Nrf2 KO Mice Research Articles

Nrf2 Ameliorates DDC-Induced Sclerosing Cholangitis and Biliary Fibrosis and Improves the Regenerative Capacity of the Liver.

The Nrf2 pathway protects against oxidative stress and induces regeneration of various tissues. Here, we investigated whether Nrf2 protects from sclerosing cholangitis and biliary fibrosis and simultaneously induces liver regeneration. Diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was fed to Nrf2-KO mice (Nrf2-/-), mice with liver-specific hyperactivated Nrf2 (HKeap1-/-) and wild-type (WT) littermates to induce cholangitis, liver fibrosis, and oval cell expansion. HKeap1-/--mice were protected from almost all DDC-induced injury compared with WT and Nrf2-/-. Liver injury in Nrf2-/- and WT mice was mostly similar, albeit Nrf2-/- suffered more from DDC diet as seen for several parameters. Nrf2 activity was especially important for the expression of the hepatic efflux transporters Abcg2 and Abcc2-4, which are involved in hepatic toxin elimination. Surprisingly, cell proliferation was more enhanced in Nrf2-/-- and HKeap1-/--mice compared with WT. Interestingly, Nrf2-/--mice failed to sufficiently activate oval cell expansion after DDC treatment and showed almost no resident oval cell population under control conditions. The resident oval cell population of untreated HKeap1-/--mice was increased and DDC treatment resulted in a stronger oval cell expansion compared with WT. We provide evidence that Nrf2 activation protects from DDC-induced sclerosing cholangitis and biliary fibrosis. Moreover, our data establish a possible role of Nrf2 in oval cell expansion.

PO-191 The effect of hypoxic training at different simulated altitude on the antioxidant activity mediated by Nrf2 in mice skeletal muscle

Objective It is generally believed that the long-term hypoxic training could impact oxidation resistance. Nrf2-Keapl signaling pathway is a key pathway of cell oxidative stress reaction. This research attempts to investigate the role and mechanism of Nrf2 in oxidation resistance to hypoxic training of different oxygen concentration.
 Methods Part one, 8-week-old Nrf2 knockout mice and wild type mice were divided into normoxic control group (NC), simulated altitude of 3500m hypoxic training group (3500HT) and simulated altitude of 5000m hypoxic training group (5000HT) randomly and respectively. The mice run on treadmill in speed of 12 m/min, 1h/day, 6day/week, for 4 weeks. Oxygen concentration in hypoxia was 13.3% and 10%. Mice were treated for 4 weeks, 8h/day. 48 h after the last training, the mice were sacrificed and skeletal muscles of legs were collected. Western Blot tested Nrf2 and antioxidant enzyme protein. Antioxidant enzymes mRNA were tested by RT-PCR. High quality fluorescence measurement was used to test ROS levels in skeletal muscle. Part two, The 30 C57BL/6J mice were divided into three groups: control group (WC), hypoxia group (WH), hypoxic training group (WHT). The hypoxic training arrangement was same as before. After both the interventions, the mice were sacrificed and collected skeletal muscle of legs. The expression of Nrf2, Keap1 and p-Nrf2 were analyzed by western blot. High quality fluorescence assay was done to detect ROS level in skeletal muscle of mice.
 Results (1) Compared with the same type mice NC group, Nrf2 protein, the mRNA and protein of CAT, GPX-1, GCLm, the mRNA of SOD1, SOD2, HO-1 were increased in wild type mice 3500HT group. And the Nrf2 protein, the mRNA and protein of SOD1, SOD2, the mRNA of CAT, NQO-1, GCLc, GCLm mRNA, the protein of HO-1 were decreased, and the ROS levels was higher in wile type mice 5000HT group. The mRNA of CAT, HO-1 in Nrf2-KO mice 3500HT group were increased, the mRNA and protein of SOD1, the mRNA of SOD2, the protein of GCLc were decreased, but the GCLc mRNA was increased in Nrf2-KO mice 5000HT group. When compared with the same intervention wild type mice, the mRNA and protein of SOD1, GPX-1, SOD2, HO-1, the mRNA of CAT, NQO-1, GCLc, GCLm were decreased in Nrf2-KO mice 3500HT group. The mRNA of GCLm, NQO-1, the protein of GCLc, HO-1 were decreased, but the GCLc mRNA was increased. (2) Nrf2/Keap1 complex contents in mice skeletal muscle of WH and WHT groups were significantly increased compared with WC group respectively. The free Nrf2 in mice skeletal muscle of WH、WHT groups were significantly reduced compared with WC group respectively. After both types of intervention, free Keap1 had no change nearly in skeletal muscle of mice. Compared with WC group, p-Nrf2 in mice skeletal muscle of WH and WHT groups were significantly reduced. The ROS level in mice skeletal muscle of WHT group significantly increased compared with WC group mice. Conclusions: Hypoxia and hypoxia training three interventions could increase Nrf2/Keap1 combination in skeletal muscle of mice, reduce the volume of free Nrf2; Phosphorylation of Nrf2 in skeletal muscle of mice in hypoxia training group was significantly lower, which may be result in marked increase in ROS level.
 Conclusions (1) Hypoxic training could affect antioxidant activity via Nrf2 in mice skeletal muscle, which is connected with the oxygen concentration. (2) Moderate hypoxia training (at the altitude of 3500m) can promote the antioxidant activity via Nrf2. However, extremely hypoxic training (at the altitude of 5000m) can restrain the antioxidant activity via Nrf2 through the inhibition of Nrf2/Keap1 dissociation.

