoperative CKD and biologically aggressive RCC with an unfavorable prognosis.

Role of RAS/Wnt/β-catenin axis activation in the pathogenesis of podocyte injury and tubulo-interstitial nephropathy

Renin-angiotensin system (RAS) plays a key role in the development and progression of chronic kidney disease (CKD). Recent studies have demonstrated activation of Wnt/β-catenin pathway by RAS in CKD. However, the underlying mechanisms of RAS and Wnt/β-catenin signaling interaction and their contribution to the pathogenesis of CKD have not been fully elucidated. Present study is designed to investigate the role of RAS/Wnt/β-catenin axis activation in tubulo-interstitial fibrosis and glomerulosclerosis by the cultured HK-2 and podocytes. HK-2 cells and podocytes are treated by angiotensin II (Ang II). Ang II up-regulates expression of various Wnt mRNA and active β-catenin protein in HK-2 cells and podocytes in the time- and dose-dependent manners. In addition, Ang II induces injury, oxidative stress and inflammation and impaired Nrf2 activation in HK-2 cells and podocytes. This was accompanied by up-regulations of RAS components as well as Wnt1, activated β-catenin and its target proteins. RAS/Wnt/β-catenin axis activation results in epithelial-to-mesenchymal transition in HK-2 cells and injuries podocytes. The effect of Ang II is inhibited by losartan and ICG-001, a Wnt/β-catenin inhibitor. We further found that treatment with natural products, ergone, alisol B 23-acetate and pachymic acid B inhibit extracellular matrix accumulation in HK-2 cells and attenuated podocyte injury, in part, by inhibiting Ang II induced RAS/Wnt/β-catenin axis activation. In summary, activation of RAS/Wnt/β-catenin axis results in podocytes and tubular epithelial cell, injury and up-regulations of oxidative, inflammatory and fibrotic pathways. These adverse effects are ameliorated by ergone, alisol B 23-acetate and pachymic acid B. Therefore, these natural products could be considered as novel Wnt/β-catenin signaling inhibitors and anti-fibrotic agents.

Ozone therapy could attenuate tubulointerstitial injury in adenine-induced CKD rats by mediating Nrf2 and NF-κB.

This study aims to determine the effects of ozone therapy on restoring impaired Nrf2 activation to ameliorate chronic tubulointerstitial injury in rats with adenine-induced CKD. Sprague-Dawley rats were fed with 0.75% adenine-containing diet to induce CKD and chronic tubulointerstitial injury. Ozone therapy was administered by rectal insufflation. After 4 weeks, serum and kidney samples were collected and analyzed. Renal function and systemic electrolyte level were detected. Pathological changes in kidney were assessed by hematoxylin-eosin staining and Masson trichrome staining. Nrf2 activation was detected by immunohistochemistry and Western blot analyses. The levels of SOD, CAT, GSH, PCO, and MDA were detected in the kidney. Immunohistochemistry, Western blot, and real-time PCR analyses were performed to evaluate the activation of the nuclear factor kappa B (NF-κB) P65 pathway and inflammation infiltration in the tubulointerstitium of the rats. Ozone therapy improved severe renal insufficiency and tubulointerstitial morphology injury as well as restored Nrf2 activation and inhibited the NF-κB pathway in rats with adenine-induced CKD. Ozone therapy also up-regulated anti-oxidation enzymes (SOD, CAT, and GSH) and down-regulated oxidation products (PCO and MDA), as well as inflammatory cytokines (IL-1β, IL-6, TNF-α, and ICAM-1) in the kidney. These findings indicated that ozone therapy could attenuate tubulointerstitial injury in rats with adenine-induced CKD by mediating Nrf2 and NF-κB.

Therapeutic Potential for Modulation of Nrf2-Keap-1 Signaling Pathway as Treatment for Diabetes and Other Disorders.

