Dichlorodihydrofluorescein Diacetate Research Articles

Adaption of in vitro and in chemico phototoxicity tests for tattoo pigments and the effect of adsorption of the phototoxic contaminant benzo[a]pyrene to carbon black

Phototoxicity plays an important role in tattoo toxicology due to the skin's daily exposure to sunlight and the permanent deposition of pigments in skin. Here, we investigated in chemico reactive oxygen species generation using 2′,7′-dichlorodihydrofluorescein diacetate and in vitro phototoxicity towards skin fibroblasts based on the OECD Test Guideline 432.Proficiency substances, titanium dioxide (TiO2, anatase 30 nm, anatase 100 nm, rutile 30 nm), pigment red 22 (PR 22) with and without purification and carbon black (CB) were investigated. Phototoxic properties of benzo[a]pyrene (B[a]P) were investigated with and without adsorption to CB. Pigments were characterised by dynamic light scattering.All proficiency substances except for norfloxacin were predicted successfully and only TiO2 anatase exhibited phototoxicity in the cell-based approach. B[a]P showed the expected phototoxicity in contrast to B[a]P bound to carbon black. Upon UVA irradiation, only TiO2 pigment anatase and PR 22 generated ROS in the cell-free assay. DLS measurements showed a high degree of polydispersity/agglomeration.OECD Test Guideline 432 should be applied with an incubation time of 24 h when testing highly insoluble pigments. The in chemico ROS assay introduced below might serve as a quick screening test but may be further adapted to predict the in vitro outcome.

Antidepressant Properties of a Four-compound Cocktail Identified from Si-Ni-San by HIF-1 Pathway Modulation.

Si-Ni-San (SNS) is the formula prescription of Traditional Chinese Medicine (TCM) with anti-depression properties, but its underlying mechanisms remain unclear. This study provides novel approaches for the study of Traditional Chinese Medicine (TCM) and offers new opportunities for exploring the pharmacological properties of SNS. The ingredients in SNS implicated in the treatment of depression were identified and studied using network pharmacology. SwissTargetPrediction and molecular docking were used to study the interaction of SNS ingredients and their targets. The protective effect of these ingredients and their cocktail in rat pheochromocytoma cells (PC12) exposed to corticosterone (Cor) were evaluated using the CCK-8 assay, Hoechst 33342 staining, 2',7'-dichlorodihydro fluorescein diacetate (H2DCFDA) staining, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and in-cell Western analysis. The network pharmacology study showed that the HIF-1 signaling pathway was the most crucial pathway implicated in the anti-depressive property of SNS. MAPK1 (ERK2), MAPK3 (ERK1), AKT1, VEGFA, STAT3, and EGF were identified as hub target proteins in the HIF-1 signaling pathway. Quercetin, naringenin, licochalcone A, and kaempferol from SNS, which targeted the six proteins mentioned above, were used to create a cocktail. This cocktail exerted protective properties, decreased the oxidative stress in PC12 exposed to Cor, and successfully regulated the expressions of AKT1, p-AKT1, ERK1, ERK2, p-ERK1/2, STAT3, p- STAT3, and VEGFA induced by Cor exposure. The SwissTargetPrediction and molecular docking study showed that the cocktail may regulate the HIF-1 signaling pathway by directly binding with AKT1 and MAPK1. The cocktail from SNS comprised of quercetin, naringenin, licochalcone A, and kaempferol exerts anti-depression potentiality by modulating the HIF-1 signaling pathway via direct interactions with AKT1 and MAPK1.

Mangiferin alleviates cisplatin-induced ototoxicity in sensorineural hearing loss

Mangiferin(MGF) exhibits crucial biological roles, including antioxidant and anti-inflammatory functions. However, how to clearly elucidate the functioning mechanism of MGF for inhibiting cisplatin-induced hearing loss requires in-depth investigation. In this work, we aimed at gaining insight into how MGF functions as the protective agent against cisplatin-triggered ototoxicity using various assays. The variation for reactive oxygen species (ROS) concentrations was determined with MitoSOX-Red and 2′,7′-Dichlorodihydrofluorescein diacetate staining (DCFH-DA). The protective function and corresponding mechanism of MGF in hair cell survival in the House Ear Institute-Organ of Corti (HEI-OC1) cell line were assessed using RNA sequencing (RNA-Seq). Our findings demonstrated that MGF significantly alleviated cisplatin-induced injury to hair cells in vitro, encompassing cell lines and cochlear explants, as well as in vivo models, including C57BL/6 J mice and zebrafish larvae. Mechanistic studies revealed that MGF reversed the increased accumulation of ROS and inhibited cell apoptosis through mitochondrial-mediated intrinsic pathway. Moreover, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting data indicated MGF protected against cisplatin-mediated ototoxicity via the mitogen-activated protein kinase pathway (MAPK). These findings demonstrated MGF has significant potential promise in combating cisplatin-induced ototoxicity, offering a foundation for expanded investigation into therapeutic approaches for auditory protection.

