mRNA Levels Of Nrf2 Research Articles

The role of TF-b in iron homeostasis and bacterial defense in common carp (Cyprinus carpio)

Transferrin (TF) is a prototypical biological macromolecule protein known for its iron-binding properties. TF proteins play a crucial role in modulating host iron homeostasis and defending against pathogen invasion. In this study, we utilized common carp (Cyprinus carpio) tissues and Epithelioma papulosum cyprinid (EPC) cells to establish experimental models of iron overload with FeCl3 or ferric amine citrate (FAC), and to establish experimental models of bacterial infection with Aeromonas hydrophila. The current research has successfully identified the CcTF-b gene in common carp, revealing an ORF of 2001 bp with N-terminal and C-terminal structures. CcTF-b exhibited inhibitory effects on the growth of LPS and LTA in vitro. In the experimental models, the upregulations of PTGS2a and PTGS2a-like mRNA and protein levels were observed. Overexpression or interference with CcTF-b levels can modulate the expression of ferroptosis-related genes, inflammatory cytokines, lipid reactive oxygen species, GSH/GSSH levels, and Fe2+ concentration. Significantly, the expression levels of Nrf2 and GPX4 mRNA and protein, as well as the bacterial load of A. hydrophila, could be also modulated either by upregulating or downregulating CcTF-b factors. In conclusion, in this study, these findings suggest that CcTF-b plays a critical role in the innate immune response of common carp.

Chlorogenic Acid Plays an Important Role in Improving the Growth and Antioxidant Status and Weakening the Inflammatory Response of Largemouth Bass (Micropterus salmoides).

This experiment evaluated the function of chlorogenic acid (CGA) in the growth, health status, and inflammation of largemouth bass (Micropterus salmoides). Over eight weeks, CGA supplementation was designed at five levels: 0, 60, 120, 180, and 240 mg/kg. The 180 and 240 mg/kg CGA-supplemented groups showed significant improvements in the FBW, SGR, and WGR compared to the control group (0 mg/kg) (p < 0.05). All the CGA-supplemented groups exhibited a significant reduction in the FCR (p < 0.05), with the 180 mg/kg CGA group showing the lowest FCR. Nonetheless, there were no appreciable differences in the plasma concentrations of TP, ALT, or AST among the treatments (p > 0.05). Compared to the control group, the 180 mg/kg CGA group exhibited significantly lower TC and TG levels (p < 0.05). The ALP levels showed no significant differences from the control group (p > 0.05). In terms of antioxidant parameters, CGA supplementation considerably reduced the MDA content (p < 0.05) and increased the GSH levels, while decreasing the CAT, SOD, and GPx activity levels Meanwhile, CGA supplementation resulted in reduced mRNA levels of SOD, CAT, Nrf2, Keap1, and NF-κB compared to the control group. In contrast, the mRNA levels of GPx, IL-8, TLR2, and RelA were elevated in the liver. Our findings indicated that CGA supplementation improved the growth performance and antioxidant status and weakened the inflammatory response of largemouth bass. These findings suggest that CGA could be a valuable dietary supplement for enhancing the health and growth of this species.

Impact of Indole Inclusion in the Design of Multi-Tactical Metal-Binding Tetra-Aza Macrocycles that Target the Molecular Features of Neurodegeneration.

Numerous small molecules have been studied for their ability to counteract oxidative stress, a key contributor to neurodegenerative diseases such as Alzheimer's. Despite these efforts, the pharmacological properties and structure-activity relationships of these compounds remain insufficiently understood, yet they are critical in evaluating a drug molecule's therapeutic potential. A modified tetra-aza macrocycle has demonstrated strong antioxidant activity through various mechanisms; however, its limited permeability presents challenges for advanced formulation studies. To enhance permeability while preserving the beneficial reactivity of the parent molecule, two synthetic modifications involving indole functionality were explored and compared to modifications using methyl groups alone. New synthetic strategies were developed to produce the indole-containing molecules, which were characterized by 1D/2D NMR techniques. Isoelectric points, metal binding, and radical scavenging activity were determined to validate that the reactivity of the parent molecules was retained. The permeability of all molecules explored was improved. Protection against oxidative stress through activation of the Nrf2 pathway was demonstrated for molecules containing indoles in cellular models by measuring ROS levels upon treatment and mRNA levels of HO-1 and Nrf2. In contrast, no protection or Nrf2 activation was observed with the methylation of the O- or N atom. These results suggest that while alkylation improves permeability overall, concomitant antioxidant protection and positive permeability are achieved with the indole congeners alone.