A novel synthetic small molecule DMFO targets Nrf2 in modulating proinflammatory/antioxidant mediators to ameliorate inflammation

Inflammation is a protective immune response against invading pathogens, however, dysregulated inflammation is detrimental. As the complex inflammatory response involves multiple mediators, including the involvement of reactive oxygen species, concomitantly targeting proinflammatory and antioxidant check-points may be a more rational strategy. We report the synthesis and anti-inflammatory/antioxidant activity of a novel indanedione derivative DMFO. DMFO scavenged reactive oxygen species (ROS) in in-vitro radical scavenging assays and in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. In acute models of inflammation (carrageenan-induced inflammation in rat paw and air pouch), DMFO effectively reduced paw oedema and leucocyte infiltration with an activity comparable to diclofenac. DMFO stabilised mast cells (MCs) in in-vitro A23187 and compound 48/80-induced assays. Additionally, DMFO stabilised MCs in an antigen (ovalbumin)-induced MC degranulation model in-vivo, without affecting serum IgE levels. In a model of chronic immune-mediated inflammation, Freund’s adjuvant-induced arthritis, DMFO reduced arthritic score and contralateral paw oedema, and increased the pain threshold with an efficacy comparable to diclofenac but without being ulcerogenic. Additionally, DMFO significantly reduced serum TNFα levels. Mechanistic studies revealed that DMFO reduced proinflammatory genes (IL1β, TNFα, IL6) and protein levels (COX2, MCP1), with a concurrent increase in antioxidant genes (NQO1, haem oxygenase 1 (HO-1), Glo1, Nrf2) and protein (HO-1) in LPS-stimulated macrophages. Importantly, the anti-inflammatory/antioxidant effect on gene expression was absent in primary macrophages isolated from Nrf2 KO mice suggesting an Nrf2-targeted activity, which was subsequently confirmed using siRNA transfection studies in RAW macrophages. Therefore, DMFO is a novel, orally-active, safe (even at 2 g/kg p.o.), a small molecule which targets Nrf2 in ameliorating inflammation.

The effects of Nrf2 knockout on regulation of benzene-induced mouse hematotoxicity

Previous studies have demonstrated that oxidative stress is implicated in benzene-induced hematotoxicity. However, the low dose-response effects and the mechanism underlying perturbation of hematopoiesis remain to be defined. This study aims to address the role of Nrf2 pathway in mediating benzene-induced hematotoxicity. Nrf2+/+ (wildtype, Nrf2-WT) and Nrf2−/− (knockout, Nrf2-KO) mice were administrated with benzene at doses of 0.1, 1.0, 10.0, 100.0 mg/kg by oral gavage for a consecutive 4 weeks (6 times/week). As a result, benzene exposure caused a decline of WBC and lymphocyte counts in a dose-dependent manner at a dose range from 1.0 to 100.0 mg/kg, while low dose benzene induced hormesis effects. Interestingly, Nrf2 deficiency seemed to relieve the decline of peripheral blood cell counts upon benzene exposure, indicating the involvement of Nrf2 in regulation of benzene-induced hematotoxicity. The suppression of phase II enzyme expression in Nrf2-KO mice resulted in considerable reduction in detoxification indicated by the decrease of urinary S-phenylmercapturic acid (SPMA), a metabolite of benzene. Ex vivo assay revealed enhanced cytotoxicity and oxidative stress were induced by benzene in Nrf2-KO mice. Notably, the depletion of Nrf2 triggered the proliferation and differentiation of hematopoietic cells, but induced aberrant morphological changes in periphery erythrocytes and bone marrow cells, implicating the compensatory effects carried on at the expense of induction of dysfunctional blood cells. Our findings provide a new insight into a low dose–response towards benzene-induced hematotoxicity and uncover the critical role of Nrf2 pathway in mediating abnormal hematopoiesis in response to oxidative stress.

