Inhibition Of Reactive Oxygen Species Generation Research Articles

Klotho relieves H2O2-induced lens epithelial cell damage via suppression of NOX4.

Age-related cataract (ARC) is a common eye disease and represents a common contributing factor to visual damage and loss. Klotho is a longevity gene and has been reported to participate in aging-related disorders. This work aims to investigate the potential role of klotho in ARC. In human lens epithelial cells (HLECs) induced by varying concentrations of hydrogen peroxide (H2O2), CCK-8 assay was used to detect cell viability. DCFH-DA probe was used to detect reactive oxygen species (ROS) level. Western blot was used to detect klotho expression. JC-1 fluorochrome assay was used to detect mitochondrial membrane potential (MMP). The concentrations of oxidative stress markers malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by related assay kits. Flow cytometry analysis, immunofluorescence staining and western blot were used to detect cell apoptosis. SA-β-gal staining and western blot were used to detect cell senescence. Klotho expression was decreased in HLECs induced by increasing concentrations of H2O2. Overexpression of klotho significantly inhibited ROS generation, decreased MDA content, increased SOD content, promoted cell viability and suppressed cell apoptosis and senescence in H2O2-induced HLECs. Furthermore, klotho down-regulated NOX4 expression and NOX4 elevation partially reversed the effects of klotho on H2O2-induced HLECs. To sum up, klotho may down-regulate NOX4 to protect against H2O2-induced HLECs damage. This finding suggested the potential therapeutic use of klotho in ARC, which needs further investigation.

Twenty-Week Dietary Supplementation with Beeswax Alcohol (BWA; Raydel®) Ameliorates High-Cholesterol-Induced Long-Term Dyslipidemia and Organ Damage in Hyperlipidemic Zebrafish in a Dose-Dependent Manner: A Comparative Analysis Between BWA and Coenzyme Q10.

Beeswax alcohol (BWA; Raydel®) is a blend of six long-chain aliphatic alcohols extracted from honeybee wax and is well known for its diverse functionality and health benefits. Herein, the efficacy of a BWA dietary intervention for 20 weeks was assessed to ameliorate high-cholesterol diet (HCD)-induced dyslipidemia and adverse effects on the vital organs of adult zebrafish. Adult zebrafish were fed different high-cholesterol diets (HCDs; final concentration of 4%, w/w) supplemented with BWA (final concentrations of 0.1%, 0.5% and 1.0%, w/w) or CoQ10 (final concentration of 1.0%). Following 20 weeks of supplementation, blood and different organs (liver, kidney, testes and ovaries) were collected, and biochemical, histological and immunohistochemical analyses were performed. The results demonstrate a dose-dependent effect of BWA of mitigating HCD-induced mortality in zebrafish over the 20-week supplementation period, which was noticeably better than the effect exerted by coenzyme Q10 (CoQ10). Consistently, a dose-dependent effect of BWA consumption of curtailing HCD-induced total cholesterol (TC) and triglyceride (TG) levels and increasing high-density-lipoprotein cholesterol (HDL-C) levels was noticed. Compared with CoQ10 (final concentration of 1.0%, w/w), BWA (final concentration of 1.0%, w/w) displayed a significantly better effect of mitigating HCD-induced dyslipidemia, as evidenced by 1.2-fold (p < 0.05) and 2.0-fold (p < 0.05) lower TC and TG levels and 2.4-fold (p < 0.01) higher HDL-C levels. The histological analysis revealed substantial prevention of fatty liver changes, reactive oxygen species (ROS) generation, cellular senescence and interleukin (IL)-6 production in the hepatic tissue of BWA zebrafish, which was significantly better than the effect exerted by CoQ10. Consistently, compared with CoQ10, significant 25% (p < 0.05) and 35% (p < 0.01) reductions in the HCD-induced elevated levels of the hepatic function biomarkers aspartate aminotransferase and alanine aminotransferase was observed in the BWA group. Likewise, BWA consumption efficiently ameliorated HCD-induced kidney, ovary and testis damage by inhibiting ROS generation, cellular senescence and lipid accumulation. Supplementation with BWA demonstrated higher therapeutic potential than that with CoQ10 to prevent dyslipidemia and organ damage associated with long-term consumption of HCDs.

Notoginsenoside R1 Attenuates Cisplatin-Induced Ototoxicity by Inducing Heme Oxygenase-1 Expression and Suppressing Oxidative Stress.

