Total Reactive Oxygen Species Research Articles

The mitochondrial uncoupler 2,4-dinitrophenol modulates inflammatory and oxidative responses in Trypanosoma cruzi-induced acute myocarditis in mice

By uncoupling oxidative phosphorylation, 2,4-dinitrophenol (DNP) attenuates reactive oxygen species (ROS) biosynthesis, which are known to aggravate infectious myocarditis in Chagas disease. Thus, the impact of DNP-based chemotherapy on Trypanosoma cruzi-induced acute myocarditis was investigated. C56BL/6 mice uninfected and infected untreated and treated daily with 100 mg/kg benznidazole (Bz, reference drug), 5 and 10 mg/kg DNP by gavage for 11 days after confirmation of T. cruzi infection were investigated. Twenty-four hours ​after the last treatment, the animals were euthanized and the heart was collected for microstructural, immunological and biochemical analyses. T. cruzi inoculation induced systemic inflammation (e.g., cytokines and anti-T. cruzi IgG upregulation), cardiac infection (T. cruzi DNA), oxidative stress, inflammatory infiltrate and microstructural myocardial damage in untreated mice. DNP treatment aggravated heart infection and microstructural damage, which were markedly attenuated by Bz. DNP (10 mg/kg) was also effective in attenuating ROS (total ROS, H2O2 and O2−), nitric oxide (NO), lipid (malondialdehyde - MDA) and protein (protein carbonyl - PCn) oxidation, TNF, IFN-γ, IL-10, and MCP-1/CCL2, anti-T. cruzi IgG, cardiac troponin I levels, as well as inflammatory infiltrate and cardiac damage in T. cruzi-infected mice. Our findings indicate that DNP aggravated heart infection and microstructural cardiomyocytes damage in infected mice. These responses were related to the antioxidant and anti-inflammatory properties of DNP, which favors infection by weakening the pro-oxidant and pro-inflammatory protective mechanisms of the infected host. Conversely, Bz-induced cardioprotective effects combined effective anti-inflammatory and antiparasitic responses, which protect against heart infection, oxidative stress and microstructural damage in Chagas disease.

Activation of the Nrf-2 pathway improves the mitochondrial phenotype in skeletal muscle cells

Aging muscle displays a reduction in mitochondrial content, along with elevated reactive oxygen species (ROS) emission and reduced levels of antioxidant enzymes. These characteristics enhance tissue susceptibility to oxidative damage, facilitating sub-optimal cellular metabolism and subsequent muscle atrophy. While these perturbations to metabolic homeostasis are a natural course of the aging process, there has been ongoing research investigating how to implement interventions to reduce this decline in muscle health. Regular exercise is one possible treatment modality, however this may be unsuitable for some populations who are unable to participate due to their health status. The objective of this study was to examine the activation of the Nrf-2 pathway using the nutraceutical Sulforaphane (SFN) and evaluate its role as a potential exercise mimetic. We hypothesized that SFN would act through similar pathways activated with chronic exercise to improve mitochondrial content, respiration, and ROS emission. To address our objective, C2C12 myotubes were treated with SFN for various timepoints basally, and in the presence of the Complex-I inhibitor rotenone to induce oxidative stress. Western blotting revealed an 8-fold increase in Nrf-2 nuclear localization following 24hrs of SFN treatment, which subsequently led to the upregulation of antioxidant enzymes NQO1, Heme Oxygenase-1, catalase, glutathione reductase and Glucose-6-Phosphate Dehydrogenase by 2-to 3-fold. These protein changes, were accompanied by dramatic reductions in both total cellular and mitochondrial ROS emission with SFN, detected with CellROX Green and MitoSOX Red respectively. Interestingly, this led to an increase in mitochondrial membrane potential, detected via MitoTracker Red (MTR), despite rotenone-induced oxidative stress. Western blotting revealed TFAM upregulation at 24hrs, suggesting an increase in mitochondrial DNA (mtDNA) replication and transcription. This was corroborated by an increase in COX subunit I protein, a mitochondrially-encoded subunit of cytochrome c oxidase, as well as MitoTracker Green and MTR staining by 48hrs. Mitochondrial respiration was evaluated using Seahorse analysis, demonstrating a marked increase in ATP production as well as basal and maximal respiration. Together these data suggest that activation of the Nrf-2 pathway via SFN may lead to adaptations similar to chronic exercise. Additionally, SFN may have applications in disorders characterized by mitochondrial loss and high ROS emission. To fully elucidate the precise mechanisms underlying this agent, SFN will be combined with chronic contractile activity to determine if these two interventions operate through independent pathways. This work was supported by funds from the Natural Science and Engineering Research Council (NSERC). Sabrina Champsi is the recipient of the NSERC Alexander Graham Bell Canada Graduate Scholarship-Master’s (CGS-M). David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of host antioxidant effectors as thioridazine targets: Impact on cardiomyocytes infection and Trypanosoma cruzi-induced acute myocarditis

