Intracellular Peroxide Levels Research Articles

Cisplatin-activated and hemoglobin-mediated injectable hydrogel system for antitumor chemodynamic and chemotherapy

Low specificity and hypoxia-induced drug resistance are significant challenges in traditional cancer treatment. To enhance the anticancer efficacy, an injectable hydrogel system is developed through the formation of dynamic covalent bonds in hyaluronic acid, allowing for localized controlled release of drugs. This system also utilizes double-stranded DNA sequences for the intercalation delivery of the chemotherapeutic drug, enabling a multifaceted approach to therapy. Cisplatin not only serves as a chemotherapy drug but also acts as a catalyst for chemodynamic therapy (CDT) to initiate CDT cascades by creating hydrogen peroxide for the Fenton reaction. Hemoglobin, enclosed in PLGA nanoparticles, provides ferrous ions that react with hydrogen peroxide in an acidic environment, yielding hydroxyl radicals that induce cancer cell death. Additionally, oxygen released from hemoglobin mitigates hypoxia-induced chemoresistance, bolstering overall anticancer efficacy. Results demonstrate the shear-thinning properties and injectability of the hydrogel. Cisplatin elevates intracellular hydrogen peroxide levels in tumor cells, while hemoglobin efficiently releases ferrous ions and generates reactive oxygen species (ROS) in the presence of hydrogen peroxide. In in vitro and in vivo study, the combinational use of chemo- and chemodynamic therapies achieves a synergistic anticancer effect on combating glioblastoma. In summary, our CDT-based hydrogel, activated by endogenous cues and mediated by chemo drugs, spontaneously produces ROS and ameliorates the adverse tumor microenvironment with rational and selective antitumor strategies.

Computational models as catalysts for investigating redoxin systems.

Thioredoxin, glutaredoxin and peroxiredoxin systems play central roles in redox regulation, signaling and metabolism in cells. In these systems, reducing equivalents from NAD(P)H are transferred by coupled thiol-disulfide exchange reactions to redoxins which then reduce a wide array of targets. However, the characterization of redoxin activity has been unclear, with redoxins regarded as enzymes in some studies and redox metabolites in others. Consequently, redoxin activities have been quantified by enzyme kinetic parameters in vitro, and redox potentials or redox ratios within cells. By analyzing all the reactions within these systems, computational models showed that many kinetic properties attributed to redoxins were due to system-level effects. Models of cellular redoxin networks have also been used to estimate intracellular hydrogen peroxide levels, analyze redox signaling and couple omic and kinetic data to understand the regulation of these networks in disease. Computational modeling has emerged as a powerful complementary tool to traditional redoxin enzyme kinetic and cellular assays that integrates data from a number of sources into a single quantitative framework to accelerate the analysis of redoxin systems.

The Role of PNPLA3_rs738409 Gene Variant, Lifestyle Factors, and Bioactive Compounds in Nonalcoholic Fatty Liver Disease: A Population-Based and Molecular Approach towards Healthy Nutrition.

This study aimed to investigate the impact of a common non-synonymous gene variant (C>G, rs738409) in patatin-like phospholipase domain-containing 3 (PNPLA3), leading to the substitution of isoleucine with methionine at position 148 (PNPLA3-I148M), on susceptibility to nonalcoholic fatty liver disease (NAFLD) and explore potential therapeutic nutritional strategies targeting PNPLA3. It contributed to understanding sustainable dietary practices for managing NAFLD, recently referred to as metabolic-dysfunction-associated fatty liver. NAFLD had been diagnosed by ultrasound in a metropolitan hospital-based cohort comprising 58,701 middle-aged and older Korean individuals, identifying 2089 NAFLD patients. The interaction between PNPLA3 and lifestyle factors was investigated. In silico analyses, including virtual screening, molecular docking, and molecular dynamics simulations, were conducted to identify bioactive compounds from foods targeting PNPLA3(I148M). Subsequent cellular experiments involved treating oleic acid (OA)-exposed HepG2 cells with selected bioactive compounds, both in the absence and presence of compound C (AMPK inhibitor), targeting PNPLA3 expression. Carriers of the risk allele PNPLA3_rs738409G showed an increased association with NAFLD risk, particularly with adherence to a plant-based diet, avoidance of a Western-style diet, and smoking. Delphinidin 3-caffeoyl-glucoside, pyranocyanin A, delta-viniferin, kaempferol-7-glucoside, and petunidin 3-rutinoside emerged as potential binders to the active site residues of PNPLA3, exhibiting a reduction in binding energy. These compounds demonstrated a dose-dependent reduction in intracellular triglyceride and lipid peroxide levels in HepG2 cells, while pretreatment with compound C showed the opposite trend. Kaempferol-7-glucoside and petunidin-3-rutinoside showed potential as inhibitors of PNPLA3 expression by enhancing AMPK activity, ultimately reducing intrahepatic lipogenesis. In conclusion, there is potential for plant-based diets and specific bioactive compounds to promote sustainable dietary practices to mitigate NAFLD risk, especially in individuals with genetic predispositions.