Cisplatin-induced ototoxicity occurs in approximately half of patients treated with cisplatin, and pediatric patients are more likely to be affected than adults. The oxidative stress elicited by cisplatin is a key contributor to the pathogenesis of ototoxicity. Notoginsenoside R1 (NGR1), the main bioactive compound of Panax notoginseng saponins, has antioxidant and antiapoptotic effects. This study investigated the ability of NGR1 to protect against cisplatin-induced damage in auditory HEI-OC1 cells and neonatal murine cochlear explants. The viability of HEI-OC1 cells treated with NGR1 and cisplatin was greater than that of cells treated with cisplatin alone. The results of Western blots and immunostaining for cleaved caspase-3 revealed that the level of cleaved caspase-3 in the cells treated with cisplatin was repressed by NGR1. NGR1 attenuated cisplatin-induced cytotoxicity in HEI-OC1 cells. Intracellular reactive oxygen species (ROS) were detected with a DCFDA assay and immunostaining for 4-HNE. The result revealed that its expression was induced by cisplatin and was significantly reduced by NGR1. Moreover, NGR1 can promote heme oxygenase-1 (HO-1) expression at both the mRNA and protein levels. ZNPPIX, an HO-1 inhibitor, was administered to cisplatin-treated cells to investigate the role of HO-1 in the protective effect of NGR1. The suppression of HO-1 activity by ZNPPIX markedly abolished the protective effect of NGR1 on cisplatin-treated cells. Therefore, NGR1 protects cells from cisplatin-induced damage by activating HO-1 and its antioxidative activity. In cochlear explants, NGR1 protects cochlear hair cells and attenuates cisplatin-induced ototoxicity by inhibiting ROS generation. In the group treated with cisplatin alone, prominent loss of outer hair cells and severe damage to the structure of the stereociliary bundles of inner and outer hair cells were observed. Compared with the group treated with cisplatin alone, less loss of outer hair cells (p = 0.009) and better preservation of the stereociliary bundles of hair cells were observed in the group treated with cisplatin and NGR1. In conclusion, these findings indicate that NGR1 can protect against cisplatin-induced ototoxicity by inducing HO-1 expression and suppressing oxidative stress.

Anti-Photoaging Effects of Antioxidant Peptide from Seahorse (Hippocampus abdominalis) in In Vivo and In Vitro Models.

Overexposure to ultraviolet (UV) radiation can lead to photoaging, which contributes to skin damage. The objective of this study was to evaluate the effects of an antioxidant peptide (SHP2) purified from seahorse (Hippocampus abdominalis) alcalase hydrolysate on UVB-irradiated skin damage in human keratinocyte (HaCaT) and human dermal fibroblast (HDF) cells and a zebrafish model. The data revealed that SHP2 significantly enhanced cell viability by attenuating apoptosis through the reduction of intracellular reactive oxygen species (ROS) levels in UVB-stimulated HaCaT cells. Moreover, SHP2 effectively inhibited ROS, improved collagen synthesis, and suppressed the secretion of matrix metalloproteinases (MMPs) in UVB-irradiated HDF cells. SHP2 restored the protein levels of HO-1, Nrf2, and SOD, while decreasing Keap1 expression in UVB-treated HDF, indicating stimulation of the Keap1/Nrf2/HO-1 signaling pathway. Furthermore, an in vivo study conducted in zebrafish confirmed that SHP2 inhibited photoaging by reducing cell death through the suppression of ROS generation and lipid peroxidation. Particularly, 200 µg/mL of SHP2 exerted a remarkable anti-photoaging effect on both in vitro and in vivo models. These results demonstrate that SHP2 possesses antioxidant properties and regulates skin photoaging activities, suggesting that SHP2 may have the potential for use in the development of cosmetic products.

Antioxidant Features of Humic Products by ABTS Assay.

Oxidative stress plays a pivotal role in driving immunosenescence by disrupting cellular homeostasis and impairing immune function. Humic substances exhibit scavenging activity against reactive oxygen species (ROS), inhibit ROS generation via metal chelation, and modulate endogenous antioxidant enzyme activity. Additionally, humic substances display anti-inflammatory effects, further supporting cellular redox balance. Given their antioxidant activity, humic substances hold promise as natural compounds for mitigating oxidative stress-associated immunosenescence. Here we describe the evaluation of antioxidant capacities of humic products by ABTS spectrophotometric assay.

Targeting the Labile Iron Pool with Engineered DFO Nanosheets to Inhibit Ferroptosis for Parkinson's Disease Therapy.