Honokiol relieves hippocampal neuronal damage in Alzheimer's disease by activating the SIRT3-mediated mitochondrial autophagy.

This work elucidated the effect of honokiol (HKL) on hippocampal neuronal mitochondrial function in Alzheimer's disease (AD). APP/PS1 mice were used as AD mice models and exposed to HKL and 3-TYP. Morris water maze experiment was performed to appraise cognitive performance of mice. Hippocampal Aβ+ plaque deposition and neuronal survival was evaluated by immunohistochemistry and Nissl staining. Hippocampal neurons were dissociated from C57BL/6 mouse embryos. Hippocampal neuronal AD model was constructed by Aβ oligomers induction and treated with HKL, CsA and 3-TYP. Neuronal viability and apoptosis were detected by cell counting kit-8 assay and TUNEL staining. mRFP-eGFP-LC3 assay, MitoSOX Red, dichlorodihydrofluorescein diacetate, and JC-1 staining were performed to monitor neuronal autophagosomes, mitochondrial reactive oxygen species (ROS), neuronal ROS, and mitochondrial membrane potential. Autophagy-related proteins were detected by Western blot. In AD mice, HKL improved cognitive function, relieved hippocampal Aβ1-42 plaque deposition, promoted hippocampal neuron survival, and activated hippocampal SIRT3 expression and mitochondrial autophagy. These effects of HKL on AD mice were abolished by 3-TYP treatment. In hippocampal neuronal AD model, HKL increased neuronal activity, attenuated neuronal apoptosis and Aβ aggregation, activated SIRT3 and mitochondrial autophagy, reduced mitochondrial and neuronal ROS, and elevated mitochondrial membrane potential. CsA treatment and 3-TYP treatment abrogated the protection of HKL on hippocampal neuronal AD model. The promotion of mitochondrial autophagy by HKL in hippocampal neuronal AD model was counteracted by 3-TYP. HKL activates SIRT3-mediated mitochondrial autophagy to mitigate hippocampal neuronal damage in AD. HKL may be effective in treating AD.

Polyethylene terephthalate nanoparticles induce oxidative damage in Chlorella vulgaris

Polyethylene Terephthalate (PET) is a type of plastic largely used for packing food and beverages. Unfortunately, it includes a major portion of the plastic distributed through aquatic systems wherever systematic collection and recycling are lacking. Although PET is known to be non-toxic, it is not obvious whether the nanoparticles (NPs) formed due to their degradation have any direct/indirect effect on aquatic organisms. In order to study the effects on aquatic environment, fresh water algae Chlorella vulgaris was subjected to incremental concentrations of the NPs. We observed a concentration and duration of exposure dependent decrease in algal growth rate along with reduced total chlorophyll content. Scanning electron microscopy revealed deformities in cell shape and the uptake of Propidium Iodide suggested membrane damage in response to NP exposure. Intracellular Reactive Oxygen Species level was also found significantly higher, evidenced by Dichlorodihydrofluorescein diacetate staining. Activity of antioxidant enzymes Superoxide dismutase (SOD), Peroxidase (POD) and Catalase (CAT) were significantly higher in the NP exposed groups suggesting the cellular response to regain homeostasis. Further, expression levels of the genes psaB, psbC, and rbcL associated with photosynthesis increased above two fold with respect to the control inferring the possibility of damage to photosynthesis and the initial molecular responses to circumvent the situation. In short, our studies provide evidence for oxidative stress mediated cellular damages in Chlorella vulgaris exposed to NPs of PET.

DHODH Alleviates Heart Failure via the Modulation of CoQ-Related Ferroptotic Inhibition.