A Novel CRISPR/Cas9‐Encapsulated Biomimetic Manganese Sulfide Nanourchins for Targeted Magnetic Resonance Contrast Enhancement and Self‐Enhanced Chemodynamics‐Gene‐Immune Synergistic Tumor Therapy

AbstractCRISPR/Cas9 gene editing systems are widely studied in cancer therapy, however, it is still challenging to improve the gene editing efficiency and enhance the anticancer efficacy. Herein, a novel self‐assembled manganese sulfide nanourchin with CRISPR/Cas9 and hybrid membrane encapsulation (MCRT) is developed to highlight the synergistic mechanism of hydrogen sulfide (H2S) and CRISPR/Cas9 in cancer therapy. The special structure and cationic surface of nanourchins facilitate the loading of CRISPR/Cas9 with the large size and the encapsulation of erythrocyte‐tumor cell hybrid membrane, endowing the nanoplatform with long circulation and precise tumor targeting. MCRT with pH‐response can effectively decompose into H2S and Mn2+, and release CRISPR/Cas9 in acidic tumor microenvironment. H2S can up‐regulate intracellular hydrogen peroxide level by inhibiting the activity of catalase, which enables self‐enhanced chemodynamic therapy (CDT) of Mn2+. In addition, H2S can synergize with the released CRISPR/Cas9, co‐downregulating the level of intracellular anti‐apoptotic protein survivin, achieving self‐enhanced gene therapy. Furthermore, systemic delivery of MCRT displays significantly tumor magnetic resonance imaging (MRI) contrast enhancement. Importantly, H2S‐enhanced CDT‐gene therapy simultaneously induces immunogenic cell death and triggered a robust anti‐tumor immune response. Thus, MCRT has a potential application for the targeted MRI and self‐enhanced CDT‐gene‐immune synergistic tumor therapy.

Oxidative replication stress induced by long-term exposure to hydroxyurea in root meristem cells of Vicia faba.

Low concentrations of hydroxyurea, an inhibitor of DNA replication, induced oxidative and replicative stress in root apical meristem (RAM) cells of Vicia faba. Plant cells are constantly exposed to low-level endogenous stress factors that can affect DNA replication and lead to DNA damage. Long-term treatments of Vicia faba root apical meristems (RAMs) with HU leads to the appearance of atypical cells with intranuclear asynchrony. This rare form of abnormality was manifested by a gradual condensation of chromatin, from interphase to mitosis (so-called IM cells). Moreover, HU-treated root cells revealed abnormal chromosome structure, persisting DNA replication, and elevated levels of intracellular hydrogen peroxide (H2O2) and superoxide anion (O2∙-). Immunocytochemical studies have shown an increased number of fluorescent foci of H3 histones acetylated at lysine 56 (H3K56Ac; canonically connected with the DNA replication process). We show that continuous 3-day exposure to low concentrations (0.75mM) of hydroxyurea (HU; an inhibitor of DNA replication) induces cellular response to reactive oxygen species and to DNA replication stress conditions.

"Multi-in-One" Yolk-Shell Structured Nanoplatform Inducing Pyroptosis and Antitumor Immune Response Through Cascade Reactions.