Ferroptosis in neurons is considered one of the key factors that induces Parkinson's disease (PD), which is caused by excessive iron accumulation in the intracellular labile iron pool (LIP). The iron ions released from the LIP lead to the aberrant generation of reactive oxygen species (ROS) to trigger ferroptosis and exacerbate PD progression. Herein, a pioneering design of multifunctional nanoregulator deferoxamine (DFO)-integrated nanosheets (BDPR NSs) is presented that target the LIP to restrict ferroptosis and protect against PD. The BDPR NSs are constructed by incorporating a brain-targeting peptide and DFO into polydopamine-modified black phosphorus nanosheets. These BDPR NSs can sequester free iron ions, thereby ameliorating LIP overload and regulating iron metabolism. Furthermore, the BDPR NSs can decrease lipid peroxidation generation by mitigating ROS accumulation. More importantly, BDPR NSs can specifically accumulate in the mitochondria to suppress ROS generation and decrease mitochondrial iron accumulation. In vivo experiments demonstrated that the BDPR NSs highly efficiently mitigated dopaminergic neuronloss and its associated behavioral disorders by modulating the LIP and inhibiting ferroptosis. Thus, the BDPR-based nanovectors holds promise as a potential avenue for advancing PD therapy.

Impact of arsenic on male and female reproductive function: a review of the pathophysiology and potential therapeutic strategies.

Arsenic is a ubiquitous metalloid and heavy metal that contributes to the global decline in human fertility. Humans are constantly exposed to arsenic through biotic and abiotic sources, especially ingestion of arsenic-contaminated food and water. Its exposure is associated with several adverse health challenges, including reproductive toxicity. In spite of its reported adverse effects, arsenic exposure remains a global challenge. Hence, this study provides a comprehensive review of the literature on the impact and mechanism of arsenic on male and female reproductive function. Additionally, a review of the potential therapeutic strategies is presented. Evidence from the literature reveals that arsenic upregulates reactive oxygen species (ROS) generation which mediates arsenic-induced suppression of the hypothalamic-pituitary-gonadal axis and inactivation of 3β-HSD and 17β-HSD activities, leading to reduced gonadal steroidogenesis. Through several oxidative stress-dependent signaling, arsenic induces the apoptosis of the germ cells, thus contributing to the development of infertility. At the moment, there is no specific treatment for arsenic-induced reproductive toxicity. However, increasing data form the scientific literature reveals the benefits of antioxidants in ameliorating arsenic-induced reproductive toxicity. These molecules suppress ROS generation and maintain optimal activities of the hypothalamic-pituitary-gonadal axis, leading to optimal steroidogenesis and gametogenesis as well as improved germ cells. Overall, this study revealed the impact and associated mechanism of arsenic-induced reproductive toxicity. It also provides evidence from the literature demonstrating potential therapeutic measures in managing arsenic-induced reproductive toxicity.

NADPH oxidase 1/4 dual inhibitor setanaxib suppresses platelet activation and thrombus formation

AimsThe production of reactive oxygen species (ROS) by NADPH oxidase (NOX) is able to induce platelet activation, making NOX a promising target for antiplatelet therapy. In this study, we examined the effects of setanaxib, a dual NOX1/4 inhibitor, on human platelet function and ROS-related signaling pathways. Materials and methodsIn collagen-stimulated human platelets, aggregometry, assessment of ROS and Ca2+, immunoblotting, ELISA, flow cytometry, platelet adhesion assay, and assessment of mouse arterial thrombosis were performed in this study. Key findingsSetanaxib inhibited both intracellular and extracellular ROS production in collagen-activated platelets. Additionally, setanaxib significantly inhibited collagen-induced platelet aggregation, P-selectin exposure from α-granule release, and ATP release from dense granules. Setanaxib blocked the specific tyrosine phosphorylation-mediated activation of Syk, LAT, Vav1, and Btk within collagen receptor signaling pathways, leading to reduced activation of PLCγ2, PKC, and Ca2+ mobilization. Setanaxib also inhibited collagen-induced activation of integrin αIIbβ3, which is linked to increased cGMP levels and VASP phosphorylation. Furthermore, setanaxib suppressed collagen-induced p38 MAPK activation, resulting in decreased phosphorylation of cytosolic PLA2 and reduced TXA2 generation. Setanaxib also inhibited ERK5 activation, affecting the exposure of procoagulant phosphatidylserine. Setanaxib reduced thrombus formation under shear conditions by preventing platelet adhesion to collagen. Finally, in vivo administration of setanaxib in animal models led to the inhibition of arterial thrombosis. SignificanceThis study is the first to show that setanaxib suppresses ROS generation, platelet activation, and collagen-induced thrombus formation, suggesting its potential use in treating thrombotic or cardiovascular diseases.