Integrating network pharmacology and experimental verification to study the mechanism of Polygonum hydropiper total flavonoids against stress-induced gastric mucosal damage

ContextPolygonum hydropiper L. (P. hydropiper) has outstanding clinical efficacy in treating both acute and chronic gastroenteritis. However, the definite mechanism remains unclear. ObjectiveThis study aimed to explore the potential mechanisms of the total flavonoid of P. hydropiper (FPH) in stress-induced gastric mucosal damage (SGMD) rats through a combination of network pharmacology, molecular docking, and animal experiments. MethodsNetwork pharmacology and molecular docking were utilized to predict the potential mechanisms of FPH against SGMD. In experimental studies, SGMD rat models were established using water-immersion restraint stress (WIRS). FPH was administered at doses of 140, 70, and 35 mg/kg, with ranitidine serving as a positive control, through gavage once daily for 6 consecutive days after model establishment. Stomach and serum specimens were analyzed using HE staining, Western blotting, qPCR, and ELISA to investigate the protective mechanism of FPH in SGMD. ResultsThe network pharmacology analysis identified 16 active ingredients and 183 common targets, with potential pathways including PI3K/Akt, MAPK and Keap1/Nrf2. In vivo experiments demonstrated that FPH intervention alleviated SGMD pathological changes, reduced elevated serum IL-6 and TNF-α levels, and enhanced SOD and GSH activity in rats. Additionally, FPH increased the protein expression of p62, Nrf2, HO-1, PI3K, and p-Akt, along with mRNA levels of Nrf2, p62, and HO-1. ConclusionsFPH exerts a gastric mucosal protective effect by upregulating antioxidant gene expression through the PI3K/Akt and Keap1/Nrf2 pathways. This study provides an experimental basis for the potential clinical treatment of SGMD with the traditional Chinese medicine P. hydropiper.

Genetic Variations in the NRF2 Microsatellite Contribute to the Regulation of Bovine Sperm-Borne Antioxidant Capacity.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor protein-coding gene, considered a master regulator of the cellular stress response. The genetic variations of the NRF2 could influence its transcriptional profile and, subsequently, the stress resilience in all cell types, including sperm cells. Therefore, the sperm-borne antioxidants abundance in association with the genetic variation of a GCC microsatellite located at the 5' upstream region of the NRF2 gene was investigated in young (n = 8) and old (n = 8) Holstein bulls' sperm cells at different seasons. The sperm DNA was sequenced using Sanger sequencing, while- the sperm-borne mRNA analysis was carried out using the synthesized cDNA and qPCR. The data were statistically analyzed using GraphPad Prism 10.0.2 software. The results showed that two bulls had a heterozygous genotype of eight and nine GCC repeats, while biallelic of eight, nine, and fifteen repeats were identified in two, ten, and two bulls, respectively. The computational in silico analysis revealed that the NRF2 upstream sequence with 15, 9, and 8 GCC repeats bound with 725, 709, and 707 DNA-binding transcription factor proteins, respectively. Lower quality of sperm DNA was detected in the spring season compared to other seasons and in young bulls compared to old ones, particularly in the summer and autumn seasons. The mRNA expression analysis revealed that the PRDX1 gene was the abundant transcript among the studied sperm-borne antioxidants and was significantly determined in old bulls' spermatozoa. Moreover, two transcripts of the NRF2 gene and antioxidant (SOD1, CAT, GPX1, TXN1, NQO1) genes displayed differential expression patterns between the age groups across seasons in an antioxidant-dependent manner. The bulls with a heterozygous GCC sequence exhibited elevated sperm-borne mRNA levels of NRF2 and PRDX1 transcripts. Taken together, the findings suggest that the NRF2-GCC microsatellite may contribute to the transcription regulation of NRF2 transcripts and their subsequent downstream antioxidants in bovine sperm cells.

Lycium barbarum polysaccharides (LBP) suppresses hypoxia/reoxygenation (H/R)-induced rat H9C2 cardiomyocytes pyroptosis via Nrf2/HO-1 signaling pathway

This study aimed to explore the mechanism that Lycium barbarum polysaccharides (LBP) suppress hypoxia/reoxygenation (H/R)-caused pyroptosis in cardiomyocytes (H9C2) via the Nrf2/HO-1 pathway. Initially, we established the cell model of H/R (6 h hypoxia plus with 24 h reoxygenation), and found that 90 μg/mL LBP was the optimal concentration. Subsequently, we confirmed that LBP reduced the apoptosis rate of cells after H/R, the activity of LDH, the inflammatory factors IL-1β and IL-18, and the levels of pyroptosis-specific markers ASC, NLRP3, and Caspase-1 (mRNAs and proteins). It increased the cell survival rate and the mRNA levels of the Nrf2/HO-1 pathway markers Nrf2 and HO-1, and allowed cytoplasmic Nrf2 protein to enter the nucleus to activate HO-1 protein. The Nrf2 siRNA2 caused the following events in H/R model: (1) the increases of the apoptosis rate, LDH activity, the levels of inflammatory factors (IL-1β and IL-18), the levels of ACS, NLRP3, and Caspase-1 (mRNAs and proteins); and (2) the decreases of the cell survival rate, the mRNA levels of Nrf2 and HO-1, and the protein levels of cytoplasm-Nrf2, nucleus-Nrf2, and HO-1. Therefore we concluded that 90 μg/mL LBP suppressed H/R-induced H9C2 cardiomyocyte pyroptosis via the Nrf2/HO-1 pathway.