Pyroptosis, a new mode of regulatory cell death, holds a promising prospect in tumor therapy. The occurrence of pyroptosis can trigger the release of damage-associated molecular patterns (DAMPs) and activate the antitumor immune response. Moreover, enhancing intracellular reactive oxygen species (ROS) generation can effectively induce pyroptosis. Herein, an integrated nanoplatform (hCZAG) based on zeolitic imidazolate framework-8 (ZIF-8) with Cu2+ and Zn2+ as active nodes and glucose oxidase (GOx) loading is constructed to evoke pyroptosis. GOx can effectively elevate intracellular hydrogen peroxide (H2 O2 ) levels to regulate the unfavorable tumor microenvironment (TME). Cu2+ can be reduced to Cu+ by endogenous overexpressed GSH and both Cu2+ and Cu+ can exert Fenton-like activity to promote ROS generation and amplify oxidative stress. In addition, the accumulation of Cu2+ leads to the aggregation of lipoylated dihydrolipoamide S-acetyltransferase (DLAT), thus resulting in cuproptosis. Notably, the outburst of ROS induced by hCZAG activates Caspase-1 proteins, leads to the cleavage of gasdermin D (GSDMD), and induces pyroptosis. Pyroptosis further elicits an adaptive immune response, leading to immunogenic cell death (ICD). This study provides effective strategies for triggering pyroptosis-mediated immunotherapy and achieving improved therapeutic effects.

Impact of amino acid substitutions in hepatitis C virus core region on the severe oxidative stress

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, leading to liver steatosis, fibrosis, and hepatocellular carcinoma (HCC). Despite the accumulation of clinical data showing the impact of amino acid substitutions at positions 70 (R70Q/H) and/or 91 (L91M) in the HCV core protein in progressive liver diseases, including HCC, the underlying mechanisms have not been elucidated.We analyzed 72 liver biopsy specimens from patients with chronic HCV genotype 1b (HCV-1b) infection prior to antiviral treatment. Levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nuclear factor erythroid 2-related factor 2 (NRF2) in the nucleus were quantified using liver tissue immunohistochemistry. The effects of amino acid substitutions in the HCV core region on hepatocellular oxidative stress were investigated using wild-type or double-mutant (R70Q/H+L91M) HCV-1b core transfection and stable expression in human hepatoma HuH-7 cells.Overall, 24, 19, 11, and 18 patients had the wild-type, R70Q/H, L91M, and R70Q/H+L91M genotypes, respectively, in the HCV core. A significantly higher accumulation of hepatocellular 8-OHdG and a lower NRF2/8-OHdG ratio were observed in patients with R70Q/H+L91M than in those with the wild-type disease. Increased levels of intracellular superoxide and hydrogen peroxide in the cytoplasm and mitochondria, mRNA expression of enzymes generating oxidative stress, and nuclear expression of nicotinamide adenine dinucleotide phosphate oxidase 4 were augmented in cells treated with R70Q+L91M.HCV core proteins harboring either or both substitutions of R70Q/H or L91M enhanced hepatocellular oxidative stress in vivo and in vitro. These amino acid substitutions may affect HCC development by enhancing hepatic oxidative stress in patients with chronic HCV-1b infection.

Abstract 13872: CD70 Modulates Vascular Smooth Muscle Phenotype and Metabolism by Regulating Cellular Redox State

Introduction: The tumor necrosis factor superfamily (TNFSF) displays a wide array of vascular effects; however, the role of the TNFSF member CD70 is still being elucidated in the vasculature. We have recently described how CD70 modulates endothelial cell function and redox status by altering nitric oxide bioavailability and intracellular reactive oxygen species (ROS). However, the role of CD70 in vascular smooth muscle cells (VSMCs) is not known. We hypothesized that CD70 can alter VSMC phenotype and mitochondrial function through regulation of intracellular ROS. Methods: Human aortic VSMCs (AoVSMCs) were transfected with control or CD70-directed siRNA (n = 5-6). Cellular phenotype was assessed by scratch assay and collagen matrix gel contraction assay. Cell lysates were analyzed by Western blot and quantitative RT-PCR. Intracellular cytosolic hydrogen peroxide levels were measured using the fluorescent biosensor Hyper7 following stimulation by auranofin (50-70 cells per group averaged over 4 experiments). Metabolic profiling was performed with Seahorse assay, and mitochondrial membrane potential was assessed using TMRM dye (120 cells per group averaged over 3 experiments). Results: Following CD70 knockdown, AoVSMCs demonstrated augmented wound closure and decreased collagen matrix contraction. Expression of synthetic/proliferative markers, including vimentin, alpha-1 type I collagen, osteopontin, caldesmon-1, and c-Myc, was increased by up to 50%, and contractile markers including smoothelin, synemin, and synaptopodin-2 were decreased by up to 50%. Cytosolic hydrogen peroxide production was increased, with expression of NADPH oxidase (NOX) 4 and gp91phox (the catalytic subunit of NOX2) both increased by over 2-fold. CD70 knockdown led to a pronounced alteration in VSMC metabolic profile, with significantly decreased oxygen consumption rate (25%), extracellular acidification rate (45%), and maximal respiratory capacity (46%), as well as a decrease in mitochondrial membrane potential (37%). Conclusions: CD70 exerts a novel regulatory effect on VSMC phenotype; loss of CD70 promotes transition to a synthetic phenotype characterized by increased ROS with consequent metabolic reprogramming and mitochondrial dysfunction.

