NRF2-mediated Oxidative Stress Response Research Articles

Quantitative Proteomics Reveal That CB2R Agonist JWH-133 Downregulates NF-κB Activation, Oxidative Stress, and Lysosomal Exocytosis from HIV-Infected Macrophages.

HIV-associated neurocognitive disorders (HAND) affect 15-55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND.

The Effect of SkQ1 on the Oxidative Stress and Inflammatory Responses in Corneal Endothelial Cells During Ex Vivo Expansion

Purpose: The purpose of this study was to characterize the oxidative stress and inflammatory responses in ex vivo corneal endothelial cells (evCEnC) during expansion and assess the impact of SkQ1, an antioxidant and anti-inflammatory compound, on measures of oxidative stress and inflammation. Methods: A CEnC line (HCEnC-21T) was cultured in media supplemented with varying SkQ1 concentrations to determine the optimal SkQ1 dose range of toxicity and protective effect on CEnC exposed to acute oxidative stress. The impact of SkQ1 treatment on intracellular free radical (IFR) levels, NRF2-mediated oxidative stress response, and NFkB-mediated inflammatory response was determined at each passage of evCEnC isolated from donor corneas and cultured in SkQ1-supplemented and untreated media. Results: HCEnC-21T cultured in media supplemented with ≤250 nM SkQ1 retained over 95% cell viability compared with untreated cells. SkQ1 provided oxidative stress protection to HCEnC-21T in a dose-dependent manner up to 500 nM. In evCEnC, 50 nM and 250 nM SkQ1 supplementation significantly reduced IFR levels across passages 0 to 3 (P-values of 0.015 and 0.023, respectively) and 50 nM SkQ1 supplementation led to decreased levels of active NRF2 in evCEnC at passage 2. However, media supplementation with SkQ1 (50 nM and 250 nM) did not alter NFkB activation at any passage. Conclusions: SkQ1 media supplementation provides oxidative stress protection to HCEnC-21T in a dose-dependent manner and decreases IFR levels and NRF2 activation in evCEnC during expansion at concentrations that do not negatively affect CEnC viability. These findings indicate that SkQ1 supplementation may increase the expansion potential of evCEnC.

The Functional Significance of McMafF_G_K in Molluscs: Implications for Nrf2-Mediated Oxidative Stress Response

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal regulator of antioxidant gene expression in mammals, forming heterodimer complexes with small Maf proteins through its BZip domain. However, the underlying mechanism of Nrf2 action in molluscs remains poorly understood. The thick shell mussel, Mytilus coruscus, represents a model organism for the marine environment and molluscs interaction research. In this study, we used in silico cloning to obtain a small Maf homologue called McMafF_G_K from M. coruscus. McMafF_G_K possesses a typical BZip domain, suggesting its affiliation with the traditional small Maf family and its potential involvement in the Nrf2 signaling pathway. Transcriptional analysis revealed that McMafF_G_K exhibited a robust response to benzo[a]pyrene (Bap) in the digestive glands. However, this response was down-regulated upon interference with McMafF_G_K-siRNA. Interestingly, the expression levels of Nrf2, NAD(P)H: quinone oxidoreductase (NQO-1), and Glutathione Peroxidase (GPx), which are key players in oxidative stress response, showed a positive correlation with McMafF_G_K in digested adenocytes of M. coruscus. Furthermore, in vitro analysis of antioxidant capacity in digestive gland cells demonstrated that Bap exposure led to an increase in reactive oxygen species (ROS) levels, accompanied by an elevation in total antioxidant capacity (T-AOC), potentially counterbalancing the excessive ROS. Strikingly, transfection of McMafF_G_K siRNA resulted in a significant rise in ROS level and a down-regulation of T-AOC level. To validate the functional relevance of McMafF_G_K, a glutathione S-transferase (GST) pull-down assay confirmed its interaction with McNrf2, providing compelling evidence of their protein interaction. This study significantly contributes to our understanding of the functional role of McMafF_G_K in the Nrf2 signaling pathway and sheds light on its potential as a target for further research in oxidative stress response.

LRRK2 Attenuates Antioxidant Response in Familial Parkinson's Disease Derived Neural Stem Cells.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, characterized by the loss of midbrain dopaminergic neurons which leads to impaired motor and cognitive functions. PD is predominantly an idiopathic disease; however, about 5% of cases are linked to hereditary mutations. The most common mutation in both familial and sporadic PD is the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2). Currently, it is not fully understood how this mutation leads to PD pathology. In this study, we isolated self-renewable, multipotent neural stem cells (NSCs) from induced pluripotent stem cells (iPSCs) harboring the G2019S LRRK2 mutation and compared them with their isogenic gene corrected counterparts using single-cell RNA-sequencing. Unbiased single-cell transcriptomic analysis revealed perturbations in many canonical pathways, specifically NRF2-mediated oxidative stress response, and glutathione redox reactions. Through various functional assays, we observed that G2019S iPSCs and NSCs exhibit increased basal levels of reactive oxygen species (ROS). We demonstrated that mutant cells show significant increase in the expression for KEAP1 and decrease in NRF2 associated with a reduced antioxidant response. The decreased viability of mutant NSCs in the H2O2-induced oxidative stress assay was rescued by two potent antioxidant drugs, PrC-210 at concentrations of 500 µM and 1 mM and Edaravone at concentrations 50 µM and 100 µM. Our data suggest that the hyperactive LRRK2 G2019S kinase activity leads to increase in KEAP1, which binds NRF2 and leads to its degradation, reduction in the antioxidant response, increased ROS, mitochondria dysfunction and cell death observed in the PD phenotype.