Summary: Theinventioninthispatentapplicationisrelatedtonaphthylsulfonamidepyrrolidinederivativesrepresentedgenerallybyformula(I). ThesecompoundsareKeap-1modulatorsandmaybeusefulforthepreventionand/ortreatmentofdiabetes,obesity,dyslipidemia, and related disorders as well as neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. The transcription factor nuclearfactor erythroid2 p45 (NF-E2)-related factor (Nrf2) is a cellular sensor ofoxidative and electrophilic stress. It is a member of the Cap ‘n’ collar family of basic leucine zipper transcription factors, and it is critical for the expression of severalcytoprotectivegenes.Nrf2isregulatedbytheKelch-likeECH-associatedprotein1(Keap-1),whichisanadaptorproteinof the cullin 3-dependent ubiquitin E3 ligase complex. Keap-1 contains functional domains, including a broad complex/tramtrack/ Bric-a-Brac (BTB) domain, an intervening region (IVR) domain, and a Kelch domain. Ubiquitin is the original member of a family of small proteins, which are found in most cellular tissues in the human body that help in regulating the processes of other cellular proteins. Ubiquitin and other relative molecules add covalently to cellular proteins in a process known as ubiquitinylation that alters their activities and the functions. This process plays a critical role in apoptosis of proteinsaswell asin severalothercellularprocesses relatedtothe regulationofproteins.Keap-1mediatesthe ubiquitinylationand subsequentdegradationofNrf2bythe26Sproteasome.Inthisinteraction,theKelchdomainonaKeap-1homodimerbindstothe DLGandETGEsequencemotivesofNrf2molecule.TheKeap-1BTBdomaincontainsacysteineresidueatthe151position,while theIVRdomaincontainstwocysteineresidues,Cys273andCys288.WhileKeap-1hasothercysteineresidues,thesethreeresidues arehighlyreactiveandplaycriticalrolesinstresssensing.Theyaremodifiedinresponsetooxidativestressorelectrophilicreagents, and that alters the conformation of Keap-1, inhibits its function, and prevents the degradation of Nrf2. This leads to the translocation of Nrf2 into the nucleus and its subsequent activation. Nrf2 binds to the antioxidant response element (ARE) in the nucleusanddrivestheexpressionofNrf2targetgenessuchasNAD(P)Hquinoneoxidoreductase1(NQO1)andhemeoxygenase 1 (HMOX1), which are upregulated by the activation of Nrf2. The activation of Nrf2 may provide a potential therapy for the prevention and treatment of several diseases associated with increased oxidative and inflammatory stress. For example, diabetes is associated with impaired activation of Nrf2, which causes a reduced oxidative-stress defense. Diabetes is also associated with low-grade inflammation. Inflammatory processes and excessive oxidative stress induce insulin resistance, endothelial dysfunction, and diabetic nephropathy. Therefore, pharmacological activation of Nrf2 may potentially improve glucose control and lipid handling as well as micro- and macrocardiovascular disease in diabetics. In addition, the activation of Nrf2 reduces cellular stress and damage in response to xenobiotics, which could be useful for the prevention of cancer. Since excessive production of reactive oxidative species is implicated in neurodegenerative diseases such as multiple sclerosis, Huntington’s and Parkinson’s disease; therefore, the activation of Nrf2 may also provide a potential therapeutic strategy for these diseases. Therefore, targeting Nrf2 may also be useful in treating chronic inflammatory diseases such as chronic obstructive pulmonary disease, arthritis, and sepsis. The modulators of KEAP-1, particularly those with antagonistic activities, such as the compounds of formula (I) described in this patent application can lead to activation of Nrf2 and may potentially provide therapies for the prevention and/or treatment of diabetes, obesity, dyslipidemia, and other related disorders.

Alteration of serum lipid profile, SRB1 loss, and impaired Nrf2 activation in CDKL5 disorder

CDKL5 mutation is associated with an atypical Rett syndrome (RTT) variant. Recently, cholesterol homeostasis perturbation and oxidative-mediated loss of the high-density lipoprotein receptor SRB1 in typical RTT have been suggested. Here, we demonstrate an altered lipid serum profile also in CDKL5 patients with decreased levels of SRB1 and impaired activation of the defensive system Nrf2. In addition, CDKL5 fibroblasts showed an increase in 4-hydroxy-2-nonenal– and nitrotyrosine–SRB1 adducts that lead to its ubiquitination and probable degradation. This study highlights a possible common denominator between two different RTT variants (MECP2 and CDKL5) and a possible common future therapeutic target.

High amylose resistant starch diet ameliorates oxidative stress, inflammation, and progression of chronic kidney disease.

