Sulfiredoxin 1 ameliorates doxorubicin-induced cardiotoxicity by suppressing oxidative stress and inflammation via the Sirt1/NLRP3 pathway.

BACKGROUND: Doxorubicin (DOX) is limited in clinical use due to its cardiotoxic side effects. Oxidative stress and inflammation are pivotal mechanisms underlying doxorubicin-induced cardiotoxicity (DIC). Sulfiredoxin 1 (Srxn1) plays a central role in antioxidant effects. However, the role of Srxn1 in DIC has not yet been fully elucidated. This study aims to explore the effects and underlying mechanisms of Srxn1 on DIC. METHODS: We overexpressed Srxn1 in the myocardium using an adeno-associated virus 9 (AAV9) system, delivered through tail vein injection. C57BL/6 mice received intraperitoneal injections of DOX (4 mg/kg) weekly for four consecutive weeks to establish a mouse model of DIC. We used echocardiography, histopathological, and molecular techniques to elucidate the effects and mechanisms. RESULTS: Our findings demonstrate that overexpression of Srxn1 significantly enhanced cardiac function and mitigated myocardial injury in mice exposed to DOX. Overexpressing Srxn1 attenuated oxidative stress and inflammation induced by DOX. Furthermore, Srxn1 overexpression led to upregulation of sirtuin 1 (Sirt1) expression and inhibited the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome. Notably, the protective effects of Srxn1 were significantly abrogated by the Sirt1 inhibitor EX527. CONCLUSION: The protective effects of Srxn1 against DOX-induced cardiac oxidative stress and inflammation operate by targeting the Sirt1/NLRP3 signaling pathway to alleviate DIC. Srxn1 could be a potential candidate for the treatment of DOX-induced myocardial injury.

Beyond Antioxidant Activity: Redox Properties of Catechins May Affect Changes in the DNA Methylation Profile-The Example of SRXN1 Gene.

The role of catechins in the epigenetic regulation of gene expression has been widely studied; however, if and how this phenomenon relates to the redox properties of these polyphenols remains unknown. Our earlier study demonstrated that exposure of the human colon adenocarcinoma HT29 cell line to these antioxidants affects the expression of redox-related genes. In particular, treatment with (-)-epigallocatechin (EGC) downregulated transcription of gene encoding sulfiredoxin-1 (SRXN1), the peroxidase involved in the protection of cells against hydrogen peroxide-induced oxidative stress. The aim of this study was to investigate whether the observed SRXN1 downregulation was accompanied by changes in the DNA methylation level of its promoter and, if so, whether it was correlated with the redox properties of catechins. The impact on DNA methylation profile in HT29 cells treated with different concentrations of five catechins, varying in chemical structures and standard reduction potentials as well as susceptibility to oxidation, was monitored by a methylation-sensitive high-resolution melting technique employing the SRXN1 promoter region as a model target. We demonstrated that catechins, indeed, are able to modulate DNA methylation of the SRXN1 gene in a redox-related manner. The nonlinear method in the statistical analysis made it possible to fish out two parameters (charge transfer in oxidation process Qox and time of electron transfer t), whose strong interactions correlated with observed modulation of DNA methylation by catechins. Based on these findings, we present a proof-of-concept that DNA methylation, which limits SRXN1 expression and thus restricts the multidirectional antioxidant action of SRXN1, may represent a mechanism protecting cells against reductive stress caused by particularly fast-reacting reductants such as EGC and (-)-epicatechin gallate (ECG) in our study.

Structure-based analysis and rational design of human peroxiredoxin-1's C-terminus-derived peptides to target sulfiredoxin-1 in pancreatic cancer.