PlWRKY47 Coordinates With Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase 2 Gene to Improve Thermotolerance Through Inhibiting Reactive Oxygen Species Generation in Herbaceous Peony.

Although WRKY transcription factors play crucial roles in plant responses to high-temperature stress, little is known about Group IIb WRKY family members. Here, we identified the WRKY-IIb protein PlWRKY47 from herbaceous peony (Paeonia lactiflora Pall.), which functioned as a nuclear-localized transcriptional activator. The expression level of PlWRKY47 was positively correlated with high-temperature tolerance. Silencing of PlWRKY47 in P. lactiflora resulted in the decreased tolerance to high-temperature stress by accumulating reactive oxygen species (ROS). Overexpression of PlWRKY47 improved plant high-temperature tolerance through decreasing ROS accumulation. Moreover, PlWRKY47 directly bound to the promoter of cytosolic glyceraldehyde-3-phosphate dehydrogenase 2 (PlGAPC2) gene and activated its transcription. PlGAPC2 was also positively regulated high-temperature tolerance in P. lactiflora by increasing NAD+ content to inhibit ROS generation. Additionally, PlWRKY47 physically interacted with itself to form a homodimer, and PlWRKY47 could also interact with one Group IIb WRKY family member PlWRKY72 to form a heterodimer, they all promoted PlWRKY47 to bind to and activate PlGAPC2. These data support that the PlWRKY47-PlWRKY47 homodimer and PlWRKY72-PlWRKY47 heterodimer can directly activate PlGAPC2 expression to improve high-temperature tolerance by inhibiting ROS generation in P. lactiflora. These results will provide important insights into the plant high-temperature stress response by WRKY-IIb transcription factors.

Maternal folic acid over-supplementation impairs cardiac function in mice offspring by inhibiting SOD1 expression.

Folic acid (FA) supplementation during pregnancy aims to protect foetal development. However, maternal over-supplementation of FA has been demonstrated to cause metabolic dysfunction and increase the risk of autism, retinoblastoma, and respiratory illness in the offspring. Moreover, FA supplementation reduces the risk of congenital heart disease. However, little is known about its possible adverse effects on cardiac health resulting from maternal over-supplementation. In this study, we assessed the detrimental effects of maternal FA over-supplementation on the cardiac health of the offspring. Eight-week-old C57BL/6J pregnant mice were randomly divided into control and over-supplemented groups. The offspring cardiac function was assessed using echocardiography. Cardiac fibrosis was assessed in the left ventricular myocardium by histological analysis. Proteomic, protein, RNA, and DNA methylation analyses were performed by liquid chromatography-tandem mass spectrometry, western blotting, real-time quantitative PCR, and bisulfite sequencing, respectively. We found that maternal periconceptional FA over-supplementation impaired cardiac function with the decreased left ventricular ejection fraction in the offspring. Biochemical indices and tissue staining further confirmed impaired cardiac function in offspring caused by maternal FA over-supplementation. The combined proteomic, RNA expression, and DNA methylation analyses suggested that key genes involved in cardiac function were inhibited at the transcriptional level possibly due to increased DNA methylation. Among these, superoxide dismutase 1 was downregulated, and reactive oxygen species (ROS) levels increased in the mouse heart. Inhibition of ROS generation using the antioxidant N-acetylcysteine rescued the impaired cardiac function resulting from maternal FA over-supplementation. Our study revealed that over-supplementation with FA during mouse pregnancy is detrimental to cardiac function with the decreased left ventricular ejection fraction in the offspring and provides insights into the mechanisms underlying the association between maternal FA status and health outcomes in the offspring.

Mycoplasma pneumoniae-induced Kawasaki disease via PINK1/Parkin-mediated mitophagy