Inflammation is a major mediator of CKD progression and is partly driven by altered gut microbiome and intestinal barrier disruption, events which are caused by: urea influx in the intestine resulting in dominance of urease-possessing bacteria; disruption of epithelial barrier by urea-derived ammonia leading to endotoxemia and bacterial translocation; and restriction of potassium-rich fruits and vegetables which are common sources of fermentable fiber. Restriction of these foods leads to depletion of bacteria that convert indigestible carbohydrates to short chain fatty acids which are important nutrients for colonocytes and regulatory T lymphocytes. We hypothesized that a high resistant starch diet attenuates CKD progression. Male Sprague Dawley rats were fed a chow containing 0.7% adenine for 2 weeks to induce CKD. Rats were then fed diets supplemented with amylopectin (low-fiber control) or high fermentable fiber (amylose maize resistant starch, HAM-RS2) for 3 weeks. CKD rats consuming low fiber diet exhibited reduced creatinine clearance, interstitial fibrosis, inflammation, tubular damage, activation of NFkB, upregulation of pro-inflammatory, pro-oxidant, and pro-fibrotic molecules; impaired Nrf2 activity, down-regulation of antioxidant enzymes, and disruption of colonic epithelial tight junction. The high resistant starch diet significantly attenuated these abnormalities. Thus high resistant starch diet retards CKD progression and attenuates oxidative stress and inflammation in rats. Future studies are needed to explore the impact of HAM-RS2 in CKD patients.

Abstract 284: Lower Nrf2 Activity And Decreased Levels Of Pdi And No In The Kidney Contributes To Hypertension In The Aging Fbns

We recently reported that age-associated oxidative stress is causal to higher renal angiotensin AT1 receptor (AT1R) function and blood pressure (BP) in the aging Fischer Brown Norway (FBN) rats. This suggests that redox homeostasis is critical for normal AT1R function and blood pressure (BP). Our objective was to understand the mechanism responsible for the higher oxidative stress in the aging kidneys contributing to impaired AT1R function and high BP in the aged FBNs. Kidneys from adult (3-month) and aged (21-month) FBN rats were isolated. Nrf2 transcription factor, involved in transcriptional activity of diverse antioxidant enzymes genes, was determined in the nuclear and cytosolic fractions of cortical tissues by Western blotting. Nuclear accumulation of Nrf2 is an index of its activation. Protein disulfide isomerase (PDI), an enzyme involved in isomerization reaction, was determined in the cortical homogenates. Moreover, nitrate/nitrite levels were determined in the urine as an index of NO levels in the kidney. We found that the levels of Nrf2 were decreased (adult vs old: 1.86±0.69 vs 0.87±0.18, -53.2%) in the nuclei but increased (adult vs old: 1.20±0.27 vs 1.51±0.36, +25.8%) in the cytosol of aging kidneys. PDI levels also were decreased (adult vs old: 5.38±2.60 vs 3.59±1.76, -33.3%) in the aging kidneys. Furthermore, nitrate/nitrite levels were lower (adult vs old: 372±73 vs 211±30, -43.3%) in the urine from aged than in adult rats. These data suggest that impaired Nrf2 activity in the aging kidneys contributes to increased oxidative stress, renal AT1R function and BP in the aged FNBs. Furthermore, these data also indicate that PDI in an NO-dependent pathway regulates Nrf2 activity contributing to redox-balance in the kidney.

Effect of Redox Modulating NRF2 Activators on Chronic Kidney Disease

Chronic kidney disease (CKD) is featured by a progressive decline of kidney function and is mainly caused by chronic diseases such as diabetes mellitus and hypertension. CKD is a complex disease due to cardiovascular complications and high morbidity; however, there is no single treatment to improve kidney function in CKD patients. Since biological markers representing oxidative stress are significantly elevated in CKD patients, oxidative stress is receiving attention as a contributing factor to CKD pathology. Nuclear factor erythroid-2 related factor 2 (NRF2) is a predominant transcription factor that regulates the expression of a wide array of genes encoding antioxidant proteins, thiol molecules and their generating enzymes, detoxifying enzymes, and stress response proteins, all of which can counteract inflammatory and oxidative damages. There is considerable experimental evidence suggesting that NRF2 signaling plays a protective role in renal injuries that are caused by various pathologic conditions. In addition, impaired NRF2 activity and consequent target gene repression have been observed in CKD animals. Therefore, a pharmacological intervention activating NRF2 signaling can be beneficial in protecting against kidney dysfunction in CKD. This review article provides an overview of the role of NRF2 in experimental CKD models and describes current findings on the renoprotective effects of naturally occurring NRF2 activators, including sulforaphane, resveratrol, curcumin, and cinnamic aldehyde. These experimental results, coupled with recent clinical experiences with a synthetic triterpenoid, bardoxolone methyl, have brought a light of hope for ameliorating CKD progression by preventing oxidative stress and maintaining cellular redox homeostasis.