Phenothiazines inhibit antioxidant enzymes in trypanosomatids. However, potential interferences with host cell antioxidant defenses are central concerns in using these drugs to treat Trypanosoma cruzi-induced infectious myocarditis. Thus, the interaction of thioridazine (TDZ) with T. cruzi and cardiomyocytes antioxidant enzymes, and its impact on cardiomyocytes and cardiac infection was investigated in vitro and in vivo. Cardiomyocytes and trypomastigotes in culture, and mice treated with TDZ and benznidazole (Bz, reference antiparasitic drug) were submitted to microstructural, biochemical and molecular analyses. TDZ was more cytotoxic and less selective against T. cruzi than Bz in vitro. TDZ-pretreated cardiomyocytes developed increased infection rate, reactive oxygen species (ROS) production, lipid and protein oxidation; similar catalase (CAT) and superoxide dismutase (SOD) activity, and reduced glutathione's (peroxidase - GPx, S-transferase – GST, and reductase – GR) activity than infected untreated cells. TDZ attenuated trypanothione reductase activity in T. cruzi, and protein antioxidant capacity in cardiomyocytes, making these cells more susceptible to H2O2-based oxidative challenge. In vivo, TDZ potentiated heart parasitism, total ROS production, myocarditis, lipid and protein oxidation; as well as reduced GPx, GR, and GST activities compared to untreated mice. Benznidazole decreased heart parasitism, total ROS production, heart inflammation, lipid and protein oxidation in T. cruzi-infected mice. Our findings indicate that TDZ simultaneously interact with enzymatic antioxidant targets in cardiomyocytes and T. cruzi, potentiating the infection by inducing antioxidant fragility and increasing cardiomyocytes and heart susceptibility to parasitism, inflammation and oxidative damage.

HSP70 protects against acute pancreatitis-elicited intestinal barrier damage in rats

Acute pancreatitis (AP) is a frequent but severe abdominal emergency in general surgery with intestinal barrier dysfunction. Heat shock protein 70 (HSP70) is a ubiquitous molecular chaperone that has been proposed to exert favorable effects on AP. Nonetheless, the detailed impacts of HSP70 on the intestinal barrier function in AP are unknown, which will be investigated here. After the injection of sodium taurocholate into the biliopancreatic duct, the rat models of AP were established. After modeling, HSP70 expression was up-regulated through lentivirus infection. Western blot was used to detect HSP70 expression. H&E staining was used to examine the histological changes in the pancreatic and intestinal tissues. The levels of pancreatic biochemical markers and oxidative stress markers were detected using corresponding assay kits. ELISA was used to detect the levels of inflammatory cytokines and gastrointestinal function indicators. Immunofluorescence staining and Western blot were used to detect the expression of tight junction proteins. DCFH-DA probe and MitoSOX Red probe were used to detect total reactive oxygen species (ROS) and mitochondrial ROS (mtROS), respectively. TUNEL assay and Western blot were used to detect apoptosis. During the model construction, severe pancreatic and abnormal intestinal tissue abnormalities were observed, inflammatory response was activated and the intestinal barrier was disrupted. HSP70 expression was down-regulated in the intestinal tissues AP rat models. HSP70 ameliorated the morphological damage of pancreatic and intestinal tissues of AP rats. In addition, HSP70 significantly reduced intestinal barrier damage, inflammatory response, oxidative stress and apoptosis in the intestinal tissues of AP rat models. Collectively, HSP70 might attenuate AP through exerting anti-inflammatory, anti-oxidant, anti-apoptotic effects and inhibiting intestinal barrier disruption.

Polydopamine-Coated Copper-Doped Co3O4 Nanosheets Rich in Oxygen Vacancy on Titanium and Multimodal Synergistic Antibacterial Study.

Medical titanium-based (Ti-based) implants in the human body are prone to infection by pathogenic bacteria, leading to implantation failure. Constructing antibacterial nanocoatings on Ti-based implants is one of the most effective strategies to solve bacterial contamination. However, single antibacterial function was not sufficient to efficiently kill bacteria, and it is necessary to develop multifunctional antibacterial methods. This study modifies medical Ti foils with Cu-doped Co3O4 rich in oxygen vacancies, and improves their biocompatibility by polydopamine (PDA/Cu-Ov-Co3O4). Under near-infrared (NIR) irradiation, nanocoatings can generate •OH and 1O2 due to Cu+ Fenton-like activity and a photodynamic effect of Cu-Ov-Co3O4, and the total reactive oxygen species (ROS) content inside bacteria significantly increases, causing oxidative stress of bacteria. Further experiments prove that the photothermal process enhances the bacterial membrane permeability, allowing the invasion of ROS and metal ions, as well as the protein leakage. Moreover, PDA/Cu-Ov-Co3O4 can downregulate ATP levels and further reduce bacterial metabolic activity after irradiation. This coating exhibits sterilization ability against both Escherichia coli and Staphylococcus aureus with an antibacterial rate of ca. 100%, significantly higher than that of bare medical Ti foils (ca. 0%). Therefore, multifunctional synergistic antibacterial nanocoating will be a promising strategy for preventing bacterial contamination on medical Ti-based implants.