Plant polyphenol extract supplementation affects performance, welfare, and the Nrf2-oxidative stress response in adipose tissue of heat-stressed dairy cows

We examined the effects of a supplement of plant polyphenols extracts of green tea, capsicum, and fenugreek, and electrolytes ([Na+, K+]; AXT, Axion ThermoPlus, CCPA, France] during summer heat load on production, welfare, and oxidative stress proteins in adipose tissue (AT) of dairy cows. A total of 42 multiparous mid-lactation cows were divided into 3 groups during summer, and were fed for 2 wk either a standard milking cow diet (CTL, n = 14) or diets supplemented with 100 g/d of AXT (100AXT, n = 14), or 150 g/d of AXT (150AXT, n = 14), while being cooled 5 times a day. Then, half of the cows from each dietary treatment were cooled (CL) or not cooled (NCL) for 2 wk, after which the cooled and uncooled groups were switched for additional 2 wk. Cows were milked 3 times a day, and milk composition was analyzed at the end of each 2-wk period. Vaginal temperature (VT) was measured for 3 consecutive days in each period. Biopsies of subcutaneous AT were taken from 10 NCL cows (5 each of CTL and 150AXT) at the end of the period and examined by liquid chromatography-tandem mass spectrometry proteomics analysis. Data were analyzed with PROC MIXED of SAS (version 9.2, SAS Institute Inc.). The model included the effects of dietary treatment, cooling regimen, period, and their interactions. Protein and mRNA abundances and proteomic data (P ≤ 0.05 and fold change [FC] ± 1.5) were analyzed by t-test. Milk yields and 4% fat-corrected milk (FCM) were higher in 100AXT than in CTL; milk components were not different. Dry matter intake (DMI) was higher in 100AXT than in CTL. The effect of cooling and the interactions of period × cooling were significant for DMI, 4% FCM, energy-corrected milk, and milk/DMI. The proportion of time that VT was >39°C was lower in 100AXT and in 150AXT than in CTL. Daily rumination time was greater in 150AXT than in CTL, and lying time was greater in 100AXT and 150AXT than in CTL. Proteomics of AT demonstrated that 150AXT had increased abundances of peroxidasin (FC = 1.6), microsomal glutathione S-transferase 2 (FC = 2.5), and heme oxygenase 1 (FC = 3.6) compared with CTL. Top enriched canonical pathways included acute phase response signaling, Nrf2-mediated oxidative stress response, and lipopolysaccharide (LPS)/IL-1-mediated inhibition of RXR function. Immunoblots of AT showed a higher abundance of the transient receptor potential vanilloid 1 and of LPS binding protein in AT of 150AXT compared with CTL. Supplementation of AXT increased DMI, milk, and 4% FCM, lowered VT, improved welfare indices, and enriched the AT with Nrf2-oxidative stress response and acute phase response proteins in heat-stressed dairy cows.