Impaired Activation of the Nrf2-ARE Signaling Pathway Undermines H2O2-Induced Oxidative Stress Response: A Possible Mechanism for Melanocyte Degeneration in Vitiligo

Vitiligo melanocytes possess higher susceptibility to oxidative insults. Consistent with this, impairment of the antioxidant defense system has been reported to be involved in the onset and progression of vitiligo. Our previous study showed that the nuclear factor E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway and its downstream antioxidant enzyme heme oxygenase-1 (HO-1) are crucial for melanocytes to cope with H2O2-induced oxidative damage. Here, we sought to determine whether the diminished Nrf2-ARE activity that contributes to reduced downstream antioxidant enzymes and increased oxidative stress could be the reason why melanocytes are more vulnerable to vitiligo. We found that vitiligo melanocytes exhibited hypersensitivity to H2O2-induced oxidative injury because of reduced Nrf2 nuclear translocation and transcriptional activity, which led to decreased HO-1 expression and aberrant redox balance. Moreover, we also found that the level of serum HO-1 was significantly decreased and that of IL-2 was markedly increased in 113 vitiligo patients when compared with healthy controls. These data demonstrate that impaired activation of Nrf2 under oxidative stress could result in decreased expression of antioxidant enzymes and increased death of vitiligo melanocytes.

Bardoxolone methyl (BARD) ameliorates aristolochic acid (AA)-induced acute kidney injury through Nrf2 pathway

Bardoxolone methyl (BARD) is an antioxidant modulator that acts through induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. This study aimed to investigate the role of BARD in protecting kidneys from aristolochic acid (AA)-induced acute kidney injury (AKI). Male C57BL/6 mice received intraperitoneal (i.p.) injections of aristolochic acid I (AAI) (5mg/kg/day) for 5 days to produce acute AA nephropathy (AAN) model. BARD (10mg/kg/day, i.p.) was applied for 7 consecutive days, starting 2 days prior to AAI administration. The mice in the AA group showed AKI as evidenced by worsening kidney function evaluated by blood urea nitrogen (BUN) and serum creatinine (SCr) levels, and severe tubulointerstitial injury marked by massive tubule necrosis in kidney tissues. BARD significantly reduced BUN and SCr levels which were elevated by AAI. Additionally, AAI-induced histopathological renal damage was ameliorated by BARD. Furthermore, the expression of Nrf2 was reduced, and its repressor Kelch-like ECH-associated protein 1 (Keap1) was increased significantly, whereas heme oxygenase-1 (HO-1) was upregulated and NAD(P)H quinone oxidoreductase-1 (NQO1) was barely increased in the cytoplasm of tubules in kidneys after treatment with AAI. BARD significantly upregulated renal Nrf2, NQO1 and HO-1 expression and downregulated Keap1 expression compared with those in the AA group. Moreover, it was found that Nrf2 was expressed both in the cytoplasm and nuclear of glomeruli and tubules, whereas NQO1 and HO-1 were localized in the cytoplasm of tubules only. In conclusion, AA-induced acute renal injury was associated with impaired Nrf2 activation and expression of its downstream target genes in renal tissues. BARD prevented renal damage induced by AAI, and this renoprotective effect may be exerted by activating the Nrf2 signaling pathway and increasing expression of the downstream target genes.

P38MAPK-regulated induction of p62 and NBR1 after photodynamic therapy promotes autophagic clearance of ubiquitin aggregates and reduces reactive oxygen species levels by supporting Nrf2–antioxidant signaling

Emerging evidence indicates that oxidative stress instigates the formation of ubiquitin (Ub) aggregates, substrates of autophagy, through a process requiring the ubiquitin binding adaptors p62/SQSTM1 and NBR1. Here, we have investigated the role of p62 and NBR1 in cell survival after hypericin-mediated photodynamic therapy (Hyp-PDT), a procedure known to incite robust reactive oxygen species (ROS)-based endoplasmic reticulum stress and autophagy pathways. We found that Hyp-PDT stimulated the formation of p62- and NBR1-associated Ub aggregates in normal and cancer cells, which were ultimately removed by autophagy, through a mechanism partially regulated by p38MAPK. In line with this, genetic or pharmacological p38MAPK inhibition reduced p62 and NBR1 levels and aggregate formation and impaired Nrf2 activation, thus increasing photo-oxidative stress and cell death. p62-deficient cells, or cells lacking p62 and with reduced levels of NBR1 (through siRNA knockdown), also displayed reduced aggregate formation but exhibited attenuated ROS levels, reduced caspase activation, and improved survival after Hyp-PDT. The increased resistance to photo-oxidative stress exhibited by cells lacking p62 and/or NBR1 was overruled by the inhibition of p38MAPK, which restored cytotoxic ROS levels, thus indicating the relevance of this signal in the control of cell viability. Taken together these findings provide evidence that in photodynamically treated cells a p38MAPK-regulated pathway coordinates the p62/NBR1-mediated clearance of cytosolic aggregates and mitigates PDT-induced proteotoxicity. They also reveal that a functional p38MAPK–Nrf2 signal is required to keep ROS levels in check and protect against PDT-induced proteotoxicity, independent of aggregate formation.

