The role of the AMPK/ERK1/2 signaling pathway in neuronal oxidative stress damage following cerebral ischemia-reperfusion.

Cerebral ischemia-reperfusion injury involves a series of pathophysiological processes that occur when blood supply is restored after cerebral vascular obstruction, leading to neuronal damage. The AMPK/ERK1/2 signaling pathway has been identified as crucial in this process, although the exact mechanisms underlying the induction of ischemia-reperfusion injury remain unclear. In this study, we investigated the involvement of the AMPK/ERK1/2 signaling pathway in neuronal oxidative stress damage following cerebral ischemia-reperfusion by establishing animal and cell models. Our experimental results demonstrated that cerebral ischemia-reperfusion leads to oxidative stress damage, including cell apoptosis and mitochondrial dysfunction. Moreover, further experiments showed that inhibition of AMPK and ERK1/2 activity, using U0126 and Compound C respectively, could alleviate oxidative stress-induced cellular injury, improve mitochondrial morphology and function, reduce reactive oxygen species levels, increase superoxide dismutase levels, and suppress apoptosis. These findings clearly indicate the critical role of the AMPK/ERK1/2 signaling pathway in regulating oxidative stress damage and cerebral ischemia-reperfusion injury. The discoveries in this study provide a theoretical basis for further research and development of neuroprotective therapeutic strategies targeting the AMPK/ERK1/2 signaling pathway.

Toxicity mechanisms of plastic nanoparticles in three terrestrial species: antioxidant system imbalance and neurotoxicity.

The detrimental impacts of plastic nanoparticles (PNPs) are a worldwide concern, although knowledge is still limited, in particular for soil mesofauna. This study investigates the biochemical impact of 44 nm polystyrene PNPs on three soil models-Enchytraeus crypticus (Oligochaeta), Folsomia candida (Collembola) and Porcellionides pruinosus (Isopoda). Exposure durations of 3, 7 and 14 days (d) were implemented at two concentrations (1.5 and 300 mg kg-1 PNPs). Results revealed PNPs impact on the activities of the glutathione-dependent antioxidative enzyme, glutathione S-transferase (GST) and on the neurotransmitter acetylcholinesterase (AChE) for all three species. Catalase (CAT) played a minor role, primarily evident in F. candida at 300 mg kg-1 PNPs (CAT and GST response after 14 d), with no lipid peroxidation (LPO) increase. Even with the antioxidant defence, P. pruinosus was the most sensitive species for lipid oxidative damage (LPO levels increased after 7 d exposure to 300 mg kg-1 PNPs). Significant AChE inhibitions were measured already after 3 d to both PNP concentrations in F. candida and E. crypticus, respectively. Significant AChE inhibitions were also found in P. pruinosus but later (7 d). Overall, the toxicity mechanisms of PNPs involved antioxidant imbalance, being (mostly) the glutathione-associated metabolism part of that defence system. Neurotoxicity, linked to AChE activities, was evident across all species. Sensitivity to PNPs varied: P. pruinosus > F. candida ≅ E. crypticus. This pioneering study on PNPs toxicity in soil invertebrates underscores its environmental relevance, shedding light on altered biochemical responses, that may compromise ecological roles and soil ecosystem fitness.

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

ETHNOPHARMACOLOGICAL RELEVANCE: The 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 STUDY: The 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 METHODS: The 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. RESULTS: The 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. CONCLUSION: These 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.

The NEDD8 activating enzyme inhibitor MLN4924 mitigates doxorubicin-induced cardiotoxicity in mice.