#2999 APABETALONE REDUCES CARDIAC EVENTS IN CKD PATIENTS BY DOWNREGULATING FIBROTIC AND INFLAMMATORY PROCESSES

Abstract Background and Aims Major adverse cardiac events (MACE) are a leading cause of mortality in patients with chronic kidney disease (CKD). Apabetalone is an orally available inhibitor of bromodomain & extraterminal (BET) proteins – epigenetic readers that modulate gene expression involved in fibrosis & inflammation. In the phase 3 BETonMACE trial in patients with CVD & diabetes mellitus (NCT02586155), apabetalone reduced MACE by 50% in the subpopulation with CKD (eGFR < 60 mL/min/1.73 m2; HR 0.50 95% CI 0.26,0.96 p = 0.04) indicating favorable response along the kidney-heart axis. This study examines effects of apabetalone in human renal mesangial cells (HRMCs) on fibrotic & inflammatory pathways. In the clinic, we assess plasma levels of fibrotic factors in CKD patients & matched controls after receiving apabetalone. Method TGF-β1 is a pro-fibrotic cytokine that activates HRMCs to over-produce extracellular matrix (ECM). HRMCs were stimulated with TGF-β1 or LPS ± 1-25 µM apabetalone, 0.15-0.5 µM JQ1 or 0.1 µM MZ1 - BET inhibitors (BETi) with chemical scaffolds different than apabetalone. Gene expression was measured by RNA-seq & real-time PCR. RNA-seq was evaluated by Gene Ontology (GO) & Ingenuity Pathway Analysis (IPA). Proteins were measured by immunofluorescence microscopy or ELISA. Collagen gel contraction was assessed in 3D culture. In human subjects, proteomic analysis of plasma was performed via SOMAscan 1.3k 12 hours after 100 mg of apabetalone in CKD patients (n = 8 eGFR < 30 stage 4 or 5) and matched controls (n = 8 eGFR > 60). Results GO analysis of RNA-seq from TGF-β1 stimulated HRMCs showed multiple gene sets associated with ECM production & remodeling in the top 20 affected by BETi. IPA predicted NfkB-RelA and NFkB (complex) were inhibited by apabetalone, consistent with suppressed inflammation. IPA also predicted apabetalone activated pathways of glucose utilization & tolerance of ROS production, including Oxidative Phosphorylation (z-score 5.7, p = 2.95 × 10−3 at 25 µM) & NRF2-Mediated Oxidative Stress Response (z score 2.3, p = 7.9 × 10−12 at 25 µM; z-score 1.6, p = 1.5 × 10−5 at 5 µM). Predicted changes may allow tolerance of elevated glucose. Mechanistically, apabetalone suppressed TGF-β1 induced α-smooth muscle actin (α-SMA) gene expression, a marker of activation, up to 89% (p = 2.8 × 10−6 at 25 µM), and abolished α-SMA filament formation. Consequently, apabetalone opposed TGF-β1 induced collagen gel contraction up to 24% (p = 0.031 at 25 µM). Further, apabetalone countered TGF-β1 induced expression & protein production of key drivers of fibrosis including (a) thrombospondin, an activator of latent TGF-β1 (b) fibronectin, a key ECM component (c) periostin, a promoter of ECM production (d) osteonectin, a regulator of TGF-β1 expression and (e) IL6, a pro-inflammatory cytokine (Table 1). In addition, apabetalone dose dependently opposed LPS stimulated expression of inflammatory genes: IL6 up to 94%, IL1B up to 95% and PTGS2 up to 94% (p<0.001 for each). In all studies, JQ1 or MZ1 had similar activity as apabetalone, confirming on-target BETi effects. In humans, plasma levels of profibrotic and inflammatory markers were reduced 12 hours after receiving apabetalone specifically in those with CKD. Only periostin was reduced in subjects without renal impairment (Table 1). Conclusion Through epigenetic regulation of transcription in HRMCs, apabetalone reduces production of fibrotic & inflammatory factors known to signal along the kidney-heart axis & exacerbate kidney dysfunction. Predicted changes in energy metabolism suggest apabetalone facilitates adaptation to high glucose in kidneys. In a clinical trial, plasma levels of fibrotic & inflammatory factors were reduced specifically in CKD patients receiving apabetalone. Our results provide mechanistic insights into reduced MACE in CKD patients receiving apabetalone in the phase 3 BETonMACE trial & will be further evaluated in an upcoming Phase 3 trial.

Metabolomic and transcriptomic analyses of Fmo5-/- mice reveal roles for flavin-containing monooxygenase 5 (FMO5) in NRF2-mediated oxidative stress response, unfolded protein response, lipid homeostasis, and carbohydrate and one-carbon metabolism.

Flavin-containing monooxygenase 5 (FMO5) is a member of the FMO family of proteins, best known for their roles in the detoxification of foreign chemicals and, more recently, in endogenous metabolism. We have previously shown that Fmo5-/- mice display an age-related lean phenotype, with much reduced weight gain from 20 weeks of age. The phenotype is characterized by decreased fat deposition, lower plasma concentrations of glucose, insulin and cholesterol, higher glucose tolerance and insulin sensitivity, and resistance to diet-induced obesity. In the present study we report the use of metabolomic and transcriptomic analyses of livers of Fmo5-/- and wild-type mice to identify factors underlying the lean phenotype of Fmo5-/- mice and gain insights into the function of FMO5. Metabolomics was performed by the Metabolon platform, utilising ultrahigh performance liquid chromatography-tandem mass spectroscopy. Transcriptomics was performed by RNA-Seq and results analysed by DESeq2. Disruption of the Fmo5 gene has wide-ranging effects on the abundance of metabolites and expression of genes in the liver. Metabolites whose concentration differed between Fmo5-/- and wild-type mice include several saturated and monounsaturated fatty acids, complex lipids, amino acids, one-carbon intermediates and ADP-ribose. Among the genes most significantly and/or highly differentially expressed are Apoa4, Cd36, Fitm1, Hspa5, Hyou1, Ide, Me1 and Mme. The results reveal that FMO5 is involved in upregulating the NRF2-mediated oxidative stress response, the unfolded protein response and response to hypoxia and cellular stress, indicating a role for the enzyme in adaptation to oxidative and metabolic stress. FMO5 also plays a role in stimulating a wide range of metabolic pathways and processes, particularly ones involved in lipid homeostasis, the uptake and metabolism of glucose, the generation of cytosolic NADPH, and in one-carbon metabolism. The results predict that FMO5 acts by stimulating the NRF2, XBP1, PPARA and PPARG regulatory pathways, while inhibiting STAT1 and IRF7 pathways.