The pH-dependent multiple nanozyme activities of copper-cerium dioxide and its application in regulating intracellular oxygen and hydrogen peroxide levels

Cu doped CeO2 (Cu-CeO2) nanoparticles were synthesized via the hydrothermal method, exhibiting multiple nanozyme activities such as good oxidase-like (OXD), peroxidase-like (POD) and catalase-like (CAT) activities. These Cu-CeO2 nanoparticles were successfully utilized to modulate the levels of oxygen and hydrogen peroxide in cells. The OXD and POD mimics showed optimal at pH of 3.5 and 4.0, but their activities significantly decreased under neutral or slightly acidic conditions. On the other hand, the CAT activity was excellent at pH 6.5, while the OXD and POD mimics were less effective. These findings demonstrated that nanozyme activity of Cu-CeO2 nanoparticles is pH-dependent. Consequently, within the microenvironment of tumor cells, the CAT can efficiently catalyze the conversion of H2O2 into O2, unaffected by H2O2/O2 consuming mimics. Both oxygen indicator and oxygen tester measurements confirmed a significant increase in O2 levels in phosphate-buffered saline (PBS). Furthermore, confocal microscopic imagings using fluorescence probes for H2O2 and O2 validated the variations of H2O2/O2 in cells. This study highlights the ability of Cu-CeO2 to regulate intracellular reactive oxygen levels and its tremendous potential in nanotherapy and alleviating tumor hypoxia.

Arctiin attenuates iron overload‑induced osteoporosis by regulating the PI3K/Akt pathway.

Iron overload is a prevalent pathological factor observed among elderly individuals and those with specific hematological disorders, and is frequently associated with an elevated incidence of osteoporosis. Although arctiin (ARC) has been shown to possess antioxidant properties and the ability to mitigate bone degeneration, its mechanism of action in the treatment of iron overload‑induced osteoporosis (IOOP) remains incompletely understood. To explore the potential molecular mechanisms underlying the effects of ARC, the MC3T3‑E1 cell osteoblast cell line was used. Cell Counting Kit was used to assess MC3T3‑E1 cell viability. Alkaline phosphatase staining and alizarin red staining were assessed for osteogenic differentiation. Calcein AM assay was used to assess intracellular iron concentration. In addition, intracellular levels of reactive oxygen species (ROS), lipid peroxides, mitochondrial ROS, apoptosis rate and mitochondrial membrane potential changes in MC3T3‑E1 cells were examined using flow cytometry and corresponding fluorescent dyes. The relationship between ARC and the PI3K/Akt pathway was then explored by western blotting and immunofluorescence. In addition, the effects of ARC on IOOP was verified using an iron overload mouse model. Immunohistochemistry was performed to evaluate expression of osteogenesis‑related proteins. Micro-CT and H&E were used to analyze bone microstructural parameters and histomorphometric indices in the bone tissue. Notably, ARC treatment reversed the decreased viability and increased apoptosis in MC3T3‑E1 cells originally induced by ferric ammonium citrate, whilst promoting the formation of mineralized bone nodules in MC3T3‑E1 cells. Furthermore, iron overload induced a decrease in the mitochondrial membrane potential, augmented lipid peroxidation and increased the accumulation of ROS in MC3T3‑E1 cells. ARC not only positively regulated the anti‑apoptotic and osteogenic capabilities of these cells via modulation of the PI3K/Akt pathway, but also exhibited antioxidant properties by reducing oxidative stress. Invivo experiments confirmed that ARC improved bone microarchitecture and biochemical parameters in a mouse model of iron overload. In conclusion, ARC exhibits potential as a therapeutic agent for IOOP by modulating the PI3K/Akt pathway, and via its anti‑apoptotic, antioxidant and osteogenic properties.

