Reactive Oxygen Species Assay Research Articles

Evaluation of cytotoxic and genotoxic potential of avobenzone and octocrylene on human skin fibroblast cells

We investigated the cytotoxic and genotoxic effects of the organic UVA filter avobenzone (AVB), and the UVB filter octocrylene (OCT), found alone or in combination in different sunscreen formulations, on normal human skin fibroblast cells (CCD-1118Sk). To achieve this purpose, we used XTT viability assay, single cell gel electrophoresis (comet) assay and reactive oxygen species (ROS) assay. For cytotoxicity, CCD-1118Sk cells were exposed to various concentrations of AVB (32–800 µM), and OCT (130–6500 μM). The IC50 values were 100.2 and 1390.95 µM for AVB and OCT, respectively. IC12.5, IC25 and IC50 concentrations were chosen for genotoxicity testing. The comet assay revealed DNA damage at the two highest concentrations of AVB and all OCT concentrations. The rate of DNA damage was significantly higher than in the control groups. Co-treatment with AVB and OCT increased the level of DNA damage and intracellular oxidative stress compared with AVB and OCT treatment alone. Finally, our study showed that the UVA filter AVB, with the UVB filter OCT, may induce cytotoxic and genotoxic effects on human skin fibroblast cells. Highlights Avobenzone and octocrylene are UV filters used in sunscreen products. Healthy human skin fibroblast cell line CCD-1118Sk was used for cytotoxic and genotoxic assays. Cotreatment with avobenzone and octocrylene caused more severe DNA damage than their treatment alone. Our findings will contribute to the limited number of cytotoxicity and genotoxicity studies and lead to more detailed studies on the use of these two chemicals.

Dietary exposure to polystyrene microplastics exacerbates liver damage in fulminant hepatic failure via ROS production and neutrophil extracellular trap formation

With the increasing influx of microplastics (MPs) into the environment, their potential toxicity represents an increasing threat to human health. However, there is a lack of relevant research surrounding the biological toxicity associated with pre-exposure to MPs under pathological conditions. To fill this gap, we established a mouse model of fulminant hepatic failure after 14 days of pre-exposure to polystyrene (PS) MPs and investigated its biological response process under combined stimulation with lipopolysaccharide (LPS)/d-galactosamine (d-GalN) and PS-MPs. The results indicated that the stress response from exposure to PS-MPs exacerbated the death induced by LPS/d-GalN and reinforced the potential of liver damage in mice. The dominant roles of inflammation promotion, reactive oxygen species (ROS), and neutrophil extracellular traps in this process were confirmed by cellular reactive oxygen species assays and experiments on oxidative stress and inflammatory responses in the liver. Transcriptomic analysis revealed that PS-MPs exacerbated the expression levels of neutrophil extracellular traps in mice treated with LPS/d-GalN, and weakened the expression of genes involved in pathways related to peroxisome, taurine, and hypotaurine metabolism, which was further validated by reverse-transcription quantitative polymerase chain reaction. This study addresses the knowledge gap regarding the adverse effects caused by a pathological state upon exposure to MPs and provides a theoretical reference for further assessment of the underlying health risks of MPs.

Synthesis, structure and biological activity of silver(I) complexes containing triphenylphosphine and non-steroidal anti-inflammatory drug ligands

Two Ag(I) complexes containing triphenylphosphine and non-steroidal anti-inflammatory drug ligands were synthesized and investigated using various spectroscopic studies and single crystal X-ray crystallography. The binding properties of tolfenamic acid, ibuprofen and the two complexes with DNA and BSA were investigated using UV or fluorescence spectroscopy. The results showed that two Ag(I) complexes bound to DNA by the intercalation mode and interacted with BSA using a static quenching procedure. Furthermore, the results of fluorescence titration suggested that the complexes had good affinity for BSA and one binding site close to BSA. The in vitro cytotoxicity of tolfenamic acid, ibuprofen, and the two complexes against four human carcinoma cell lines (MCF-7, HepG-2, A549, and HeLa cells) was tested using an MTT assay. Complex 1 had higher cytotoxicity against HeLa cells. The intracellular reactive oxygen species (ROS) assay showed complex 1 induced the ROS generation in HeLa cells in a concentration dependent manner. Flow cytometry analysis showed complex 1 could suppress the HeLa cells growth during the G0/G1 phase and induce apoptosis in dose-depended manner.

Astaxanthin protects against pirarubicin-induced H9c2 cardiomyocytes by adjusting microRNA-494-3p-mediated MDM4/p53 signalling pathway.