LncRNA Tuna is activated in cadmium-induced placental insufficiency and drives the NRF2-mediated oxidative stress response.

Cadmium (Cd) is a toxic heavy metal found throughout the environment and one of the top ten toxicants of major public health concern identified by the World Health Organization. In utero Cd exposure causes fetal growth restriction, malformation, and spontaneous abortion; however, the mechanisms by which Cd impacts these outcomes are poorly understood. Cd accumulates in the placenta, suggesting that these negative outcomes may be a consequence of disrupted placental function and placental insufficiency. To understand the impact of Cd on gene expression within the placenta, we developed a mouse model of Cd-induced fetal growth restriction through maternal consumption of CdCl2 and performed RNA-seq on control and CdCl2 exposed placentae. The top differentially expressed transcript was the Tcl1 Upstream Neuron-Associated (Tuna) long non-coding RNA, which was upregulated over 25-fold in CdCl2 exposed placentae. Tuna has been shown to be critical for neural stem cell differentiation. However, within the placenta, there is no evidence that Tuna is normally expressed or functional at any developmental stage. To determine the spatial expression of Cd-activated Tuna within the placenta, we used in situ hybridization as well as placental layer-specific RNA isolation and analysis. Both methods confirmed the absence of Tuna expression in control samples and determined that Cd-induced Tuna expression is specific to the junctional zone. Since many lncRNAs regulate gene expression, we hypothesized that Tuna forms part of the mechanism of Cd-induced transcriptomic changes. To test this, we over-expressed Tuna in cultured choriocarcinoma cells and compared gene expression profiles to those of control and CdCl2 exposed cells. We demonstrate significant overlap between genes activated by Tuna overexpression and genes activated by CdCl2 exposure, with enrichment in the NRF2-mediated oxidative stress response. Herein we analyze the NRF2 pathway and show that Tuna increases NRF2/NRF2 both at the transcript and protein levels. Tuna drives increased NRF2 target gene expression, a result that is abrogated with the use of an NRF2 inhibitor, confirming that Tuna activates oxidative stress response genes through this pathway. This work identifies the lncRNA Tuna as a potential novel player in Cd-induced placental insufficiency.

Proteomics and secreted lipidomics of mouse-derived bone marrow cells exposed to a lethal level of ionizing radiation

High doses of ionizing radiation (IR) exposure can lead to the development of severe acute radiation syndrome with bone marrow failure. Defining risk factors that predict adverse events is a critical mission to guide patient selection for personalized treatment protocols. Since non-hematopoietic stem cells act as feeder cells in the niche and their secreted lipids may regulate hematopoietic stem cells, we focused on non-hematopoietic stem cells and aimed to discover biomarkers that can assess radiation exposure from their secreted lipids. Bone marrow stromal cells (BMSCs) and osteoblast differentiation-inducing cells (ODICs) isolated from mouse femurs were exposed to lethal doses of IR and the proteomic differences between BMSC and ODIC cell layers were compared. We observed an increased Nrf2-mediated oxidative stress response and IL6 expression in ODICs and decreased expression of mitochondrial proteins in BMSCs. To elucidate secreted factors, lipidomics of the cultures were profiled; the relevant lipids distinguishing IR-exposed and control groups of BMSC were acyl-acyl phosphatidylcholine (PC aa C34:1 and PC aa C34:4), lysophosphatidylcholine (lyso-PC a C18:0 and lyso PC a C17:0) and sphingomyelin (SM C20:2). These analyses suggest that certain lipids are candidate markers for the toxic effects of IR.

Abstract 5257: Activation of NRF2-mediated oxidative stress response pathway by curcumin analog FLLL12: Implication for chemoprevention