Increased susceptibility of human limbal aniridia fibroblasts to oxidative stress

Aniridia-associated keratopathy originates from a haploinsufficiency of the transcription factor PAX6 (PAX6+/−). In the corneal epithelium of PAX6+/− mice, a significant increase in oxidized proteins was observed, accompanied by impaired compensation for elevated oxidative stress (OS). The extent to which limbal fibroblast cells (LFCs) are affected by an increased susceptibility to OS in cases of congenital aniridia (AN) has not been determined, yet. Our aim was to examine the impact of OS on antioxidant enzyme expression in normal and AN-LFCs. Following isolation and culture of primary LFCs (n = 8) and AN-LFCs (n = 8), cells were treated with cobalt chloride for 48 h to chemically induce hypoxic conditions and OS. Subsequently, HIF-1α/-2α, PHD1/2, Nrf2, CAT, SOD1, PRDX6, and GPX1 gene expression was examined by qPCR. SOD1, PRDX6, and GPX1 protein levels were assessed from the cell lysate by Western blot. The induction of hypoxia led to reduced HIF-1α gene expression in both fibroblast groups (p≤0.008), while the decrease in PHD1 was limited to AN-LFCs (p = 0.0007). On the other hand, under hypoxic conditions, PHD2 showed higher mRNA expression in AN-LFCs compared to normal LFCs (p = 0.013). As a result of OS, the mRNA levels of Nrf2 (p<0.0001) and the antioxidant enzymes CAT (p = 0.005), SOD1 (p = 0.005), GPX1 (p = 0.002) decreased in AN-LFCs. This was accompanied by an increased protein expression of SOD1 (p = 0.019) and PRDX6 (p=0.0009). In the normal LFC group, the induced extent of OS had no impact on the gene (p≥0.151) and protein expression (p ≥ 0.629) of antioxidant enzymes, except for the GPX1 mRNA level (p = 0.027). AN-LFCs exhibit higher susceptibility to OS than normal LFCs. Therefore, in AN-LFCs, there are sustained alterations in gene and protein expression of antioxidative enzymes even after 48 h of CoCl2 treatment.

Ameliorative Effect of Lycopene on Follicular Reserve Depletion, Oxidative Damage, Apoptosis Rate, and Hormonal Profile during Repeated Superovulations in Mice.

Superovulation is a crucial step in assisted reproductive technology that involves the administration of gonadotrophins. Repeated superovulations result in severe ovarian damage. The present study investigated the effect of in vivo administration of lycopene on ovarian damage induced by four successive cycles of superovulation. Superovulated mice were simultaneously administered intraperitoneally with saline (R4) or 5 mg/kg lycopene (R4-Lyc). The evaluated parameters were the count of different types of follicles, expression of ovarian antioxidant- and apoptosis-related genes, and serum concentrations of estradiol, progesterone, and inhibin-B. Increased numbers of healthy follicles and a decreased count of atretic follicles were observed in mice of the R4-Lyc group compared to those of the R4 group. Moreover, significantly higher mRNA levels of Sod3, Cat, and Nrf2 and lower mRNA levels of Keap1, Tnf, Nfkb, and Casp3, together with decreased H2O2 concentrations and increased total antioxidant capacity, were detected in the ovaries of lycopene-treated mice. Regarding serum reproductive hormones, elevated concentrations of estradiol, progesterone, and inhibin-B were evident in lycopene-administered mice. The present study reports a significant role of lycopene in alleviating the ovarian damage induced by multiple hormonal superstimulations, which could help to improve the outcomes of in vitro embryo production.

Lactiplantibacillus plantarum JM113 alleviates deoxynivalenol induced intestinal damage by microbial modulation in broiler chickens

Deoxynivalenol (DON) contamination causes the grievous injury in public and animal health, poultry suffer from the greater toxin challenge. Probiotic have been considered as a potential way to mitigate the deleterious effects of DON. In this study, a total of 144 1-day-old Arbor Acres chickens were randomly assigned into 3 groups: control group, DON group (5 mg/kg DON diet), DJ group (1×109 cfu Lactiplantibacillus plantarum JM113/kg DON diet). The results showed that Lactiplantibacillus plantarum JM113 (L. plantarum JM113) increased the growth performance of 21-day-old broilers that challenged by the DON (P < 0.05), and the DON-induced disorder of jejunal morphology was recovered in DJ group (P < 0.05). Compared with the DON group, the mRNA and protein levels of Nrf2 and NQO-1 were upregulated in jejunum of DJ group broilers (P < 0.05). Meanwhile, administration of L. plantarum JM113 effectively increased the expression level of barrier-related genes, and the protein abundance of occludin and claudin1 (P < 0.05). L. plantarum JM113 restored the mRNA and protein abundance of PCNA, and proliferation-linked gene (Lgr5 and Bmi1) expression levels in jejunum of DON-insulted broilers (P < 0.05). Furthermore, administration of L. plantarum JM113 significantly enhanced the relative abundance of s_Limosilactobacillus_reuteri in jejuna of DON-challenged broilers (P < 0.05). Spearman correlation analysis showed that s_Limosilactobacillus_reuteri was positively associated with the jejunal barrier related genes (P < 0.05). In conclusion, L. plantarum JM113 alleviated the toxic effects of DON by regulating the jejunal function through microbial adjustment. Our findings proposed a viable approach to mitigating the adverse effects of deoxynivalenol exposure in broilers.