Pirarubicin (THP) is an antitumour drug widely used in clinical practice, but its cardiotoxicity limits its application. THP cardiotoxicity must be treated as soon as possible. There is an urgent need to find drugs that alleviate THP cardiotoxicity. The purpose of this study was to investigate the effects and mechanisms of Astaxanthin (AST) on THP-induced cardiomyocytes. Rat cardiomyocytes H9c2 were induced with THP. The effects of AST on THP-induced H9c2 and its mechanism were investigated by CCK8, reactive oxygen species assay, tunnel assay, flow cytometry, RT-qPCR, and Western blot. AST increased cell viability, inhibited apoptosis and accelerated cell cycle progression, reduced oxidative damage and inflammatory response in THP-induced H9c2; down-regulated miR-494-3p expression, promoted MDM4 expression, inhibited p53 activation, and suppressed apoptosis-related protein expression. Overexpression of MiR-494-3p reversed the above effects of AST. AST can inhibit H9c2 apoptosis induced by THP and attenuate H9c2 damage by THP, which may be achieved by downregulating miR-494-3p, upregulating MDM4, and inhibiting p53.

Procyanidin A1 acts as an antioxidant stressor in gestational diabetes

Purpose: To evaluate the mechanism and effect of Procyanidin A1 (PCA1) in gestational diabetes mellitus (GDM).Methods: Human trophoblast cell line HTR-8/Svneo was treated with 25 mM glucose for 24 h, and the effect of PCA1 on HTR-8/SVneo cell viability and proliferation was determined by CCK-8 assay and EdU proliferation kit. The expression of BAX, Bcl-2, cleaved Caspase-3 and cleaved caspase-9 was determined by western blotting. Cell apoptosis was assessed by Annexin V-FITC/ PI staining. Cellular generation of reactive oxygen species (ROS) was determined by ROS assay kit while superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were determined by the corresponding assay kits. The effect of PCA1 on Nrf2/HO-1 pathway was evaluated by determining the expression of Keap1, Nfr2, and HO-1.Results: Procyanidin A1 (PCA1) increased the viability of high glucose (HG) -induced HTR-8/SVneo cells and promoted cell proliferation. Furthermore, PCA1 significantly inhibited HG-induced BAX, cleaved caspase-3, and cleaved caspase-9 expression, resulting in further reduction in HG-induced cell apoptosis. High glucose (HG) induced a significant increase in intracellular ROS levels, and this HGinduced oxidative stress was inhibited by PCA1. Furthermore, PCA1 activated Nrf2/HO-1 pathway and this was responsible for its proliferative, anti-apoptosis and anti-oxidative effects.Conclusion: Procyanidin A1 promotes proliferation, and inhibits apoptosis and oxidative stress induced by high glucose in trophoblast cells by activating Nrf2/HO-1 signaling pathway. Therefore, it is a potential drug for the treatment of GDM.

Investigating anti-inflammatory and apoptotic actions of fucoidan concentrating on computational and therapeutic applications.

Fucoidan is linked to a variety of biological processes. Differences in algae species, extraction, seasons, and locations generate structural variability in fucoidan, affecting its bioactivities. Nothing is known about fucoidan from the brown alga Dictyota bartayresiana, its anti-inflammatory properties, or its inherent mechanism. This study aimed to investigate the anti-inflammatory properties of fucoidan isolated from D. bartayresiana against LPS-induced RAW 264.7 macrophages and to explore potential molecular pathways associated with this anti-inflammatory effects. Fucoidan was first isolated and purified from D. bartayresiana, and then, MTT assay was used to determine the effect of fucoidan on cell viability. Its effects on reactive oxygen species (ROS) formation and apoptosis were also studied using the ROS assay and acridine orange/ethidium bromide fluorescence labelling, respectively. Molecular docking and molecular dynamics simulation studies were performed on target proteins NF-κB and TNF-α to identify the route implicated in these inflammatory events. It was observed that fucoidan reduced LPS-induced inflammation in RAW 264.7 cells. Fucoidan also decreased the LPS-stimulated ROS surge and was found to induce apoptosis in the cells. Molecular docking and molecular dynamics simulation studies revealed that fucoidan's potent anti-inflammatory action was achieved by obstructing the NF-κB signalling pathway. These findings were particularly noteworthy and novel because fucoidan isolated from D. bartayresiana had not previously been shown to have anti-inflammatory properties in RAW 264.7 cells or to exert its activity by obstructing the NF-κB signalling pathway. Conclusively, these findings proposed fucoidan as a potential pharmaceutical drug for inflammation-related diseases.