Doxorubicin (DOX) is a widely utilized chemotherapeutic agent in clinical oncology for treating various cancers. However, its clinical use is constrained by its significant side effects. Among these, the development of cardiomyopathy, characterized by cardiac remodeling and eventual heart failure, stands as a major concern following DOX chemotherapy. In our current investigation, we have showcased the efficacy of MLN4924 in mitigating doxorubicin-induced cardiotoxicity through direct inhibition of the NEDD8-activating enzyme, NAE. MLN4924 demonstrated the ability to stabilize mitochondrial function post-doxorubicin treatment, diminish cardiomyocyte apoptosis, alleviate oxidative stress-induced damage in the myocardium, enhance cardiac contractile function, mitigate cardiac fibrosis, and impede cardiac remodeling associated with heart failure. At the mechanistic level, MLN4924 intervened in the neddylation process by inhibiting the NEDD8 activating enzyme, NAE, within the murine cardiac tissue subsequent to doxorubicin treatment. This intervention resulted in the suppression of NEDD8 protein expression, reduction in neddylation activity, and consequential manifestation of cardioprotective effects. Collectively, our findings posit MLN4924 as a potential therapeutic avenue for mitigating doxorubicin-induced cardiotoxicity by attenuating heightened neddylation activity through NAE inhibition, thereby offering a viable and promising treatment modality for afflicted patients.

Bu zhong Yiqi Decoction ameliorates mild cognitive impairment by improving mitochondrial oxidative stress damage via the SIRT3/MnSOD/OGG1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu-Zhong-Yi-Qi Decoction(BZYQD) is a traditional formula commonly used in China, known for its effects in tonifying Qi and raising Yang. It can relieve symptoms of cognitive impairment such as forgetfulness and lack of concentration caused by qi deficiency, which is common in aging and debilitating. However, much of the current research on BZYQD has been focused on its impact on the digestive system, leaving its molecular mechanisms in improving cognitive function largely unexplored. AIM OF THE STUDY: Cognitive decline in the aging central nervous system is intrinsically linked to oxidative damage. This study aims to investigate the therapeutic mechanism of BZYQD in treating mild cognitive impairment caused by qi deficiency, particularly through repair of mitochondrial oxidative damage. MATERIALS AND METHODS: A rat model of mild cognitive impairment (MCI) was established by administering reserpine subcutaneously for two weeks, followed by a two-week treatment with BZYQD/GBE. In vitro experiments were conducted to assess the effects of BZYQD on neuronal cells using a H2O2-induced oxidative damage model in PC12 cells. The open field test and the Morris water maze test evaluated the cognitive and learning memory abilities of the rats. HE staining and TEM were employed to observe morphological changes in the hippocampus and its mitochondria. Mitochondrial activity, ATP levels, and cellular viability were measured using assay kits. Protein expression in the SIRT3/MnSOD/OGG1 pathway was analyzed in tissues and cells through western blotting. Levels of 8-OH-dG in mitochondria extracted from tissues and cells were quantified using ELISA. Mitochondrial morphology in PC12 cells was visualized using Mito Red, and mitochondrial membrane potential was assessed using the JC-1 kit. RESULTS: BZYQD treatment significantly improved cognitive decline caused by reserpine in rats, as well as enhanced mitochondrial morphology and function in the hippocampus. Our findings indicate that BZYQD mitigates mtDNA oxidative damage in rats by modulating the SIRT3/MnSOD/OGG1 pathway. In PC12 cells, BZYQD reduced oxidative damage to mitochondria and mtDNA in H2O2-induced conditions and was associated with changes in the SIRT3/MnSOD/OGG1 pathway. CONCLUSION: BZYQD effectively counteracts reserpine-induced mild cognitive impairment and ameliorates mitochondrial oxidative stress damage through the SIRT3/MnSOD/OGG1 pathway.