Changes in redox network expression during Caco-2 cell differentiation into enterocytes

Graphical abstract Abstract Objective This work characterized fluctuations in cell components involved in the regulation of cell redox homeostasis during Caco-2 cell differentiation into enterocytes. Methods Caco-2 cells were differentiated for 10 days. Gene expression of NADPH oxidases; enzymes that metabolize superoxide anion and hydrogen peroxide, proteins involved in the production and/or regeneration of glutathione, thioredoxin, and in NADPH production, and NRF2-dependent genes were measured by qPCR at 0, 1, 4, 7, and 10 days post-confluence. Results NADPH oxidase 1 mRNA levels decreased with Caco-2 cell differentiation, in agreement with its role in regulating cell proliferation. NADPH oxidase 4, DUOX2, superoxide dismutase 1, and catalase mRNA levels increased with differentiation. NRF2 mRNA levels increased with differentiation up to day 4 post-confluence, reaching a plateau until day 10. A similar pattern was observed for the NRF2-regulated genes: NAD(P)H quinone dehydrogenase 1, glutathione reductase 1, and thioredoxin reductase 1. On the contrary, glutamate-cysteine ligase catalytic subunit mRNA levels decreased after reaching a maximum 4 days post-confluence. This and the finding of a correlation between glutathione reductase 1 and thioredoxin reductase 1 mRNA levels suggest that recycling of glutathione and thioredoxin is more relevant than their synthesis during Caco-2 cell differentiation. Conclusion Results support the relevance of redox homeostasis for cell fate decisions and in preparing enterocytes to interact with their environment. Significance statement Current findings resemble changes in redox components previously characterized in vivo. This stresses the concept that Caco-2 cells are an appropriate model to be used to evaluate redox-regulated mechanisms in human enterocytes.

Acute waterborne cadmium exposure induces liver ferroptosis in Channa argus

The impact of cadmium (Cd) toxicity on fish liver injury has received much attention in recent years. Currently, autophagy, apoptosis and endoplasmic reticulum stress were reported in Cd exposed fish liver, and if there are other mechanisms (such as ferroptosis) and relevant signaling pathways involved in fish remains unknown. An experiment was conducted to investigate Cd toxicity in Channa argus (Cantor, 1842) exposed to 0, 1.0, and 2.0 mg Cd/L of water for 96 h. Cd disrupted the structure of mitochondria in the liver. Besides, Cd induced ferroptosis by significantly increasing the level of Fe2+, ROS, MDA and significantly decreasing the level of Ferritin, GSH, GSH-Px, GPX4, GST and SOD (p < 0.05 in all cases). In addition, the mRNA expression of ferroptosis related genes, gpx4 and slc7a11, were significantly downregulated by Cd. Moreover, Cd exposure significantly inhibited the Nrf2/Keap1 signaling pathway, one of the pathways involved in ferroptosis, by upregulating the mRNA levels of keap1a and keap1b, and downregulating the mRNA levels of nrf2 and its target genes (ho-1, nqo1 and cat). Cd exposure also caused extensive accumulation of vacuoles and lipid droplets in liver, as well as an increase in triglyceride content. Cd significantly affected lipid metabolism related enzyme activity and gene expression, which were also regulated by Nrf2/Keap1 signaling pathway. In summary, these results indicate that ferroptosis is a mechanism in waterborne Cd exposed fish liver injury via the Nrf2/Keap1 signaling pathway and the Cd induced hepatic steatosis is also modulated by Nrf2/Keap1 pathway at the whole-body level in fish. These findings provide new insights into the fish liver injury and molecular basis of Cd toxicity.

Berberine Inhibits Ferroptosis and Stabilizes Atherosclerotic Plaque through NRF2/SLC7A11/GPX4 Pathway.

