Pharmacological effects of methysticin and L-sulforaphane through the Nrf2/ARE signaling pathway in MLO-Y4 osteocytes: in vitro study.

BACKGROUND: Oxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway. METHODS: MLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining. RESULTS: The experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions. CONCLUSIONS: Nrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

Hepatocellular loss of mTOR aggravates tumor burden in nonalcoholic steatohepatitis-related HCC.

Obesity and associated nonalcoholic steatohepatitis (NASH) are on the rise globally. NASH became an important driver of hepatocellular carcinoma (HCC) in recent years. Activation of the central metabolic regulator mTOR (mechanistic target of rapamycin) is frequently observed in HCCs. However, mTOR inhibition failed to improve the outcome of HCC therapies, demonstrating the need for a better understanding of the molecular and functional consequences of mTOR blockade. We established a murine NASH-driven HCC model based on long-term western diet feeding combined with hepatocellular mTOR-inactivation. We evaluated tumor load and whole-body fat percentage via µCT-scans, analyzed metabolic blood parameters and tissue proteome profiles. Additionally, we used a bioinformatic model to access liver and HCC mitochondrial metabolic functions. The tumor burden was massively increased via mTOR-knockout. Several signs argue for extensive metabolic reprogramming of glucose, fatty acid, bile acid and cholesterol metabolism. Kinetic modeling revealed reduced oxygen consumption in KO-tumors. NASH-derived HCC pathogenesis is driven by metabolic disturbances and should be considered separately from those caused by other etiologies. We conclude that mTOR functions as tumor suppressor in hepatocytes especially under long-term western diet feeding. However, some of the detrimental consequences of this diet are attenuated by mTOR blockade.

The Contribution of the Nrf2/ARE System to Mechanotransduction in Musculoskeletal and Periodontal Tissues.

Mechanosensing plays an essential role in maintaining tissue functions. Across the human body, several tissues (i.e., striated muscles, bones, tendons, ligaments, as well as cartilage) require mechanical loading to exert their physiological functions. Contrary, mechanical unloading triggers pathological remodeling of these tissues and, consequently, human body dysfunctions. At the cellular level, both mechanical loading and unloading regulate a wide spectrum of cellular pathways. Among those, pathways regulated by oxidants such as reactive oxygen species (ROS) represent an essential node critically controlling tissue organization and function. Hence, a sensitive balance between the generation and elimination of oxidants keeps them within a physiological range. Here, the Nuclear Factor-E2-related factor 2/Antioxidant response element (Nrf2/ARE) system plays an essential role as it constitutes the major cellular regulation against exogenous and endogenous oxidative stresses. Dysregulations of this system advance, i.a., liver, neurodegenerative, and cancer diseases. Herein, we extend our comprehension of the Nrf2 system to the aforementioned mechanically sensitive tissues to explore its role in their physiology and pathology. We demonstrate the relevance of it for the tissues' functionality and highlight the imperative to further explore the Nrf2 system to understand the physiology and pathology of mechanically sensitive tissues in the context of redox biology.

SULFORAPHANE ADMINISTRATION AFTER HEMORRHAGIC SHOCK/RESUSCITATION IN MICE REDUCES THE SECRETION OF INFLAMMATORY CYTOKINES AND INCREASES THE IMMUNOCOMPETENCE OF SPLENIC MACROPHAGES.