Cold atmospheric plasma attenuates skin cancer via ROS induced apoptosis.

Cold atmospheric plasma (CAP) has been widely used in biomedical research, especially in vitro cancer therapy. Cutaneous squamous cell carcinoma (CSCC) is a malignant tumor originating from epidermal keratinocytes. However, the mechanism of CAP therapy on CSCC remains unclear. The animal models of CSCC induced by 7,12-dimethylbenz(a) anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) were constructed. For the CAP treatment group, after each TPA application, CAP was administered for 3min twice weekly after drying. HE staining were used to detect the pathological status of tumor tissue in each group. The levels of PCNA, Bcl-2, Bax, MMP2 and MMP9 were evaluated by western blot and qPCR. TUNEL staining were used to detect apoptosis in tumor tissues. In vivo, serum samples were used for ELISA of total ROS. MTT assay was used to detect the viability of A431 cells. Western blot and qPCR were used to detect the levels of PCNA, Bcl-2, Bax, MMP2 and MMP9 in A431 cells. A431 cell proliferation was examined by colony formation assay. The proportions of apoptosis of A431 cells were detected by flow cytometry. Transwell assessed the ability of A431 cells migration and proliferation. We found that CAP could induce skin cancer cells apoptosis and inhibit the progress of skin cancer. Through experiments in vitro, reactive oxygen species (ROS) generated by N-acetylcysteine (NAC) and CAP inhibited the proliferation and migration of A431 skin cancer cells while promoting apoptosis. These evidences suggest the protective effect of CAP in CSCC, and CAP has the potential clinical application of CSCC.

ALKBH5 Modulates Asthma Progression by Downregulating N6-methyladenosine Methylation

Asthma is a chronic respiratory disease that is characterized by airway inflammation, excessive mucus production, and airway remodeling. Prevention and treatment for asthma is an urgent issue in clinical studies. In recent years, N6-methyladenosine methylation (m6A) has emerged as a promising regulatory approach involved in multiple diseases. ALKBH5 (alkB homolog 5) is a demethylase widely studied in disease pathologies. This work aimed to explore the regulatory mechanisms underlying the ALKBH5-regulated asthma.
 We established an interleukin-13 (IL-13)-stimulated cell model to mimic the in vitro inflammatory environment of asthma. ALKBH5 knockdown in bronchial epithelial cells was performed using siRNAs, and the knockdown efficacy was analyzed by quantitative PCR (qPCR). Cell viability and proliferation were measured by cell counting kit 8 (CCK-8) and colony formation assay. The ferroptosis was assessed by measuring the total iron, Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. The enrichment of N6-methyladenosine methylation (m6A) modification was detected by the MeRIP assay.
 Knockdown of ALKBH5 significantly elevated the survival and colony formation ability of bronchial epithelial cells in the IL-13 induction model. The levels of total iron, Fe2+, lipid ROS, and MDA were remarkedly elevated, and the SOD level was reduced in IL-13-induced bronchial epithelial cells, and depletion of ALKBH5 reversed these effects. Knockdown of ALKBH5 elevated the enrichment of m6A modification and expression of glutathione peroxidase 4 (GPX4). Knockdown of GPX4 abolished the pro-proliferation and anti-ferroptosis effects of siALKBH5.
 Knockdown of ALKBH5 improved the proliferation of bronchial epithelial cells and alleviated cell ferroptosis. 

Dihydroquercetin improves experimental acute liver failure by targeting ferroptosis and mitochondria-mediated apoptosis through the SIRT1/p53 axis