Kawasaki disease (KD) is a systemic vasculitis with an unknown cause that primarily affects children. The objective of this study was to explore the function and underlying mechanism of mitophagy in Mycoplasma pneumoniae (MP)-induced KD.To create MP-induced KD models, Human coronary endothelial cells (HCAECs) and DBA/2 mice were employed and treated with Mp-Lipid-associated membrane proteins （LAMPs）. Lactate dehydrogenase (LDH) levels were tested to determine cellular damage or death. The inflammatory cytokines tumor necrosis factor (TNF)--α and interleukin (IL)-6 were measured using the Enzyme-Linked Immunosorbent Assay (ELISA) method. RT-qPCR and Western blotting were used to determine the expression of Intercellular Adhesion Molecule(ICAM)-1, vascular cell adhesion molecule (VCAM)-1, inducible nitric oxide synthase(iNOS), LC3, p62, PINK1(a mitochondrial serine/threonine-protein kinase), and PARKIN(a cytosolic E3-ubiquitin ligase). The adenosine triphosphate （ATP）, reactive oxygen species （ROS）, and mitochondrial membrane potential（MMP） levels were measured to determine mitochondrial function. Mitophagy was investigated using immunofluorescence and a mitophagy detection test. Autophagosome and mitochondrial morphology were examined using transmission electron microscopy. To identify inflammatory cell infiltration, hematoxylin and eosin staining was utilized. Mp-LAMPs increased the levels of TNF-α, IL-6, ICAM-1, VCAM-1, and iNOS in an HCAEC cell model, along with LDH release. After Mp-LAMPs exposure, there was a rise in LC3 and a reduction in p62. Meanwhile, the expression of PINK1 and Parkin was increased. Cyclosporin A dramatically increased ATP synthesis and MMP in HCAEC cells treated with Mp-LAMPs, while suppressing ROS generation, demonstrating excessive mitophagy-related mitochondrial dysfunction. Additionally, neither body weight nor artery tissue were affected due to PINK1 and Parkin suppression Cyclosporin A in Mp-LAMPs-treated mice. These findings indicated that PINK1/Parkin-mediated mitophagy inhibition may be a therapeutic target for MP-induced KD.

Licochalcone A mitigates aflatoxin B1-induced immunotoxicity via ferroptosis in bursa of broilers and macrophages

Aflatoxin B1 (AFB1) is a mycotoxin which is responsible for severe damage to the immune system of humans and livestock. Licochalcone A (Lico A), a polyphenol derived from turmeric, has attracted great attention due to its wonderful antioxidant properties. Ferroptosis, an iron-dependent cell death related to oxidative stress, which plays a crucial role in the resistance of phytochemical to immune-associated injury. Nevertheless, effects of Lico A on the bursa of broilers exposed to AFB1 remain unclear. In this work, broilers were fed diets supplemented with 2 mg/kg of AFB1 and 50 mg/kg of Lico A. Meanwhile, various concentrations of Lico A and AFB1 (15 μM) were used to stimulate macrophages. These results revealed that AFB1 resulted in more severe bursa atrophy and relative weight reduction; the expression of pro-ferroptosis protein ACSL4 and the content of malondialdehyde (MDA) were significantly elevated, while the expression of anti-ferroptosis proteins GPX4, xCT, FSP1 and the content of Glutathione (GSH) was obviously reduced. However, Lico A treatment effectively reversed these effects in the bursa of broilers. Meanwhile, in bursa and macrophages, Lico A mitigated the expression of AFB1-induced apoptosis-associated protein (Caspase-3, Bax, Bcl-2) as well as antioxidant protein (Nrf2, GCLM, HO-1). Importantly, ferroptosis was also observed in macrophages induced by AFB1. Lico A efficaciously alleviated AFB1-induced mitochondrial membrane potential decrease and reactive oxygen species (ROS) production in macrophages; in contrast, Lico A evidently inhibited AFB1-triggered ROS generation and cytotoxicity, which was disabled by the addition of Erastin. Moreover, Liproxstatin-1 significantly inhibited ROS generation induced by AFB1. In summary, the present study elucidates that the main mechanism by which Lico A attenuates AFB1-induced immunotoxicity is through the suppression of ferroptosis, apoptosis, mitochondrial damage and oxidative stress, which is promising for the improvement of immunotoxic effects of AFB1.

Neuroprotective Effects of Probucol against Rotenone-Induced Toxicity via Suppression of Reactive Oxygen Species Production in SH-SY5Y Cells.

Probucol is a hyperlipidemic drug with antioxidant properties. It has been reported to prevent mitochondrial dysfunction, reduce oxidative stress, and suppress neurotoxicity in neurodegenerative disease models, including Parkinson's disease models. However, the molecular mechanisms underlying the neuroprotective effects of probucol have been not examined yet. Thus, in this study, we investigated whether probucol can alleviate the effects of a mitochondrial complex I inhibitor, rotenone, on a human neuroblastoma cell line (SH-SY5Y). We evaluated the cell viability and cytotoxicity and apoptosis rates of SH-SY5Y cells treated with rotenone and probucol or edaravone, a known free-radical scavenger. Subsequently, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels in the cells were evaluated to determine the effects of probucol on mitochondrial function. We found that rotenone caused cytotoxicity, cell apoptosis, and mitochondrial dysfunction, enhanced ROS generation, and impaired MMP. However, probucol could inhibit this rotenone-induced decrease in cell viability, MMP loss, intracellular ROS generation, and apoptosis. These results suggest that probucol exerts neuroprotective effects via MMP stabilization and the inhibition of ROS generation. Additionally, this effect of probucol was equal to or greater than and more persistent than that of edaravone. Thus, we believe probucol may be a promising drug for the treatment of neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases.