Abstract 4051: Different immune signatures in advanced lung tumors of Nrf2 wildtype vs. knockout mice

Abstract Activation of Nrf2 and its downstream target genes serves as a master defense mechanism, protecting cells against oxidative and electrophilic stress. Numerous activators of Nrf2 inhibit carcinogenesis. However, genetic alterations resulting in constitutive Nrf2 activation in human lung tumors promotes tumor growth and chemoresistance. The dual role of Nrf2 in cancer has generated a high level of interest, but little is known regarding the effect of Nrf2 on immune cells during lung carcinogenesis. In this study, we used the potent carcinogen vinyl carbamate to induce lung tumors in mice. Tumor burden, the immunophenotype, and genetic signatures were compared in the lungs of Nrf2 knockout (-/-, KO) vs. wildtype (+/+, WT) mice. Nrf2 KO mice were more susceptible to vinyl carbamate; these mice developed tumors earlier than the WT mice and exhibited more and larger tumors over times ranging from 20-32 weeks after initiation. By 32 weeks, WT mice have an average of 6.5 ± 0.95 tumors per lung compared to 12.25 ± 2.6 tumors in the KO mice. Immune cell populations including T cells (CD4+ and CD8+), B cells, macrophages and myeloid derived suppressor cells were investigated in the lungs and spleens of Nrf2 WT and KO mice challenged with vinyl carbamate. T cell populations were significantly (P < 0.05) reduced in the lungs of Nrf2 KO mice, while tumor-promoting macrophages and myeloid derived suppressor cells were elevated in the lung and spleen, respectively, of Nrf2 KO mice compared to WT mice. Moreover, immune response genes, especially a series of cytokines (Cxcl1, Cxcl12, Csf1, and Ccl9) and MHC antigens that promote tumor growth, were significantly upregulated in tumors from Nrf2-/- mice. Importantly, this expression profile is conserved in patients with lung cancer. Overall, our results confirmed a tumor protective role of Nrf2 in carcinogenesis and suggest an important regulatory role for Nrf2 in immune cells in both mouse and human lung tumors. Citation Format: Di Zhang, Jonathan Rennhack, Eran Andrechek, Cheryl Rockwell, Karen Liby. Different immune signatures in advanced lung tumors of Nrf2 wildtype vs. knockout mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4051.

Critical Regulation of Angiogenesis by Nrf2 Signaling Is Absent in Diabetic Wound Healing

Diabetes-associated ulcers account for 11% of non-traumatic limb and extremity amputations. Tailoring therapies for chronic diabetic wounds is challenging, however, as specific molecular/cellular events responsible for the extreme delays or lack of wound healing mostly remain at large. We previously identified dysfunction of the cytoprotective Nrf2/Keap1 signaling pathway induced by chronic hyperglycemia and diabetes. In the diabetic wound bed, we found detrimental impact on angiogenesis which is a requisite for tissue repair. Exogenous activation of Nrf2 in the whole diabetic wound bed accelerated healing and illustrated the promise of targeting Nrf2 activity for reversing diabetes-associated impairments. Here, we explored the specific role of Nrf2 in endothelial cells of the wound tissue repair niche, and corresponding deficiencies in Nrf2 activity during diabetic wound healing. We generated Cadherin5-CreER;Nrf2fl/fl mice to deplete Nrf2 specifically in endothelial cells, upon tamoxifen administration (Nrf2KO mice). Stented excisional wounds (10mm diameter) on Nrf2KO mice, induced prior to wounding, required 33±1.23 days for closure. This delayed repair phenotype was significantly higher than that of wild type wounds requiring 14.5±0.29 days (p<0.05), but very similar to the 31.5±0.65 days required by wounds on Leprdb/db diabetic mice. Successful inhibition of Nrf2 in microvasculature-derived endothelial cells in vitro demonstrated compromised proliferation (7.12±1.19 units), compared to cells transfected with non-sense siRNA (11.37±0.96 units, p<0.05). Endothelial cells with silenced Nrf2 also demonstrated less angiogenic capacity in vitro, unable to branch or form nodes. Our data prove that Nrf2 activity regulates endothelial cell proliferation and angiogenesis among features that are vital during wound repair. Our results reinforce the necessity of Nrf2 activation in skin for wound repair and underscore the impact of its deficiency in diabetic skin. Disclosure P.S. Rabbani: None. J.A. David: None. D.L. Sultan: None. A.P. Villarreal-Ponce: None. J. Kwong: None. J. Lee: None. C. Shen: None. S.A. Abdou: None. D. Ceradini: None.