BackgroundFerroptosis and mitochondria-mediated apoptosis are both involved in the pathogenesis of acute liver failure (ALF). Ferroptosis-produced reactive oxygen species (ROS) trigger the chain oxidation of polyunsaturated phospholipids and promote mitochondrial apoptosis. Dihydroquercetin (DHQ) also plays an important protective role against liver injury. PurposeHere, we aimed to investigate the protective effects of DHQ on ALF. We also explored the underlying mechanism. MethodsWe established a Lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced ALF mouse model and tumor necrosis factor-α (TNF-α)/D-Gal-induced ALF LO2 cell model. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) and Dihydroethidium (DHE) were used to detect total ROS levels. Lipid ROS was assessed using C11-BODIPY flow cytometry. Lipid peroxidative products levels were detected using MDA ELISA assay and 4-hydroxynonenal (4-HNE) immunohistochemistry. QRT-PCR and western blots were used to test mRNA and protein expression levels, respectively. Cell viability was evaluated with CCK8 assay, and apoptosis was analyzed using flow cytometry. ResultsDHQ treatment improved LPS/D-Gal-induced ALF, as well as TNF-α/D-Gal-induced reductions in LO2 viability and increased sirtuin 1 (SIRT1) expression. DHQ pretreatment also reduced the accumulation of ROS, reduced lipid peroxidation, elevated mitochondrial membrane potentials (ΔΨm), and decreased liver cell apoptosis both in vivo and in vitro. Additionally, the knockdown of SIRT1 and p53 activator (Tenovin-6) treatment reversed DHQ's inhibitory effects on ferroptosis and mitochondria-mediated apoptosis in vitro. DHQ enhanced p53 deacetylation by both up-regulating SIRT1 expression and directly bonding to SIRT1. We also found that Tenovin-6′s stimulatory effects on ferroptosis and mitochondria-mediated apoptosis in the DHQ-treated LO2 ALF cell model were partially attenuated by overexpression of solute carrier family 7member 11 (SLC7A11), as well as by apoptotic protease activating factor 1 (Apaf-1) knockdown. ConclusionOur results suggest that DHQ alleviated ALF by inhibiting both ferroptosis and mitochondria-mediated apoptosis by regulating the SIRT1/p53 axis. Thus, DHQ may serve as a novel therapy for ALF.

Effects of reproductive condition on haemocyte parameters in the Pacific abalone Haliotis discus hannai during the spawning period

ABSTRACT The assessment of morphology and immunology of specimens at different maturity stages is essential for the determination of spawning strategy and reducing the risk of mortality. In this paper, female and male Pacific abalones (Haliotis discus hannai) at different statuses (pre-spawning, spawning and post-spawning) were collected to estimate the effects of spawning on the immune response. The haemocyte parameters, including total haemocyte counts (THC), haemocyte mortality, and reactive oxygen species production (ROS) were analysed using flow cytometry. THC decreased after spawning, which was lowest 3 days post-spawning. Haemocyte mortality was higher during spawning, and was highest 3 days post-spawning. Abalone also reduced their metabolic activity after spawning, reflected by an increase in ROS in both females and males. Moreover, our results show that female abalone reach the spawning period with a higher degree of activities of haemocytes and immune responses than male. The results confirmed that variability in abalone haemocytes during the spawning period might related to individual metabolic conditions.

An in vitro analysis of an innovative standardized phospholipid carrier-based Melissa officinalis L. extract as a potential neuromodulator for emotional distress and related conditions.

Background: Melissa officinalis L. (MO), commonly known as lemon balm, a member of the mint family, is considered a calming herb. In various traditional medicines, it has been utilized to reduce stress and anxiety and promote sleep. A growing body of clinical evidence suggests that MO leaf extract supplementation possesses considerable neuropharmacological properties. However, its possible mechanism of action largely remains unknown. Objective: In the present in vitro studies, we comparatively investigated the central nervous system (CNS)-calming and antioxidative stress properties of an innovative standardized phospholipid carrier-based (Phytosome™) MO extract (Relissa™) vs. an unformulated dry MO extract. Methods: The neuropharmacological effect of the extract was studied in the anti-depressant enzymes γ-aminobutyrate transaminase (GABA-T) and monoamine oxidase A (MAO-A) assays and SH-SY5Y cells brain-derived neurotrophic factor (BDNF) expression assay. The neuroprotective effect of the extract against oxidative stress was assessed in SH-SY5Y cell-based (H2O2-exposed) Total Antioxidant Status (TAS) and Total Reactive Oxygen Species (ROS) assays. The cytotoxic effect of the extract was evaluated using MTT and LDH assays. The extract antioxidant effect was also evaluated in cell-free chemical tests, including TEAC-ABTS, DPPH, Ferric Reducing Antioxidant Power (FRAP), Oxygen Radical Antioxidant Capacity (ORAC), and Hydroxyl Radical Antioxidant Capacity (HORAC) assays. Results: Relissa™ exhibited high GABA-T inhibitory activity, IC50 (mg/mL) = 0.064 vs. unformulated dry MO extract, IC50 (mg/mL) = 0.27. Similar inhibitory effects were also observed for MAO-A. Relissa™ demonstrated an improved neuroprotective antioxidant effect on SH-SY5Y cells against H2O2-induced oxidative stress. Compared to unformulated dry MO extract, Relissa™ exerted high protective effect on H2O2-exposed SH-SY5Y cells, leading to higher cells BDNF expression levels. Moreover, cell-free chemical tests, including TEAC-ABTS, DPPH radical scavenging, FRAP, ORAC, and HORAC assays, validated the improved antioxidant effect of Relissa™ vs. unformulated dry MO extract. Conclusion: The results of the present study support the neuromodulating and neuroprotective properties of Relissa™, and its supplementation may help in the amelioration of emotional distress and related conditions.