Heart failure (HF) is a clinical syndrome that seriously endangers human health and quality of life as the terminal stage of cardiovascular diseases. Ferroptosis as a new iron-dependent programmed cell death mode that is closely related to the occurrence and development of cardiovascular diseases. Dihydroorotate dehydrogenase (DHODH) has been found to play a crucial role in inhibiting ferroptosis and improving mitochondrial function, and its expression can be upregulated by estradiol (E2). Recent studies have found that DHODH can inhibit ferroptosis by reducing coenzyme Q (CoQ) to CoQH2. Therefore, this study aims to explore the effect of up-regulation of DHODH on the pathological hypertrophy and fibrosis of heart failure and its mechanisms. The mouse heart failure model was established by transverse aortic constriction (TAC), surgery in mice. Two days after the operation, a subcutaneous injection of E2 or the same volume of sesame oil was given for 8 weeks. Then, the left ventricular systolic function related indicators of mice were measured by echocardiography, and the degree of myocardial fibrosis of mice was detected by histological analysis; the expression levels of heart failure markers were detected by quantitative polymerase chain reaction (q-PCR) and western blot (WB) analysis; the morphological changes of mitochondria in cardiac cells of mice were observed by transmission electron microscopy. Cell model were established by stimulating with phenylephrine for 96 hours. Ferroptosis markers were detected by kits and WB analysis. Mitochondrial function was verified by a JC-1 fluorescent probe, and 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The knockdown results were detected by WB analysis after transfection of small interfering RNA (siRNA) of CoQ. Fer-1 was added as a positive control to verify the ferroptosis-related changes of myocardial cells. In the animal model, we found that E2 treatment alleviates TAC-induced cardiac hypertrophy and fibrosis and suppresses cardiomyocyte ferroptosis by promotes DHODH upregulation in murine cardiomyocytes. In the cell model, DHODH upregulation protects against phenylephrine-induced cardiomyocytes with failure. However, the effect on up-regulating DHODH was inhibited by transfection to down-regulate CoQ expression. The up-regulation of DHODH could effectively ameliorate the manifestations of heart failure such as myocardial hypertrophy and fibrosis in mice after TAC surgery, inhibit ferroptosis of cardiac myocytes, and ameliorate mitochondrial function. The mechanism involves CoQ-related biological processes.

Combination of Vismodegib and Paclitaxel Enhances Cytotoxicity via Bak-mediated Mitochondrial Damage in EGFR-Mutant Non-Small Cell Lung Cancer Cells.

Half of NSCLC patients harbor epidermal growth factor receptor (EGFR) mutations, and their therapeutic responses are remarkably different from patients with wild-type EGFR (EGFR-WT) NSCLC. We previously demonstrated that the hedgehog inhibitor vismodegib (Vis) potentiates paclitaxel (PTX)-induced cytotoxicity via suppression of Bax phosphorylation, which promotes accumulation of mitochondrial damage and apoptosis in EGFR-WT NSCLC cells. In this study, we further delineated the anticancer activity and underlying mechanisms of this combination treatment in EGFR-mutant NSCLC cells. MTS/PMS activity and trypan blue exclusion assays were used to assess cell viability. Apoptosis was monitored by chromosome condensation, annexin V staining, and cleavage of PARP and caspase-3. Western blots were conducted to track proteins of interest after treatment. Reactive oxygen species (ROS) level was monitored by 2',7'-dichlorodihydrofluorescein diacetate. Mitochondrial status was analyzed by tetramethylrhodamine, ethyl ester. Hedgehog signaling was induced by PTX, which rendered H1975 and PC9 cells insensitive to PTX-induced mitochondrial apoptosis via suppression of Bak. However, Vis enhanced PTX-induced Bak activation, leading to mitochondrial damage, ROS accumulation, and subsequent apoptosis. Our findings suggest that the combination of Vis and PTX could be a potential therapeutic strategy to increase PTX sensitivity of EGFR-mutant NSCLC.

Protective Effect of Arzanol against H2O2-Induced Oxidative Stress Damage in Differentiated and Undifferentiated SH-SY5Y Cells.

Oxidative stress can damage neuronal cells, greatly contributing to neurodegenerative diseases (NDs). In this study, the protective activity of arzanol, a natural prenylated α-pyrone-phloroglucinol heterodimer, was evaluated against the H2O2-induced oxidative damage in trans-retinoic acid-differentiated (neuron-like) human SH-SY5Y cells, widely used as a neuronal cell model of neurological disorders. The pre-incubation (for 2 and 24 h) with arzanol (5, 10, and 25 μM) significantly preserved differentiated SH-SY5Y cells from cytotoxicity (MTT assay) and morphological changes induced by 0.25 and 0.5 mM H2O2. Arzanol reduced the generation of reactive oxygen species (ROS) induced by 2 h oxidation with H2O2 0.5 mM, established by 2',7'-dichlorodihydrofluorescein diacetate assay. The 2 h incubation of differentiated SH-SY5Y cells with H2O2 determined a significant increase in the number of apoptotic cells versus control cells, evaluated by propidium iodide fluorescence assay (red fluorescence) and NucView® 488 assay (green fluorescence). Arzanol pre-treatment (2 h) exerted a noteworthy significant protective effect against apoptosis. In addition, arzanol was tested, for comparison, in undifferentiated SH-SY5Y cells for cytotoxicity and its ability to protect against H2O2-induced oxidative stress. Furthermore, the PubChem database and freely accessible web tools SwissADME and pkCSM-pharmacokinetics were used to assess the physicochemical and pharmacokinetic properties of arzanol. Our results qualify arzanol as an antioxidant agent with potential neuroprotective effects against neuronal oxidative stress implicated in NDs.

Therapeutic Effect of Donepezil on Neuroinflammation and Cognitive Impairment after Moderate Traumatic Brain Injury.