ABSTRACT: Objective : The purpose of this study was to investigate the immunomodulatory effects of sulforaphane (SFN), a nuclear factor erythroid 2-related factor (Nrf2) pathway activator, on splenic macrophages' immunocompetence after hemorrhagic shock/resuscitation (HS/R). Methods : Male C57/BL6 wild-type mice (n = 6 per group) were subjected to either pressure-controlled HS (MAP, 35-45 mm Hg) or a sham procedure surgery (without HS). After 90 minutes of HS, fluid resuscitation with withdrawn blood and 0.9% NaCl was performed. Sulforaphane (50 mg/kg of body weight) was applied intraperitoneally immediately after the resuscitation phase as well as 24 and 48 h thereafter, depending on group allocation. The mice were killed at 6, 24, and 72 h after resuscitation. After killing, spleens were harvested to perform Nrf2 immunofluorescence histology. Splenic macrophages were isolated and cultured to measure cytokine secretion in the cell culture supernatant. Furthermore, macrophages isolated after 24-hour resuscitation were treated with 100 ng/mL of bacterial LPS to measure immunocompetence. Matrix-assisted laser desorption/ionization mass spectrometry imaging was performed to verify the distribution of SFN in the spleen after intraperitoneal injection. Results : We showed that administered SFN reached the spleen within the first hour after administration. Furthermore, we identified that SFN increased splenic Nrf2 activation and decreased cytokine expression in splenic macrophages after HS/R. In addition, we showed that SFN exhibited splenic anti-inflammatory properties of macrophages in vitro (IL-6/IL-10-ratio of the HS/R group: 51.79 ± 9.99 [at 6 h] and 15.70 ± 3.35 [at 24 h] vs. HS/R + SFN group: 20.54 ± 5.35 [at 6 h] and 8.60 ± 2.37 [at 24 h], P < 0.05). Furthermore, SFN improved in vitro splenic macrophage immunocompetence after HS/R, as evidenced by the increased secretion of inflammatory cytokines in response to LPS stimulation in vitro . Conclusions : Our study shows that SFN can reduce inflammatory cytokines secreted by splenic macrophages after HS/R and increase their immunocompetence toward a more anti-inflammatory profile.

Nuclear factor erythroid 2-related factor 2 (Nrf2) deficiency causes age-dependent progression of female osteoporosis.

BACKGROUND: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial transcription factor for cellular redox homeostasis. The association of Nrf2 with elderly female osteoporotic has yet to be fully described. The aim was to elucidate a potential age-dependent Nrf2 contribution to female osteoporosis in mice. METHODS: Eighteen female wild type (WT) and 16 Nrf2-knockout (KO) mice were sacrificed at different ages (12 weeks = young mature adult and 90 weeks = old) to analyze their femurs. The morphological properties (trabecular and cortical) were evaluated by micro-computed tomography (µCT) and compared to gold standard histochemistry analysis. The quasi-static compression tests were performed to calculate the mechanical properties of bones. Additionally, the population of bone resorbing cells and aromatase expression by osteocytes was immunohistochemically evaluated and empty osteocyte lacunae was counted in cortical bone. RESULTS: Old Nrf2-KO mice revealed a significantly reduced trabecular bone mineral density (BMD), cortical thickness, cortical area, and bone fraction compared to old WT mice, regardless of no significant difference in skeletally mature young adult mice between WT and KO. Specifically, while all old WT mice showed thin metaphyseal trabeculae, trabecular bone was completely absent in 60% of old KO mice. Additionally, old KO mice showed significantly more osteoclast-like cells and fewer aromatase-positive osteocytes than WT mice, whereas the occurrence of empty osteocyte lacunae did not differ between both groups. Nrf2-KO mice further showed an age-dependently reduced fracture resilience compared to age-matched WT mice. CONCLUSION: Our results suggest that chronic Nrf2 loss can lead to age-dependent progression of female osteoporosis.

Nrf2 induces malignant transformation of hepatic progenitor cells by inducing ß-catenin expression.

The Nrf2 signaling pathway prevents cancer initiation, but genetic mutations that activate this pathway are found in various types of cancer. The molecular mechanisms underlying this Janus-headed character are still not understood. Here, we show that sustained Nrf2 activation induces proliferation and dedifferentiation of a Wnt-responsive perivenular hepatic progenitor cell population, transforming them into metastatic cancer cells. The neoplastic lesions display many histological features known from human hepatoblastoma. We describe an Nrf2-induced upregulation of ß-catenin expression and its activation as the underlying mechanism for the observed malignant transformation. Thus, we have identified the Nrf2-ß-catenin axis promoting proliferation of hepatic stem cells and triggering tumorigenesis. These findings support the concept that different functional levels of Nrf2 control both the protection against various toxins as well as liver regeneration by activating hepatic stem cells. Activation of the hepatic stem cell compartment confers the observation that unbridled Nrf2 activation may trigger tumorigenesis.