Propyl gallate induces cell death in human pulmonary fibroblast through increasing reactive oxygen species levels and depleting glutathione

Propyl gallate (PG) exhibits an anti-growth effect on various cell types. The present study investigated the impact of PG on the levels of reactive oxygen species (ROS) and glutathione (GSH) in primary human pulmonary fibroblast (HPF) cells. Moreover, the effects of N-acetyl cysteine (NAC, an antioxidant), l-buthionine sulfoximine (BSO, a GSH synthesis inhibitor), and small interfering RNA (siRNAs) against various antioxidant genes on ROS and GSH levels and cell death were examined in PG-treated HPF cells. PG (100–800 μM) increased the levels of total ROS and O2·− at early time points of 30–180 min and 24 h, whereas PG (800–1600 μM) increased GSH-depleted cell number at 24 h and reduced GSH levels at 30–180 min. PG downregulated the activity of superoxide dismutase (SOD) and upregulated the activity of catalase in HPF cells. Treatment with 800 μM PG increased the number of apoptotic cells and cells that lost mitochondrial membrane potential (MMP; ΔΨm). NAC treatment attenuated HPF cell death and MMP (ΔΨm) loss induced by PG, accompanied by a decrease in GSH depletion, whereas BSO exacerbated the cell death and MMP (ΔΨm) loss without altering ROS and GSH depletion levels. Furthermore, siRNA against SOD1, SOD2, or catalase attenuated cell death in PG-treated HPF cells, whereas siRNA against GSH peroxidase enhanced cell death. In conclusion, PG induced cell death in HPF cells by increasing ROS levels and depleting GSH. NAC was found to decrease HPF cell death induced by PG, while BSO enhanced cell death. The findings shed light on how manipulating the antioxidant system influence the cytotoxic effects of PG in HPF cells.

The cadmium tolerance enhancement through regulating glutathione conferred by vacuolar compartmentalization in Aspergillus sydowii

Aspergillus was found to be a vital hyperaccumulation species for heavy metal removal with admirable tolerance capacity. But the potential tolerance mechanism has not been completely studied. This study quantified the amounts of total cadmium (Cd), Cd2+, glutathione (GSH), and reactive oxygen species (ROS) in the protoplasts and vacuoles of mycelium. We modulated GSH synthesis using buthionine sulfoximine (BSO) and 2-oxothiazolidine-4-carboxylic acid (OTC) to investigate the subcellular regulatory mechanisms of GSH in the accumulation of Cd. The results confirmed that GSH plays a crucial role in vacuolar compartmentalization under Cd stress. GSH and GSSG as a redox buffer to keep the cellular redox state in balance and GSH as a metal chelating agent to reduce toxicity. When regulating the decreased GSH content with BSO, and increased GSH content with OTC, the system of Cd-GSH-ROS can change accordingly, this also supported that vacuolar compartmentalization is a detoxification strategy that can modulate the transport and storage of substances inside and outside the vacuole reasonably. Interestingly, GSH tended to be distributed in the cytoplasm, the battleground of redox takes place in the cytoplasm but not in the vacuole. These finding potentially has implications for the understanding of tolerance behavior and detoxification mechanisms of cells. In the future bioremediation of Cd in soil, the efficiency of soil remediation can be improved by developing organisms with high GSH production capacity.

Identification and characterization of lettuce cultivars with high inhibitory activity against the human pathogen Escherichia coli O157:H7: Toward a plant-intrinsic hurdle approach to microbial safety

Foodborne illness linked to fruit and vegetables poses a major challenge to public health and horticulture production. Processed lettuce has been implicated in recurrent outbreaks of pathogenic Shiga toxin-producing Escherichia coli (STEC) infection. We hypothesized that plant defenses elicited by mechanical injury may effect STEC inhibition in cut leaves. Intact and cut leaves of 31 lettuce cultivars (Lactuca spp.) selected from >500 accessions based on their resistance to common insect pests and the necrotrophic lettuce drop pathogen were assessed for STEC serovar O157:H7 (EcO157) survival under cold storage conditions. The cultivars were previously ranked also for phenotypes such as resistance to other phytopathogens and physiological traits. Total leaf phenolic compounds, anthocyanins, and reactive oxygen species (ROS); and phenylalanine lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) activity were quantified in each cultivar. Principal Component Analysis (PCA) and K-means cluster analysis were applied to group phenotypes and EcO157 population decline on lettuce. In five cultivars, high resistance to herbivorous insects (aphids, leaf miners and thrips), diseases (lettuce drop, downy mildew, and Impatiens Necrotic Spot Virus), and the disorder tipburn was associated with up to 42-fold EcO157 population decline on cold-stored cut (shredded) leaves, and with up to 11-fold difference in decline on cold-stored intact vs cut leaves (ΔD). The decline on these cold-stored cut cultivars also was up to 24-fold greater than on the cultivar with the lowest decline, and up to 8-fold greater than the mean decline for all 31 cultivars (P < 0.01). Resistance to the hemibiotrophic pathogen causing lettuce bacterial leaf spot, Xanthomonas hortorum pv. vitians, was not indicative of high EcO157 inhibition in this system. High PAL activity, and phenolic and anthocyanin content (PPA); and high PAL and POD activity, were associated with high EcO157 population decline (P < 0.05). One cultivar with high susceptibility to herbivorous insects and phytopathogens, and low levels of defense metabolism nevertheless showed high EcO157 inhibition, suggesting other plant inhibitory characteristics at play. Basal plant immunity and pathways associated with a strong wound response may inform about enteric pathogen survival on minimally processed postharvest lettuce. The related traits identified in this study may serve to breed new genotypes with intrinsically enhanced microbial safety.