Assessing neutrophil-derived ROS production at the bedside: a potential prognostic tool in severe COVID-19 cases

PurposeA prompt and effective immune response is required for clearance of pathogens but exaggerated states of inflammation can cause extensive collateral damage to the host. We have previously used a rapid near-patient assay that measures the functional capacity of neutrophils to produce reactive oxygen species (ROS) to show that values are elevated in patients with severe COVID-19 or sepsis. Here, we assess the utility of longitudinal ROS measurements to monitor and predict mortality outcome for patients with COVID-19 infection being treated in an ICU setting.MethodsWe used the Leukocyte ImmunoTest™ (LIT™) to quantify neutrophil ROS release using a small volume (10 µL) of capillary blood in a portable, rapid (10-min) format.ResultsROS values (LIT score) and ROS levels assessed in relation to neutrophil count (LIT/N) were both markedly elevated in the patient group. Furthermore, these correlated strongly with peripheral neutrophil count and CRP value. Serial measurement of neutrophil or CRP values were not able to reliably predict mortality within the study. In contrast, LIT and LIT/N values started to decline at 7 and 5 days, respectively, in patients who survived ICU admission and this increment increased further thereafter.ConclusionsThis study raises the possibility of LIT and LIT/N to be used as a predictive clinical tool for patients with severe COVID-19 and argues for its assessment to inform on prognosis, and potentially guide treatment pathways, in other disorders associated with neutrophil activation.Take-home messageA longitudinal study of 44 severe COVID-19 patients in the ICU of a leading teaching hospital has demonstrated the prognostic potential of a rapid bedside assay of neutrophil-derived reactive oxygen species (ROS). Assessment of changes in ROS production, as measured using the Leukocyte ImmunoTest™, shows that ROS production generally declined back to normal levels for patients who survived, but remained elevated for those patients who did not survive.

Radiosensitizing Effects of Lithium Ascorbate on Normal and Tumor Lymphoid Cells under X-ray Irradiation

Abstract: The study aimed to assess the radiosensitizing effect of lithium ascorbate on tumor cells. Background: Cancer cells radioresistance is an important factor restraining the success of X-ray therapy. Radiosensitizing drugs make tumor cells more sensitive to ionizing radiation and improve the effectiveness of radiotherapy. Although many chemical substances can potentiate the cytotoxic effects of X-ray radiation, their clinical applications are limited due to possible adverse reactions. Recently, several approaches have been proposed to develop new radiosensitizers that are highly effective and feature low toxicity. Among new enhancers of X-ray therapy, ascorbic acid, and its derivates demonstrate very low toxicity along with a wide therapeutic range. Lithium ascorbate is a promising X-ray therapy enhancer, but its mechanism of action is unknown. This research focuses on the radiosensitizing properties of lithium ascorbate and its effects on both tumor and normal irradiated cells. Methods: The viability of the radiosensitized cells was evaluated by fluorescence flow cytometry using Annexin V-FITC Apoptosis Detection Kit and Cellular ROS Assay Kit (Abcam, UK). The test cell cultures included normal human mononuclear and Jurkat cells. Results: Lithium ascorbate sensitizes normal human mononuclear and Jurkat cells towards ionizing radiation. The combined cytotoxic effect of X-ray irradiation (3 Gy) and lithium ascorbate (1,2 mmol/L) substantially exceeds the effects of the individual factors, i.e. synergetic action appears. The major types of cell death were late apoptosis and necrosis caused by excessive production of reactive oxygen species. Conclusion: Lithium ascorbate in combination with X-ray irradiation exhibited the cytotoxic effect on both normal and cancer lymphoid cells by activating reactive oxygen species (ROS)-induced apoptosis. These findings indicate that lithium ascorbate is a promising substance to develop a new radiosensitizing drug.