Bioenergetic profile and redox tone modulate in vitro osteogenesis of human dental pulp stem cells: new perspectives for bone regeneration and repair

BackgroundRedox signaling and energy metabolism are known to be involved in controlling the balance between self-renewal and proliferation/differentiation of stem cells. In this study we investigated metabolic and redox changes occurring during in vitro human dental pulp stem cells (hDPSCs) osteoblastic (OB) differentiation and tested on them the impact of the reactive oxygen species (ROS) signaling.MethodshDPSCs were isolated from dental pulp and subjected to alkaline phosphatase and alizarin red staining, q-RT-PCR, and western blotting analysis of differentiation markers to assess achievement of osteogenic/odontogenic differentiation. Moreover, a combination of metabolic flux analysis and confocal cyto-imaging was used to profile the metabolic phenotype and to evaluate the redox tone of hDPSCs.ResultsIn differentiating hDPSCs we observed the down-regulation of the mitochondrial respiratory chain complexes expression since the early phase of the process, confirmed by metabolic flux analysis, and a reduction of the basal intracellular peroxide level in its later phase. In addition, dampened glycolysis was observed, thereby indicating a lower energy-generating phenotype in differentiating hDPSCs. Treatment with the ROS scavenger Trolox, applied in the early-middle phases of the process, markedly delayed OB differentiation of hDPSCs assessed as ALP activity, Runx2 expression, mineralization capacity, expression of stemness and osteoblast marker genes (Nanog, Lin28, Dspp, Ocn) and activation of ERK1/2. In addition, the antioxidant partly prevented the inhibitory effect on cell metabolism observed following osteogenic induction.ConclusionsAltogether these results provided evidence that redox signaling, likely mediated by peroxide species, influenced the stepwise osteogenic expansion/differentiation of hDPSCs and contributed to shape its accompanying metabolic phenotype changes thus improving their efficiency in bone regeneration and repair.

Cyclic amplification of intracellular ROS boosts enzymatic prodrug activation for enhanced chemo-immunotherapy.

Tumor-associated enzyme activated prodrug is a potential strategy to overcome the limitations of chemotherapeutic agents. However, the efficiency of enzymatic prodrug activation is limited by the inability to reach adequate enzyme levels in vivo. Herein, we report an intelligent nanoplatform with cyclic amplification of intracellular reactive oxygen species (ROS) that significantly up-regulates the expression of tumor-associated enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to efficiently activate the prodrug of doxorubicin (DOX) for enhanced chemo-immunotherapy. The nanoplatform termed as CF@NDOX was fabricated by self-assembly of the amphiphilic cinnamaldehyde (CA) containing poly(thioacetal) conjugated with ferrocene (Fc) and poly(ethylene glycol) (PEG) (TK-CA-Fc-PEG), which further encapsulated the NQO1 responsive prodrug of DOX (NDOX). After CF@NDOX accumulates in tumors, the TK-CA-Fc-PEG with ROS responsive thioacetal group responds to endogenous ROS in tumor to release CA, Fc or NDOX. CA induces mitochondria dysfunction and elevates the intracellular hydrogen peroxide (H2O2) levels, which react with Fc to generate highly oxidative hydroxyl radical (•OH) through Fenton reaction. The •OH not only promotes ROS cyclic amplification but also increase the expression of NQO1 through Keap1-Nrf2 pathway regulation, which further boost the prodrug activation of NDOX for enhanced chemo-immunotherapy. Overall, our well-designed intelligent nanoplatform provides a tactic to enhance the antitumor efficacy of tumor-associated enzyme activated prodrug. STATEMENT OF SIGNIFICANCE: In this work, a smart nanoplatform CF@NDOX with intracellular ROS cyclic amplification for continuous upregulation of NQO1 enzyme expression was innovatively designed. It could utilize Fenton reaction of Fc to increase the level of NQO1 enzyme and CA to increase the level of intracellular H2O2, thereby facilitating the continuous Fenton reaction. This design allowed for a sustained elevation of the NQO1 enzyme, and a more complete activation of the NQO1 enzyme in response to the prodrug NDOX. This smart nanoplatform can achieve a desirable anti-tumor effect with the combined therapy of chemotherapy and ICD effects.