P-198 - Relationship between aspirin-induced small intestinal mucosal injury and oxidative stress

Background Endoscopic observation has clearly demonstrated the intestinal mucosal damage induced by aspirin, however, its mechanism remains unclear. We have reported that aspirin induces oxidative stress in the small intestinal epithelial cells. Aim The aim of this study is to investigate the involvement of oxidative stress on aspirin-induced small intestinal mucosal damage by using nuclear factor-erythroid 2-related factor 2 knock out (nrf2KO) mice, in which anti-oxidative defense system is disturbed. Method Under the anesthesia, aspirin (2–10 mg/body) suspended in 0.5% methylcellulose was injected into the proximal duodenum of C57Bl/6 wild type (WT) and nrf2KO mice (male, 8 weeks old). At 1.5 h after treatment, the mice were killed with ether and the small intestine was removed for histological examination. Result Aspirin significantly caused small intestinal mucosal injury in WT mice as assessed histologically in a concentration-dependent manner. Although low concentration of aspirin did not cause small intestinal mucosal injury in WT mice, it caused significant mucosal damage in nrf2KO mice. Conclusion The deficient of nrf2-driven oxidative stress detoxification pathway is important for the protection against aspirin-induced small intestinal mucosal injury.

Protective Role of Nuclear Factor Erythroid 2-Related Factor 2 Against Respiratory Syncytial Virus and Human Metapneumovirus Infections.

The pathogenesis of respiratory syncytial virus (RSV) infections is characterized by lower airway obstruction driven at great extent by the exuberant production of inflammatory cytokines. We have previously shown that RSV infection in vitro and in vivo results in production of reactive oxygen species along with reduction in the expression of antioxidant enzymes (AOEs), which are involved in maintaining the cellular oxidant–antioxidant balance. These events were associated with the concomitant reduction in nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor that controls AOE expression. The objective of the current study was to establish the role of Nrf2 in shaping innate immune responses, clinical disease, airway inflammation, and viral replication in established experimental models of intranasal RSV and human metapneumovirus (hMPV) infections, by employing mice genetically deficient for the Nrf2 gene. Compared to control wild type (WT), mice genetically deficient in Nrf2 (Nrf2 KO) developed enhanced clinical disease, airway inflammation and pathology, and significantly greater lung viral titers following experimental infection with either RSV or hMPV. In particular, compared to control mice, RSV-infected Nrf2 KO mice lost more body weight and had increased airway obstruction at time points characterized by a remarkable increase in inflammatory cytokines and airway neutrophilia. Airway levels of AOEs and enzymes that regulate synthesis of the endogenous hydrogen sulfide (H2S) pathway, which we showed to play an important antiviral function, were also decreased in RSV-infected Nrf2 KO compared to WT. In conclusion, these results suggest that Nrf2 is a critical regulator of innate, inflammatory, and disease-associated responses in the airways of mice infected with viruses that are members of the Pneumoviridae family. Importantly, the results of this study suggest that Nrf2-dependent genes, including those controlling the cellular antioxidant and H2S-generating enzymes and cytokines can affect several aspects of the antiviral response, such as airway neutrophilia, clinical disease, airway obstruction, and viral replication.

NSMase2 (Type 2-Neutral Sphingomyelinase) Deficiency or Inhibition by GW4869 Reduces Inflammation and Atherosclerosis in Apoe-/- Mice.