Roflumilast attenuates neuroinflammation post retinal ischemia/reperfusion injury by regulating microglia phenotype via the Nrf2/STING/NF-κB pathway

PurposeThe abnormal polarisation of microglial cells (MGs) following retinal ischemia/reperfusion (RIR) initiates neuroinflammation and progressive death of retinal ganglion cells (RGCs), causing increasingly severe and irreversible visual dysfunction. Roflumilast (Roflu) is a promising candidate for treating neuroinflammatory diseases. This study aimed to explore whether Roflu displayed a cytoprotective effect against RIR-induced neuroinflammation and to characterise the underlying signalling pathway. MethodsThe effects and mechanism of Roflu against RIR injury were investigated in C57BL/6J mice and the BV2 cell line. We used quantitative real-time PCR and enzyme-linked immunosorbent assay to examine the levels of inflammatory factors. Furthermore, haematoxylin and eosin and immunofluorescence (IF) stainings were used to assess the morphology of the retina and the states of MGs and RGCs. Reactive oxygen species (ROS) levels were examined using a ROS assay kit, while whole-genome sequencing analysis was conducted to identify altered pathways and molecules. Western blotting and IF staining were used to quantify the proteins associated with the nuclear factor erythroid 2-related factor 2 (Nrf2)/stimulator of interferon gene (STING)/nuclear factor kappa beta (NF-κB) pathway. ResultsMG polarisation includes the pro-inflammatory and neurotoxic M1 phenotype as well as the anti-inflammatory and neuroprotective M2 phenotype. Roflu significantly attenuated MG activation and contributed to a shift in the MG phenotype from M1 to M2. Moreover, Roflu decreased ROS release and increased heme oxygenase 1 and NAD(P)H quinone oxidoreductase 1 expression. In vitro and in vivo experiments validated that Roflu exerted its neuroprotective effects primarily by upregulating the Nrf2/STING/NF-κB pathway. However, these effects were abrogated when the Nrf2 expression was inhibited by pharmacological or genetic manipulation. ConclusionsRoflu suppressed RIR-induced neuroinflammation by driving the shift of MG polarisation from M1 to M2 phenotype, which was mediated by the upregulation of the Nrf2/STING/NK-κB pathway.

The SP/NK1R system promotes the proliferation of breast cancer cells through NF-κB-mediated inflammatory responses.

Numerous molecules have been introduced to participate in the formation of breast cancer, the most common malignancy in women. Among them, neuropeptide substance P (SP) and its related receptor neurokinin-1 receptor (NK1R) have attracted unprecedented attention in tumorigenesis processes. In this study, we investigated the effect of the SP/NK1R pathway on the induction of oxidative stress in breast cancer and examine the therapeutic potential of NK1R inhibition in this malignancy. MCF-7 cells were treated with varying concentrations of SP and aprepitant, an FDA-approved NK1R antagonist, either as a single drug or in a combined modality. Resazurin assay was used to evaluate the anti-cancer ability of aprepitant. The alteration in the intracellular levels of reactive oxygen species (ROS) and gene expression were determined using ROS assay and the qRT-PCR analysis, respectively. The stimulation of the SP/NK1R axis in the MCF-7 cells was coupled with the accumulation of ROS as well as upregulation of NF-κB and its related pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and IL-6. In contrast, the suppression of NK1R by aprepitant halted the viability of MCF-7 cells, at least partly due to p53-mediated upregulation of p21. Moreover, aprepitant attenuated the oncogenic properties of SP by preventing the oxidative property of this neuropeptide. Overall, our results suggest that the SP/NK1R pathway might play a critical role in breast cancer pathogenesis, probably through inducing ROS/NF-κB-mediated inflammatory responses. Moreover, it seems that blockage of the axis has promising therapeutic value against breast cancer cells. Schematic representation proposed for the plausible mechanism by which the stimulation of the SP/NK1R might induce oxidative stress in breast cancer-derived MCF-7 cells. Once SP interacts with NK1R, this signaling axis could disturb the balance between the expression of p53 and NF-κB, an event that leads to the accumulation of ROS within MCF-7 cells. The produced ROS, in turn, elevates the expression of pro-inflammatory cytokines (TNF-α and IL-6) and downregulates the expression of p21. On the other hand, aprepitant, an antagonist of NK1R, could reduce the survival of proliferative capacity of MCF-7 cells by decreasing the intracellular levels of ROS and p53-mediated up-regulation of p21. Along with the effect on p53, aprepitant could also reduce the expression of NF-κB and its related pro-inflammatory cytokines.