An Assessment of Ankle Function, Oxidative Damage, and Inflammatory Factor Levels in Minimally Invasive Ankle Joint Fusion Surgery for the Treatment of Traumatic Ankle Osteoarthritis

SUMMARY: Traumatic ankle osteoarthritis is a degenerative condition resulting from traumatic injuries. The objective of this study was to evaluate the impact of minimally invasive ankle joint fusion surgery on ankle function, oxidative damage, and inflammatory factor levels in traumatic ankle osteoarthritis patients. A total of 112 traumatic ankle osteoarthritis patients treated in our hospital from January 2022 to January 2023 were enrolled. They were randomly rolled into a control group (Group C) and an experimental group (Group E), with the former undergoing conventional open ankle joint fusion surgery and the latter receiving minimally invasive ankle joint fusion surgery. A comparison was made between the two groups based on American Orthopedic Foot and Ankle Society (AOFAS), bony fusion rates, and visual analog scale (VAS) scores at pre-operation, and at 1, 2, and 3 months post-operation. Additionally, serum oxidative damage indicators and inflammatory factor levels were measured to evaluate the recovery effects in both groups. Relative to Group C, Group E showed drastically increased AOFAS scores and bony fusion rates (P<0.05), as well as greatly decreased VAS scores (P<0.05). Moreover, Group E exhibited more pronounced improvements in oxidative damage indicators and inflammatory factors versus Group C (P<0.05). Minimally invasive ankle joint fusion surgery drastically improves ankle function in traumatic ankle osteoarthritis patients and reduces levels of oxidative damage and inflammatory response. This provides an important clinical treatment option.

La osteoartritis traumática del tobillo es una afección degenerativa resultante de lesiones traumáticas. El objetivo de este estudio fue evaluar el impacto de la cirugía mínimamente invasiva de fusión de la articulación talocrural sobre la función del tobillo, el daño oxidativo y los niveles de factor inflamatorio en pacientes con osteoartritis traumática del tobillo. Se inscribieron un total de 112 pacientes con artrosis traumática de tobillo tratados en nuestro hospital desde enero de 2022 hasta enero de 2023. Fueron divididos aleatoriamente en un grupo de control (Grupo C) y un grupo experimental (Grupo E), donde el primero se sometió a una cirugía de fusión de la articulación talocrural abierta convencional y el segundo recibió una cirugía de fusión de la articulación talocrural mínimamente invasiva. Se realizó una comparación entre los dos grupos según la Sociedad Estadounidense de Ortopedia de Pie y Tobillo (AOFAS), las tasas de fusión ósea y las puntuaciones de la escala visual analógica (EVA) antes de la operación y 1, 2 y 3 meses después de la operación. Además, se midieron los indicadores de daño oxidativo sérico y los niveles de factor inflamatorio para evaluar los efectos de la recuperación en ambos grupos. En relación con el grupo C, el grupo E mostró puntuaciones AOFAS y tasas de fusión ósea drásticamente aumentadas (P <0,05), así como puntuaciones VAS muy disminuidas (P <0,05). Además, el grupo E exhibió mejoras más pronunciadas en los indicadores de daño oxidativo y factores inflamatorios en comparación con el grupo C (P <0,05). La cirugía de fusión de la articulación talocrural mínimamente invasiva mejora drásticamente la función del tobillo en pacientes con osteoartritis traumática del tobillo y reduce los niveles de daño oxidativo y la respuesta inflamatoria. Esto proporciona una importante opción de tratamiento clínico.

Aronia melanocarpa Flavonol Extract-Antiradical and Immunomodulating Activities Analysis.

The study of Aronia melanocarpa's (A. melanocarpa) biological activity is focused on obtaining the crude extract and separation of the flavonol compounds. The extraction and fractionation of A. melanocarpa fruits, followed by quantitative analysis, were accomplished using high-performance liquid chromatography and Darco G-60 filtering. This approach enabled the quantification of flavonoids within each fraction. The antioxidative, immunomodulating activities and cytotoxicity with respect to the lymphoblast cell line RPMI-1788 were studied. The flavonol extract of A. melanocarpa has been shown to have a high capacity to neutralize free DPPH and AAPH radicals in vitro. It also caused an accelerated 'respiratory burst' formation of neutrophils and an increase in the metabolic reserves of cells in rats exposed to cyclophosphamide. The reference solution (an equivalent quercetin-rutin blend) contributed to a decrease in lipid peroxidation, intensifying phagocytosis processes. The studied compounds demonstrated their low influence on the leukocyte blood profile in animals.