Physiological Mineralization during In Vitro Osteogenesis in a Biomimetic Spheroid Culture Model.

Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in α-MEM with 10% FCS, at 37 °C and 5% CO2 for up to 28 days, in non-adherent V-shaped plates to form uniformly sized 3D spheroids. Osteogenic differentiation was induced by 10 mM ß-glycerophosphate and 50 µg/mL ascorbic acid. Mineralization stages were assessed through studying expression of marker genes, alkaline phosphatase activity, and calcium deposition by histochemistry. Mineralization quality was evaluated by Fourier transformed infrared (FTIR) and scanning electron microscopic (SEM) analyses and quantified by micro-CT analyses. Expression profiles of selected early- and late-stage osteoblast differentiation markers indicated a well-developed 3D biomineralization process with strongly upregulated Col1a1, Bglap and Alpl mRNA levels and type I collagen- and osteocalcin-positive immunohistochemistry (IHC). A dynamic biomineralization process with increasing mineral densities was observed during the second half of the culture period. SEM-Energy-Dispersive X-ray analyses (EDX) and FTIR ultimately confirmed a native bone-like hydroxyapatite mineral deposition ex vivo. We thus established a robust and versatile biomimetic, and high-throughput compatible, cost-efficient spheroid culture model with a native bone-like mineralization for improved pharmacological ex vivo screenings.

Siponimod ameliorates metabolic oligodendrocyte injury via the sphingosine-1 phosphate receptor 5.

Multiple sclerosis (MS), an autoimmune-driven, inflammatory demyelinating disease of the central nervous system (CNS), causes irreversible accumulation of neurological deficits to a variable extent. Although there are potent disease-modifying agents for its initial relapsing-remitting phase, immunosuppressive therapies show limited efficacy in secondary progressive MS (SPMS). Although modulation of sphingosine-1 phosphate receptors has proven beneficial during SPMS, the underlying mechanisms are poorly understood. In this project, we followed the hypothesis that siponimod, a sphingosine-1 phosphate receptor modulator, exerts protective effects by direct modulation of glia cell function (i.e., either astrocytes, microglia, or oligodendrocytes). To this end, we used the toxin-mediated, nonautoimmune MS animal model of cuprizone (Cup) intoxication. On the histological level, siponimod ameliorated cuprizone-induced oligodendrocyte degeneration, demyelination, and axonal injury. Protective effects were evident as well using GE180 translocator protein 18-kDa (TSPO) imaging with positron emission tomography (PET)/computed tomography (CT) imaging or next generation sequencing (NGS). Siponimod also ameliorated the cuprizone-induced pathologies in Rag1-deficient mice, demonstrating that the protection is independent of T and B cell modulation. Proinflammatory responses in primary mixed astrocytes/microglia cell cultures were not modulated by siponimod, suggesting that other cell types than microglia and astrocytes are targeted. Of note, siponimod completely lost its protective effects in S1pr5-deficient mice, suggesting direct protection of degenerating oligodendrocytes. Our study demonstrates that siponimod exerts protective effects in the brain in a S1PR5-dependent manner. This finding is not just relevant in the context of MS but in other neuropathologies as well, characterized by a degeneration of the axon-myelin unit.

Astrocytic Nrf2 expression protects spinal cord from oxidative stress following spinal cord injury in a male mouse model.