Atherosclerosis is a chronic multifactorial and inflammatory disease of large and medium arteries and the leading cause of cardiovascular diseases worldwide. The aim of this study was to investigate whether and how the nSMase2 (type 2-neutral sphingomyelinase), a key enzyme of sphingolipid metabolism, may contribute to the development of atherosclerotic lesions. The role of nSMase2 in atherosclerosis was investigated in Apoe-/-;Smpd3fro/fro mice, mutant for nSMase2, and in Apoe-/-;Smpd3+/+ mice intraperitoneally injected with GW4869, a pharmacological nSMase2 inhibitor. The defect or inhibition of nSMase2 resulted in a reduction of atherosclerotic lesions and a decrease in macrophage infiltration and lipid deposition, although cholesterolemia remained unchanged. nSMase2 inhibition decreased the inflammatory response of murine endothelial cells to oxLDL (oxidized low-density lipoprotein), as assessed by the significant reduction of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) mRNA expressions and macrophage recruitment. Likewise, in RAW264.7 or in macrophages isolated from Apoe-/-/Smpd3fro/fro or Apoe-/-/Smpd3+/+ mice stimulated by lipopolysaccharides, nSMase2 inhibition resulted in a decrease in the expression of inflammatory molecules. Mechanistically, the anti-inflammatory response resulting from nSMase2 inhibition involves Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or NF-E2-related factor-2) activation in both endothelial cells and macrophages, as assessed by the lack of protective effect of GW4869 in endothelial cells silenced for Nrf2 by small interfering RNAs, and in lipopolysaccharide-stimulated macrophages issued from Nrf2-KO mice. The genetic deficiency or inhibition of nSMase2 strongly decreases the development of atherosclerotic lesions in Apoe-/- mice, by reducing inflammatory responses through a mechanism involving the Nrf2 pathway. Inhibitors of nSMase2 may, therefore, constitute a novel approach to slow down atherosclerosis progression.

Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 via stimulating the Akt/GSK-3ß/Fyn pathway

AimsActivation of nuclear factor erythroid 2-related factor 2 (Nrf2) by sulforaphane (SFN) protects from, and deletion of the Nrf2 gene exaggerates, diabetic cardiomyopathy. Angiotensin II (Ang II) plays a critical role in the development of diabetic cardiomyopathy. Therefore, whether SFN prevents Ang II-induced cardiomyopathy through activation of Nrf2 was examined using wild-type, global deletion of Nrf2 gene (Nrf2-KO) and cardiomyocyte-specific overexpression of Nrf2 gene (Nrf2-TG) mice. Methods and resultsAdministration of a subpressor dose of Ang II to wild-type mice induced cardiac oxidative stress, inflammation, remodeling and dysfunction, all of which could be prevented by SFN treatment with Nrf2 up-regulation and activation. Nrf2-KO mice are susceptible, and Nrf2-TG mice are resistant, respectively, to Ang II-induced cardiomyopathy. Meanwhile, the ability of SFN to protect against Ang II-induced cardiac damage was lost in Nrf2-KO mice. Up-regulation and activation of Nrf2 by SFN is accompanied by activation of Akt, inhibition of glycogen synthase kinase (GSK)-3β, and accumulation of Fyn in nuclei. In vitro up-regulation of Nrf2 by SFN was abolished and nuclear Fyn accumulation was increased when cardiac cells were exposed to a PI3K inhibitor or GSK-3β-specific activator. ConclusionThese results suggest that Nrf2 plays a central role in the prevention of Ang II-induced cardiomyopathy, and SFN prevents Ang II-induced cardiomyopathy partially via the Akt/GSK-3β/Fyn-mediated Nrf2 activation.

Nrf2 Deficiency Unmasks the Significance of Nitric Oxide Synthase Activity for Cardioprotection.

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch that controls the expression of antioxidant and cytoprotective enzymes, including enzymes catalyzing glutathione de novo synthesis. In this study, we aimed to analyze whether Nrf2 deficiency influences antioxidative capacity, redox state, NO metabolites, and outcome of myocardial ischemia reperfusion (I/R) injury. In Nrf2 knockout (Nrf2 KO) mice, we found elevated eNOS expression and preserved NO metabolite concentrations in the aorta and heart as compared to wild types (WT). Unexpectedly, Nrf2 KO mice have a smaller infarct size following myocardial ischemia/reperfusion injury than WT mice and show fully preserved left ventricular systolic function. Inhibition of NO synthesis at onset of ischemia and during early reperfusion increased myocardial damage and systolic dysfunction in Nrf2 KO mice, but not in WT mice. Consistent with this, infarct size and diastolic function were unaffected in eNOS knockout (eNOS KO) mice after ischemia/reperfusion. Taken together, these data suggest that eNOS upregulation under conditions of decreased antioxidant capacity might play an important role in cardioprotection against I/R. Due to the redundancy in cytoprotective mechanisms, this fundamental antioxidant property of eNOS is not evident upon acute NOS inhibition in WT mice or in eNOS KO mice until Nrf2-related signaling is abrogated.