Despite the important clinical issue of cognitive impairment after moderate traumatic brain injury (TBI), there is currently no suitable treatment. Here, we used in vitro and in vivo models to investigate the effect of Donepezil-an acetylcholinesterase (AChE) inhibitor-on cognitive impairment in the acute period following injury, while focusing on neuroinflammation and autophagy- and mitophagy-related markers. The purpose of the in vitro study was to investigate potential neuroprotective effects in TBI-induced cells after donepezil treatment, and the in vivo study, the purpose was to investigate therapeutic effects on cognitive impairment in the acute period after injury by analyzing neuroinflammation and autophagy- and mitophagy-related markers. The in vitro TBI model involved injuring SH-SY5Y cells using a cell-injury controller and then investigating the effect of donepezil at a concentration of 80 μM. The in vivo TBI model was made using a stereotaxic impactor for male C57BL/6J mice. Immuno-histochemical markers and cognitive functions were compared after 7 days of donepezil treatment (1 mg/kg/day). Mice were divided into four groups: sham operation with saline treatment, sham operation with donepezil treatment, TBI with saline treatment, and TBI with donepezil treatment (18 mice in each group). Donepezil treatment was administered within 4 h post-TBI. In vitro, donepezil was found to lead to increased cell viability and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), along with decreased reactive oxygen species (ROS), lactate-dehydrogenase (LDH), 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA)-positive cells, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. The mRNA and protein expressions of neuroinflammation (Cyclooxygenase-2, COX-2; NOD-like receptor protein 3, NLRP3; Caspase-1; and Interleukin-1 beta, IL-1β), as well as autophagy- and mitophagy-related markers (death-associated protein kinase 1, DAPK1; PTEN-induced kinase 1, PINK1; BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like, BNIP3L; Beclin-1, BECN1; BCL2-associated X protein, BAX; microtubule-associated protein 1A/1B-light chain 3B (LC3B); Sequestosome-1; and p62) were all found to decrease after donepezil treatment. The in vivo study also showed that donepezil treatment resulted in decreased levels of cortical tissue losses and brain swelling in TBI compared to the TBI group without donepezil treatment. Donepezil treatment was also shown to decrease the mRNA and Western blotting expressions of all markers, and especially COX-2 and BNIP3L, which showed the most significant decreases. Moreover, TBI mice showed an decreased escape latency, increased alteration rate, and improved preference index, altogether pointing to better cognitive performance after donepezil treatment. Donepezil treatment may be beneficial in improving cognitive impairment in the early phase of moderate traumatic brain injury by ameliorating neuroinflammation, as well as autophagy and mitophagy.

Chlorogenic Acid Alleviates High Glucose-induced HK-2 Cell Oxidative Damage through Activation of KEAP1/NRF2/ARE Signaling Pathway.

To investigate the alleviating effect of chlorogenic acid (CGA) on oxidative damage in high glucose (HG)-induced HK-2 cells and to explore its potential mechanisms. We cultured the human proximal tubular cell line HK-2 and divided them into the control group and different concentrations of CGA groups (0, 5, 10, 25, 50, 100, 200 μM). The trypan blue dye test was used to detect CGA's potential cytotoxicity on HK-2 cells. Then, we treated HK-2 with HG and CGA; the Cell Counting Kit-8 (CCK-8) method was used to detect the cell viability of HK-2 cells in each group. Flow cytometry was employed to measure the apoptosis rate of cells. Western blot was performed to detect the expression of apoptosis proteins B-cell lymphoma-2 (BCL-2), BCL-2-associated X protein (BAX), cysteinyl aspartate specific proteinase (CASPASE)-9, and CASPASE-3. In addition, enzymatic activities, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and lipid peroxide (LPO), were measured with the corresponding detection kits. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) assay and flow cytometry were performed to detect reactive oxygen species (ROS) production. Western blot analysis and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were conducted to evaluate protein and mRNA expressions of the Kelch-like ECH-associated protein-1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2)/Antioxidant Response Elements (ARE) signaling pathway. The outcomes showed that, in a dose-dependent way, CGA dramatically increased the vitality of HK-2 induced by HG. Furthermore, CGA significantly reduced the HG-stimulated HK-2 cell apoptosis, which may be linked to the promotion of BCL-2 and the suppression of BAX, cleaved-CASPASE-3, and cleaved-CASPASE-9 expression. In HK-2 cells, CGA reduced the formation of ROS generated by HG levels and markedly boosted the activity of the antioxidant enzymes SOD, GSH-Px, and CAT. Furthermore, compared with the HG group, CGA significantly raised NRF2 nuclear expression and downregulated NRF2 cytosolic expression and increased the mRNA expression of NRF2 and its target genes, heme oxygenase-1 (HO-1), KEAP1, and NAD(P)H dehydrogenase quinone 1 (NQO1). These results show that CGA might be useful in managing oxidative damage in HG-induced HK-2 cells.