BACKGROUND: Spinal cord injury (SCI) induces a multitude of deleterious processes, including neuroinflammation and oxidative stress (OS) which contributed to neuronal damage and demyelination. Recent studies have suggested that increased formation of reactive oxygen species (ROS) and the consequent OS are critical events associated with SCI. However, there is still little information regarding the impact of these events on SCI. Astrocytes are key regulators of oxidative homeostasis in the CNS and astrocytic antioxidant responses promote the clearance of oxidants produced by neurons. Therefore, dysregulation of astrocyte physiology might largely contribute to oxidative damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the main transcriptional regulator of cellular anti-oxidative stress responses. METHODS: In the current study, we hypothesized that astrocytic activation of Nrf2 protects the spinal cord post injury via suppression of neuroinflammation. Thus, using mice line with a GFAP-specific kelch-like ECH-associated protein 1 (Keap1)-deletion, we induced a hyperactivation of Nrf2 in astrocytes and further its effects on SCI outcomes. SCI-induction was performed in mice using the Infinite Horizon Spinal Cord Impactor with a force of 60 kdyn. To assess the quantitative pattern of Nrf2/ARE-activation, we included transgenic ARE-Luc mice. Data were analyzed with GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Brown-Forsythe test was performed to test for equal variances and normal distribution was tested with Shapiro-Wilk. RESULTS: In ARE-Luc mice, a significant induction of luciferase-activity was observed as early as 1 day post-injury, indicating a functional role of Nrf2-activity at the epicenter of SCI. Furthermore, SCI induced loss of neurons and oligodendrocytes, demyelination and inflammation in wild type mice. The loss of myelin and oligodendrocytes was clearly reduced in Keap1 KO mice. In addition, Keap-1 KO mice showed a significantly better locomotor function and lower neuroinflammation responses compared to wild type mice. CONCLUSIONS: In summary, our in vivo bioluminescence data showed Nrf2-ARE activation during primary phase of SCI. Furthermore, we found that cell specific hyperactivation of Nrf2 was sufficient to protect the spinal cord against injury which indicate a promising therapeutic approach for SCI-treatment.

Neuroprotective effect of the Nrf2/ARE/miRNA145-5p signaling pathway in the early phase of spinal cord injury.

AIMS: Spinal cord injury (SCI) is a debilitating neurological condition often associated with chronic neuroinflammation and redox imbalance. Oxidative stress is one of the main hallmark of secondary injury of SCI which is tightly regulated by nuclear factor E2-related factor 2/antioxidant response element (Nrf2/ARE) signaling. In this study, we aimed at investigating the interplay between inflammation-related miRNAs and the Nrf2 pathway in animal model of SCI. MATERIALS AND METHODS: The expression of selected four validated miRNA-target pairs (miRNA223-3p, miRNA155-5p, miRNA145-5p, and miRNA124-3p) was examined at different time points (6 h, 12 h, 1 day, 3 day and 7 day) after SCI. Further, using GFAP-specific kelch-like ECH-associated protein 1 deletion (Keap1-/-) and whole-body Nrf2-/- knockout mice, we investigated the potential interplay between each miRNA and the Keap1/Nrf2 signaling system. KEY FINDINGS: The expression of all miRNAs except miRNA155-5p significantly increased 24 h after SCI and decreased after 7 days. Interestingly, Keap1-/- mice only showed significant increase in the miRNA145-5p after 24 h SCI compared to the WT group. In addition, Keap1-/- mice showed significant decrease in CXCL10/12 (CXCL12 increased in Nrf2-/- mice), and TNF-α, and an increase in Mn-SOD and NQO-1 (Mn-SOD and NQO-1 decreased in Nrf2-/- mice) compared to WT mice. SIGNIFICANCE: Our results suggest that astrocytic hyperactivation of Nrf2 exert neuroprotective effects at least in part through the upregulation of miRNA145-5p, a negative regulator of astrocyte proliferation, and induction of ARE in early phase of SCI. Further studies are needed to investigate the potential interplay between Nrf2 and miRNA145-5p in neuroinflammatory condition.

Sulforaphane Exerts Beneficial Immunomodulatory Effects on Liver Tissue via a Nrf2 Pathway-Related Mechanism in a Murine Model of Hemorrhagic Shock and Resuscitation.