SP600125 suppresses Keap1 expression and results in NRF2-mediated prevention of diabetic nephropathy.

c-Jun N-terminal kinase (JNK) contributes to the pathogenesis of diabetic nephropathy (DN). The JNK inhibitor SP600125 was reported to ameliorate DN. However, the mechanism remained unclear. We previously reported that SP600125 activated nuclear factor erythroid 2-related factor 2 (NRF2), a governor of the cellular antioxidant defense system, in the aortas of the diabetic mice. Given the critical role of NRF2 in preventing DN, the present study aimed to test whether or not NRF2 is required for SP600125's protection against DN. To test the role of NRF2 in SP600125's effect, streptozotocin-induced C57BL/6 wild-type (WT) and Nrf2-knockout (KO) diabetic mice were treated in the presence or absence of SP600125, for 24 weeks. To explore the mechanism by which SP600125 activates NRF2, mouse mesangial cells (MMCs) were treated with high glucose (HG), in the presence or absence of either SP600125 or JNK siRNA. SP600125 significantly attenuated the diabetes-induced renal oxidative stress, inflammation, fibrosis, pathological change and dysfunction in the WT, but not the Nrf2 KO mice. SP600125 inactivated JNK, inhibited kelch-like ECH-associated protein 1 expression, preserved NRF2 protein and facilitated its nuclear translocation in the kidneys of the WT mice, the effects of which were similarly produced by either SP600125 or JNK siRNA in HG-treated MMCs. Further, both SP600125 and JNK siRNA alleviated HG-induced mesangial oxidative stress and expression of inflammatory and fibrotic genes. The present study demonstrates that NRF2 is required for SP600125's protection against DN. SP600125 activates NRF2 possibly via inhibition of JNK-induced Keap1 expression.

Effects of Nrf2 deficiency on arsenic metabolism in mice

Inorganic arsenic (iAs) is a known toxicant and carcinogen. Worldwide arsenic exposure has become a threat to human health. The severity of arsenic toxicity is strongly correlated with the speed of arsenic metabolism (methylation) and clearance. Furthermore, oxidative stress is recognized as a major mechanism for arsenic-induced toxicity. Nuclear factor-E2-related factor 2 (Nrf2), a key regulator in cellular adaptive antioxidant response, is clearly involved in alleviation of arsenic-induced oxidative damage. Multiple studies demonstrate that Nrf2 deficiency mice are more vulnerable to arsenic-induced intoxication. However, what effect Nrf2 deficiency might have on arsenic metabolism in mice is still unknown. In the present study, we measured the key enzymes involved in arsenic metabolism in Nrf2-WT and Nrf2-KO mice. Our results showed that basal transcript levels of glutathione S-transferase omega 2 (Gsto2) were significantly higher and GST mu 1 (Gstm1) lower in Nrf2-KO mice compared to Nrf2-WT control. Arsenic speciation and methylation rate in liver and urine was then studied in mice treated with 5mg/kg sodium arsenite for 12h. Although there were some alterations in arsenic metabolism enzymes between Nrf2-WT and Nrf2-KO mice, the Nrf2 deficiency had no significant effect on arsenic methylation. These results suggest that the Nrf2-KO mice are more sensitive to arsenic than Nrf2-WT mainly because of differences in adaptive antioxidant detoxification capacity rather than arsenic methylation capacity.

Inhibition of valproic acid-induced neural tube defects through Nrf2 signaling

Valproic acid (VPA) is an effective antiepileptic drug but is also a known human teratogen that causes neural tube defects (NTDs). The mechanisms responsible for VPA-induced embryopathy remain poorly understood, but various studies suggest that VPA-induced oxidative stress is central to its involvement in its developmental toxicity. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor that activates the cellular antioxidant response and upregulates detoxification enzymes, potentially preventing the disruption of redox-sensitive signaling. The aim of this study is to elucidate if VPA-induced NTDs can be attenuated though Nrf2 induction using both in vivo and ex vivo mouse models. Gestational day 8.0 mice were treated with or without 5 μM D3T, a Nrf2 inducer, and were then treated with 600 μM VPA on GD 8.6 and collected periodically over an 18 h period for glutathione (GSH) redox potential (Eh) and morphology assessment on GD 9.0. In embryos treated with VPA only, GSH Eh increased by over +20 mV at 2 h post VPA treatment and then slowly rebounded, nearing untreated control GSH Eh levels by 18 h. Pretreatment with D3T prevented any changes to GSH Eh. At 24 h, 58% of embryos treated with only VPA exhibited NTDs, but NTD rates dropped to 5% with D3T pretreatment. Embryos from WT mice treated on GD 8.0 in utero and evaluated on GD 9.0 demonstrated similar NTD rates, but the prevalence of NTDs nearly doubled in Nrf2 KO embryos. To further understand the role of Nrf2 in NTDs, both WT and Nrf2 KO mice were treated with D3T (i.p. 5 mg/kg) on GD 7.12, followed by treatment with VPA (s.c. 400mg/kg) on gestational day 8.0. D3T pretreatments significantly reduced NTDs in WT embryos but failed to reduce rates in KO mice treated with VPA. These data suggest that mitigation of VPA-induced redox imbalance in the early organogenesis stage mouse embryo is critical for NTD prevention and that Nrf2 is a protective factor during development that requires further investigation.