Protective Effect of Apple Polyphenols on H2O2-Induced Oxidative Stress Damage in Human Colon Adenocarcinoma Caco-2 Cells.

Apple is an important dietary agent for human and apple polyphenols (AP) are the main secondary metabolites of apples. In this study, the protective effects of AP on hydrogen peroxide (H2O2)-induced oxidative stress damage in human colon adenocarcinoma Caco-2 cells were investigated by cell viability, oxidative stress change as well as cell apoptosis. Pre-adding AP could significantly increase the survival rate of H2O2-treated Caco-2 cells. Besides, the activities of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) were elevated. While the malondialdehyde (MDA) content which is the major oxidant products of polyunsaturated fatty acids (PUFA) reduced after AP treatment. In addition, AP also suppressed the emergence of DNA fragment and decreased the expression of apoptosis-related protein Caspase-3. These results demonstrated that AP could ameliorate H2O2-induced oxidative stress damage in Caco-2 cells, which could serve as a reference for further studies of apple natural active products and deep study of the anti-oxidative stress mechanism.

Sericin/crocetin micro/nanoparticles for nucleus pulposus cells regeneration: An "active" drug delivery system.

Introduction: Initiation and progression of intervertebral disk degeneration are linked to oxidative stress, with reactive oxygen species being a key factor. Therefore, as a potentially novel approach able to regenerate the damaged intervertebral disk, this work aimed to prepare an "active per sé" drug delivery system by combining sericin and crocetin: both are bioactive compounds with antioxidant, anti-inflammatory, immunomodulant and regenerative properties. Methods: In detail, sericin nanoparticles were prepared using crocetin as a cross-linker; then, the nanoparticle dispersions were dried by spray drying as it is (NP), with an excess of sericin (NPS) or crocin/crocetin (NPMix), obtaining three microparticle formulations. Results and Discussion: Before drying, the nanoparticles were nanometric (about 250 nm), with a negative surface charge, and appeared spherical and smooth. Following the drying process, spherical and smooth microparticles were obtained, with a mean diameter of about 1.7-2.30 µm. NPMix was the most active in antioxidant and anti-tyrosinase activities, likely due to the excess of crocin/crocetin, while NPS had the best anti-elastase activity, likely due to sericin in excess. Furthermore, all the formulations could prevent oxidative stress damage on nucleus pulposus cells, with NPMix being the best. Overall, the intrinsic anti-tyrosinase and anti-elastase activities and the ability to protect from oxidative stress-induced damages justify future investigations of these "active per sé" formulations in treating or preventing intervertebral disk degeneration.

Lycopene ameliorates DEHP exposure-induced renal pyroptosis through the Nrf2/Keap-1/NLRP3/Caspase-1 axis.

Di (2-ethylhexyl) phthalate (DEHP) is commonly used as a plasticizer in plastic products, and due to its unique chemical composition, it frequently dissolves and enters the environment. Lycopene as a natural carotenoid has been shown to have powerful antioxidant capacity and strong kidney protection. This study aimed to investigate the role of the interplay between oxidative stress and the classical pyroptosis pathway in LYC alleviating DEHP-induced renal injury. ICR mice were given DEHP (500 mg/kg/d or 1000 mg/kg/d) and/or LYC (5 mg/kg/d) for 28 days to explore the underlying mechanisms of this hypothesis. Our results indicated that DEHP caused the shedding of renal tubular epithelial cells, increased the content of kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL) in the tissue, the decrease of antioxidant activity markers and the increase of oxidative stress indexes. It is gratifying that LYC alleviates DEHP-induced renal injury. The expression of nuclear factor erythrocyte 2-related factor 2 (Nrf2) and its downstream target genes is improved in DEHP induced renal injury through LYC mediated protection. Meanwhile, LYC supplementation can inhibit DEHP-induced Caspase-1/NLRP3-dependent pyroptosis and inflammatory responses. Taken together, DEHP administration resulted in nephrotoxicity, but these changes ameliorated by LYC may through crosstalk between the Nrf2/Keap-1/NLRP3/Caspase-1 pathway. Our study provides new evidence that LYC protects against kidney injury caused by DEHP.