Human peroxiredoxin (PRX) family of antioxidant enzymes reduces hydrogen peroxide and alkyl hydroperoxide involved in the redox signaling, among which the widely documented PRX1 is a versatile molecule regulating cell growth, differentiation and apoptosis, and has been implicated in the tumorigensis of pancreatic cancer. In this study, we systematically examined the complex crystal structure of PRX1 with its cognate interacting partner sulfiredoxin-1 (SRX1) at molecular level, and found that the PRX1-SRX1 association is a typical peptide-mediated protein-protein interaction, where a 18-mer C-terminal tail (CTT) segment of PRX1 was identified to be primarily responsible for the interaction, which contributes ~80% and ~ 55% to the total binding potency of SRX1 to PRX1 monomer and homodimer, respectively. We also demonstrated that the SRX1 exhibits a strong global selectivity for PRX1 CTT tail over other PRX family proteins. Next, the intermolecular interaction between PRX1 CTT tail and SRX1 was investigated at structural, energetic and dynamic levels, from which a 9-mer core region of PRX1 CTT tail was defined as the SRX1-binding hotspot. Biophysical assays substantiated that the CTT and CTTc peptides (out of PRX1 protein context) can bind in an independent manner and possess a close affinity to SRX1. Based on the CTTc sketch a computational combinatorial library consisting of 216 designed peptide derivatives was rationally generated, from which the top-5 hits were found to have comparable affinity with CTT peptide and improved affinity relative to CTTc peptide. They can be used as structurally reduced lead molecular entities to further develop new peptidic agents for therapeutic purpose to disrupt the native PRX1-SRX1 interaction by competing with PRX1 CTT tail for the peptide-binding pocket of SRX1.

Increased sulfiredoxin-1 levels as compensatory mechanism against reactive oxygen species in women with gestational diabetes mellitus

Objective: This study aimed to investigate the correlation between serum Sulfiredoxin-1 (Srx-1) levels and gestational diabetes mellitus (GDM). Materials and Methods: A total of 40 patients diagnosed with GDM according to the American Diabetes Association Criteria and 40 age matched and gestational age-matched healthy pregnant women as a control group were included in this cross-sectional study. Serum Srx-1 levels and other demographic and laboratory variables were analyzed. Results: Fasting plasma glucose, first and second-hour plasma glucose levels, fasting insulin levels, homeostasis model assessment of insulin resistance (HOMA-IR), and Srx-1 levels were significantly different in patients with GDM than control (p<0.05). Plasma Srx-1 levels significantly correlated with fasting plasma glucose, first and second-hour plasma glucose levels, fasting insulin levels, and HOMA-IR of patients with GDM (p<0.05), whereas no correlation in the control group. Conclusion: This is the first study demonstrating an association between serum Srx-1 levels and GDM. Our results suggest increased serum Srx-1 levels may be a novel predictive marker for GDM. More randomized-controlled trials are needed to evaluate Srx-1 as a marker for adverse fetal results; closer monitoring is warranted with high Srx-1 levels.

Sulfiredoxin-1 protects spinal cord neurons against oxidative stress in the oxygen-glucose deprivation/reoxygenation model through the bax/cytochrome c/caspase 3 apoptosis pathway.

BACKGROUND: Spinal cord ischemia/reperfusion injury is a common clinical, pathophysiological phenomenon with complex molecular mechanisms. Currently, there are no therapeutics available to alleviate the same. This study investigates the protective effects of sulfiredoxin-1 (Srxn 1) on spinal cord neurons following exposure to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. MATERIALS AND METHODS: Primary spinal cord neurons were cultured, detected by anti-tubulin ßⅢ, and transfected with adeno-associated virus (AAV)-Srxn 1 to overexpress Srxn 1. They were identified by their morphology and CCK-8 assay. The superoxide dismutase level was measured by superoxide dismutase assay. Malondialdehyde level was measured by malondialdehyde assay. The apoptosis ratio was calculated by Hoechst 33342 and Annexin V-PE/7-AAD staining. Mitochondrial transmembrane potential (Δψm) was detected by tetramethylrhodamine-methyl ester-perchlorate (TMRM) staining. The mRNA expression levels of Srxn 1 and caspase 3 were detected by quantitative reverse transcription-polymerase chain reaction, and the protein expression levels of Srxn 1, bax, bcl-2, cytosolic cytochrome c, and caspase 3 were detected by western blotting. RESULTS: AAV-Srxn 1 up-regulated mRNA and protein levels of Srxn 1 in spinal cord neurons. Following exposure to OGD/R, overexpression of Srxn 1 improved the neuronal viability, alleviated the neuron apoptosis, enhanced the mitochondrial transmembrane potential, increased the SOD level, decreased the MDA level, inhibited the expression of cytosolic cytochrome c, bax, and caspase 3, and promoted the expression of bcl-2. CONCLUSION: Srxn 1 plays a significant role in anti-apoptosis of spinal cord neurons, and Srxn 1 may be a potential therapeutic target for spinal cord I/R injury.

Sulfiredoxin-1 is a promising novel prognostic biomarker for hepatocellular carcinoma.