Abstract Purpose: Carcinogenesis is a multi-step complex process initiated with irreversible genetic mutation due to endogenously derived or carcinogen-induced oxidative stress. Activation of the antioxidant pathways ameliorates oxidative stress, protects cells from carcinogenic insults, and plays a pivotal role in chemoprevention. We have previously reported that FLLL12 is a potent curcumin analog, possesses in vitro and in vivo anticancer activity, and has better pharmacokinetic profiles than curcumin. The purpose of the current study is to identify novel pathways activated by FLLL12 using RNASeq analysis. Methods: MDA686, a head and neck cancer cell line, were treated with 2 µM FLLL12 for 24h. Total RNA was isolated and used for RNASeq analysis. 2-fold differentially expressed genes were used for Ingenuity Pathway Analysis to identify the most significantly affected pathways. Real-time qPCR and western blotting were used to confirm the expression of the genes associated with the most significantly affected pathways and their protein products respectively in normal, premalignant, and malignant cell lines. Results: Whole transcriptome analysis using RNASeq identified 641 genes that were either upregulated or downregulated by 2-folds after FLLL12 treatment. Enrichment of this gene set with Ingenuity Pathway Analysis identified the ferroptosis signaling pathway, the tumor microenvironment pathway, and the NRF2-mediated oxidative response pathway as the top three most significantly affected pathways by FLLL12. We confirmed the activation of HMOX-1, NQO1, SLC7A11, and GCLC mRNA and proteins in HOK (normal), MSK-LEUK1 (premalignant), and MDA686TU (malignant) cells. Although these genes are common for the ferroptosis signaling pathway and the NRF2-mediated oxidative response pathway, treatment of cells with ferroptosis inhibitors, ferrostatin 1, and deferoxamine, had no effect on FLLL12-induced cell death suggesting that these genes are associated with oxidative stress response pathway. Conclusions: Our results strongly suggest that FLLL12 activates the oxidative stress response pathway in normal, premalignant, and malignant head and neck cancer cell lines and has strong promise for chemoprevention. Citation Format: Raji Lukmon, Adeoluwa A. Adeluola, A.R.M. Ruhul Amin. Activation of NRF2-mediated oxidative stress response pathway by curcumin analog FLLL12: Implication for chemoprevention. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5257.

Sex-Specific Alterations of the Lung Transcriptome at Birth in Mouse Offspring Prenatally Exposed to Vanilla-Flavored E-Cigarette Aerosols and Enhanced Susceptibility to Asthma.

Pregnant mice were exposed throughout gestation to either filtered air or vanilla-flavored e-cig aerosols containing 18 mg/mL of nicotine. Male and female exposed mouse offspring were sacrificed at birth, and then the lung transcriptome was evaluated. Additionally, once sub-groups of male offspring mice reached 4 weeks of age, they were challenged with house dust mites (HDMs) for 3 weeks to assess asthmatic responses. The lung transcriptomic responses of the mouse offspring at birth showed that in utero vanilla-flavored e-cig aerosol exposure significantly regulated 88 genes in males (62 genes were up-regulated and 26 genes were down-regulated), and 65 genes were significantly regulated in females (17 genes were up-regulated and 48 genes were down-regulated). Gene network analyses revealed that in utero e-cig aerosol exposure affected canonical pathways associated with CD28 signaling in T helper cells, the role of NFAT in the regulation of immune responses, and phospholipase C signaling in males, whereas the dysregulated genes in the female offspring were associated with NRF2-mediated oxidative stress responses. Moreover, we found that in utero exposures to vanilla-flavored e-cig aerosol exacerbated HDM-induced asthma in 7-week-old male mouse offspring compared to respective in utero air + HDM controls. Overall, these data demonstrate that in utero e-cig aerosol exposure alters the developing mouse lung transcriptome at birth in a sex-specific manner and provide evidence that the inhalation of e-cig aerosols is detrimental to the respiratory health of offspring by increasing the offspring' susceptibility to developing lung diseases later in life.

Atmospheric aging increases the cytotoxicity of bare soot particles in BEAS-2B lung cells

Soot particles (SP) are ubiquitous components of atmospheric particulate matter and have been shown to cause various adverse health effects. In the atmosphere, freshly emitted SP can be coated by condensed low-volatility secondary organic and inorganic species. In addition, gas-phase oxidants may react with the surface of SP. Due to the chemical and physical resemblance of SP carbon backbone with polyaromatic hydrocarbon species and their potent oxidation products, we investigated the biological responses of BEAS-2B lung epithelial cells following exposure to fresh- and photochemically aged-SP at the air–liquid interface. A comprehensive physical and chemical aerosol characterization was performed to depict the atmospheric transformations of SP, showing that photochemical aging increased the organic carbon fraction and the oxidation state of the SP. RNA-sequencing and qPCR analysis showed varying gene expression profiles for fresh- and aged-SP. Exposure to aged-SP increased DNA damage, oxidative damage, and upregulation of NRF2-mediated oxidative stress response genes compared to fresh-SP. Furthermore, aged-SP augmented inflammatory cytokine secretion and activated AhR-response, as evidenced by increased expression of AhR-responsive genes. These results indicate that oxidative stress, inflammation, and DNA damage play a key role in the cytotoxicity of SP in BEAS-2B cells, where aging leads to higher toxic responses. Collectively, our results suggest that photochemical aging may increase SP toxicity through surface modifications that lead to an increased toxic response by activating different molecular pathways.

Nrf2 Is Involved in Hyperglycemia-induced Abnormal Lung Development through both Antioxidation-Dependent and Antioxidation-Independent Activation.