Our research explores the immunomodulatory effects of sulforaphane (SFN), a well-known nuclear factor erythroid 2-related factor 2 (Nrf2) pathway agonist, on the sterile inflammation of and ischemia-reperfusion injuries to the liver after hemorrhagic shock (HS) followed by resuscitation (R). Male C57/BL6 wild-type and transgenic ARE-luc mice were exposed to mean arterial pressure-controlled HS. Fluid resuscitation was performed after 90 min of HS, and SFN was administrated intraperitoneally after that. The animals were sacrificed at 6 h, 24 h, and 72 h after resuscitation, and their livers were extracted to perform H&E staining and myeloperoxidase (MPO) activity analysis. The Kupffer cells were isolated for cytokines profile measurements and Nrf2 immunofluorescence staining. Further, the ARE-luc mice were used to assess hepatic Nrf2 activity in vivo. We identified that SFN-activated Kupffer cells' Nrf2 pathway and modulated its cytokines expression, including TNF-α, MCP-1, KC/CXCL1, IL-6, and IL-10. Furthermore, SFN mitigated liver ischemia-reperfusion injury, as evidenced by the downregulation of the Suzuki score and the enhanced hepatic Nrf2 activity. The in vivo SFN treatment decreased neutrophils infiltration, as shown by the decreased MPO levels. Our study shows that SFN can decrease HS/R-induced hepatic ischemia-reperfusion injury and modulate the activity of Kupffer cells via an Nrf2-dependent pathway.

Nrf2/ARE Signaling Directly Regulates SOX9 to Potentially Alter Age-Dependent Cartilage Degeneration.

Oxidative stress is implicated in osteoarthritis, and nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway maintains redox homeostasis. We investigated whether Nrf2/ARE signaling controls SOX9. SOX9 expression in human C-28/I2 chondrocytes was measured by RT-qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap ''n'' collar homology-associated protein 1 (Keap1). To verify whether Nrf2 transcriptionally regulates SOX9, putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL-TK for dual-luciferase assays. SOX9 promoter activities without and with Nrf2-inducer methysticin were compared. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Nrf2-specific RNAi significantly decreased SOX9 expression, whereas Keap1-specific RNAi increased it. Putative ARE sites (ARE1, ARE2) were identified in the SOX9 promoter region. ARE2 mutagenesis significantly reduced SOX9 promoter activity, but ARE1 excision did not. Functional ARE2 site was essential for methysticin-mediated induction of SOX9 promoter activity. Young Nrf2-knockout mice revealed significantly lower Sox9-positive chondrocytes, and old Nrf2-knockout animals showed thinner cartilage and more cartilage degeneration. Our results suggest Nrf2 directly regulates SOX9 in articular cartilage, and Nrf2-loss can develop mild osteoarthritis at old age. Pharmacological Nrf2 induction may hold the potential to diminish age-dependent cartilage degeneration through improving SOX9 expression.

The effect of radiofrequency electromagnetic fields (RF-EMF) on biomarkers of oxidative stress in vivo and in vitro: A protocol for a systematic review.