The role of ayahuasca in cell viability and oxidative stress in gastric adenocarcinoma cell line

Ayahuasca, a psychoactive beverage native to the Amazon, originally derived from Banisteriopsis caapi stem scrapings and Psychotria viridis leaves, exhibits hallucinogenic properties due to N,N-dimethyltryptamine. When combined with β-carbolines, it enters the bloodstream and central nervous system, inhibiting monoamine oxidase-A. Over time, therapeutic effects have been associated to ayahuasca consumption. This study assessed the impact of extracts from three plant decoctions used in ayahuasca preparation on the gastric adenocarcinoma cell line (AGS). MTT reduction assays selected B. caapi, Mimosa hostilis, and Peganum harmala samples as most effective. Lactate dehydrogenase activity evaluated membrane integrity loss, while oxidative stress induction was measured using dihydroethidium and 2′,7′-dichlorodihydrofluorescein diacetate probes. Results revealed apoptosis induction in AGS cells, with all three samples significantly reducing oxidative stress.

Nrf2 mediates the effects of shionone on silica-induced pulmonary fibrosis

BackgroundExtended contact with silica particles can lead to Silicosis, a chronic lung condition lacking established treatment protocols or clear mechanisms of development. The urgency for innovative treatments arises from the unavailability of effective treatment methodologies. The origin of silica-induced pulmonary fibrosis includes essential processes such as macrophage activation and the conversion of fibroblasts into myofibroblasts, with oxidative stress playing a pivotal role. Shionone (SHI), a triterpenoid extracted from the Aster tataricus plant, is recognized for its extensive health benefits. This study explores the capability of SHI to alleviate the effects of silica-induced lung fibrosis in mice.MethodsThis investigation explored the impact of SHI on lung inflammation and fibrosis at different stages (early and late) triggered by silica in mice, focusing specifically on the initial and more developed phases. It comprised an analysis of isolated peritoneal macrophages and fibroblasts extracted from mice to elucidate SHI's therapeutic potential and its underlying mechanism. The methodology employed encompassed quantitative PCR, immunofluorescence, flow cytometry, and western blotting to examine macrophage activity and their transition into myofibroblasts. The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by SHI was confirmed via immunofluorescence and western blot studies. SHI's antioxidative properties were evidenced by the measurement of reactive oxygen species (ROS) and mitochondrial ROS within both macrophages and fibroblasts, using 2′, 7′-dichlorodihydrofluorescein diacetate and MitoSOX, respectively. The relevance of SHI was further underscored by applying ML385 and Nrf2 siRNA to gauge its effectiveness.ResultsStarting SHI treatment early countered the harmful effects of lung inflammation and fibrosis caused by silica, while initiating SHI at a later phase decelerated the advancement of fibrosis. SHI's action was linked to the activation of the Nrf2 signaling pathway, a boost in antioxidant enzyme levels, and a decrease in oxidative stress and inflammation in macrophages affected by silica. Furthermore, SHI prevented the conversion of fibroblasts into myofibroblasts prompted by TGF-β, along with the resultant oxidative stress. The beneficial outcomes of SHI were negated when ML385 and Nrf2 siRNA were applied, highlighting the pivotal role of the Nrf2 pathway in SHI's efficacy.ConclusionSHI plays a significant role in stimulating the Nrf2 pathway, thereby defending against silica-induced oxidative stress and inflammatory reactions in macrophages, and inhibiting the conversion of fibroblasts to myofibroblasts due to TGF-β. This suggests that SHI is a viable option for treating lung inflammation and fibrosis in mice suffering from silicosis.

Improving the dichloro-dihydro-fluorescein (DCFH) assay for the assessment of intracellular reactive oxygen species formation by nanomaterials

To facilitate Safe and Sustainable by Design (SSbD) strategies during the development of nanomaterials (NMs), quick and easy in vitro assays to test for hazard potential at an early stage of NM development are essential. The formation of reactive oxygen species (ROS) and the induction of oxidative stress are considered important mechanisms that can lead to NM toxicity. In vitro assays measuring oxidative stress are therefore commonly included in NM hazard assessment strategies. The fluorescence-based dichloro-dihydro-fluorescein (DCFH) assay for cellular oxidative stress is a simple and cost-effective assay, making it a good candidate assay for SSbD hazard testing strategies. It is however subject to several pitfalls and caveats. Here, we provide further optimizations to the assay using 5-(6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA-AE, referred to as DCFH probe), known for its improved cell retention.We measured the release of metabolic products of the DCFH probe from cells to supernatant, direct reactions of CM-H2DCFDA-AE with positive controls, and compared the commonly used plate reader-based DCFH assay protocol with fluorescence microscopy and flow cytometry-based protocols. After loading cells with DCFH probe, translocation of several metabolic products of the DCFH probe to the supernatant was observed in multiple cell types. Translocated DCFH products are then able to react with test substances including positive controls. Our results also indicate that intracellularly oxidized fluorescent DCF is able to translocate from cells to the supernatant. In either way, this will lead to a fluorescent supernatant, making it difficult to discriminate between intra- and extra-cellular ROS production, risking misinterpretation of possible oxidative stress when measuring fluorescence on a plate reader.The use of flow cytometry instead of plate reader-based measurements resolved these issues, and also improved assay sensitivity. Several optimizations of the flow cytometry-based DCFH ISO standard (ISO/TS 19006:2016) were suggested, including loading cells with DCFH probe before incubation with the test materials, and applying an appropriate gating strategy including live-death staining, which was not included in the ISO standard.In conclusion, flow cytometry- and fluorescence microscopy-based read-outs are preferred over the classical plate reader-based read-out to assess the level of intracellular oxidative stress using the cellular DCFH assay.