To investigate potential mechanisms of anti-atherosclerosis by berberine (BBR) using ApoE-/- mice. Eight 8-week-old C57BL/6J mice were used as a blank control group (normal), and 56 8-week-old AopE-/- mice were fed a high-fat diet for 12 weeks, according to a completely random method, and were divided into the model group, BBR low-dose group (50 mg/kg, BBRL), BBR medium-dose group (100 mg/kg, BBRM), BBR high-dose group (150 mg/kg, BBRH), BBR+nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor group (100 mg/kg BBR+30 mg/kg ML385, BBRM+ML385), NRF2 inhibitor group (30 mg/kg, ML385), and positive control group (2.5 mg/kg, atorvastatin), 8 in each group. After 4 weeks of intragastric administration, samples were collected and serum, aorta, heart and liver tissues were isolated. Biochemical kits were used to detect serum lipid content and the expression levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in all experimental groups. The pathological changes of atherosclerosis (AS) were observed by aorta gross Oil Red O, aortic sinus hematoxylin-eosin (HE) and Masson staining. Liver lipopathy was observed in mice by HE staining. The morphology of mitochondria in aorta cells was observed under transmission electron microscope. Flow cytometry was used to detect reactive oxygen species (ROS) expression in aorta of mice in each group. The content of ferrous ion Fe2+ in serum of mice was detected by biochemical kit. The mRNA and protein relative expression levels of NRF2, glutathione peroxidase 4 (GPX4) and recombinant solute carrier family 7 member 11 (SLC7A11) were detected by quantitative real time polymerase chain reaction (RT-qPCR) and Western blot, respectively. BBRM and BBRH groups delayed the progression of AS and reduced the plaque area (P<0.01). The characteristic morphological changes of ferroptosis were rarely observed in BBR-treated AS mice, and the content of Fe2+ in BBR group was significantly lower than that in the model group (P<0.01). BBR decreased ROS and MDA levels in mouse aorta, increased SOD activity (P<0.01), significantly up-regulated NRF2/SLC7A11/GPX4 protein and mRNA expression levels (P<0.01), and inhibited lipid peroxidation. Compared with the model group, the body weight, blood lipid level and aortic plaque area of ML385 group increased (P<0.01); the morphology of mitochondria showed significant ferroptosis characteristics; the serum Fe2+, MDA and ROS levels increased (P<0.05 or P<0.01), and the activity of SOD decreased (P<0.01). Compared with BBRM group, the iron inhibition effect of BBRM+ML385 group was significantly weakened, and the plaque area significantly increased (P<0.01). Through NRF2/SLC7A11/GPX4 pathway, BBR can resist oxidative stress, inhibit ferroptosis, reduce plaque area, stabilize plaque, and exert anti-AS effects.

Sulodexide protects endothelial cells against 4-hydroxynonenal-induced oxidative stress and glutathione-dependent redox imbalance by modulation of sestrin2/nuclear factor erythroid 2-related factor 2 pathway.

The lipid peroxidation product 4-hydroxynonenal (HNE) may be involved in vascular endothelial cell damage by induction of oxidative stress, apoptosis, and loss of redox homeostasis. There is evidence that stimulation of endothelial cells with 4-HNE induces the activation of the nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap-1) pathway. Sestrin2 protein (SESN2) is one of the key regulators of Nrf2 and is involved in the cellular response to oxidative stress. However, the function of SESN2 in HNE-induced endothelial injury is not yet understood. Sulodexide (SDX) is a mixture of glycosaminoglycans used in clinical practice in the treatment of chronic venous and arterial diseases. While SDX has well-documented endothelial protective properties, little is known about its antioxidant effects. The aim of this study was to elucidate the molecular mechanisms activated by SDX in human umbilical endothelial cells (HUVECs) under HNE-induced oxidative stress. In this experimental model, we decided to evaluate the anti-apoptotic and antioxidant potential of SDX and its effect on the SESN2/Nrf2/GSH pathway. HUVECs were treated with 25 _M HNE or HNE combined with 0.5 LRU/mL SDX for 4 hours. Cell viability, apoptosis and intracellular reactive oxygen species (ROS) production were assessed by MTT assay and fluorescence microscopy. The expressions of Bax, cleaved caspase-3, Keap-1 and Nrf2 were determined by Western blot analysis. The intracellular concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by colorimetric assay. SESN2, glutamate-cysteine ligase catalytic subunit (GCLc) and glutathione synthase (GSS) were assessed using ELISA. RT-qPCR was performed to detect Nrf2, GCLc and GSS mRNA levels. Transient Nrf2 silencing was obtained by short interfering RNA (siRNA). We have demonstrated that SDX can reduce the negative impact of HNE on HUVECs. SDX significantly protected HNE-treated HUVECs from apoptosis (p<0.001) and oxidative stress (p<0.001). SDX treatment significantly reduced Bax (p<0.05) and cleaved caspase-3 (p<0.01) expression. Co-administration of HNE and SDX increased GSH content (p<0.001) and GSH:GSSG ratio (p<0.001) as well as decreased SESN2 concentration (p<0.001) and Nrf2 (p<0.01), GCLc (p<0.05) and GSS (p<0.01) gene expression compared with the HNE group. Moreover, we revealed a negative correlation between SESN2 levels and GSH concentrations (p<0.001). Nrf2 silencing significantly decreased the effect of HNE and SDX on the induction of GCLc and GSS genes. SDX also significantly ameliorated the increase of nuclear Nrf2 in response to HNE (p<0.05). The results confirmed that SDX may protect against HNE-induced endothelial damage through its antioxidant effect and modulation of the SESN2/Nrf2/GSH signaling pathway.