BACKGROUND: Oxidative stress is conjectured to be related to many diseases. Furthermore, it is hypothesized that radiofrequency fields may induce oxidative stress in various cell types and thereby compromise human and animal health. This systematic review (SR) aims to summarize and evaluate the literature related to this hypothesis. OBJECTIVES: The main objective of this SR is to evaluate the associations between the exposure to radiofrequency electromagnetic fields and oxidative stress in experimental models (in vivo and in vitro). METHODS: The SR framework has been developed following the guidelines established in the WHO Handbook for Guideline Development and the Handbook for Conducting a Literature-Based Health Assessment). We will include controlled in vivo and in vitro laboratory studies that assess the effects of an exposure to RF-EMF on valid markers for oxidative stress compared to no or sham exposure. The protocol is registered in PROSPERO. We will search the following databases: PubMed, Embase, Web of Science Core Collection, Scopus, and the EMF-Portal. The reference lists of included studies and retrieved review articles will also be manually searched. STUDY APPRAISAL AND SYNTHESIS METHOD: Data will be extracted according to a pre-defined set of forms developed in the DistillerSR online software and synthesized in a meta-analysis when studies are judged sufficiently similar to be combined. If a meta-analysis is not possible, we will describe the effects of the exposure in a narrative way. RISK OF BIAS: The risk of bias will be assessed with the NTP/OHAT risk of bias rating tool for human and animal studies. We will use GRADE to assess the certainty of the conclusions (high, moderate, low, or inadequate) regarding the association between radiofrequency electromagnetic fields and oxidative stress. FUNDING: This work was funded by the World Health Organization (WHO). REGISTRATION: The protocol was registered on the PROSPERO webpage on July 8, 2021.

Sulforaphane-Dependent Up-Regulation of NRF2 Activity Alleviates Both Systemic Inflammatory Response and Lung Injury After Hemorrhagic Shock/Resuscitation in Mice.

ABSTRACT: Hemorrhagic shock/resuscitation (HS/R) is closely associated with overwhelming oxidative stress and systemic inflammation. As an effective activator of the nuclear factor-erythroid factor 2 related factor 2 (Nrf2) pathway, sulforaphane (SFN) exerts antioxidant and anti-inflammatory effects. We explored SFN's effects on alveolar macrophages (AMs), systemic inflammation, and pulmonary damage in an isolated murine HS/R model. Male C57/BL6 wild type and transgenic antioxidant response element (ARE)-luciferase (luc) mice (both n = 6 per group) were exposed to either pressure-controlled HS/R (mean arterial pressure 35-45 mm Hg for 90 min) or sham procedure (surgery without HS/R) or were sacrificed without intervention (control group). Fluid resuscitation was performed via the reinfusion of withdrawn blood and 0.9% saline. Sulforaphane or 0.9% saline (vehicle) was administrated intraperitoneally. Mice were sacrificed 6, 24, or 72 h after resuscitation. Bioluminescence imaging of ARE-luc mice was conducted to measure pulmonary Nrf2 activity. Plasma was collected to determine systemic cytokine levels. Alveolar macrophages were isolated before measuring cytokines in the supernatant and performing immunofluorescence staining, as well as Western blot for intracellular Nrf2. Histological damage was assessed via the acute lung injury score and wet/dry ratio.Hemorrhagic shock/resuscitation was associated with pulmonary Nrf2 activation. Sulforaphane enhanced pulmonary Nrf2 activity and the Nrf2 activation of AM, while it decreased lung damage. Sulforaphane exerted down-regulatory effects on AM-generated and systemic pro-inflammatory mediators, while it did not have such effects on IL-10.In conclusion, SFN beneficially enhances pulmonary Nrf2 activity and promotes Nrf2 accumulation in AMs' nuclei. This may exert not only local protective effects but also systemic effects via the down-regulation of pro-inflammatory cytokines. The administration of Nrf2 activator post-HS/R may represent an innovative treatment strategy.

Reference Gene Selection for Gene Expression Analyses in Mouse Models of Acute Lung Injury.