Antioxidant and Neuroprotective Effects of Fucoxanthin and Its Metabolite Fucoxanthinol: A Comparative In Vitro Study.

Fucoxanthin is the most abundant carotenoid found in marine brown algae that exhibits several healthy properties. Dietary fucoxanthin is metabolized in the intestine, plasma, and other tissues to various metabolites, including fucoxanthinol. In this regard, the contribution of fucoxanthinol to the healthy properties of its precursor, fucoxanthin, against pathogenetic events associated with neurodegenerative diseases remains unexplored. Here, we evaluated and compared the antioxidant and neuroprotective effects of the carotenoids fucoxanthin and fucoxanthinol in in vitro models of Alzheimer's (AD) and Parkinson's (PD) disease. Neuronal SH-SY5Y cells were used to evaluate the antioxidant properties of the carotenoids against ABTS radical in the membrane and cytoplasm and oxidative stress elicited by tert-butyl hydroperoxide using the 2',7'-dichlorodihydrofluorescein diacetate probe. We also assessed the ability of the carotenoids to increase the glutathione (GSH) and activate the Nrf2/Keap1/ARE pathway using the monochlorobimane probe and western blotting method, respectively. The neuroprotective effects of the carotenoids against the neurotoxicity generated by oligomers of Beta-Amyloid (1-42) peptide (OAβ) and 6-hydroxydopamine (6-OHDA), which are neurotoxins of AD and PD, respectively, were finally evaluated in the same neuronal cells using the thiazolyl blue tetrazolium bromide assay. Both carotenoids could reach the cytoplasm, which explains the mainly free radical scavenging activity at this level. Notably, fucoxanthinol had higher and lower antioxidant activity than fucoxanthin at extracellular and cellular levels. Although studied carotenoids exerted the ability to activate the Nrf2/Keap1/ARE pathway, leading to an increase of intracellular GSH, our results suggested that the antioxidant activity of the carotenoids could be mainly attributed to their radical scavenging activity in neuronal membrane and cytoplasm, where they accumulate. Fucoxanthinol also shared similar neuroprotective effects as fucoxanthin against the neurotoxicity generated by OAβ and 6-OHDA, suggesting a potential neuroprotective contribution to the action of fucoxanthin administered as a food supplement in in vivo experimental models. These results encourage further research to evaluate the bioavailability of fucoxanthinol and other metabolites of fucoxanthin at the brain level to elucidate the dietary neuroprotective potential of fucoxanthin.

Multiomics Analyses Reveal MsC3H29 Positively Regulates Flavonoid Biosynthesis to Improve Drought Resistance of Autotetraploid Cultivated Alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa subsp. sativa), the "queen of forage," is the most important perennial legume, with high productivity and an excellent nutritional profile. Medicago sativa subsp. falcata is a subspecies of the alfalfa complex and exhibits better drought tolerance. However, drought stress significantly hampers their development and yield. The molecular mechanisms underlying the aboveground and underground tissues of sativa and falcata responding to drought stress remain obscure. Here, we performed a comprehensive comparative analysis of the physiological and transcriptomic responses of sativa and falcata under drought stress. The results showed that photosynthesis was inhibited, and antioxidant enzymes were activated under drought stress. MsC3H29, a CCCH-type zinc finger protein, was identified as a hub gene through weighted gene coexpression network analysis (WGCNA) and was significantly induced by drought in underground tissue. The MsC3H29 protein was localized in the nucleus. Overexpression (OE) of MsC3H29 can increase the primary root length and fresh weight of transgenic alfalfa hairy roots, while RNA interference (RNAi) decreases them under drought stress. The 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining revealed that MsC3H29 promoted drought tolerance of alfalfa hairy roots through decreasing ROS accumulation. The targeted metabolome analysis showed that the overexpression of MsC3H29 resulted in higher levels of accumulation for flavonoid monomers, including vicenin, daidzein, apigenin, isorhamnetin, quercetin, and tricin, in transgenic alfalfa hairy roots before and after drought stress, while RNAi led to a reduction. Our study provided a key candidate gene for molecular breeding to improve drought resistance in alfalfa.