Effects of H9N2 avian influenza virus infection on metabolite content and gene expression in chick DF1 cells

After viral infection, the virus relies on the host cell's complex metabolic and biosynthetic machinery for replication. However, the impact of avian influenza virus (AIV) on metabolites and gene expression in poultry cells remains unclear. To investigate this, we infected chicken embryo fibroblasts DF1 cells with H9N2 AIV at an MOI of 3. Our aim was to explore how H9N2 AIV alters DF1 cells metabolic pathways to facilitate its replication. We employed metabolomics and transcriptomics techniques to analyze changes in metabolite content and gene expression. Metabolomics analysis revealed a significant increase in glutathione-related metabolites, including reduced glutathione (GSH), oxidized glutathione (GSSG) and total glutathione (T-GSH) upon H9N2 AIV infection in DF1 cells. Elisa results confirmed elevated levels of GSH, GSSG, and T-GSH consistent with metabolomics findings, noting a pronounced increase in GSSG compared to GSH. Transcriptomics showed significant alterations in genes involved in glutathione synthesis and metabolism post-H9N2 infection. However, adding the glutathione synthesis inhibitor BSO exogenously significantly promoted H9N2 replication in DF1 cells. This was accompanied by increased mRNA levels of pro-inflammatory cytokines (IL-1β, IFN-γ) and decreased mRNA levels of anti-inflammatory cytokines (TGF-β, IL-13). BSO also reduced catalase (CAT) gene expression and inhibited its activity, leading to higher reactive oxygen species (ROS) and malondialdehyde (MDA) level in DF1 cells. qPCR results indicated decreased mRNA levels of Nrf2, NQO1, and HO-1 with BSO, ultimately increasing oxidative stress in DF1 cells. Therefore, the above results indicated that H9N2 AIV infection in DF1 cells activated the glutathione metabolic pathway to enhance the cell's self-defense mechanism against H9N2 replication. However, when GSH synthesis is inhibited within the cells, it leads to an elevated oxidative stress level, thereby promoting H9N2 replication within the cells through Nrf2/HO-1 pathway. This study provides a theoretical basis for future rational utilization of the glutathione metabolic pathway to prevent viral replication.

Sex similarities and divergences in systemic and muscle iron metabolism adaptations to extreme physical inactivity in rats.

Previous data in humans suggest that extreme physical inactivity (EPI) affects iron metabolism differently between sexes. Our objective was to deepen the underlying mechanisms by studying rats of both sexes exposed to hindlimb unloading (HU), the reference experimental model mimicking EPI. Eight-week-old male and female Wistar rats were assigned to control (CTL) or hindlimb unloading (HU) conditions (n=12/group). After 7days of HU, serum, liver, spleen, and soleus muscle were removed. Iron parameters were measured in serum samples, and ICP-MS was used to quantify iron in tissues. Iron metabolism genes and proteins were analysed by RT-qPCR and Western blot. Compared with control males, control females exhibited higher iron concentrations in serum (+43.3%, p<0.001), liver (LIC; +198%, P<0.001), spleen (SIC; +76.1%, P<0.001), and transferrin saturation (TS) in serum (+53.3%, P<0.001), contrasting with previous observations in humans. HU rat males, but not females, exhibited an increase of LIC (+54% P<0.001) and SIC (+30.1%, P=0.023), along with a rise of H-ferritin protein levels (+60.9% and +134%, respectively, in liver and spleen; P<0.05) and a decrease of TFRC protein levels (-36%; -50%, respectively, P<0.05). HU males also exhibited an increase of splenic HO-1 and NRF2 mRNA levels, (p<0.001), as well as HU females (P<0.001). Concomitantly to muscle atrophy observed in HU animals, the iron concentration increased in soleus in females (+26.7, P=0.004) while only a trend is observed in males (+17.5%, P=0.088). In addition, the H-ferritin and myoglobin protein levels in soleus were increased in males (+748%, P<0.001, +22%, P=0.011, respectively) and in females (+369%, P<0.001, +21.9%, P=0.007, respectively), whereas TFRC and ferroportin (FPN) protein levels were reduced in males (-68.9%, P<0.001, -76.8%, P<0.001, respectively) and females (-75.9%, P<0.001, -62.9%, P<0.001, respectively). Interestingly, in both sexes, heme exporter FLVCR1 mRNA increased in soleus, while protein levels decreased (-39.9% for males P=0.010 and -49.1% for females P<0.001). Taken together, these data support that, in rats (1) extreme physical inactivity differently impacts the distribution of iron in both sexes, (2) splenic erythrophagocytosis could play a role in this iron misdistribution. The higher iron concentrations in atrophied soleus from both sexes are associated with a decoupling between the increase in iron storage proteins (i.e., ferritin and myoglobin) and the decrease in levels of iron export proteins (i.e., FPN and FLVCR1), thus supporting an iron sequestration in skeletal muscle under extreme physical inactivity.

Excessive Replacement of Fish Meal by Soy Protein Concentrate Resulted in Inhibition of Growth, Nutrient Metabolism, Antioxidant Capacity, Immune Capacity, and Intestinal Development in Juvenile Largemouth Bass (Micropterus salmoides).