Accumulating evidence has shown that hyperglycemia during pregnancy negatively affects lung development. However, the pathological mechanism of lung dysplasia caused by hyperglycemia remains unclear. In this study, we demonstrated the phenotypes of the impaired lung epithelial cell differentiation of mouse lungs in pregestational diabetes mellitus (PGDM) and gestational diabetes mellitus (GDM), and increased levels of oxidative stress and activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways occurred. Nrf2 deficiency during pregnancy led to the aforementioned similar and aggravated phenotypes of the poor saccular process as in diabetes, implying the Nrf2 signaling pathway played a very important role in both physiological and pathological conditions. Based on RNA sequencing and luciferase reporter gene analysis, we revealed that Nrf2 could regulate Wnt signaling by targeting Ctnnd2. In summary, we revealed the pathological mechanism of how diabetes affected late lung development during embryogenesis, especially elucidating the bilateral roles of Nrf2-mediated oxidative stress responses and Wnt signaling. This finding also indicated that Nrf2 could potentially be used in preventing or treating pulmonary anomalies induced by hyperglycemia during pregnancy.

The Effect of Carnosine on UVA-Induced Changes in Intracellular Signaling of Human Skin Fibroblast Spheroids

Dermis fibroblasts are very sensitive to penetrating UVA radiation and induce photo-damage. To protect skin cells against this environmental damage, there is an urgent need for effective compounds, specifically targeting UVA-induced mitochondrial injury. This study aimed to analyze the effect of carnosine on the proteome of UVA-irradiated human skin fibroblast, cultured in a three-dimensional (3D) biological system recapitulating dermal compartment as a test system to investigate the altered cellular pathways after 48 h and 7 days of culture with or without carnosine treatment. The obtained results indicate that UVA dysregulates Oxidative Phosphorylation, the Fibrosis Signaling Pathway, Glycolysis I and Nrf2-mediated Oxidative Stress Response. Carnosine exercises provide a protective function against the harmful effects of UVA radiation by activating the Nrf2 pathway with the upregulations of some ROS-detoxifying enzymes such as the glutathione S-transferase (GST) protein family. Additionally, carnosine regulates the activation of the Epithelial Adherens Junction and Wound Healing Signaling Pathway by mediating the activation of structural proteins such as vinculin and zyxin as well as fibronectin 1 and collagen type XVIII alpha 1 chain against UVA-induced changes.

The Role of Superoxide Dismutase 1 in Amyotrophic Lateral Sclerosis: Identification of Signaling Pathways, Regulators, Molecular Interaction Networks, and Biological Functions through Bioinformatics.

Mutations in superoxide dismutase 1 (SOD1) result in misfolding and aggregation of the protein, causing neurodegenerative amyotrophic lateral sclerosis (ALS). In recent years, several new SOD1 variants that trigger ALS have been identified, making it increasingly crucial to understand the SOD1 toxicity pathway in ALS. Here we used an integrated bioinformatics approach, including the Ingenuity Pathway Analysis (IPA) tool to analyze signaling pathways, regulators, functions, and network molecules of SOD1 with an emphasis on ALS. IPA toxicity analysis of SOD1 identified superoxide radicals' degradation, apelin adipocyte, ALS, NRF2-mediated oxidative stress response, and sirtuin signaling as the key signaling pathways, while the toxicity of SOD1 is exerted via mitochondrial swelling and oxidative stress. IPA listed CNR1, APLN, BTG2, MAPK, DRAP1, NFE2L2, SNCA, and CG as the upstream regulators of SOD1. IPA further revealed that mutation in SOD1 results in hereditary disorders, including ALS. The exploration of the relationship between SOD1 and ALS using IPA unveiled SOD1-ALS pathway molecules. The gene ontology (GO) analysis of SOD1-ALS pathway molecules with ShinyGO reaffirmed that SOD1 toxicity results in ALS and neurodegeneration. The GO analysis further identified enriched biological processes, molecular functions, and cellular components for SOD1-ALS pathway molecules. The construction of a protein-protein interaction network of SOD1-ALS pathway molecules using STRING and further analysis of that network with Cytoscape identified ACTB followed by TP53, IL6, CASP3, SOD1, IL1B, APP, APOE, and VEGFA as the major network hubs. Taken together, our study provides insight into the molecular underpinning of SOD1's toxicity in ALS.

Infectious spleen and kidney necrosis virus induces the reactive oxidative species/Nrf2-mediated oxidative stress response for the regulation of mitochondrion-mediated Bax/Bak cell death signals in GF-1 cells.