Controlled delivery of procyanidin through magnesium oxide nanoparticles (MgO NPs) to improve the activity and mineralization of osteoblasts under oxidative stress in vitro

Excessive reactive oxygen species (ROS) in the microenvironment of osteoporosis (OP) not only accelerate the bone absorption, but also affect the osteogenic and mineralized effect of osteoblasts. Procyanidins (PC) have been reported to have anti-oxidation effects, but low bioavailability. This study aimed to explore the effect of magnesium oxide nanoparticles (MgO-PC NPs)-loaded PC on the osteogenesis and mineralization of osteoblasts that stimulated by H2O2. PC was loaded onto MgO NPs and characterized by transmission electron microscopy, energy dispersive spectroscopy, dynamic light scattering, and Fourier transform infrared spectroscopy. After primary screening by cytotoxicity assay, MgO-PC NPs containing 20 μM of PC were chosen for further studies. In H2O2-stimulated osteoblasts, dichlorodihydrofluorescein diacetate probe, Cell Counting Kit-8, quantitative real-time polymerase chain reaction, alkaline phosphatase staining/activity and Alizarin red staining were used to detect the ROS production, cell viability and osteogenic and mineralized markers of osteoblasts. PC was loaded onto MgO NPs to successfully receive MgO-PC NPs with a diameter of about 144 nm and negative potential. PC can sustain release from MgO-PC NPs for at least 16 d. The controlled release of PC from MgO-PC NPs can effectively eliminate ROS and thereby promoted the cell activity. Most importantly, the osteogenesis and mineralization of osteoblasts under oxidative stress were also significantly reversed by MgO-PC NPS. Thus, these findings indicate that MgO-PC NPs may be developed as a potential therapeutic strategy for OP.

GIP attenuates neuronal oxidative stress by regulating glucose uptake in spinal cord injury of rat.

Glucose-dependent insulinotropic polypeptide (GIP) is a ligand of glucose-dependent insulinotropic polypeptide receptor (GIPR) that plays an important role in the digestive system. In recent years, GIP has been regarded as a hormone-like peptide to regulate the local metabolic environment. In this study, we investigated the antioxidant role of GIP on the neuron and explored the possible mechanism. Cell counting Kit-8 (CCK-8) was used to measure cell survival. TdT-mediated dUTP Nick-End Labeling (TUNEL) was used to detect apoptosis invitro and invivo. Reactive oxygen species (ROS) levels were probed with2', 7'-Dichloro dihydrofluorescein diacetate (DCFH-DA), and glucose intake was detected with 2-NBDG. Immunofluorescence staining and western blot were used to evaluate the protein level in cells and tissues. Hematoxylin-eosin (HE) staining, immunofluorescence staining and tract-tracing were used to observe the morphology of the injured spinal cord. Basso-Beattie-Bresnahan (BBB) assay was used to evaluate functional recovery after spinal cord injury. GIP reduced the ROS level and protected cells from apoptosis in cultured neurons and injured spinal cord. GIP facilitated wound healing and functional recovery of the injured spinal cord. GIP significantly improved the glucose uptake of cultured neurons. Meanwhile, inhibition of glucose uptake significantly attenuated the antioxidant effect of GIP. GIP increased glucose transporter 3 (GLUT3) expression via up-regulating the level of hypoxia-inducible factor 1α (HIF-1α) in an Akt-dependent manner. GIP increases GLUT3 expression and promotes glucose intake in neurons, which exerts an antioxidant effect and protects neuronal cells from oxidative stress both invitro and invivo.

Sorghum Grain Polyphenolic Extracts Demonstrate Neuroprotective Effects Related to Alzheimer's Disease in Cellular Assays.

Sorghum grain contains high levels and a diverse profile of polyphenols (PPs), which are antioxidants known to reduce oxidative stress when consumed in the diet. Oxidative stress leading to amyloid-β (Aβ) aggregation, neurotoxicity, and mitochondrial dysfunction is implicated in the pathogenesis of Alzheimer's disease (AD). Thus, PPs have gained attention as possible therapeutic agents for combating AD. This study aimed to (a) quantify the phenolic compounds (PP) and antioxidant capacities in extracts from six different varieties of sorghum grain and (b) investigate whether these PP extracts exhibit any protective effects on human neuroblastoma (BE(2)-M17) cells against Aβ- and tau-induced toxicity, Aβ aggregation, mitochondrial dysfunction, and reactive oxygen species (ROS) induced by Aβ and tert-butyl hydroperoxide (TBHP). PP and antioxidant capacity were quantified using chemical assays. Aβ- and tau-induced toxicity was determined using the 3-(4,5-dimenthylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTS) assay. The thioflavin T (Th-T) assay assessed anti-Aβ aggregation. The dichlorodihydrofluorescein diacetate (DCFDA) assay determined the levels of general ROS and the MitoSOX assay determined the levels of mitochondrial superoxide. Sorghum varieties Shawaya short black-1 and IS1311C possessed the highest levels of total phenolics, total flavonoids, and antioxidant capacity, and sorghum varieties differed significantly in their profile of individual PPs. All extracts significantly increased cell viability compared to the control (minus extract). Variety QL33 (at 2000 µg sorghum flour equivalents/mL) showed the strongest protective effect with a 28% reduction in Aβ-toxicity cell death. The extracts of all sorghum varieties significantly reduced Aβ aggregation. All extracts except that from variety B923296 demonstrated a significant (p ≤ 0.05) downregulation of Aβ-induced and TBHP-induced ROS and mitochondrial superoxide relative to the control (minus extract) in a dose- and variety-dependent manner. We have demonstrated for the first time that sorghum polyphenolic extracts show promising neuroprotective effects against AD, which indicates the potential of sorghum foods to exert a similar beneficial property in the human diet. However, further analysis in other cellular models and in vivo is needed to confirm these effects.