Tumor-targeted glycogen nanoparticles loaded with hemin and glucose oxidase to promote tumor synergistic therapy

Strategies which are used to address the low levels of intracellular hydrogen peroxide and the development of biocompatible catalysts still need to be fulfilled in tumor chemodynamic therapy. Therefore, a novel tumor-targeted glycogen-based nanoparticle system (GN/He/GOx/HA) was developed to co-deliver hemin (He) and GOx, which can self-supply glucose formed upon degradation of glycogen by α-glycosidase in the lysosome environment, in order to achieve synergistic antitumor therapy. Hyaluronic acid (HA) was selected as the outer shell to protect the activity of GOx, and to increase the uptake by tumor cells via CD44 receptor-mediated endocytosis. GN/He/GOx/HA NPs had a good stability in the blood circulation, but fast release of the therapeutic cargos upon intracellular uptake. Hemin had a cascade catalytic reaction with GOx. Furthermore, GN/He/GOx/HA NPs had the strongest cytotoxicity in Hela cells in a glucose concentration dependent manner. The NPs could efficiently produce reactive oxygen species in tumor cells, resulting in a decrease in the mitochondrial membrane potential and apoptosis of tumor cells. The in vivo results showed that the drug-loaded nanoparticles had good safety, biocompatibility, and efficacious antitumor effect. Therefore, the glycogen-based nanoparticle delivery system provides potential application for self-enhancing CDT, which can be used for effective antitumor therapy.

Expression of the H2O2 Biosensor roGFP-Tpx1.C169S in Fission and Budding Yeasts and Jurkat Cells to Compare Intracellular H2O2 Levels, Transmembrane Gradients, and Response to Metals

Intracellular hydrogen peroxide (H2O2) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H2O2 have been developed in recent decades. In particular, the redox-sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins are among the most sensitive H2O2 biosensors. Using fission yeast as a model system, we recently demonstrated that the gradient of extracellular-to-intracellular peroxides through the plasma membrane is around 300:1, and that the concentration of physiological H2O2 is in the low nanomolar range. Here, we have expressed the very sensitive probe roGFP2-Tpx1.C169S in two other model systems, budding yeast and human Jurkat cells. As in fission yeast, the biosensor is ~40-50% oxidized in these cell types, suggesting similar peroxide steady-state levels. Furthermore, probe oxidation upon the addition of extracellular peroxides is also quantitatively similar, suggesting comparable plasma membrane H2O2 gradients. Finally, as a proof of concept, we have applied different concentrations of zinc to all three model systems and have detected probe oxidation, demonstrating that an excess of this metal can cause fluctuations of peroxides, which are moderate in yeasts and severe in mammalian cells. We conclude that the principles governing H2O2 fluxes are very similar in different model organisms.

Silencing the Catalase Gene with SiRNA for Enhanced Chemodynamic Therapy.

Chemodynamic therapy (CDT) has been emerging as a promising strategy for cancer treatment. But the CDT efficiency is restricted by the insufficient intracellular hydrogen peroxide (H2O2) level. Herein, we present a method for H2O2 accumulation in tumor cells by silencing the catalase (CAT) gene with siRNA to achieve enhanced CDT. Cu-siRNA nanocomposites are fabricated by self-assembly of Cu2+ and CAT siRNA and then modified with hyaluronic acid (HA) for active tumor targeting. After tumor cell uptake, the released Cu2+ is reduced by highly expressed glutathione (GSH) to Cu+, which then catalyzes H2O2 to produce toxic hydroxyl radicals (•OH) to kill tumor cells. CAT siRNA can efficiently silence the CAT mRNA to inhibit the consumption of H2O2, resulting in H2O2 accumulation. The Cu2+-mediated GSH elimination and siRNA-induced endogenous H2O2 enrichment both potentiate CDT. Cu-siRNA@HA exhibits good biocompatibility and therapeutic efficiency. This work thus paves a new way to supply H2O2 in CDT and may hold potential for clinical application.