Role of impaired Nrf2 activation in the pathogenesis of oxidative stress and inflammation in chronic tubulo-interstitial nephropathy

Tubulo-interstitial nephropathy (TIN) is a common cause of chronic kidney disease (CKD). Consumption of an adenine-containing diet causes the accumulation of 2,8-dihydroxyadenine in the renal tubules triggering intense chronic TIN and progressive CKD in rats. CKD in this model is associated with, and largely driven by, oxidative stress and inflammation. Oxidative stress and inflammation in rats with spontaneous focal segmental glomerulosclerosis and rats with CKD induced by 5/6 nephrectomy are associated with an impaired activation of nuclear factor-erythroid-2-related factor 2 (Nrf2) which is the master regulator of genes encoding many antioxidant and detoxifying enzymes. The effect of TIN on the Nrf2 pathway and its key target genes is unknown and was investigated here. Sprague-Dawley rats were randomized to control and adenine-treated (rat chow-containing 0.7% adenine for 2 weeks) groups and followed up for 4 weeks. The adenine-treated animals exhibited marked azotemia, impaired urinary concentrating capacity, intense tubular and glomerular damage, interstitial inflammation and fibrosis. This was associated with an increased expression of NAD(P)H oxidase, cyclooxygenase-2 and 12-lipoxygenase, and activation of NF-κB, the master regulator of pro-inflammatory cytokines and chemokines. Oxidative stress and inflammation in the kidneys of adenine-treated animals was accompanied by an impaired activation of Nrf2 and down-regulation of its target gene products including, catalase, heme oxygenase-1 and glutamate-cysteine ligase. Chronic TIN is associated with impaired Nrf2 activity which contributes to the pathogenesis of oxidative stress and inflammation and amplifies their damaging effects on the kidney.

The synthetic triterpenoid RTA dh404 (CDDO-dhTFEA) restores endothelial function impaired by reduced Nrf2 activity in chronic kidney disease

Chronic kidney disease (CKD) is associated with endothelial dysfunction and accelerated cardiovascular disease, which are largely driven by systemic oxidative stress and inflammation. Oxidative stress and inflammation in CKD are associated with and, in part, due to impaired activity of the cytoprotective transcription factor Nrf2. RTA dh404 is a synthetic oleanane triterpenoid compound which potently activates Nrf2 and inhibits the pro-inflammatory transcription factor NF-κB. This study was designed to test the effects of RTA dh404 on endothelial function, inflammation, and the Nrf2-mediated antioxidative system in the aorta of rats with CKD induced by 5/6 nephrectomy. Sham-operated rats served as controls. Subgroups of CKD rats were treated orally with RTA dh404 (2mg/kg/day) or vehicle for 12 weeks. The aortic rings from untreated CKD rats exhibited a significant reduction in the acetylcholine-induced relaxation response which was restored by RTA dh404 administration. Impaired endothelial function in the untreated CKD rats was accompanied by significant reduction of Nrf2 activity (nuclear translocation) and expression of its cytoprotective target genes, as well as accumulation of nitrotyrosine and upregulation of NAD(P)H oxidases, 12-lipoxygenase, MCP-1, and angiotensin II receptors in the aorta. These abnormalities were ameliorated by RTA dh404 administration, as demonstrated by the full or partial restoration of the expression of all the above analytes to sham control levels. Collectively, the data demonstrate that endothelial dysfunction in rats with CKD induced by 5/6 nephrectomy is associated with impaired Nrf2 activity in arterial tissue, which can be reversed with long term administration of RTA dh404.

Immune-Regulatory Mechanisms in Systemic Autoimmune and Rheumatic Diseases

Immune-Regulatory Mechanisms in Systemic Autoimmune and Rheumatic Diseases