Ilex pubescens inhibits pyroptosis post-myocardial infarction through suppression of the ROS/NLRP3 pathway

IntroductionIlex pubescens (IPES), a traditional Chinese herb widely used in cardiovascular diseases, has shown potential anti-inflammatory capabilities in myocardial infarction. Reactive Oxygen Species (ROS) and the NOD-like Receptor Protein 3 (NLRP3) pathway are significant contributors to aseptic inflammation in heart diseases. This study aims to elucidate the primary mechanism by which IPES inhibits pyroptosis post-myocardial infarction. MethodsBy ligating the left coronary artery in C57BL/6 mice, a myocardial infarction model was established to be conducted in vivo. To establish pyroptosis in vitro, primary neonatal cardiomyocytes, extracted from the hearts of Sprague-Dawley rats, were treated in an oxygen-glucose deprivation way. ROS scavenger, NLRP3 inhibitor, and NLRP3 was overexpressed by adenovirus to confirm IPES inhibiting myocardial pyroptosis through the ROS/NLRP3 pathway. ResultsIn vivo, IPES exerted significant cardioprotective effects, as evidenced by reducing heart injury, improving cardiac function, and decreasing serum markers of cardiac damage. Furthermore, IPES treatment significantly inhibits ROS generation and reduces the expression levels of NLRP3 and its downstream pyroptosis-related proteins. In vitro, IPES therapy significantly decreased cell damage and pyroptosis in a concentration-dependent manner in an oxygen-glucose deprivation/re-oxygenation (OGD/R) cell model. Additionally, IPES demonstrates synergistic cardiomyocyte protection with the ROS scavenger NAC, whereas its inhibition of pyroptosis is not significantly different from that of the NLRP3 inhibitor. More importantly, the inhibitory impacts of IPES on pyroptosis were partially reversed by NLRP3 overexpression. The active components of IPES exhibit the ability to stably and efficiently bind with NLRP3. DiscussionThese results demonstrate that IPES inhibit pyroptosis post-MI by suppressing the ROS/NLRP3 pathway, providing a new insight into its potential application in treating MI.

Resveratrol targeting NRF2 disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage in mice pulmonary infection

Ethnopharmacological relevanceThe root of Polygonum cuspidatum Sieb. et Zucc (PC), known as 'Huzhang' in the Chinese Pharmacopoeia, has been traditionally employed for its anti-inflammatory, antiviral, antimicrobial, and other biological activities. Polydatin (PD) and its aglycone, resveratrol (RES), are key pharmacologically active components responsible for exerting anti-inflammatory and antioxidant effects. However, its specific targets and action mechanisms remain unclear. Aim of the studyThe equilibrium of the KEAP1-NRF2 system serves as the primary protective response to oxidative and electrophilic stresses within the body, particularly in cases of acute lung injury caused by pathogenic microbial infection. In this study, the precise mechanisms by which RES alleviates oxidative stress damage in conjunction with NRF2 activators are discussed. Materials and methodsThe active components from PC were screened to evaluate their potential to inhibit reactive oxygen species (ROS) and activate antioxidant activity dependent on antioxidant response elements (ARE). RES was evaluated for its potential to alleviate the oxidative stress caused by pathogenic microbial infection. Functional probes were designed to study the RES distribution and identify its targets. A lipopolysaccharide (LPS)-induced oxidative injury model was used to evaluate the effects of RES on the KEAP1-NRF2/ARE pathway in RAW 264.7 cells. The interaction between RES and NRF2 was elucidated using drug-affinity responsive target stability (DARTS), cellular thermal shift assays (CETSA), co-immunoprecipitation (Co-IP), and microscale thermophoresis (MST) techniques. The key binding sites were predicted using molecular docking and validated in NRF2-knockdownand reconstructed cells. Finally, protective effects against pulmonary stress were verified in a mouse model of pathogenic infection. ResultsThe accumulation of RES in lung macrophages disrupted the binding between KEAP1 and NRF2, thereby preventing the ubiquitination degradation of NRF2 through its interaction with Ile28 on the NRF2-DLG motif. The activation of NRF2 resulted in the upregulation of nuclear transcription, enhances the expression of antioxidant genes dependent on ARE, suppresses ROS generation, and ameliorates oxidative damage both in vivo and in vitro. ConclusionThese findings shed light on the potential of RES to mitigate oxidative stress damage caused by pathogenic microorganism-induced lung infections and facilitate the discovery of novel small molecule modulators targeting the KEAP1-NRF2 DLG motif interaction.