Contamination profiles and potential health risks of environmentally persistent free radicals in PM2.5 over typical central Chinese megacity

As one of the most important transportation hubs and industrial bases in China, Zhengzhou has suffered from serious PM2.5 pollution for a long time. However, the investigation of contamination status and possible exposure risks of environmentally persistent free radicals (EPFRs) in PM2.5 from Zhengzhou is rare. In this work, a comprehensive study of pollution levels, seasonal variations, sources, and potential health risks of PM2.5-bound EPFRs in Zhengzhou was conducted for the first time. The atmospheric concentrations of EPFRs in PM2.5 from Zhengzhou ranged from 1.732 × 1012 spin m−3 to 7.182 × 1014 spin m−3 between 2019 and 2020. Relatively serious contamination was noticed in winter and spring. Primary fossil fuel combustion and Fe-mediated secondary formation were apportioned as possible sources of PM2.5-bound EPFRs in Zhengzhou. Moreover, to avert the bias of the toxicity assessment induced by utilization of incompletely extracted EPFRs from sample filter, simulatively generated EPFRs were applied to toxicological evaluations (cell viability and reactive oxygen species assays). Corresponding experimental dosages were based on the estimated adults’ annual exposure amounts of EPFRs in real PM2.5 samples. The results elucidated that EPFRs might cause growth inhibition and oxidative stress of human lung cells, suggesting the possible exposure-induced health concerns for local people in Zhengzhou. This study provides practical information of real contamination status of PM2.5-bound EPFRs in Zhengzhou, which is favorable to local air pollution control and reduction of exposure risks on public health in central China.

Rosiglitazone inhibits acyl-CoA synthetase long-chain family number 4 and improves secondary brain injury in a rat model of surgical brain injury.

Ferroptosis is a recently discovered non-apoptotic form of cellular death. Acyl-CoA synthetase long-chain family number 4 (ACSL4) is necessary for iron-dependent cellular death, and reactive oxygen species (ROS) produced by ACSL4 are the executioners of ferroptosis. Rosiglitazone improves ferroptosis by inhibiting ACSL4. There is no research indicating whether ACSL4 plays a role in cell death after surgical brain injury (SBI). This study aimed to investigate the role of ACSL4 in SBI via the ferroptosis pathway. Ninety male Sprague-Dawley rats were examined using a model of SBI. Subsequently, the inhibitory effect of rosiglitazone on ACSL4 was assessed via western blot, real-time polymerase chain reaction (PCR), immunofluorescence, fluoro-jade C staining, Perl's staining, ROS assay, and neurological scoring. The results showed that compared with the Sham group, the protein levels of ACSL4 and transferrin were significantly increased after SBI. Administration of rosiglitazone significantly reduced neuronal necrosis, iron deposition, brain water content and ROS in brain tissue and ameliorated neurological deficits at 48 h after SBI, which was concomitant with decreased transferrin expression. These findings demonstrate that SBI-induced upregulation of ACSL4 may be partly mediated by the ferroptosis pathway, which can be reversed by rosiglitazone administration.

In vitro analysis of antibacterial activity against wound pathogens, potential for wound healing, and anti‐melanoma properties of biosynthesized zinc oxide nanoparticles

AbstractStaphylococcus aureus is the most common cause of wound infections. Infected wounds increase wound severity and have a slower rate of healing. Moreover, emergence of multiple‐drug resistant bacteria such as methicillin‐resistant S. aureus (MRSA) limited treatment options. This study was therefore aimed to evaluate antibacterial activity against wound pathogen and wound‐healing properties of green synthesized ZnO nanoparticles derived from mangosteen peel crude extract (ZnO‐Gm). Moreover, their anti‐skin cancer activity was also investigated in vitro. As a result, the ZnO‐Gm particles significantly inhibited growth of S. aureus and MRSA with the IC50 values at 0.44 and 0.51 mg/mL, respectively. By performing quantitative reactive oxygen species (ROS) assay, the intracellular ROS in both treated S. aureus and MRSA with ZnO‐Gm was found to be significantly elevated. Furthermore, ZnO‐Gm exhibited cytotoxic effects via induction of apoptosis on the A375 melanoma cancer cell line, with an IC50 value of 8.91 µg/mL, while not affecting the normal cell line (Vero). In addition, 30 µg/mL of ZnO‐Gm could strongly promote wound healing of an epidermal keratinocyte cell line (HaCaT). Consequently, the findings of this study demonstrated that the green synthesized ZnO nanoparticles have potential as antibacterial agents, wound‐healing materials, and anti‐melanoma agents.