Xanthoangelol alleviates ischemic stroke injury by inhibiting the inflammatory activation of microglia

BackgroundLocal inflammation induced by microglial activation plays a significant role in the pathological process of cerebral ischemia. Angelica keiskei (Miq.) Koidz., a traditional botanical drug, can be used as a diuretic, laxative or galactagogue. Xanthoangelol (XA), an active chalcone compound from the aerial part of Angelica keiskei, has anti-inflammatory effects in the peripheral tissues. However, its effect against neuroinflammation is yet unclear. ObjectiveThe present study aims to investigate whether XA could mitigate ischemic stroke damage through attenuating neuroinflammation due to microglia activation. MethodsMiddle cerebral artery occlusion/reperfusion (MCAO/R) induced cerebral ischemia and oxygen-glucose deprivation/reperfusion (OGD/R) or lipopolysaccharide (LPS)-stimulated BV2 microglia cells were utilized to evaluate XA's protection against ischemic injury and neuroinflammation. The severity of brain injury was assessed using 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and neurological assessment. The expressions of inflammatory cytokines were quantified by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Reactive oxygen species (ROS) were assessed using dichlorodihydrofluorescein diacetate (DCF-DA) staining. NF-κB p65 nuclear translocation was confirmed by immunofluorescence (IF) staining. The expressions of proteins were quantified by Western blotting. ResultsXA was efficacious in reducing infarct size and improving neurological function in MCAO/R mice. In ischemic brain tissue, XA reduced microglial activation and proinflammatory cytokine expression. In lipopolysaccharide (LPS) and OGD/R-induced cell models, XA suppressed the production of ROS and decreased the secretion of inflammatory cytokines. Additionally, XA suppressed the nuclear translocation and phosphorylation of NF-κB p65 and blocked the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome. The protection of XA against MCAO/R-induced damages was not attenuated in TLR4−/− and MD2−/− mice. Mito-TEMPO treatment reversed XA's anti-inflammatory properties in OGD/R-induced BV2 cells. ConclusionXA attenuates ischemic stroke injury by suppressing microglial inflammatory responses. This efficacy is tied to its antioxidant activity and is independent of Toll-like receptor 4 (TLR4) or myeloid differentiation protein 2 (MD2).

Protective Effects of Chitosan Nanoparticles against Hepatic and Renal Damage in Rats with Chronic Lead Poisoning

Background: Lead exposure inflicts severe damage to various body organs. The present study sought to assess the effect of chitosan nanoparticles (NPs) on the prevention of hepatic and renal damage in rats with chronic lead poisoning. The indicators were the levels of oxidative stress and proinflammatory cytokines expression. Methods: We used Rattus norvegicus as the animal model. They were assigned to three groups: negative control, positive control, and treatment (n=8 each). The negative control and treatment groups were the models for chronic lead poisoning, and the serum lead levels were determined by atomic absorption spectrometry. The treatment group was orally administered chitosan NPs at 64 mg/kg for 30 days. The reactive oxygen species (ROS) were measured using 2',7’-dichlorodihydrofluorescein diacetate flow cytometry. The hepatic and renal TNF-α and IL-6 gene expressions were also analyzed. Results: The mean serum lead level was 0.52 mg/L, validating the rats as the lead poisoning model. The results indicated that the liver and kidneys in the treatment group had the lowest ROS and TNF-α levels compared to those in other groups. The treatment rats had a lower hepatic IL-6 level compared to those in positive controls, although this was higher than that in the negative controls. The renal IL-6 level in the treatment group was the highest among all groups. Conclusion: As evidenced by the results of this study, chitosan NPs had a protective effect on the liver and kidneys of rats during chronic lead poisoning by reducing the ROS and TNF-α levels; nonetheless, it did not suppress the renal IL-6 expression.