Identifying novel prognostic biomarkers for hepatocellular carcinoma (HCC) and then, develop an effective individualized treatment strategy remain extremely warranted. The prognostic role of sulfiredoxin-1(SRXN1), an antioxidant enzyme, remains unknown in HCC. This study aimed to explore the prognostic implications of SRXN1 in HCC patients after partial hepatectomy. The expression of SRXN1 in HCC and normal tissue were analyzed using the patients from the public databases and Zhongshan Hospital. The Cox regression, Kaplan-Meier survival analysis, and time-dependent receiver operating characteristic curves were performed to identify the predictive role of SRXN1 expression on HCC patients. A prognostic nomogram based on SRXN1 expression was constructed and validated to further confirm the predictive power of SRXN1 as a prognostic biomarker. Finally, functional enrichment analysis and protein-protein interaction network analysis of SRXN1 and its associated genes were conducted. The results showed that SRXN1 was upregulated in HCC samples compared with the normal liver tissues. Patients with SRXN1 upregulation had shorter survival time. SRXN1 overexpression was significantly correlated with advanced clinicopathological parameters. The prognostic nomogram based on SRXN1 expression was proved to be more accurate than routine staging systems for the prediction of overall survival. Protein-protein interaction network analysis demonstrated the first neighbor genes of SRXN1 mainly participated in response to oxidative stress. In brief, SRXN1 could be a prognostic biomarker for the management of HCC.

Bacteroides fragilis Enterotoxin Induces Sulfiredoxin-1 Expression in Intestinal Epithelial Cell Lines Through a Mitogen-Activated Protein Kinases- and Nrf2-Dependent Pathway, Leading to the Suppression of Apoptosis.

Enterotoxigenic Bacteroides fragilis is a causative agent of colitis and secrets enterotoxin (BFT), leading to the disease. Sulfiredoxin (Srx)-1 serves to protect from oxidative damages. Although BFT can generate reactive oxygen species in intestinal epithelial cells (IECs), no Srx-1 expression has been reported in ETBF infection. In this study, we explored the effects of ETBF-produced BFT on Srx-1 induction in IECs. Treatment of IECs with BFT resulted in increased expression of Srx-1 in a time-dependent manner. BFT treatment also activated transcriptional signals including Nrf2, AP-1 and NF-κB, and the Srx-1 induction was dependent on the activation of Nrf2 signals. Nrf2 activation was assessed using immunoblot and Nrf2-DNA binding activity and the specificity was confirmed by supershift and competition assays. Suppression of NF-κB or AP-1 signals did not affect the upregulation of Srx-1 expression. Nrf2-dependent Srx-1 expression was associated with the activation of p38 mitogen-activated protein kinases (MAPKs) in IECs. Furthermore, suppression of Srx-1 significantly enhanced apoptosis while overexpression of Srx-1 significantly attenuated apoptosis during exposure to BFT. These results imply that a signaling cascade involving p38 and Nrf2 is essential for Srx-1 upregulation in IECs stimulated with BFT. Following this upregulation, Srx-1 may control the apoptosis in BFT-exposed IECs.

Effects of Srxn1 on growth and Notch signalling of astrocyte induced by hydrogen peroxide.

OBJECTIVE: To investigate the effect of Sulfiredoxin-1 (Srxn1) on astrocyte injury induced by hydrogen peroxide (H2O2). METHODS: Observing the changes of H2O2 on contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) and apoptosis after transfected Srxn1 siRNA into astrocytes. The protein expression of Notch 1, NICD and Hes1, the content of LDH and MDA, the activity of SOD and apoptosis rate of astrocytes after inhibiting or activation of Notch signalling pathway were detected by Western blot, ELISA and flow cytometry, respectively. RESULTS: Knockdown of Srxn1 could promote the secretion of LDH and MDA, decrease the activity of SOD and aggravate apoptosis of astrocytes induced by H2O2. The results of Western blot, ELISA assay and flow cytometry indicated that activation of the Notch signalling pathway attenuated the effect of Srxn1 on H2O2-induced oxidative damage and apoptosis of astrocytes. CONCLUSION: Srxn1 may protect astrocytes from oxidative stress injury induced by H2O2 by activation of Notch signalling pathway.

Sulfiredoxin-1 enhances cardiac progenitor cell survival against oxidative stress via the upregulation of the ERK/NRF2 signal pathway.