Infectious spleen and kidney necrosis virus (ISKNV) infections can trigger host cell death and are correlated with viral replication; however, they have rarely been considered in terms of the host organelle involvement. In the present study, we demonstrated that ISKNV triggered an oxidative stress signal in the Nrf2-mediated oxidative stress response and induced stress signals for Bax/Bak-mediated host cell death in fish GF-1 cells. The results showed that after ISKNV infection, the levels of reactive oxidative species (ROS) increased by 60–80% from day 3 to day 5, as assessed by an H2DCFDA assay for tracing hydrogen peroxide (H2O2), which was correlated with up to a one-fold change in the fish GF-1 cells. Furthermore, we found that ISKNV infection induced Nrf2-mediated ROS stress signals from D1 to D5, which were correlated with the upregulation of antioxidant enzymes, such as catalase, SOD1, and SOD2; these effects were blocked by the antioxidants GSH and NAC. By analyzing Nrf2-mediated ROS stress signals for cell death regulation via an apoptotic assay, we found that treatment with antioxidants reduced annexin-V-positive signals by 10% (GSH) to 15% (NAC); moreover, necrotic-positive signals were reduced by 6% (GSH) and 32% (NAC) at day 5 (D5) in GF-1 cells, as indicated by PI staining. Furthermore, we found that Nrf2-mediated ROS stress regulated mitochondrion-mediated Bax/Bak death signals at D3 and D5; this was effectively blocked by antioxidant treatment in the GF-1 cells, as demonstrated by a JC1 assay (ΔΨm) and western blot analysis. In addition, we found that downstream signals for caspase-9 and -3 activation were apparently blocked by antioxidant treatment at D3 and D5. Finally, we found that treatment with GSH and NAC reduced major capsid protein (MCP) expression and virus titer (TCID50%) by up to 15-fold at D5 in GF-1 cells. Thus, our data suggest that ISKNV can induce ROS production, which triggers Nrf2-mediated stress signals. Then, these stress signals can regulate mitochondrion-mediated Bax/Bak apoptotic signaling, which is connected to downstream caspase-9 and -3 activation. If ISKNV-induced Nrf2-mediated stress signaling is blocked, then the antioxidants GSH and NAC can also suppress apoptotic signals or reduce viral replication. These findings may provide insights into the control and treatment of double-stranded DNA viruses.

Identification of novel biomarkers in prostate cancer diagnosis and prognosis.

Prostate cancer (PCa) is a common urinary malignancy. The lack of specific and sensitive biomarkers for the early diagnosis and prognosis of PCa makes it important to seek alternatives.R software was used to analyze the PCa expression profile from data sets in Gene Expression Omnibus. Core differential genes were identified by String and Cytoscape and further validated by Gene Expression Profiling Interactive Analysis (GEPIA) and The Human Protein Atlas(HPA). Gene Ontologyanalysis was done in the DIVID database and visualization analysis was conducted by Hiplot. Pathway enrichment was analyzed by IPA. To identify potential competitive endogenous RNAs(ceRNA) networks, the experimentally validated microRNA-target interactions database(miRTarBase), The Encyclopedia of RNA Interactomes (StarBase), lncBase, and GEPIA were used. The lncLocator was utilized to perform subcellular localization of longnoncoding RNAs(lncRNAs). Both miRTarBase and StarBase were used to find the binding site of mRNAs-miRNAs and miRNAs-lncRNAs. Visualization of the ceRNA network was performed with Cytoscape.Nine genes closely related to the diagnosis and prognosis of PCa were obtained, including four identified biomarkers by HPA, CENPF, TPX2, TK1, and CCNB1, and five novel PCa biomarkers, RRM2, UBE2C, TOP2A, BIRC5, and ZWINT. Pathway analysis indicated that PCa carcinogenesis was highly correlated with liver fibrosis pathways, ILK signaling, and NRF2-mediated oxidative stress response. Two sets of ceRNA networks,BIRC5/hsa-miR-218-5p/NEAT1and UBE2C/hsa-miR-483-3p/NEAT1 were found to be novel biomarkers for the identification of PCa. The quantitative real-time polymerase chain reactionresults verified that UBE2C, BIRC5, and NEAT1 were upregulated and hsa-miR-218-5p and hsa-miR-483-3p were downregulated in human PCa cells compared with normal prostate epithelial cells.The novel identified biomarkers in this study would be valuable for the diagnosis and prognosis of PCa.

Transgenerational bone toxicity in F3 medaka (Oryzias latipes) induced by ancestral benzo[a]pyrene exposure: Cellular and transcriptomic insights