qRT-PCR still remains the most widely used method for quantifying gene expression levels, although newer technologies such as next generation sequencing are becoming increasingly popular. A critical, yet often underappreciated, problem when analysing qRT-PCR data is the selection of suitable reference genes. This problem is compounded in situations where up to 25% of all genes may change (e.g., due to leukocyte invasion), as is typically the case in ARDS. Here, we examined 11 widely used reference genes for their suitability in commonly used models of acute lung injury (ALI): ventilator-induced lung injury (VILI), in vivo and ex vivo, lipopolysaccharide plus mechanical ventilation (MV), and hydrochloric acid plus MV. The stability of reference gene expression was determined using the NormFinder, BestKeeper, and geNorm algorithms. We then proceeded with the geNorm results because this is the only algorithm that provides the number of reference genes required to achieve normalisation. We chose interleukin-6 (Il-6) and C-X-C motif ligand 1 (Cxcl-1) as the genes of interest to analyse and demonstrate the impact of inappropriate normalisation. Reference gene stability differed between the ALI models and even within the subgroup of VILI models, no common reference gene index (RGI) could be determined. NormFinder, BestKeeper, and geNorm produced slightly different, but comparable results. Inappropriate normalisation of Il-6 and Cxcl1 gene expression resulted in significant misinterpretation in all four ALI settings. In conclusion, choosing an inappropriate normalisation strategy can introduce different kinds of bias such as gain or loss as well as under- or overestimation of effects, affecting the interpretation of gene expression data.

Adverse Effects of Oxidative Stress on Bone and Vasculature in Corticosteroid-Associated Osteonecrosis: Potential Role of Nuclear Factor Erythroid 2-Related Factor 2 in Cytoprotection.

Significance: Osteonecrosis (ON) is characterized by bone tissue death due to disturbance of the nutrient artery. The detailed process leading to the necrotic changes has not been fully elucidated. Clinically, high-dose corticosteroid therapy is one of the main culprits behind osteonecrosis of the femoral head (ONFH). Recent Advances: Numerous studies have proposed that such ischemia concerns various intravascular mechanisms. Of all reported risk factors, the involvement of oxidative stress in the irreversible damage suffered by bone-related and vascular endothelial cells during ischemia simply cannot be overlooked. Several articles also have sought to elucidate oxidative stress in relation to ON using animal models or in vitro cell cultures. Critical Issues: However, as far as we know, antioxidant monotherapy has still not succeeded in preventing ONFH in humans. To provide this desideratum, we herein summarize the current knowledge about the influence of oxidative stress on ON, together with data about the preventive effects of administering antioxidants in corticosteroid-induced ON animal models. Moreover, oxidative stress is counteracted by nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent cytoprotective network through regulating antioxidant expressions. Therefore, we also describe Nrf2 regulation and highlight its role in the pathology of ON. Future Directions: This is a review of all available literature to date aimed at developing a deeper understanding of the pathological mechanism behind ON from the perspective of oxidative stress. It may be hoped that this synthesis will spark the development of a prophylactic strategy to benefit corticosteroid-associated ONFH patients. Antioxid. Redox Signal. 35, 357-376.

Effects of Strontium-Doped ß-Tricalcium Scaffold on Longitudinal Nuclear Factor-Kappa Beta and Vascular Endothelial Growth Factor Receptor-2 Promoter Activities during Healing in a Murine Critical-Size Bone Defect Model.

It was hypothesized that strontium (Sr)-doped ß-tricalcium phosphate (TCP)-based scaffolds have a positive effect on the regeneration of large bone defects (LBD). Readouts in our mice models were nuclear factor-kappa beta (NF-κB) activity and vascular endothelial growth factor receptor-2 (VEGFR-2) promoter activity during the healing process. A 2-mm critical-size femoral fracture was performed in transgenic NF-κB- and VEGFR-2-luciferase reporter mice. The fracture was filled with a 3D-printed ß-TCP scaffold with or without Sr. A bioluminescence in-vivo imaging system was used to sequentially investigate NF-κB and VEGFR-2 expression for two months. After sacrifice, soft and osseous tissue formation in the fracture sites was histologically examined. NF-κB activity increased in the ß-TCP + Sr group in the latter stage (day 40-60). VEGFR-2 activity increased in the + Sr group from days 0-15 but decreased and showed significantly less activity than the ß-TCP and non-scaffold groups from days 40-60. The new bone formation and soft tissue formation in the + Sr group were significantly higher than in the ß-TCP group, whereas the percentage of osseous tissue formation in the ß-TCP group was significantly higher than in the ß-TCP + Sr group. We analyzed longitudinal VEGFR-2 promoter activity and NF-κB activity profiles, as respective agents of angiogenesis and inflammation, during LBD healing. The extended inflammation phase and eventually more rapid resorption of scaffold caused by the addition of strontium accelerates temporary bridging of the fracture gaps. This finding has the potential to inform an improved treatment strategy for patients who suffer from osteoporosis.