Evaluating the Protective Effects of Thymoquinone on Methamphetamine-induced Toxicity in an In Vitro Model Based on Differentiated PC12 Cells.

Methamphetamine (Meth) is a highly addictive stimulant. Its potential neurotoxic effects are mediated through various mechanisms, including oxidative stress and the initiation of the apoptotic process. Thymoquinone (TQ), obtained from Nigella sativa seed oil, has extensive antioxidant and anti-apoptotic properties. This study aimed to investigate the potential protective effects of TQ against Meth-induced toxicity by using an in vitro model based on nerve growth factor-differentiated PC12 cells. Cell differentiation was assessed by detecting the presence of a neuronal marker with flow cytometry. The effects of Meth exposure were evaluated in the in vitro neuronal cell-based model via the determination of cell viability (in an MTT assay) and apoptosis (by annexin/propidium iodide staining). The generation of reactive oxygen species (ROS), as well as the levels of glutathione (GSH) and dopamine, were also determined. The model was used to determine the protective effects of 0.5, 1 and 2 μM TQ against Meth-induced toxicity (at 1 mM). The results showed that TQ reduced Meth-induced neurotoxicity, possibly through the inhibition of ROS generation and apoptosis, and by helping to maintain GSH and dopamine levels. Thus, the impact of TQ treatment on Meth-induced neurotoxicity could warrant further investigation.

Potassium Ions Decrease Mitochondrial Matrix pH: Implications for ATP Production and Reactive Oxygen Species Generation.

Potassium (K+) is the most abundant cation in the cytosol and is maintained at high concentrations within the mitochondrial matrix through potassium channels. However, many effects of K+ at such high concentrations on mitochondria and the underlying mechanisms remain unclear. This study aims to elucidate these effects and mechanisms by employing fluorescence imaging techniques to distinguish and precisely measure signals inside and outside the mitochondria. We stained the mitochondrial matrix with fluorescent dyes sensitive to K+, pH, reactive oxygen species (ROS), and membrane potential in plasma membrane-permeabilized C6 cells and isolated mitochondria from C6 cells. Fluorescence microscopy facilitated the accurate measurement of fluorescence intensity inside and outside the matrix. Increasing extramitochondrial K+ concentration from 2 mM to 127 mM led to a reduction in matrix pH and a decrease in the generation of highly reactive ROS. In addition, elevated K+ levels electrically polarized the inner membrane of the mitochondria and promoted efficient ATP synthesis via FoF1-ATPase. Introducing protons (H+) into the matrix through phosphate addition led to further mitochondrial polarization, and this effect was more pronounced in the presence of K+. K+ at high concentrations, reaching sub-hundred millimolar levels, increased H+ concentration within the matrix, suppressing ROS generation and boosting ATP synthesis. Although this study does not elucidate the role of specific types of potassium channels in mitochondria, it does suggest that mitochondrial K+ plays a beneficial role in maintaining cellular health.

Bovine umbilical vein endothelial cells conditioned medium prevents TMT-induced neurotoxicity mediated by the upregulation of brain-derived neurotropic factors on the human neuroblastoma SH-SY5Y cells

Background: Neurodegenerative diseases are characterized by damage to the central nervous system through the progressive decrease in the structure and function of neurons, followed by neuronal death. Current therapy for these diseases is still limited. Bovine umbilical vein endothelial cells conditioned medium (BUVEC-CM) is a medium harvested from culture of bovine umbilical vein endothelial cells (BUVEC). This study aimed to evaluate the capability of BUVEC-CM to prevent neurotoxicity induced by trimethyltin (TMT) on human neuroblastoma SH-SY5Y cells. Methods: The tests carried out were a viability test with MTT and CCK-8 assay, scratch wound assay to determine cell migration, Hoechst 33342 and AO/PI staining to detect apoptosis, ELISA test to measure the concentration of BDNF, and RT-qPCR to determine the expression of CD68, caspase-7 and caspase-9, and DCFH-DA staining to detect reactive oxygen species (ROS). Results: BUVEC-CM maintained the viability of cells, increase cell migration, prevent cell apoptosis, increase BDNF levels, decrease the expression of CD68, as well as the caspase-7 and -9 genes, and suppress ROS generation. Conclusions: BUVEC-CM can protect from neurodegeneration in SH-SY5Y cells through upregulation of BDNF and downregulation of CD68, caspase-7 and caspase-9.