S100A1 expression is increased in spinal cord injury and promotes inflammation, oxidative stress and apoptosis of PC12 cells induced by LPS via ERK signaling.

Spinal cord injury (SCI) is a severe neurological disorder and the molecular mechanisms leading to its poor prognosis remain to be elucidated. S100A1, a mediator of Ca2+ handling of sarcoplasmic reticulum and mitochondrial function, operates as an endogenous danger signal (alarmin) associated with inflammatory response and tissue injury. The aim of the present study was to investigate the expression and biological effects of S100A1 in SCI. A rat model of SCI and a PC12 cell model of lipopolysaccharide (LPS)­induced inflammation were established to examine S100A1 expression at the mRNA and protein levels. The inflammation level, which was mediated by S100A1, was determined based on inflammatory factor (IL­1ß, IL­6 and TNF­α) and anti­inflammatory factor (IL­10) expression. The effects of S100A1 on cellular oxidation and anti­oxidation levels were observed by detecting the levels of reactive oxygen species, superoxide dismutase, catalase activities and nuclear factor erythroid 2­related factor 2 expression. The protein levels of Bax, Bcl2 and cleaved caspase­3 were used for the evaluation of the effects of S100A1 on apoptosis. Phosphorylated (p­)ERK1/2 expression was used to evaluate the effects of S100A1 on ERK signaling. The results revealed that S100A1 expression was significantly upregulated in vivo and in vitro in the PC12 cell model of LPS­inflammation. The silencing and overexpression of S100A1 helped alleviate and aggravate LPS­induced inflammation, oxidative stress and apoptosis levels, respectively. S100A1 was found to regulate the ERK signaling pathway positively. An inhibitor of ERK signaling (MK­8353) partially abolished the promoting effects of the overexpression of S100A1 on inflammation, oxidative stress damage and apoptosis. In conclusion, S100A1 expression was elevated in model of SCI and in the PC12 cell model of LPS­induced inflammation. Furthermore, the overexpression/silencing S100A1 aggravated/mitigated the inflammation, oxidative stress damage and the apoptosis of LPS­stimulated PC12 cells via the ERK signaling pathway. The present study revealed the mechanism of S100A1 in SCI, which provided a new theoretic reference for future research on SCI.

Cerium oxide-based nanozyme suppresses kidney calcium oxalate crystal depositions via reversing hyperoxaluria-induced oxidative stress damage.

Oxidative stress damage to renal epithelial cells is the main pathological factor of calcium oxalate calculi formation. The development of medicine that could alleviate oxidative damage has become the key to the prevention and treatment of urolithiasis. Herein, porous nanorods CeO2 nanoparticles (CNPs) were selected from CeO2 with different morphologies as an antioxidant reagent to suppress kidney calcium oxalate crystal depositions with excellent oxidation resistance due to its larger specific surface area. The reversible transformation from Ce3+ to Ce4+ could catalyze the decomposition of excess free radicals and act as a biological antioxidant enzyme basing on its strong ability to scavenge free radicals. The protection capability of CNPS against oxalate-induced damage and the effect of CNPS on calcium oxalate crystallization were studied. CNPS could effectively reduce reactive oxygen species production, restore mitochondrial membrane potential polarity, recover cell cycle progression, reduce cell death, and inhibit the formation of calcium oxalate crystals on the cell surface in vitro. The results of high-throughput sequencing of mRNA showed that CNPs could protect renal epithelial cells from oxidative stress damage caused by high oxalate by suppressing the expression gene of cell surface adhesion proteins. In addition, CNPS can significantly reduce the pathological damage of renal tubules and inhibit the deposition of calcium oxalate crystals in rat kidneys while having no significant side effect on other organs and physiological indicators in vivo. Our results provide a new strategy for CNPS as a potential for clinical prevention of crystalline kidney injury and crystal deposition.