Cardiac stem/progenitor cells (CPCs) have recently emerged as a potentially transformative regenerative medicine to repair the infarcted heart. However, the limited survival of donor cells is one of the major challenges for CPC therapy. Our recent research effort on preconditioning human CPCs (hCPCs) with cobalt protoporphyrin (CoPP) indicated that sulfiredoxin-1 (SRXN1) is upregulated upon preconditioning aldehyde dehydrogenase bright hCPCs (ALDHbr-hCPCs) with CoPP. Further studies demonstrated that overexpressing SRXN1 enhanced the survival capacity for ALDHbr-hCPCs. This was associated with the up-regulation of anti-apoptotic factors, including BCL2 and BCL-xL. Meanwhile, overexpressing SRXN1 decreased the ROS generation and mitochondrial membrane potential, concomitant with the up-regulated primary antioxidant systems, such as PRDX1, PRDX3, TXNRD1, Catalase and SOD2. It was also observed that overexpressing SRXN1 increased the migration, proliferation, and cardiac differentiation of ALDHbr-hCPCs. Interestingly, SRXN1 activated the ERK/NRF2 cell survival signaling pathway, which may be the underlying mechanism through which overexpressing SRXN1 lead to protection of hCPCs against oxidative stress-induced apoptosis. Taken together, these results provide a rationale for the exploration of SRXN1 as a novel molecular target that can be used to enhance the effectiveness of cardiac stem/progenitor cell therapy for ischemic heart disease.

Neuroprotective effects of sulfiredoxin-1 during cerebral ischemia/reperfusion oxidative stress injury in rats.

As an endogenous antioxidant protein, Sulfiredoxin1 (Srxn1) can prevent cell oxidative stress damage. However, its role in cerebral ischemia/reperfusion (I/R) injury and the underlying signaling mechanisms remain largely unknown. Here, we explored effects of Srxn1 knockdown on oxidative stress using in vitro and in vivo I/R models and investigated related neuroprotective mechanisms. For in vitro studies, primary cortical neuronal cultures were transfected with an interfering lentivirus targeting Srxn1. Oxygen-glucose deprivation (OGD) was conducted after Srxn1 knockdown. MTS and lactate dehydrogenase assays indicated that knockdown of Srxn1 increased cell death and reduced cell viability. Similarly, superoxide dismutase (SOD) and reduced glutathionekits assays showed that knockdown of Srxn1 worsened oxidative stress injury. For in vivo studies, siRNA for Srxn1 or negative control siRNA was injected intracerebroventricularly 24h before middle cerebral artery occlusion (MCAO). Data shows silencing Srxn1 resulted in a significant increase in cerebral infarction, neurological deficits, histological injury, and oxidative stress injury 24h after ischemic stroke. Moreover, immunoblot analysis assessed the relationship between Srxn1 levels and Prdx1-4 as well as Prdx-SO3 activity both in vitro and in vivo models. We found that decreased Srxn1 reduced Prdx1-4 and enhanced Prdx-SO3 protein levels. In addition, knockdown of Nrf2 was performed; immunoblot analysis was used to measure Srxn1 and NQO1 protein levels. We further found that interference of Nrf2 reduced Srxn1 and NQO1 protein levels. In summary, Srxn1 can protect neurons from I/R oxidative stress injury and the mechanism involves Prdx activity. Srxn1, which might be downstream of Nrf2, can prevent cerebral ischemia reperfusion by reversing overoxidized Prdx and restoring antioxidant activity of Prdx.

Oxidative stress and altered expression of peroxiredoxin genes family (PRDXS) and sulfiredoxin-1 (SRXN1) in human lung tissue following exposure to sulfur mustard.

BACKGROUND: Sulfur mustard (SM) is a potent and mutagenic agent that targets human lung tissue. PURPOSE OF THE STUDY: The purpose of this investigation is to characterize the expression of sulfiredoxin-1 (SRXN1) and peroxiredoxin (PRDXs) genes and oxidative stress (OS) status in human lung after exposure to SM. MATERIALS AND METHODS: Lung biopsy specimens bronchoalveolar lavage (BAL) fluids were provided from SM-exposed patients (n = 6) and controls (n = 5). Changes in gene expression were measured using RT(2) Profiler PCR Array. OS was considered by measuring BAL fluid levels of malondialdehyde (MDA) and protein carbonyls (PC). RESULTS: Mean of MDA and PC values in BAL fluid of patients (0.6467 ± 0.05922 nmol/l and 1.391 ± 0.421 nmol/mg, respectively) was higher than in controls (0.486 ± 0.04615 nmol/l and 0.949 ± 0.149 nmol/mg, respectively). Expression of all examined genes was in the order PRDX1> PRDX3> PRDX6> SRXN1> PRDX2> PRDX4> PRDX5. Among the most upregulated genes was the PRDX1, which was overexpressed by 10.1029-fold (p = 0.000634). SM-exposed individuals demonstrated expression of PRDX3 4.6231 (p = 0.000134), PRDX6 3.4964 (p = 0.001102), SRXN1 3.3719 (p < 0.0001) and PRDX2 2.7725-folds (p = 0.000383) higher than those of controls that reveal. CONCLUSIONS: Upregulation of PRDXs and SRXN1 genes may be because of reactive oxygen species (ROS) production and OS in lung tissue of patients after SM exposure. Expression of SRXN1 and PRDXNs genes, especially I, II, III, and VI is increased in SM-injured lungs, suggesting the induction of cellular responses to increased production of ROS and OS in lung of the patients. Therefore, sulfiredoxin and peroxiredoxins can be targeted as biomarkers of OS in these patients.