Nrf2 deficiency increases oligodendrocyte loss, demyelination, neuroinflammation and axonal damage in an MS animal model.

Oxidative stress is a pathophysiological hallmark of many CNS diseases, among multiple sclerosis (MS). Accordingly, boosting the astrocytic transcription factor nuclear factor E2-related factor 2 (Nrf2) system in an MS mouse model efficiently ameliorates oligodendrocyte loss, neuroinflammation and axonal damage. Moreover, Dimethylfumarate, an efficient activator of Nrf2, has recently been approved as therapeutic option in MS treatment. Here, we use the cuprizone mouse model of MS to induce oxidative stress, selective oligodendrocyte loss, microglia and astrocyte activation as well as axonal damage in both wild type and Nrf2-deficient mice. We found increased oligodendrocyte apoptosis and loss, pronounced neuroinflammation and higher levels of axonal damage in cuprizone-fed Nrf2-deficient animals when compared to wild type controls. In addition, Nrf2-deficient animals showed a higher susceptibility towards cuprizone within the commissura anterior white matter tract, a structure that is relatively insensitive to cuprizone in wild type animals. Our data highlight the cuprizone model as a suitable tool to study the complex interplay of oxidative stress, neuroinflammation and axonal damage. Further studies will have to show whether distinct expression patterns of Nrf2 are involved in the variable susceptibility towards cuprizone in the mouse.

Kinetic modelling of quantitative proteome data predicts metabolic reprogramming of liver cancer.

BACKGROUND: Metabolic alterations can serve as targets for diagnosis and cancer therapy. Due to the highly complex regulation of cellular metabolism, definite identification of metabolic pathway alterations remains challenging and requires sophisticated experimentation. METHODS: We applied a comprehensive kinetic model of the central carbon metabolism (CCM) to characterise metabolic reprogramming in murine liver cancer. RESULTS: We show that relative differences of protein abundances of metabolic enzymes obtained by mass spectrometry can be used to assess their maximal velocity values. Model simulations predicted tumour-specific alterations of various components of the CCM, a selected number of which were subsequently verified by in vitro and in vivo experiments. Furthermore, we demonstrate the ability of the kinetic model to identify metabolic pathways whose inhibition results in selective tumour cell killing. CONCLUSIONS: Our systems biology approach establishes that combining cellular experimentation with computer simulations of physiology-based metabolic models enables a comprehensive understanding of deregulated energetics in cancer. We propose that modelling proteomics data from human HCC with our approach will enable an individualised metabolic profiling of tumours and predictions of the efficacy of drug therapies targeting specific metabolic pathways.

Nrf2 expression driven by Foxp3 specific deletion of Keap1 results in loss of immune tolerance in mice.

The transcription factor Nrf2 regulates oxidative stress responses. However, the specific function of Nrf2 in Tregs, the central regulators of immune homeostasis, is unclear. Here, we report an unexpected but important role of Nrf2 in Tregs. Nrf2 expression driven by Foxp3 specific deletion of Keap1 resulted in an autoinflammatory phenotype with enhanced effector T cell activation and immune cell infiltrates in the lung. While early postnatal death of mice with Foxp3 specific deletion of Keap1 was most probably due to ectopic Foxp3cre expression and subsequent Keap1 deletion in epithelial cells, bone marrow chimeras suggest that Nrf2 activation intrinsically in Tregs contributes to a loss of Treg cells and diminished peripheral tolerance. Moreover, Nrf2 activation was associated with a loss of Foxp3 expression, but an enhanced glucose uptake and mTOR activity in Tregs, thus mimicking a metabolic phenotype that is associated with impaired lineage stability and cell functioning.