Antioxidative and Immunomodulating Properties of Aronia melanocarpa Extract Rich in Anthocyanins.

The fruits of Aronia melanocarpa are well known due to their high anthocyanin content that may be effective in preventing certain health disorders arising from oxidative stress. Various polyphenolic compounds such as anthocyanins and flavonoids are responsible for the multiple effects of chokeberry. The aim of this study was to determine in vitro how active the black chokeberry anthocyanins are in scavenging radicals and to evaluate in vivo their immunomodulating capacity. Using the method of column chromatography, we extracted the anthocyanins of black chokeberries, i.e., cyanidin-3-O-galactoside with a purity of over 93.7%. Using HPLC and spectrophotometric analysis, the flavonoid content was determined. Following the analysis of the tests with AAPH and DPPH, the chokeberry cyanidin-3-O-galactoside was found much better than individual anthocyanins in regard to antioxidant capacity. The range of concentrations was revealed, showing the protective effect of anthocyanins on the RPMI-1788 cell culture against cyclophosphamide, as well as against osmotic and peroxide hemolysis. An immunomodulating effect on the functional activity of phagocytes was revealed in vivo as a result of oral administration of chokeberry cyanidin-3-O-galactoside and a mixture composed of cyanidin-3-O-glucoside and cyanidin-3-O-galactoside standards. Consequently, anthocyanins, in particular cyanidin-3-O-galactoside, play an important role, demonstrating immunomodulating effects when chokeberries are consumed.

Inhibition of the integrated stress response reverses oxidative stress damage-induced postoperative cognitive dysfunction.

Postoperative cognitive dysfunction (POCD) is a common complication following anesthesia and surgery that might lead to a decline in learning and memory. Oxidative stress damage is one of the pathogenic mechanisms underlying POCD. Recent studies had shown that the integrated stress response (ISR) is closely related to oxidative stress. The core response of the ISR is phosphorylation of eIF2α. Various cellular stress stimuli trigger activation of eIF2α kinases, thus causing phosphorylation of eIF2α. ISR is associated with many neurodegenerative diseases; however, the relationship between POCD and ISR has not been defined. In the present study, the tibias in 4-month-old male C57BL/6 mice were fractured under isoflurane anesthesia to establish the POCD animal model. Cognitive function was assessed by fear conditioning tests and the Y-maze from 3 to 14 days post-surgery. Western blot was used to determine the levels of PeIF2α, eIF2α, ATF4, GADD34, CHOP, BDNF, proBDNF, and p-NR2B expression. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured to determine oxidative stress in hippocampal tissues. After tibial fracture surgery in mice, the hippocampus had increased levels of PeIF2α, ATF4, GADD34, and CHOP protein, ROS-positive cells, and average fluorescence intensity, SOD activity was decreased, and the MDA level was increased. The ISR inhibitor, ISRIB, reduced the levels of PeIF2α, ATF4, GADD34, and CHOP protein, and alleviated oxidative stress in the hippocampus of POCD mice. Moreover, ISRIB ameliorated cognitive dysfunction in POCD mice. Our findings suggested that targeting ISR may represent an effective approach to combat POCD.

A Plasmid-Based Fluorescence Reporter System for Monitoring Oxidative Damage in E. coli.