Sulfiredoxin-1 protects against simulated ischaemia/reperfusion injury in cardiomyocyte by inhibiting PI3K/AKT-regulated mitochondrial apoptotic pathways.

Reactive oxygen species (ROS)-triggered cardiac cell injury is recognized as the major contributor for the pathogenesis progression of ischaemic cardiovascular diseases. Sulfiredoxin-1 (Srx-1) is an endogenous antioxidant and exerts the crucial neuroprotective effects in cerebral ischaemia. However, its function and the underlying mechanism in ischaemic heart diseases remain poorly defined. Here, a dramatical decrease in Srx-1 was validated in H9c2 cardiomyocytes upon simulated ischaemia-reperfusion (SI/R) injury. Moreover, Srx-1 protected H9c2 cells from SI/R-injured injury as the evidences that Srx-1 up-regulation attenuated the inhibitory effects on cell viability, lactate dehydrogenase (LDH) and cell apoptosis upon SI/R treatment. Knockdown of Srx-1 accelerated cell injury upon SI/R. Mechanism assay corroborated that SI/R treatment noticeably aggravated the loss of mitochondrial membrane potential (Δψm), which was remarkably abated in Ad-Srx-1 groups. Importantly, Srx-1 elevation substantially reduced cytochrome c release, the activity of caspase-9 and caspase-3, accompany with the subsequent decrease in the cleavage of poly (ADP ribose) polymerase (PARP). Concomitantly, overexpression of Srx-1 also decreased the expression of pro-apoptosis protein Bax and increased anti-apoptotic Bcl-2 expression. Further analysis substantiated that Srx-1 treatment remarkably induced the activation of PI3K/AKT signalling. Preconditioning with LY294002 dramatically decreased Srx-1-enhanced cell resistance to SI/R injury. Importantly, LY294002 mitigated the inhibitory effects of Srx-1 on Δψm loss, cytochrome c release, caspase-9/3 activity, and the expression of Bcl-2 family. Together, these results suggested that Srx-1 might protect cardiomyocyte injury upon SI/R by suppressing PI3K/AKT-mediated mitochondria dependent apoptosis, revealing a promising therapeutic agent against ischaemic cardiovascular diseases.

Sulfiredoxin-1 protects primary cultured astrocytes from ischemia-induced damage.

Astrocytes appear to be important regulators of the inflammatory events that occur in stroke. Sulfiredoxin-1 (Srxn1), an endogenous antioxidant protein, exhibits neuroprotective effects. Although the mechanism by which Srxn1 negatively regulates oxidative and apoptotic pathways has been extensively characterized, the impact of Srxn1 on inflammation has not been well studied. In this study, we used oxygen-glucose deprivation followed by recovery (OGD/R) and hydrogen peroxide (H2O2) to mimic stress from cerebral ischemic damage on primary cultured astrocytes. We found that knockdown of Srxn1 by two shRNAs resulted in decreased cell viability of astrocytes. Decreased level of Srxn1 also correlated with excessive levels of proinflammatory cytokines and chemokines such as TNF-α, MPO, IL-1ß, and IL-6. In addition, Srxn1 appeared to influence the strength of TLR4 signaling pathway; the expression of COX-2, IL-6, and NOS2 were strongly induced by OGD/R and H2O2 in astrocyte cultures with Srxn1-shRNAs. Our results suggested that loss of Srxn1 expression in astrocytes may cause excessive activation of inflammatory responses which contribute to OGD/R- and H2O2-induced cell death. Restoring Srxn1 function by gene therapy and/or pharmacology emerges as a promising strategy for the treatment of stroke and other chronic neurodegenerative diseases.