Protective effects of different doses of MitoQ separately and combined with trehalose on oxidative stress and sperm function of cryopreserved Markhoz goat semen

The mitochondria-targeted antioxidant MitoQ has been regarded as an effective antioxidant agent against cryo-induced oxidative cellular damage. This study aimed to evaluate the use of different doses of MitoQ combined with trehalose to minimize mitochondrial impairment and oxidative stress during sperm cryopreservation of Markhoz goat. For this, semen samples (n = 50) were collected by electroejaculation every 5 days from 5 bucks in 10 replicates. On each collection day, 5 ejaculates (one ejaculate for each buck) were pooled and then diluted in eight different Tris-based extenders as follows: no additives (control), 20, 200, 2000 nM of MitoQ (MT20, MT200, MT 2000, respectively), 150 mM of trehalose (Tr), MT20+Tr, MT200+Tr, MT2000+Tr. The semen samples were frozen using a standard protocol, and sperm function and oxidative stress were evaluated after thawing. The semen extender supplemented with MT200+Tr had higher (P < 0.05) total and progressive motility, acrosome and membrane integrity, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and lower (P < 0.05) DNA fragmentation, malondialdehyde and intracellular hydrogen peroxide levels than the all other groups except MT200; meanwhile, MT200 was also improved (P < 0.05) in these parameters than in the control group. Furthermore, MT200 and MT200+Tr showed higher (P < 0.05) percentages of live cryopreserved sperm with high mitochondrial activity than other groups. However, abnormality percentage and catalase activity of frozen-thawed sperm were not affected by treatments (P > 0.05). To conclude, we have found that supplementation of 200 nM MitoQ alone or in combination with 150 mM trehalose to semen extender improved the quality of cryopreserved sperm in goats, which is associated with enhanced antioxidant enzymatic defense and mitochondrial activity and reduced DNA fragmentation.

Green rooibos extract attenuates high glucose induced oxidative stress in a human derived (HepG2) liver cell line

• Oxidative stress biomarkers are attenuated by REE in a glucotoxic HepG2 cell model. • REE enhances antioxidant enzyme profiles of HepG2 cells exposed to high glucose. • REE activates NRF2 antioxidant pathway in HepG2 cells exposed to high glucose. The exposure of hepatocytes to high concentrations of glucose results in a glucotoxic environment and ensuing hepatocellular injury, often due to oxidative stress. The popularly consumed herbal tea, Rooibos ( Aspalathus linearis ), demonstrates pleiotropic pharmacological and antioxidant activities. Our study investigated the molecular events regulating the antioxidant effects of a Rooibos ethanolic extract (REE) in a glucotoxic HepG2 liver cell model. It is hypothesized that REE enhances antioxidant enzyme profiles of HepG2 cells exposed to high glucose to attenuate glucotoxic damage. Glucotoxicity was induced by treating the cells with 25 mM glucose. For this study, colorimetric, luminometric and western blot analyses were used to determine the potential of the REE to reduce biomarkers associated with oxidative damage and its potential to enhance the intracellular antioxidant defense system. The REE was found to be hepatoprotective as evidenced by reduced levels of oxidative damage biomarkers (MDA and protein carbonylation) and enhanced antioxidant capacity under glucotoxic conditions as evidenced by elevated GSH (reduced form). We also observed decreased intracellular hydrogen peroxide levels and caspase activity in the REE treated cells and further showed that the REE enhanced SOD2, CAT, and GPx1 protein expression. These effects were dependent on the transcriptional activity of NRF2. Collectively, our results show that the REE enhances the cellular antioxidant defense capacity and may provide a relevant supportive therapeutic option for treating diabetic complications associated with oxidative stress.