Quantitating intracellular oxidative damage caused by reactive oxygen species (ROS) is of interest in many fields of biological research. The current systems primarily rely on supplemented oxygen-sensitive substrates that penetrate the target cells, and react with ROS to produce signals that can be monitored with spectroscopic or imaging techniques. The objective here was to design a new non-invasive analytical strategy for measuring ROS-induced damage inside living cells by taking advantage of the native redox sensor system of E. coli. The developed plasmid-based sensor relies on an oxygen-sensitive transcriptional repressor IscR that controls the expression of a fluorescent marker in vivo. The system was shown to quantitatively respond to oxidative stress induced by supplemented H2O2 and lowered cultivation temperatures. Comparative analysis with fluorescence microscopy further demonstrated that the specificity of the reporter system was equivalent to the commercial chemical probe (CellROX). The strategy introduced here is not dependent on chemical probes, but instead uses a fluorescent expression system to detect enzyme-level oxidative damage in microbial cells. This provides a cheap and simple means for analysing enzyme-level oxidative damage in a biological context in E. coli.

Single and Mixture Toxicity of Boron and Vanadium Nanoparticles in the Soil Annelid Enchytraeus crypticus: A Multi-Biomarker Approach.

The increased use and production of new materials has contributed to Anthropocene biodiversity decrease. Therefore, a careful and effective toxicity evaluation of these new materials is crucial. However, environmental risk assessment is facing new challenges due to the specific characteristics of nanomaterials (NMs). Most of the available ecotoxicity studies target the aquatic ecosystems and single exposures of NMs. The present study evaluated Enchytraeus crypticus survival and reproduction (28 days) and biochemical responses (14 days) when exposed to nanoparticles of vanadium (VNPs) and boron (BNPs) (single and mixture; tested concentrations: 10 and 50 mg/kg). Although at the organism level the combined exposures (VNPs + BNPs) did not induce a different toxicity from the single exposures, the biochemical analysis revealed a more complex picture. VNPs presented a higher toxicity than BNPs. VNPs (50 mg/kg), independently of the presence of BNPs (additive or independent effects), caused a decrease in survival and reproduction. However, acetylcholinesterase, glutathione S-transferase, catalase, glutathione reductase activities, and lipid peroxidation levels revealed alterations in neurotoxicity, detoxification and antioxidant responses, depending on the time and type of exposure (single or mixture). The results from this study highlight different responses of the organisms to contaminants in single versus mixture exposures, mainly at the biochemical level.

Protective effect of polysaccharides from Ostrea rivularis against reproductive injury via regulating autophagy induced by oxidative stress.

The objective of this study was to investigate the effect of polysaccharides from Ostrea rivularis (ORP) relieving reproductive damage by regulating autophagy. The results showed that ORP intervention could alleviate the pathological changes of the testis and alleviate oxidative stress which were caused by cyclophosphamide (CTX) in vivo, including improve sperm symptoms and rise testosterone level. Reduced level of autophagy after ORP intervention was observed by transmission electron microscopy (TEM), which implied that ORP might regulate cell autophagy. In vitro experiments showed that ORP could alleviate the damage of TM4 cells induced by H2O2, reduce the level of intracellular ROS and the content of MDA. Autophagy-related protein expressions of p62, LC3, Beclin-1 before and after 3-MA inhibitor intervention were also proved that ORP could regulate autophagy. Overall, these results confirmed that ORP could reduce reproductive damage related to autophagy.

Health consequences of disinfection against SARS-CoV-2: Exploring oxidative stress damage using a biomonitoring approach.