In vitro evaluation of the antiglycation and antioxidant potential of the dietary supplement L-citrulline

Diabetes mellitus (DM) represents a significant global public health concern. It is a metabolic condition characterized by abnormal glucose levels in the bloodstream, known as hyperglycemia. This condition arises due to irregular insulin secretion, defective insulin receptivity, or a combination of both factors. The primary contributors to diabetic complications are protein glycation and oxidative stress resulting from chronic hyperglycemia.
 The aim. The increasing incidence of diabetes mellitus has prompted a quest for a novel, cost-effective, and efficacious medication. The objective of the study generally intends to explore and investigate the antiglycation and antioxidant potential of the dietary supplement L-Citrulline
 Materials and methods. A two-reaction model system was carried out to study and monitor the inhibitory impact of the dietary supplement L-Citrulline against advanced glycation end products (AGEs) formation. This system involved the in vitro glucose bovine serum albumin (BSA-glucose assay) and methylglyoxal bovine serum albumin (BSA-MGO assay). The antioxidant activity of the supplement was assessed by measuring its capacity to chelate metal ions and scavenge reactive oxygen species. The iron chelating activity was evaluated through absorbance measurements, while fluorescence measurements were employed for the remaining assays.
 Results. According to the findings of the antiglycation assays, it was observed that the dietary supplement L-Citrulline demonstrated inhibitory properties against the development of advanced glycation end products (AGEs) in the BSA-Glucose model at a concentration of 100 ppm. The degree of inhibition with respect to glycation was ascertained to be 52.19 ± 0.39 % through observation. The BSA-MGO model has exhibited inhibitory properties with an observed activity of 49.64 ± 0.27 % at 100ppm concentration with respect to glycation. On the other hand, the supplement demonstrates antioxidant characteristics through the chelation of Fe ions, leading to a percentage difference in activity of 68.58 ± 0.45 % compared to the control at 100 ppm. The utilization of Glucolypotoxixity (GLT) media during the reactive oxygen species assay yielded a significant rise of 173.48 ± 9.37 % in the reactive species levels compared to the control, with statistical significance. The addition of 10 mM dietary supplement L-Citrulline resulted in a noteworthy reduction of 98.42 ± 5.04 % in the escalation. Therefore, it can be deduced that utilizing L-Citrulline as a dietary supplement exhibits potential for its therapeutic applications in eliminating reactive oxygen species (ROS) within skeletal muscle cells.
 Conclusion. The study results suggest that the dietary supplement L-Citrulline has demonstrated inhibitory capabilities against glycation at varying concentration levels. Furthermore, it was noted to exhibit significant efficacy in both sets of antioxidant tests. Therefore, the supplement exhibits potential in the treatment of diabetes mellitus

The Combination of Upconversion Nanoparticles and Photodynamic Therapy for Alzheimer’s Disease Treatment in Real-Time Monitoring

Alzheimer’s Disease (AD) consumes many medical resources in disease caring and delays symptoms. The aggregation of amyloid-beta (Aβ) is regarded as one of the foci of AD. It will form oligomers and polymerize into fibrotic neurofibrillary tangles leading to AD. Many research has focused on the diagnosis and risk factors of Aβ aggregation, trying to reverse the situation of Aβ aggregation by various approaches. However, there are no advantageous solutions that have been developed so far. Establishing a system from monitoring to controlling Aβ aggregation is urgently needed as a new approach for AD treatment. Once Aβ aggregation can be monitored and controlled to oxide and degrade it before forming oligomers. It will bring huge benefits to AD treatment. Therefore, this study combines photodynamic therapy (PDT) and quartz crystal micro-scale (QCM) techniques to realize the degradation of Aβ and monitor whether Aβ is degraded, and establish a complete system.PDT has been applied in the medical field, especially for cancer treatment which induced oxidative stress results in different levels of protein oxidation. However, the limited excitation source limits the application of PDT, for example, the insufficient penetration depth of the excitation source prevents in vivo treatment of AD. Therefore, it needs to be combined with other materials to achieve in-depth treatment. This study proposes to modify photosensitizers on upconverting nanoparticles (UCNPs), and use 980 nm near-infrared to excite and then emit visible light to perform PDT. The combination of PDT and UCNPs generates the reactive oxygen species (ROS) to oxide Aβ and further degrade Aβ aggregation. However, most of the lanthanide metals and photosensitizers doped UCNPs are cytotoxic. Thus, Rose Bengal (RB) was selected as the reactive photosensitizer in this system owing to it being a metal-free, non-toxic, and widely available material. Also, it has been confirmed through research that RB can effectively transfer energy to form 1O2. To further ensure that material can be used in AD treatment, modify the SiO2 shell on the surface of UCNP to improve biocompatibility. The experimental results show that the cell survival rate can be maintained at about 80% when the concentration of UCNP reaches 25mg/mL.The degradation of the Aβ peptide can be monitored using the QCM technique. QCM was applied for monitoring the differences in the oscillation frequency by the degradation of the Aβ peptide attached to UCNPs. Meanwhile, the DCFH-DA ROS assay kit was applied to estimate the Aβ peptide oxidation level. The results of this study show that the combination of UCNPs for PDT can effectively oxidize and degrade Aβ, and utilizing QCM and ROS assay kit can monitor the level of Aβ oxidation and degradation in real-time. The establishment of this system can help more potential AD patients, providing a diagnostic and treatment method. Figure 1