This study investigated the effects of replacing 0% (SPC0), 25% (SPC25), 50% (SPC50), 75% (SPC75), and 100% (SPC100) of fish meal (FM) with soy protein concentrate (SPC) on the growth, nutritional metabolism, antioxidant capacity, and inflammatory factors in juvenile largemouth bass (Micropterus salmoides) (17.03 ± 0.01 g). After 56 days of culturing, various growth parameters including FW, WGR, and SGR were not significantly different among SPC0, SPC25, and SPC50 groups; however, they were significantly higher than those in SPC75 and SPC100 groups. Conversely, significantly lower FCR were determined for the SPC0, SPC25, and SPC50 groups compared with that for the SPC100 group; specifically, no significant difference among SPC0, SPC25, and SPC50 groups was found. Moreover, compared with SPC75 and SPC100 groups, a significantly higher FI was observed in the SPC0 group, whereas a significantly lower SR was observed in SPC100 compared with that in SPC0 and SPC25 groups. Compared with the SPC0 group, significantly lower mRNA levels of tor, rps6, 4ebp1, pparγ, and fas were found in SPC75 and SPC100. Additionally, the mRNA levels of cpt were significantly higher in SPC0, SPC25, and SPC50 groups than in SPC75 and SPC100 groups. Moreover, the mRNA levels of scd and acc remained unchanged for all the groups. Replacement of FM with SPC did not significantly affect the mRNA levels of gk, pk, and pepck. Compared with the SPC0 group, significantly decreased activities of CAT were observed in the SPC50, SPC75, and SPC100 groups, and significantly decreased activities of GSH-Px were observed in the SPC75 and SPC100 groups. In addition, significantly lower activity of SOD was observed in SPC100 compared with the other groups. Moreover, compared with the other groups, the SPC75 and SPC100 groups had significantly decreased and increased contents of GSH and MDA, respectively, while significantly lower mRNA levels of nrf2, cat, sod, and gsh-px were found in SPC50, SPC75, and SPC100; however, significantly higher mRNA levels of keap1 were observed in SPC75 and SPC100 groups. Additionally, significantly higher mRNA levels of il-8 and nf-κb were found in the SPC50, SPC75, and SPC100 groups compared with the SPC0 group. Conversely, significantly lower mRNA levels of il-10 and significantly higher mRNA levels of tnf-α were found in the SPC75 and SPC100 groups compared with the other groups. Compared with the SPC0 group, mucosal thickness and villus height were significantly decreased in the SPC75 and SPC100 groups. Collectively, SPC replacing 50% FM did not affect its growth of juvenile largemouth bass. However, SPC replacing 50% or more FM might inhibit antioxidant capacity and immune capacity to even threaten the SR, resulting in impaired intestinal development in replacing FM level of 75% or more.

Sitagliptin Alleviates Obesity in Immature Mice by Inhibiting Oxidative Stress and Inflammation.

To investigate the impact of Sitagliptin against obesity and the underlying mechanism. Obese immature mice were treated with 10, 30, and 90mg/kg Sitagliptin, respectively. The body weights were recorded and the level of serum biochemical indexes were detected. The visceral fat ratio of each mouse was determined. The pathological change in adipose tissues was determined by HE staining, while F4/80 and CD206 levels in adipose tissues were determined by the immunohistochemical analysis. Lipid formation was evaluated by Oil red O staining assay. RAW264.7 cells were stimulated using oxLDL, followed by being incubated with different concentrations of Sitagliptin. The release of ADPN, IL-6, IL-1β, TNF-α, and the activity of SOD, was measured by ELISA assay. Western blotting was applied to determine adipsin, Nrf2, Keap1, and HO-1 protein levels. ROS level was checked using the DCFH-DA assay. RT-PCR assay was utilized to detect the mRNA levels of IL-6, IL-1β, TNF-α, Nrf2, Keap1, and HO-1. The body weight gain, infiltration of multinucleated cells, enlarged size of adipocytes, increased lipid accumulation, elevated visceral fat ratio, declined ADPN level, upregulated adipsin, and disordered serum biochemical indexes in obese immature mice were statistically significantly reversed by Sitagliptin. Excessive release of inflammatory factors and upregulated F4/80 and CD206 were observed in obese immature mice, which were statistically significantly repressed by Sitagliptin. Furthermore, the elevated MDA level, increased SOD activity, and inhibited Nrf2 pathway in obese immature mice were significantly reversed by Sitagliptin. In oxLDL stimulated RAW264.7 cells, increased release of inflammatory factors, ROS, and MDA, elevated SOD activity, and inactivated Nrf2 pathway were observed, which were statistically significantly abolished by the treatment of Sitagliptin. Sitagliptin alleviated obesity in immature mice by inhibiting inflammation and oxidative stress.

Fucoxanthin Induces Ferroptosis in Cancer Cells via Downregulation of the Nrf2/HO-1/GPX4 Pathway.