Effects of prenatal dexamethasone exposure on adult C57BL/6J mouse metabolism and oxidative stress.

Prenatal glucocorticoid exposure has been shown to alter hypothalamic-pituitary-adrenal axis function resulting in altered fetal development that can persist through adulthood. Fetal exposure to excess dexamethasone, a synthetic glucocorticoid, has been shown to alter adult behaviour and metabolism. This study investigated the effects prenatal dexamethasone exposure had on adult offspring cardiac and liver metabolism and oxidative stress. Pregnant C57BL/6 mice received a dose of 0.4 mg/kg dexamethasone on gestational days 15-17. Once pups were approximately 7 months old, glucose uptake was determined using positron emission tomography and insulin resistance (IR) was determined by homeostatic model assessment (HOMA) IR calculation. Oxidative stress was assessed by measuring 4-hydroxynonenal protein adduct formation and total reactive oxygen species. Female dexamethasone group had significantly increased glucose uptake when insulin stimulated compared to vehicle-treated mice. HOMA IR revealed no evidence of IR in either male or female offspring. There was also no change in oxidative stress markers in either cardiac or liver tissues of male or female offspring. These data suggest that prenatal dexamethasone exposure in male mice does not alter oxidative stress or metabolism. However, prenatal dexamethasone exposure increased glucocorticoids, cardiac glucose uptake, and pAkt signaling in female heart tissues in adult mice, suggesting there are sex differences in prenatal dexamethasone exposure.

TFR1 knockdown alleviates iron overload and mitochondrial dysfunction during neural differentiation of Alzheimer’s disease-derived induced pluripotent stem cells by interacting with GSK3B

Iron metabolism disorders are implicated in the pathogenesis of Alzheimer’s disease (AD). It was previously reported that transferrin receptor (TFR1) expression was upregulated in AD mouse model. However, the precise biological functions of TFR1 in AD progression remains unclear. Herein, we observed a gradual increase in TFR1 protein expression during the differentiation of AD patient-derived induced pluripotent stem cells (AD-iPS). TFR1 knockdown inhibited the protein expression of ferritin and ferritin heavy chain 1 (FTH1), enhanced the expression of ferroportin 1 (FPN1), and decreased intracellular levels of total iron, labile iron, and reactive oxygen species (ROS). Moreover, TFR1 knockdown improved mitochondrial membrane potential (MMP), increased adenosine triphosphate (ATP) content, downregulated mitochondrial fission proteins, and upregulated mitochondrial fusion proteins. TFR1 knockdown alleviated iron overload and mitochondrial dysfunction in neural cells differentiated from AD-iPS, while TFR1 overexpression showed the opposite results. Additionally, TFR1interacted with glycogen synthase kinase 3 beta (GSK3B) and promoted GSK3B expression. GSK3B overexpression reversed the inhibitory effects of TFR1 knockdown on iron overload and mitochondrial dysfunction in AD-iPS differentiated neural cells. In conclusion, TFR1 knockdown alleviated iron overload and mitochondrial dysfunction in neural cells differentiated from AD-iPS by promoting GSK3B expression. Our findings provide a potential therapeutic target for the treatment of AD.Graphical

POU6F1 promotes ferroptosis by increasing lncRNA-CASC2 transcription to regulate SOCS2/SLC7A11 signaling in gastric cancer

ObjectiveThis study investigated the effect and mechanism of POU6F1 and lncRNA-CASC2 on ferroptosis of gastric cancer (GC) cells.MethodsGC cells treated with erastin and RSL3 were detected for ferroptosis, reactive oxygen species (ROS) level, and cell viability. The expression levels of POU6F1, lncRNA-CASC2, SOCS2, and ferroptosis-related molecules (GPX4 and SLC7A11) were also measured. The regulations among POU6F1, lncRNA-CASC2, FMR1, SOCS2, and SLC7A11 were determined. Subcutaneous tumor models were established, in which the expressions of Ki-67, SOCS2, and GPX4 were detected by immunohistochemistry.ResultsGC patients with decreased expressions of POU6F1 and lncRNA-CASC2 had lower survival rate. Overexpression of POU6F1 or lncRNA-CASC2 decreased cell proliferation and GSH levels in GC cells, in addition to increasing total iron, Fe2+, MDA, and ROS levels. POU6F1 directly binds to the lncRNA-CASC2 promoter to promote its transcription. LncRNA-CASC2 can target FMR1 and increase SOCS2 mRNA stability to promote SLC7A11 ubiquitination degradation and activate ferroptosis signaling. Knockdown of SOCS2 inhibited the ferroptosis sensitivity of GC cells and reversed the effects of POU6F1 and lncRNA-CASC2 overexpression on ferroptosis in GC cells.ConclusionTranscription factor POU6F1 binds directly to the lncRNA-CASC2 promoter to promote its expression, while upregulated lncRNA-CASC2 increases SOCS2 stability and expression by targeting FMR1, thereby inhibiting SLC7A11 signaling to promote ferroptosis in GC cells and inhibit GC progression.