Hydrogen Gas Protects Against Intestinal Injury in Wild Type But Not NRF2 Knockout Mice With Severe Sepsis by Regulating HO-1 and HMGB1 Release.

The intestine plays an important role in the pathogenesis of sepsis. Hydrogen gas (H2), which has anti-oxidative, anti-inflammatory, and anti-apoptotic effects, can be effectively used to treat septic mice. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive master switch that regulates the expression of antioxidant and protective enzymes. This study investigated the effects of 2% H2 on intestinal injuries and the underlying mechanisms in a mouse model of severe sepsis. Male Nrf2 knockout mice (Nrf2-KO) and wild-type (WT) mice were randomized into four groups: sham, sham+H2, cecal ligation and puncture (CLP), and CLP+H2. The survival rate was observed and recorded within 7 days, and pro-inflammatory cytokines (TNF-α, IL-6, HMGB1), anti-inflammatory cytokine (IL-10), antioxidant enzymes (superoxide dismutase, and catalase ), and oxidative products (MDA, 8-iso-PGF2α) were detected in the serum and intestine using an enzyme-linked immunosorbent assay. In addition, the protein and mRNA levels of heme oxygenase-1 (HO-1) and high mobility group box 1 (HMGB1) were measured by Western blotting and quantitative PCR, respectively. Immunofluorescence and immunohistochemistry were used to measure HMGB1 and HO-1 release into the intestine, respectively. The results showed that therapy with 2% H2 increased the survival rate, alleviated the injuries caused by oxidative stress and inflammation, reduced HMGB1 levels but increased HO-1 levels in WT septic mice, but not in Nrf2-KO mice. These data demonstrate that 2% H2 inhalation may be a promising therapeutic strategy for intestinal injuries caused by severe sepsis through the regulation of HO-1 and HMGB1 release. In addition, Nrf2 plays a key role in the protective effects of H2 against intestinal damage in this disease.

P 089 - NRF2/KEAP1-mediated antioxidant defence pathway regulates skeletal muscle circadian clock function

The circadian clock is an intrinsic timing mechanism which regulates most physiological processes within the body. Disruption of circadian rhythms has been identified as a risk factor for several chronic diseases with altered redox control. We investigated the hypothesis that NRF2, a master regulator of antioxidant defence, is a clock-controlled gene in skeletal muscle, which can modulate the circadian clock function. Using genetic, pharmacological and real-time imaging approaches, we identified a feedback mechanism between the circadian clock machinery and the NRF2/KEAP1 antioxidant pathway. Pharmacological manipulation of NRF2 exerted robust effects on both the amplitude and periodicity of circadian clock oscillations. Moreover, single muscle fibre isolation has confirmed cell-autonomous changes in core clock gene expression in fibres from Nrf2 KO mice. Interestingly, similar effects on clock gene expression were also evident in muscle fibres isolated from old wild-type mice. Loss of Nrf2, or ageing led to diminished clock gene cycles but activated rhythmic gene cycles of genes involved in inflammation and stress resistance. All together, these findings implicate NRF2 as an important therapeutic target which may be utilised in future to reset or re-align disrupted circadian rhythms seen in several chronic diseases associated with muscle wasting including COPD and sarcopenia.

Loss of Nrf2 promotes rapid progression to heart failure following myocardial infarction

Nrf2 gene encodes a transcription factor regulating the expression of antioxidant and detoxification genes. We test here whether Nrf2 plays a role for cardiac protection during ischemic injury in an effort to establish Nrf2 as a target for cardiac protection therapies. Cardiac ischemia induced by the left anterior descending (LAD) coronary artery ligation results in myocardial infarction (MI). Young mice surviving MI show minimal signs of heart failure. Mice lacking Nrf2 experience an accelerated progression to heart failure that occurs within 10days following induction of MI. Nrf2 knockout (Nrf2 KO) mice have a survival rate similar to wild type (WT) mice at 24h after MI, but a significantly higher mortality rate within 10days after MI (50% vs 86%). Morphological examination revealed maladaptive remodeling, including cardiac hypertrophy and dilated left ventricle in Nrf2 KO mice, which were absent in WT mice. Measurements of cardiac function revealed increased left ventricular mass and decreases in cardiac output in Nrf2 KO mice. In addition, Nrf2 KO mice show biomarkers of heart failure, such as elevated levels of β-MHC, ANF, and BNP mRNA in the myocardium. These data support that Nrf2 plays an important role in protecting the myocardium from ischemic injury. Lack of Nrf2 leads to rapid development of heart failure.