Individuals who get involved in the disinfection of public settings using sodium hypochlorite might suffer adverse health effects. However, scarce information is available on the potential oxidative stress damage caused at low concentrations typically used for disinfection. We aimed to assess whether exposure to sodium hypochlorite during the COVID-19 pandemic causes oxidative stress damage in workers engaged in disinfection tasks. 75 operators engaged in the disinfection of public places were recruited as the case group, and 60 individuals who were not exposed to disinfectant were chosen as the control group. Spot urine samples were collected before (BE) and after exposure (AE) to disinfectants in the case group. Likewise, controls provided two spot urine samples in the same way as the case group. Urinary malondialdehyde (MDA) levels were quantified by forming thiobarbituric acid reactive substances in the urine. In addition, the concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the urine was determined using an ELISA kit. Results showed significant differences in the urinary levels of oxidative stress markers, where median 8-OHdG (AE case: 3.84 ± 2.89 µg/g creatinine vs AE control 2.54 ± 1.21 µg/g creatinine) and MDA (AE case: 169 ± 89 µg/g creatinine vs AE control 121 ± 47 µg/g creatinine) levels in case group AE samples were 1.55 and 1.35-times higher than the control group AE samples (P < 0.05), respectively. Besides, urinary levels of oxidative stress markers in AE samples of the case group were significantly higher than in BE samples (8-OHdG BE 3.40 ± 1.95 µg/g creatinine, MDA BE 136 ± 51.3 µg/g creatinine, P < 0.05). Our results indicated that exposure to even low levels of sodium hypochlorite used in disinfection practices might cause oxidative stress related damage. With this in mind, implementing robust protective measures, such as specific respirators, is crucial to reduce the health burdens of exposure to disinfectants.

Enzymatic acylation of raspberry anthocyanin: Evaluations on its stability and oxidative stress prevention.

Raspberry anthocyanins were isolated and purified by XAD-7HP macroporous resin and silica gel column chromatography. Anthocyanins were then acylated with methyl salicylate as catalyzed by lipase under reduced pressure, and the conversion rate was 84.26%. LC-MS and NMR were used to identify the structure, and the stability, antioxidant capacity and protective ability of the acylated anthocyanins against oxidative damage were determined. The results showed that cyanindin-3-O-glucoside (C3G) was the primary anthocyanin in raspberry, and the binding site of acylation was on the glucoside C-6, and the product was cyanidin-3-(6-salicyloyl) glucoside (C3-6(S) G). After acylation, its stability in light, heat and oxidation environments could be significantly improved, and acylated ACN showed insignificant changes in antioxidant capacities to scavenge DPPH and ABTS free radicals, as well as oxygen free radical absorptive capacity (ORAC). And it could also effectively prevent the release of ROS caused by oxidative damage and alleviate oxidative stress damage.

Human Retinal Pigment Epithelial Cells Overexpressing the Neuroprotective Proteins PEDF and GM-CSF to Treat Degeneration of the Neural Retina.

BACKGROUND: Non-viral transposon-mediated gene delivery can overcome viral vectors' limitations. Transposon gene delivery offers the safe and life-long expression of genes such as Pigment Epithelium-Derived Factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to counteract retinal degeneration by reducing oxidative stress damage. OBJECTIVE: The study aimed at using Sleeping Beauty transposon to transfect human Retinal Pigment Epithelial (RPE) cells with the neuroprotective factors PEDF and GM-CSF to investigate the effect of these factors on oxidative stress damage. METHODS: Human RPE cells were transfected with PEDF and GM-CSF by electroporation, using the hyperactive Sleeping Beauty transposon gene delivery system (SB100X). Gene expression was determined by RT-qPCR, and protein level by Western Blot as well as ELISA. The cellular stress level and the neuroprotective effect of the proteins were determined by measuring the concentrations of the antioxidant glutathione in human RPE cells, and conducting immunohistochemical examination of retinal integrity, inflammation, and apoptosis of rat Retina-Organotypic Cultures (ROC) exposed to H2O2. RESULTS: Human RPE cells were efficiently transfected showing a significantly augmented gene expression and protein secretion. Human RPE cells overexpressing PEDF and/or GM-CSF or pretreated with recombinant proteins presented significantly increased glutathione levels post- H2O2 incubation than non-transfected/untreated controls. rPEDF and/or rGM-CSF-treated ROC exhibited decreased inflammatory reactions and cell degeneration. CONCLUSION: GM-CSF and/or PEDF could be delivered successfully to RPE cells with combined use of SB100X and electroporation. PEDF and/or GM-CSF reduced H2O2-mediated oxidative stress damage in RPE cells and ROC offering an encouraging technique to re-establish a cell protective environment to halt age-related retinal degeneration.