Benzo[a]pyrene (BaP), a ubiquitous pollutant, raises environmental health concerns due to induction of bone toxicity in the unexposed offspring. Exposure of F0 ancestor medaka (Oryzias latipes) to 1 µg/L BaP for 21 days causes reduced vertebral bone thickness in the unexposed F3 male offspring. To reveal the inherited modifications, osteoblast (OB) abundance and molecular signaling pathways of transgenerational BaP-induced bone thinning were assessed. Histomorphometric analysis showed a reduction in OB abundance. Analyses of the miRNA and mRNA transcriptomes revealed the dysregulation of Wnt signaling (frzb/ola-miR-1–3p, sfrp5/ola-miR-96–5p/miR-455–5p) and bone morphogenetic protein (Bmp) signaling (bmp3/ola-miR-96–5p/miR-181b-5p/miR-199a-5p/miR-205–5p/miR-455–5p). Both pathways are major indicators of impaired bone formation, while the altered Rank signaling in osteoclasts (c-fos/miR-205–5p) suggests a potentially augmented bone resorption. Interestingly, a typical BaP-responsive pathway, the Nrf2-mediated oxidative stress response (gst/ola-miR-181b-5p/miR-199a-5p/miR-205), was also affected. Moreover, mRNA levels of epigenetic modification enzymes (e.g., hdac6, hdac7, kdm5b) were found dysregulated. The findings indicated that epigenetic factors (e.g., miRNAs, histone modifications) may directly regulate the expression of genes associated with transgenerational BaP bone toxicity and warrants further studies. The identified candidate genes and miRNAs may serve as potential biomarkers for BaP-induced bone disease and as indicators of historic exposures in wild fish for conservation purposes.

Targeting Nrf2-Mediated Oxidative Stress Response in Traumatic Brain Injury: Therapeutic Perspectives of Phytochemicals.

Traumatic brain injury (TBI), known as mechanical damage to the brain, impairs the normal function of the brain seriously. Its clinical symptoms manifest as behavioral impairment, cognitive decline, communication difficulties, etc. The pathophysiological mechanisms of TBI are complex and involve inflammatory response, oxidative stress, mitochondrial dysfunction, blood-brain barrier (BBB) disruption, and so on. Among them, oxidative stress, one of the important mechanisms, occurs at the beginning and accompanies the whole process of TBI. Most importantly, excessive oxidative stress causes BBB disruption and brings injury to lipids, proteins, and DNA, leading to the generation of lipid peroxidation, damage of nuclear and mitochondrial DNA, neuronal apoptosis, and neuroinflammatory response. Transcription factor NF-E2 related factor 2 (Nrf2), a basic leucine zipper protein, plays an important role in the regulation of antioxidant proteins, such as oxygenase-1(HO-1), NAD(P)H Quinone Dehydrogenase 1 (NQO1), and glutathione peroxidase (GPx), to protect against oxidative stress, neuroinflammation, and neuronal apoptosis. Recently, emerging evidence indicated the knockout (KO) of Nrf2 aggravates the pathology of TBI, while the treatment of Nrf2 activators inhibits neuronal apoptosis and neuroinflammatory responses via reducing oxidative damage. Phytochemicals from fruits, vegetables, grains, and other medical herbs have been demonstrated to activate the Nrf2 signaling pathway and exert neuroprotective effects in TBI. In this review, we emphasized the contributive role of oxidative stress in the pathology of TBI and the protective mechanism of the Nrf2-mediated oxidative stress response for the treatment of TBI. In addition, we summarized the research advances of phytochemicals, including polyphenols, terpenoids, natural pigments, and otherwise, in the activation of Nrf2 signaling and their potential therapies for TBI. Although there is still limited clinical application evidence for these natural Nrf2 activators, we believe that the combinational use of phytochemicals such as Nrf2 activators with gene and stem cell therapy will be a promising therapeutic strategy for TBI in the future.

RNA-sequencing Reveals a Gene Expression Signature in Skeletal Muscle of a Mouse Model of Age-associated Postoperative Functional Decline.

This study aimed to characterize the effects of laparotomy on postoperative physical function and skeletal muscle gene expression in male C57BL/6N mice at 3, 20, and 24 months of age to investigate late-life vulnerability and resiliency to acute surgical stress. Pre and postoperative physical functioning was assessed by forelimb grip strength on postoperative day (POD) 1 and 3 and motor coordination on POD 2 and 4. Laparotomy-induced an age-associated postoperative decline in forelimb grip strength that was the greatest in the oldest mice. While motor coordination declined with increasing age at baseline, it was unaffected by laparotomy. Baseline physical function as stratified by motor coordination performance (low functioning vs high functioning) in 24-month-old mice did not differentially affect postlaparotomy reduction in grip strength. RNA sequencing of soleus muscles showed that laparotomy-induced age-associated differential gene expression and canonical pathway activation with the greatest effects in the youngest mice. Examples of such age-associated, metabolically important pathways that were only activated in the youngest mice after laparotomy included oxidative phosphorylation and NRF2-mediated oxidative stress response. Analysis of lipid mediators in serum and gastrocnemius muscle showed alterations in profiles during aging and confirmed an association between such changes and functional status in gastrocnemius muscle. These findings demonstrate a mouse model of laparotomy which recapitulated some features of postoperative skeletal muscle decline in older adults, and identified age-associated, laparotomy-induced molecular signatures in skeletal muscles. Future research can build upon this model to study molecular mechanisms of late-life vulnerability and resiliency to acute surgical stress.