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism.

SummarySenescent cells contribute to age‐related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild‐type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA‐β‐galactosidase (β‐gal) staining, senescence‐associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β‐gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β‐gal staining and pro‐inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.

Abstract WP98: Role of Hydrogen Peroxide in Preconditioning of Microglia to Achieve Improved Hematoma Cleanup After ICH

Background: Red blood cells ( RBC ) and other blood components deposited in brain parenchyma during intracerebral hemorrhage ( ICH ) are the source of secondary brain injury, inflammation, and oxidative stress. Therefore, a fast and efficient removal of the blood from the brain is essential for ameliorating secondary injury and for recovery and repair process. Microglia/macrophages ( MΦ )-mediated phagocytosis is a key component of hematoma clearance after ICH. However, the high levels of pro-oxidative molecules (including H 2 O 2 ) generated by MΦ during phagocytosis could be highly cytotoxic not only to brain cells, but also to MΦ themselves. Thus, an efficient coupling between MΦ-mediated phagocytosis and anti-oxidative processes is essential for the safe and efficient hematoma cleanup. Methods and Results: H 2 O 2 at higher concentrations is known to be neurotoxic. In this study, we established that H 2 O 2 at submicromolar, biologically relevant concentrations, acts as a pro-survival factor for MΦ. By activating transcription factor nuclear factor-erythroid 2 p45-related factor 2 ( Nrf2 ), a master regulator of anti-oxidative regulation, H 2 O 2 stimulates the expression of many antioxidant proteins, which protects MΦ from oxidative injury and damaging components of inflammation. We established that Nrf2-deficient MΦ are more susceptible to H 2 O 2 -mediated or toxic blood components-mediated damage (“ICH-like” injury). Nrf2-KO mice subjected to ICH experienced more severe brain edema and delayed hematoma resolution. In addition, the phagocytosis of RBCs (in vitro model of hematoma clearance) was significantly diminished in MΦ treated with Nrf2 decoy or in MΦ that were harvested from Nrf2-KO mice. On the other hand, pharmacologic activation of Nrf2 or administration of low dosages of H 2 O 2 improved phagocytic capacity of MΦ toward RBC. Furthermore, low levels of H 2 O 2 protected MΦ from ICH-like injury, suggesting that improved phagocytic function could involve preservation of MΦ’ integrity by activating Nrf2. Conclusion: H 2 O 2 at low doses could protect MΦ for oxidative injury and facilitate their phagocytic function, which could benefit hematoma clearance, inflammation resolution, and improve neurological recovery after ICH.

Prevention of Streptozotocin-Induced Diabetic Nephropathy by MG132: Possible Roles of Nrf2 and IκB.

Our previous study showed that proteasomal inhibitor MG132 can prevent diabetic nephropathy (DN) along with upregulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The present study was to investigate whether MG132 can prevent DN in wild-type and Nrf2-KO mice. Type 1 diabetes was induced in wild-type and Nrf2-KO mice by multiple low doses of streptozotocin. Two weeks after streptozotocin injection, both wild-type and Nrf2-KO mice were randomly divided into four groups: control, MG132, DM, and DM/MG132. MG132 (10 μg/kg/day) or vehicle was administered intraperitoneally for 4 months. Renal function, morphology, and biochemical changes were measured after 4-month treatment with MG132. MG132 treatment suppressed proteasomal activity in the two genotypes. In wild-type mice, MG132 attenuated diabetes-induced renal dysfunction, fibrosis, inflammation, and oxidative damage along with increased Nrf2 and IκB expression. Deletion of Nrf2 gene resulted in a partial, but significant attenuation of MG132 renal protection in Nrf2-KO mice compared with wild-type mice. MG132-increased IκB expression was not different between wild-type and Nrf2-KO mice. This work indicates that MG132 inhibits diabetes-increased proteasomal activity, resulting in Nrf2 and IκB upregulation and renal protection, which could be used as a strategy to prevent diabetic nephropathy.