Quercetin stimulates trophoblast fusion via the mitochondrial function

The fusion of mononuclear trophoblasts into multinucleate syncytiotrophoblasts is the critical event in the process of syncytialization, and its dysregulation can lead to pregnancy complications, notably hypertensive disorders of pregnancy (HDP). Oxidative stress may disrupt trophoblast syncytialization in HDP. Specifically, placentas with HDP exhibit impaired mitochondria, giving rise to the generation of reactive oxygen species (ROS) and subsequent oxidative stress. Quercetin, a bioflavonoid known for its antioxidant and anti-aging properties, has the potential to mitigate oxidative stress during trophoblast syncytialization. However, the precise mechanism underlying the action of quercetin in these processes remains to be elucidated. To explore the impact of quercetin on syncytialization, mitochondrial function, and ROS generation, cyclic AMP-stimulated BeWo cells were treated with quercetin. The expression of markers associated with cell fusion, mitochondrial function, and oxidative stress was determined using qPCR and western blotting. Additionally, morphological syncytialization and mitophagy (mitochondrial degradation) were assessed by immunofluorescence analysis. Our results revealed that quercetin increased the expression of syncytialization markers and promoted cell fusion. Furthermore, this compound also upregulated markers associated with mitophagy and mitochondrial fusion, which are corroborated by visual evidence of mitophagy through the fluorescence microscope. Cell fusion naturally stimulated ROS generation, which was attenuated by quercetin. Quercetin downregulated the expression of NRF2 and HO-1 during syncytialization, while increasing the expression of sirtuin1/3/6, which are known to play essential roles in antioxidant responses. In conclusion, quercetin effectively regulates mitochondrial function through its antioxidant properties and the suppression of ROS generation, ultimately promoting trophoblast fusion, suggesting that the flavonoid has the potential to ameliorate pregnancy-related disorder stemming from placental dysplasia.

Withaferin A, a natural thioredoxin reductase 1 (TrxR1) inhibitor, synergistically enhances the antitumor efficacy of sorafenib through ROS-mediated ER stress and DNA damage in hepatocellular carcinoma cells

BackgroundSorafenib (Sora), a multi-target tyrosine kinase inhibitor, is widely recognized as a standard chemotherapy treatment for advanced hepatocellular carcinoma (HCC). However, drug resistance mechanisms hinder its anticancer efficacy. Derived from Withania somnifera, Withaferin A (WA) exhibits remarkable anti-tumor properties as a natural bioactive compound. This study aimed to examine the mechanisms that underlie the impacts of Sora and WA co-treatment on HCC. MethodsCell proliferation was evaluated through colony formation and MTT assays. Flow cytometry was employed to determine cellular apoptosis and reactive oxygen species (ROS) levels. The evaluation of apoptosis-related protein levels, DNA damage, and endoplasmic reticulum stress was conducte utilizing IHC staining and western blotting. Moreover, the caspase inhibitor Z-VAD-FMK, ATF4 siRNA, ROS scavenger N-acetyl cysteine (NAC), and TrxR1 shRNA were used to elucidate the underlying signaling pathways. To validate the antitumor effects of Sora/WA co-treatment, in vivo experiments were ultimately executed using Huh7 xenografts. ResultsSora/WA co-treatment demonstrated significant synergistic antitumor impacts both in vivo and in vitro. Mechanistically, the enhanced antitumor impact of Sora by WA was achieved through the inhibition of TrxR1 activity, resulting in ROS accumulation. Moreover, ROS generation induced the activation of DNA damage and endoplasmic reticulum (ER) stress pathways, eventually triggering cellular apoptosis. Pre-treatment with the antioxidant NAC significantly inhibited ROS generation, ER stress, DNA damage, and apoptosis induced by Sora/WA co-treatment. Additionally, the inhibition of ATF4 by small interfering RNA (siRNA) attenuated Sora/WA co-treatment-induced apoptosis. In vivo, Sora/WA co-treatment significantly suppressed tumor growth in HCC xenograft models and decreased TrxR1 activity in tumor tissues. ConclusionOur study suggests that WA synergistically enhances the antitumor effect of Sora, offering promising implications for evolving treatment approaches for HCC.