Assessment of wood smoke induced pulmonary toxicity in normal- and chronic bronchitis-like bronchial and alveolar lung mucosa models at air–liquid interface

BackgroundChronic obstructive pulmonary disease (COPD) has the highest increased risk due to household air pollution arising from biomass fuel burning. However, knowledge on COPD patho-mechanisms is mainly limited to tobacco smoke exposure. In this study, a repeated direct wood smoke (WS) exposure was performed using normal- (bro-ALI) and chronic bronchitis-like bronchial (bro-ALI-CB), and alveolar (alv-ALI) lung mucosa models at air–liquid interface (ALI) to assess broad toxicological end points.MethodsThe bro-ALI and bro-ALI-CB models were developed using human primary bronchial epithelial cells and the alv-ALI model was developed using a representative type-II pneumocyte cell line. The lung models were exposed to WS (10 min/exposure; 5-exposures over 3-days; n = 6–7 independent experiments). Sham exposed samples served as control. WS composition was analyzed following passive sampling. Cytotoxicity, total cellular reactive oxygen species (ROS) and stress responsive NFkB were assessed by flow cytometry. WS exposure induced changes in gene expression were evaluated by RNA-seq (p ≤ 0.01) followed by pathway enrichment analysis. Secreted levels of proinflammatory cytokines were assessed in the basal media. Non-parametric statistical analysis was performed.Results147 unique compounds were annotated in WS of which 42 compounds have inhalation toxicity (9 very high). WS exposure resulted in significantly increased ROS in bro-ALI (11.2%) and bro-ALI-CB (25.7%) along with correspondingly increased NFkB levels (bro-ALI: 35.6%; bro-ALI-CB: 18.1%). A total of 1262 (817-up and 445-down), 329 (141-up and 188-down), and 102 (33-up and 69-down) genes were differentially regulated in the WS-exposed bro-ALI, bro-ALI-CB, and alv-ALI models respectively. The enriched pathways included the terms acute phase response, mitochondrial dysfunction, inflammation, oxidative stress, NFkB, ROS, xenobiotic metabolism of AHR, and chronic respiratory disorder. The enrichment of the ‘cilium’ related genes was predominant in the WS-exposed bro-ALI (180-up and 7-down). The pathways primary ciliary dyskinesia, ciliopathy, and ciliary movement were enriched in both WS-exposed bro-ALI and bro-ALI-CB. Interleukin-6 and tumor necrosis factor-α were reduced (p < 0.05) in WS-exposed bro-ALI and bro-ALI-CB.ConclusionFindings of this study indicate differential response to WS-exposure in different lung regions and in chronic bronchitis, a condition commonly associated with COPD. Further, the data suggests ciliopathy as a candidate pathway in relation to WS-exposure.

ROS/mtROS promotes TNTs formation via the PI3K/AKT/mTOR pathway to protect against mitochondrial damages in glial cells induced by engineered nanomaterials

BackgroundAs the demand and application of engineered nanomaterials have increased, their potential toxicity to the central nervous system has drawn increasing attention. Tunneling nanotubes (TNTs) are novel cell–cell communication that plays a crucial role in pathology and physiology. However, the relationship between TNTs and nanomaterials neurotoxicity remains unclear. Here, three types of commonly used engineered nanomaterials, namely cobalt nanoparticles (CoNPs), titanium dioxide nanoparticles (TiO2NPs), and multi-walled carbon nanotubes (MWCNTs), were selected to address this limitation.ResultsAfter the complete characterization of the nanomaterials, the induction of TNTs formation with all of the nanomaterials was observed using high-content screening system and confocal microscopy in both primary astrocytes and U251 cells. It was further revealed that TNT formation protected against nanomaterial-induced neurotoxicity due to cell apoptosis and disrupted ATP production. We then determined the mechanism underlying the protective role of TNTs. Since oxidative stress is a common mechanism in nanotoxicity, we first observed a significant increase in total and mitochondrial reactive oxygen species (namely ROS, mtROS), causing mitochondrial damage. Moreover, pretreatment of U251 cells with either the ROS scavenger N-acetylcysteine or the mtROS scavenger mitoquinone attenuated nanomaterial-induced neurotoxicity and TNTs generation, suggesting a central role of ROS in nanomaterials-induced TNTs formation. Furthermore, a vigorous downstream pathway of ROS, the PI3K/AKT/mTOR pathway, was found to be actively involved in nanomaterials-promoted TNTs development, which was abolished by LY294002, Perifosine and Rapamycin, inhibitors of PI3K, AKT, and mTOR, respectively. Finally, western blot analysis demonstrated that ROS and mtROS scavengers suppressed the PI3K/AKT/mTOR pathway, which abrogated TNTs formation.ConclusionDespite their biophysical properties, various types of nanomaterials promote TNTs formation and mitochondrial transfer, preventing cell apoptosis and disrupting ATP production induced by nanomaterials. ROS/mtROS and the activation of the downstream PI3K/AKT/mTOR pathway are common mechanisms to regulate TNTs formation and mitochondrial transfer. Our study reveals that engineered nanomaterials share the same molecular mechanism of TNTs formation and intercellular mitochondrial transfer, and the proposed adverse outcome pathway contributes to a better understanding of the intercellular protection mechanism against nanomaterials-induced neurotoxicity.Graphical abstract