Complex intestinal and hepatic in vitro barrier models reveal information on uptake and impact of micro-, submicro- and nanoplastics

Plastic particles are found almost ubiquitously in the environment and can get ingested orally by humans. We have used food-relevant microplastics (2 µm polylactic acid), submicroplastics (250 nm polylactic acid and 366 nm melamine formaldehyde resin) and nanoplastics (25 nm polymethylmethacrylate) to study material- and size-dependent uptake and transport across the human intestinal barrier and liver. Therefore, different Transwell™-based in vitro (co-)culture models were used: Differentiated Caco-2 cells mimicking the intestinal enterocyte monolayer, an M-cell model complementing the Caco-2 monoculture with antigen uptake-specialized cells, a mucus model complementing the barrier with an intestinal mucus layer, and an intestinal-liver co-culture combining differentiated Caco-2 cells with differentiated HepaRG cells. Using these complex barrier models, uptake and transport of particles were analyzed based on the fluorescence of the particles using confocal microscopy and a fluorescence-based quantification method. Additionally, the results were verified by Time-of-Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) analysis. Furthermore, an effect screening at the mRNA level was done to investigate oxidative stress response, inflammation and changes to xenobiotic metabolism in intestinal and hepatic cells after exposure to plastic particles. Oxidative stress and inflammation were additionally analyzed using a flow-cytometric assay for reactive oxygen species and cytokine measurements. The results reveal a noteworthy uptake into and transport of microplastic and submicroplastic particles across the intestinal epithelium. Particularly, we show a pronounced uptake of particles into liver cells after crossing of the intestinal epithelium, using the intestinal-liver co-culture. The particles evoke some alterations in xenobiotic metabolism, but did not cause increased oxidative stress or inflammatory response on protein level. Taken together, these complex barrier models can be applied on micro-, submicro- and nanoplastics and reveal information in particle uptake, transport and cellular impact.

Synthesis, Structural Investigations, DNA/BSA Interactions, Molecular Docking Studies, and Anticancer Activity of a New 1,4-Disubstituted 1,2,3-Triazole Derivative.

We report herein a new 1,2,3-triazole derivative, namely, 4-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-hydroxybenzaldehyde, which was synthesized by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The structure of the compound was analyzed using Fourier transform infrared spectroscopy (FTIR), 1H NMR, 13C NMR, UV-vis, and elemental analyses. Moreover, X-ray crystallography studies demonstrated that the compound adapted a monoclinic crystal system with the P21/c space group. The dominant interactions formed in the crystal packing were found to be hydrogen bonding and van der Waals interactions according to Hirshfeld surface (HS) analysis. The volume of the crystal voids and the percentage of free spaces in the unit cell were calculated as 152.10 Å3 and 9.80%, respectively. The evaluation of energy frameworks showed that stabilization of the compound was dominated by dispersion energy contributions. Both in vitro and in silico investigations on the DNA/bovine serum albumin (BSA) binding activity of the compound showed that the CT-DNA binding activity of the compound was mediated via intercalation and BSA binding activity was mediated via both polar and hydrophobic interactions. The anticancer activity of the compound was also tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using human cell lines including MDA-MB-231, LNCaP, Caco-2, and HEK-293. The compound exhibited more cytotoxic activity than cisplatin and etoposide on Caco-2 cancer cell lines with an IC50 value of 16.63 ± 0.27 μM after 48 h. Annexin V suggests the induction of cell death by apoptosis. Compound 3 significantly increased the loss of mitochondrial membrane potential (MMP) levels in Caco-2 cells, and the reactive oxygen species (ROS) assay proved that compound 3 could induce apoptosis by ROS generation.

Stromal cell-derived factor-1 downregulation contributes to neuroprotection mediated by CXC chemokine receptor 4 interactions after intracerebral hemorrhage in rats.

Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) have a substantial role in neuronal formation, differentiation, remodeling, and maturation and participate in multiple physiological and pathological events. In this study, we investigated the role of SDF-1/CXCR4 in neural functional injury and neuroprotection after intracerebral hemorrhage (ICH). Western blot, immunofluorescence and immunoprecipitation were used to detect SDF-1/CXCR4 expression and combination respectively after ICH. TUNEL staining, Lactate dehydrogenase assay, Reactive oxygen species assay, and Enzyme-linked immunosorbent assay to study neuronal damage; Brain water content to assay brain edema, Neurological scores to assess short-term neurological deficits. Pharmacological inhibition and genetic intervention of SDF-1/CXCR4 signaling were also used in this study. ICH induced upregulation of SDF-1/CXCR4 and increased their complex formation, whereas AMD3100 significantly reduced it. The levels of TNF-α and IL-1β were significantly reduced after AMD3100 treatment. Additionally, AMD3100 treatment can alleviate neurobehavioral dysfunction of ICH rats. Conversely, simultaneous SDF-1/CXCR4 overexpression induced the opposite effect. Moreover, immunoprecipitation confirmed that SDF-1/CXCR4 combined to initiate neurodamage effects. This study indicated that inhibition of SDF-1/CXCR4 complex formation can rescue the inflammatory response and alleviate neurobehavioral dysfunction after ICH. SDF-1/CXCR4 may have applications as a therapeutic target after ICH.

The biocompatibility and the metabolic impact of thermoresponsive, bile acid-based nanogels on auditory and macrophage cell lines

Deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) are bile acids that may serve as permeation enhancers when incorporated within the nanogel matrix for drug delivery in the inner ear. In this study, thermoresponsive nanogels were formulated with DCA, LCA and UDCA and their rheological properties and biocompatibility were assessed. The impact of nanogel on cellular viability was evaluated via cell viability assay, the impact of nanogels on cellular bioenergetic parameters was estimated by Seahorse mito-stress test and glycolysis-stress test, while the presence of intracellular free radicals was assessed by reactive oxygen species assay. Nanogels showed a high level of biocompatibility after 24-hour exposure to auditory and macrophage cell lines, with minimal cytotoxicity compared to untreated control. Incubation with nanogels did not alter cellular respiration and glycolysis of the auditory cell line but showed possible mitochondrial dysfunction in macrophages, suggesting tissue-dependent effects of bile acids. Bile acid-nanogels had minimal impact on intracellular reactive oxygen species, with LCA demonstrating the most pro-oxidative behaviour. This study suggests that thermoresponsive nanogels with bile acid, particularly DCA and UDCA, may be promising candidates for inner ear drug delivery.

Proton and Electron Ultrahigh-Dose-Rate Isodose Irradiations Produce Differences in Reactive Oxygen Species Yields

Investigations into ultra-high dose rate (UHDR) radiotherapy have dramatically risen because of the observed normal tissue sparing FLASH effect without sacrificing tumor control. The purpose of this study was to provide a direct beamline comparison of protons and electrons to determine where UHDR to conventional dose rates (CDR) changes affect the resultant radiochemistry. We used well characterized assays of reactive oxygen species (ROS) and oxygen consumption to assess the radiolysis in protein solutions. Three optical reporters related to ROS (CellROX Deep Red, reflects highly reactive radicals; Amplex Red reflects H2O2; and Oxyphor reflects partial pressure loss (ΔpO2)). A Varian ProBeam proton cyclotron and a converted Varian Trilogy electron linac were used for irradiation at both their CDR and UHDR capable level, to assess the assay change per unit dose. For both protons and electrons an expected reduction in ROS was noted going from CDR to UHDR, and results interpreted as a reduction in the ratio of UHDR/CDR yield. The CellROX assay showed no difference between beamlines, each showing ∼80% reduction in ROS from CDR to UHDR. The Amplex assay showed the largest inter-beamline difference, with ∼5% loss using protons vs ∼69% loss with electrons, in protein solution. The Oxyphor assay of ΔpO2 showed a small difference in CDR to UHDR with a 23% loss with protons and 43% loss with electrons. Interpretation of ROS assays and oxygen consumption is notoriously challenging. These assays might be interpreted by their most activating species' lifetime. The assay for highly reactive OH●, appeared independent of beamline, whereas the assays for the longer lived H2O2 species and DpO2 assay showed differences between beamlines via the UHDR/CDR ratio. This work can be used for FLASH hypothesis testing by comparing these assays to isodose biological FLASH effects in vivo.