Biodegradable doxorubicin-loaded ferric phosphate nanosheets for specific tumor elimination through autophagy inhibition-enhanced apoptosis/ferroptosis pathway

Killing tumor cells through apoptosis and/or ferroptosis pathways has been extensively explored for tumor therapy. However, the therapeutic efficacy is always impaired by the autophagy-mediated self-repairing capacity of tumor cells. Herein, a novel autophagy inhibition-enhanced apoptosis/ferroptosis strategy was developed for synergistic tumor elimination, which was realized through the doxorubicin (DOX)-loaded ferric phosphate nanosheets. When located in tumor regions, these nanosheets can be specifically degraded under the stimulation of tumor microenvironment. The released DOX can not only damage DNA to induce cell apoptosis directly but also up-regulate intracellular hydrogen peroxide level. Free Fe2+ can be generated from Fe3+ through intracellular over-expressed glutathione-participated redox reaction, which can further react with hydrogen peroxide through the Fenton reaction to produce highly oxidative hydroxyl radicals. This redox process simultaneously breaks the antioxidant capacity of tumor cells through the depletion of glutathione and the inactivation of intracellular antioxidase glutathione peroxidase 4, promoting the generation and accumulation of lipid peroxidation to cause tumor ferroptosis. Especially, PO43− disrupts the pH balance in lysosomes by forming conjugated acid anions to disable the self-repairing capacity of tumor cells, enhancing the antitumor therapeutic effect. This work offers a promising method with desirable biocompatibility for specific tumor elimination through autophagy inhibition-enhanced apoptosis/ferroptosis strategy.

An Enzyme-Engineered Nonporous Copper(I) Coordination Polymer Nanoplatform for Cuproptosis-Based Synergistic Cancer Therapy.

Cuproptosis, a newly identified form of regulated cell death that is copper-dependent, offers great opportunities for exploring the use of copper-based nanomaterials inducing cuproptosis for cancer treatment. Here, a glucose oxidase (GOx)-engineered nonporous copper(I) 1,2,4-triazolate ([Cu(tz)]) coordination polymer (CP) nanoplatform, denoted as GOx@[Cu(tz)], for starvation-augmented cuproptosis and photodynamic synergistic therapy is developed. Importantly, the catalytic activity of GOx is shielded in the nonporous scaffold but can be "turned on" for efficient glucose depletion only upon glutathione (GSH) stimulation in cancer cells, thereby proceeding cancer starvation therapy. The depletion of glucose and GSH sensitizes cancer cells to the GOx@[Cu(tz)]-mediated cuproptosis, producing aggregation of lipoylated mitochondrial proteins, the target of copper-induced toxicity. The increased intracellular hydrogen peroxide (H2 O2 ) levels, due to the oxidation of glucose, activates the type I photodynamic therapy (PDT) efficacy of GOx@[Cu(tz)]. The in vivo experimental results indicate that GOx@[Cu(tz)] produces negligible systemic toxicity and inhibits tumor growth by 92.4% in athymic mice bearing 5637 bladder tumors. This is thought to be the first report of a cupreous nanomaterial capable of inducing cuproptosis and cuproptosis-based synergistic therapy in bladder cancer, which should invigorate studies pursuing rational design of efficacious cancer therapy strategies based on cuproptosis.

Congenital sideroblastic anemia model due to ALAS2 mutation is susceptible to ferroptosis

X-linked sideroblastic anemia (XLSA), the most common form of congenital sideroblastic anemia, is caused by a germline mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. In XLSA, defective heme biosynthesis leads to ring sideroblast formation because of excess mitochondrial iron accumulation. In this study, we introduced ALAS2 missense mutations on human umbilical cord blood-derived erythroblasts; hereafter, we refer to them as XLSA clones. XLSA clones that differentiated into mature erythroblasts showed an increased frequency of ring sideroblast formation with impaired hemoglobin biosynthesis. The expression profiling revealed significant enrichment of genes involved in ferroptosis, which is a form of regulated cell death induced by iron accumulation and lipid peroxidation. Notably, treatment with erastin, a ferroptosis inducer, caused a higher proportion of cell death in XLSA clones. XLSA clones exhibited significantly higher levels of intracellular lipid peroxides and enhanced expression of BACH1, a regulator of iron metabolism and potential accelerator of ferroptosis. In XLSA clones, BACH1 repressed genes involved in iron metabolism and glutathione synthesis. Collectively, defective heme biosynthesis in XLSA clones could confer enhanced BACH1 expression, leading to increased susceptibility to ferroptosis. The results of our study provide important information for the development of novel therapeutic targets for XLSA.