Unraveling the neuroprotective mechanisms of naltrexone against aluminum-induced neurotoxicity

Aluminum (Al) is a known neurotoxic trace element linked to Alzheimer’s disease (AD). Naltrexone, an opioid antagonist, has shown promising effects in reducing neuroinflammation at lower doses than those prescribed for addiction. This study aimed to determine the neuroprotective effects of naltrexone on Al-induced neurotoxicity in an in vitro AD model. The SH-SY5Y cells were first cultivated in a standard growth medium. Subsequently, the cells were induced to differentiate by decreasing the concentration of fetal bovine serum and introducing retinoic acid (RA) into the culture media. Subsequently, the inclusion of brain-derived neurotrophic factor (BDNF) was implemented in conjunction with RA. The process of differentiation was concluded on the seventh day. Study groups (n = 3) were designed as the control group, naltrexone group, Al group, Al-Nal group, Alzheimer’ model (AD) group, Alzheimer model + Al-exposed group (AD-Al), Alzheimer model + Nal applied group (AD-Nal) and Alzheimer model + Al-exposed + Nal applied group (AD-Al-Nal). Hyperphosphorylated Tau protein as the specific marker of AD was measured in all groups. Glycogen synthase kinase-3 (GSK-3)β, Protein phosphatase 2A (PP2A), Akt and Wnt signaling pathways were analyzed comparatively. In addition, oxidative stress parameters (total antioxidant capacity, lipid peroxidase, protein carbonyl and reactive oxygen species) were measured comparatively in the study groups. The results showed that naltrexone reduced hyperphosphorylated tau protein levels by regulating GSK-3β, PP2A, Akt and Wnt signaling. Also, exposure to naltrexone decreased oxidative stress parameters. Based on these results, naltrexone shows promise as a potential therapy for AD, subject to additional clinical assessments.

SB431542 partially inhibits high glucose-induced EMT by restoring mitochondrial homeostasis in RPE cells

BackgroundThe epithelial–mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells participated in the development of retinal fibrosis. SB431542 is a small molecule inhibitor with inhibitory effects on the ALK4, ALK5 and ALK7. Our study aimed to explore the effect of SB431542 on the EMT of RPE cells and to provide new ideas for the treatment of retinal fibrosis.MethodsWe performed fundus fluorescein angiography, optical coherence tomography and hematoxylin–eosin staining in vivo to observe the effect of SB431542 on choroidal neovascularization (CNV)-induced retinopathy. The proliferation, migration, cytoskeleton, adhesion, reactive oxygen species (ROS), mitochondrial morphology and membrane potential of RPE cells were observed in vitro through fluorescein diacetate staining, Cell Counting Kit-8 experiment, wound healing assay, phalloidin staining, immunofluorescence, MitoSOX, DCFH-DA, MitoTracker and JC-10 staining. Western blot, reverse transcription quantitative and immunofluorescence were used to detect the expression of EMT–related markers, pERK1/2, pGSK3β and β-catenin.ResultsSB431542 significantly alleviated retinopathy in the CNV model. The proliferation, migration and adhesion in RPE cells decreased to a certain extent in SB431542 treatment. SB431542 partially normalized the structure of RPE cells. The expression levels of E-cadherin increased, while the expression levels of laminin and N-cadherin decreased with SB431542 treatment. SB431542 reduced the production of total ROS, mitochondrial SOX and recovered the mitochondrial membrane potential to a certain degree. In addition, our study showed that SB431542 downregulated the phosphorylation of ERK1/2, GSK3β and the expression of β-catenin.ConclusionSB431542 improved EMT in RPE cells by maintaining mitochondrial homeostasis via the ERK1/2 and GSK3β/β-catenin pathways.1HzEs7TmoETCuNYu8FcEmbVideo Graphical SB431542 inhibits EMT in RPE cells under high glucose conditions.