This study investigated the mechanism by which fucoxanthin acts as a novel ferroptosis inducer to inhibit tongue cancer. The MTT assay was used to detect the inhibitory effects of fucoxanthin on SCC-25 human tongue squamous carcinoma cells. The levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and total iron were measured. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to assess glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (Nrf2), Keap1, solute carrier family 7 member 11 (SLC7A11), transferrin receptor protein 1 (TFR1), p53, and heme oxygenase 1 (HO-1) expression. Molecular docking was performed to validate interactions. Compared with the control group, the activity of fucoxanthin-treated SCC-25 cells significantly decreased in a dose- and time-dependent manner. The levels of MMP, GSH, and SOD significantly decreased in fucoxanthin-treated SCC-25 cells; the levels of ROS, MDA, and total iron significantly increased. mRNA and protein expression levels of Keap1, GPX4, Nrf2, and HO-1 in fucoxanthin-treated cells were significantly decreased, whereas levels of TFR1 and p53 were significantly increased, in a concentration-dependent manner. Molecular docking analysis revealed that binding free energies of fucoxanthin with p53, SLC7A11, GPX4, Nrf2, Keap1, HO-1, and TFR1 were below -5 kcal/mol, primarily based on active site hydrogen bonding. Our findings suggest that fucoxanthin can induce ferroptosis in SCC-25 cells, highlighting its potential as a treatment for tongue cancer.

Metformin-induced oxidative stress inhibits LNCaP prostate cancer cell survival.

Preclinical and clinical studies over the past several decades have indicated the potential value of metformin, a widely utilized treatment for Type 2 diabetes, in prostate cancer therapy. Notably, these studies demonstrated metformin's pleiotropic effects on several molecular and metabolic pathways, such as androgen signaling, cell cycle, and cellular bioenergetics. In this study we investigated the role of metformin in regulating intracellular redox status and cell survival in LNCaP prostate cancer cells. The cytotoxic effects of metformin with or without the presence of SBI0206965 (AMPK inhibitor) on LNCaP cells were determined using MTT and trypan blue exclusion assays. Seahorse XP extracellular analysis, Liquid Chromatography/ Mass Spectrophotometry (LC/MS), and 2,7- and Dichlorofluoresin diacetate (DCFDA) assay were used to assess the effects of metformin on cellular bioenergetics, redox status, and redox-related metabolites. mRNA expression and protein concentration of redox-related enzymes were measured using Real Time-qPCR and ELISA assay, respectively. Independently of AMP-activated protein kinase, metformin exhibited a dose- and time-dependent inhibition of LNCaP cell survival, a response mitigated by glutathione or N-acetylcysteine (ROS scavengers) treatment. Notably, these findings were concomitant with a decline in ATP levels and the inhibition of oxidative phosphorylation. The results further indicated metformin's induction of reactive oxygen species, which significantly decreased glutathione levels and the ratio of reduced to oxidized glutathione, as well as the transsulfuration metabolite, cystathionine. Consistent with an induction of oxidative stress condition, metformin increased mRNA levels of the master redox transcription factor Nrf-2 (nuclear factor erythroid-derived 2-like), as well as transsulfuration enzymes cystathionine beta-synthase and cystathionase and GSH synthesis enzymes γ-glutamylcysteine synthetase and glutathione synthetase. Our findings highlight multiple mechanisms by which metformin-induced formation of reactive oxygen species may contribute to its efficacy in prostate cancer treatment, including promotion of oxidative stress, Nrf2 activation, and modulation of redox-related pathways, leading to its anti-survival action.

Isobavachalcone induces apoptosis of colorectal cancer cells mediated by interaction with survivin: Experimental and theoretical analyses

Isobavachalcone (IBC) as a bioactive chalcone isolated from the seeds of Psoralea corylifolia Linn has shown promising anticancer activities. However, its main anticancer mechanism in colorectal cancer cells has not been well-explored. Recently, the interaction of therapeutic compounds with survivin, an antiapoptotic marker, has been indicated to be a potential approach for advancing anticancer compounds. Therefore, in this study, IBC was evaluated for its anticancer activities against metastatic colorectal adenocarcinoma, LoVo, followed by exploring its interaction with survivin. It was observed that incubation of the human LoVo colorectal cancer cells with the IBC at 24 h resulted in a significant reduction in cell viability with an IC50 of about 35 µM as well as a significant membrane damage. Also, it was deduced that IBC could result in the formation of excessive ROS, lipid peroxidation, reduction of SOD/CAT activity, as well as downregulation of Nrf2 and HO-1 mRNA levels. Further studies showed that IBC could induce MMP reduction, cytochrome c (Cyt c) release, overexpression of Bax/Bcl-2 mRNA ratio as well as caspase-9 and −3. Moreover, it was deduced that IBC mitigated the expression of survivin at mRNA and protein levels. Then, binding parameters indicated that IBC strongly binds to one molecule of survivin at physiological temperature. Also, it was revealed that IBC substantially induced the conformational changes of survivin. Ultimately, through hydrogen bonding and hydrophobic forces, IBC has a substantial binding affinity with survivin (–9.80 kcal/mol) at a region that is critical to the dimerization process, according to molecular docking studies. Overall, this study showed that IBC may be used in future research to assess its clinical and in vivo behaviors in the modulation of colorectal cancer mediated by deactivation and downregulation of antiapoptotic survivin.