MRNA and miRNA expression patterns associated to pathways linked to metal mixture health effects.

Metals are a threat to human health by increasing disease risk. Experimental data have linked altered miRNA expression with exposure to some metals. MiRNAs comprise a large family of non-coding single-stranded molecules that primarily function to negatively regulate gene expression post-transcriptionally. Although several human populations are exposed to low concentrations of As, Cd and Pb as a mixture, most toxicology research focuses on the individual effects that these metals exert. Thus, this study aims to evaluate global miRNA and mRNA expression changes induced by a metal mixture containing NaAsO2, CdCl2, Pb(C2H3O2)2·3H2O and to predict possible metal-associated disease development under these conditions. Our results show that this metal mixture results in a miRNA expression profile that may be responsible for the mRNA expression changes observed under experimental conditions in which coding proteins are involved in cellular processes, including cell death, growth and proliferation related to the metal-associated inflammatory response and cancer.

Prolonging in utero-like oxygenation after birth diminishes oxidative stress in the lung and brain of mice pups.

BACKGROUND: Fetal-to-neonatal transition is associated with oxidative stress. In preterm infants, immaturity of the antioxidant system favours supplemental oxygen-derived morbidity and mortality. OBJECTIVES: To assess if prolonging in utero-like oxygenation during the fetal-to-neonatal transition limits oxidative stress in the lung and brain, improving postnatal adaptation of mice pups. MATERIAL AND METHODS: Inspiratory oxygen fraction (FiO2) in pregnant mice was reduced from 21% (room air) to 14% (hypoxia) 8-12 h prior to delivery and reset to 21% 6-8 h after birth. The control group was kept at 21% during the procedure. Reduced (GSH) and oxidized (GSSG) glutathione and its precursors [γ-glutamyl cysteine (γ-GC) and L-cysteine (CySH)] content and expression of several redox-sensitive genes were evaluated in newborn lung and brain tissue 1 (P1) and 7 (P7) days after birth. RESULTS: As compared with control animals, the GSH/GSSG ratio was increased in the hypoxic group at P1 and P7 in the lung, and at P7 in the brain. In the hypoxic group a significant increase in the mRNA levels of NAD(P)H:quinone oxidoreductase 1 (noq1), Sulfiredoxin 1 (srnx1) and Glutathione Peroxidase 1 (gpx) was found in lung tissue at P1, as well as a significant increase in gpx in brain tissue at P7. CONCLUSIONS: Delaying the increase in tissue oxygenation to occur after birth reduces short-and-long-term oxidative stress in the lung. Similar yet more subtle effects were found in the brain. Apparently, the fetal-to-neonatal transition under hypoxic conditions appears to have protective qualities.

Nrf2 in bone marrow-derived cells positively contributes to the advanced stage of atherosclerotic plaque formation.

Atherosclerosis is the major etiology underlying myocardial infarction and stroke, and strategies for preventing atherosclerosis are urgently needed. In the context of atherosclerosis, the deletion of the Nrf2 gene, which encodes a master regulator of the oxidative stress response in mammals, reportedly attenuates atherosclerosis formation. However, the precise mechanisms of protection against atherosclerosis are largely unknown. To further clarify the role of Nrf2 in atherosclerosis in vivo, we performed a time course analysis of atherosclerosis development utilizing an ApoE knockout (KO) mouse model. The results demonstrate that oil red O-stainable lesions were similar in size 5 weeks after the initiation of an HFC (high fat and high cholesterol) diet, but the lesions were markedly attenuated in the Nrf2 and ApoE double KO mice (A0N0 mice) compared with the lesions in the ApoE KO mice (A0N2 mice) at 12 weeks. Consistent with these results, the immunohistochemical analysis revealed that Nrf2 activation is observed in late-stage atherosclerotic plaques but not in earlier lesions. The RT-qPCR analysis of 12-week atherosclerotic plaques revealed that Nrf2 target genes, such as Ho-1 and SLPI, are expressed at significantly lower levels in the A0N0 mice compared with the A0N2 mice, and this change was associated with a decreased expression of macrophage M1-subtype genes Arginase II and inducible NO synthase in the A0N0 mice. Furthermore, the bone marrow (BM) transplantation (BMT) analysis revealed that the Nrf2 activity in the BM-derived cells contributed to lesion formation. Therefore, our study has characterized the positive role of Nrf2 in the BM-derived cells during the development of atherosclerosis, which suggests that Nrf2 may influence the inflammatory reactions in the plaques.