Intracellular GSH Levels Research Articles

Aldo-Keto Reductase 1C3 Mediates Chemotherapy Resistance in Esophageal Adenocarcinoma via ROS Detoxification.

Simple SummaryThe multidrug resistance of EAC is one of the major obstacles to chemotherapeutic efficiency. Our study aims to explore the molecular mechanism of AKR1C3 as a novel therapeutic target to overcome chemotherapy resistance for EAC patients. We demonstrate that AKR1C3 renders chemotherapy resistance through controlling cellular ROS levels via AKT signaling in EAC cells. Modulation of intracellular GSH levels by AKR1C3 could scavenge the intracellular ROS, thus regulating apoptosis. Targeting AKR1C3 may represent a novel strategy to sensitize EAC cells to conventional chemotherapy treatment and benefit the overall survival of patients diagnosed with EAC.Esophageal adenocarcinoma (EAC) is one of the most lethal malignancies, and limits promising treatments. AKR1C3 represents a therapeutic target to combat the resistance in many cancers. However, the molecular mechanism of AKR1C3 in the chemotherapy resistance of EAC is still unclear. We found that the mRNA level of AKR1C3 was higher in EAC tumor tissues, and that high AKR1C3 expression might be associated with poor overall survival of EAC patients. AKR1C3 overexpression decreased cell death induced by chemotherapeutics, while knockdown of AKR1C3 attenuated the effect. Furthermore, we found AKR1C3 was inversely correlated with ROS production. Antioxidant NAC rescued chemotherapy-induced apoptosis in AKR1C3 knockdown cells, while the GSH biosynthesis inhibitor BSO reversed a protective effect of AKR1C3 against chemotherapy. AKT phosphorylation was regulated by AKR1C3 and might be responsible for eliminating over-produced ROS in EAC cells. Intracellular GSH levels were modulated by AKR1C3 and the inhibition of AKT could reduce GSH level in EAC cells. Here, we reported for the first time that AKR1C3 renders chemotherapy resistance through controlling ROS levels via AKT signaling in EAC cells. Targeting AKR1C3 may represent a novel strategy to sensitize EAC cells to conventional chemotherapy.

PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis.

Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide-sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the first time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to fight CSC chemoresistance.

Biodegradable Nanosonosensitizers with the Multiple Modulation of Tumor Microenvironment for Enhanced Sonodynamic Therapy.

BackgroundThe specific microenvironment of solid tumors, which is characterized by hypoxia, overexpression of glutathione (GSH), and high accumulation of anti-inflammatory tumor-associated macrophages (TAMs), limits the efficiency of sonodynamic therapy (SDT).Method and ResultsHerein, a multifunctional nanoplatform was engineered to modulate the tumor microenvironment for highly efficient SDT. In this system, sonosensitizers and catalase were encapsulated in disulfide-bridged mesoporous organosilicon nanoparticles with high loading, which protected the activity of catalase and ensure the stability of sonosensitizers and enzyme. Subsequently, hyaluronic acid was grafted onto the nanoplatform to reeducate TAMs and induce the secretion of exogenous hydrogen peroxide. Due to the good protection of enzyme, the catalase within the nanoplatform efficiently produced the mount of O2 through decomposing the hydrogen peroxide in tumor tissues, which remarkably alleviated tumor hypoxia. Furthermore, degradation of the nanoparticles was observed in response to GSH, which effectively decreased the intracellular GSH level, further favoring SDT-triggered anticancer effect.ConclusionBased on the multiple adjustments to tumor microenvironment, our nanoplatform displayed extraordinary sonodynamic therapeutic effect with low systemic toxicity.

Feasibility Study on Facile and One-step Colorimetric Determination of Glutathione by Exploiting Oxidase-like Activity of Fe3O4-MnO2 Nanocomposites.

A facile and one-step colorimetric assay is described for the determination of glutathione (GSH). It is based on the use of manganese dioxide-decorated magnetic (Fe3O4@MnO2) nanocomposite that was prepared by an in-situ redox reaction. It exhibits oxidase-mimicking activity and can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) without H2O2to form a blue colored product (oxTMB) with an absorption maximum at 651 nm. Once GSH is introduced, the component of MnO2 can be rapidly reduced to Mn2+ ions, which leads to inhibit the formation of oxTMB. Based on these findings, a one-step colorimetric assay was developed for the detection GSH in the range of 0.2 to 25 μM with a low detection limit of 0.2 μM without using any procedures of separation and washing. Importantly, the proposed approach is also used to accurately evaluate the intracellular GSH levels. In our perception, the assay is rapid, sensitive and specific.

Effect of Interleukin-7 on In Vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Developmental Potential after Parthenogenetic Activation.

Simple SummaryOocyte-secreted factors play an essential role in oogenesis and fertility through bidirectional crosstalk between oocytes and somatic cells. Interleukin-7, known as an oocyte-secreted factor, has recently been shown to improve oocyte developmental competence through interaction with cumulus cells around the oocytes. This study aimed to investigate the effects of interleukin-7 on porcine cumulus-oocyte complexes during in vitro maturation. Our results showed that supplementation with interleukin-7 during in vitro maturation exerted beneficial effects on porcine oocyte meiotic maturation by upregulating antioxidant-related genes and enhanced the subsequent developmental potential of porcine embryos after parthenogenetic activation.Interleukin-7 (IL-7) is a cytokine essential for cell development, proliferation and survival. However, its role in oocyte maturation is largely unknown. To investigate the effects of IL-7 on the in vitro maturation (IVM) of porcine oocytes, we analyzed nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic developmental competence after parthenogenetic activation (PA) under several concentrations of IL-7. After IVM, IL-7 treated groups showed significantly higher nuclear maturation and significantly decreased intracellular ROS levels compared with the control group. All IL-7 treatment groups exhibited significantly increased intracellular GSH levels compared with the control group. All oocytes matured with IL-7 treatment during IVM exhibited significantly higher cleavage and blastocyst formation rates after PA than the non-treatment group. Furthermore, significantly higher mRNA expression levels of developmental-related genes (PCNA, Filia, and NPM2) and antioxidant-related genes (GSR and PRDX1) were observed in the IL-7-supplemented oocytes than in the control group. IL-7-supplemented cumulus cells showed significantly higher mRNA expression of the anti-apoptotic gene BCL2L1 and mitochondria-related genes (TFAM and NOX4), and lower transcript levels of the apoptosis related-gene, Caspase3, than the control group. Collectively, the present study suggests that IL-7 supplementation during porcine IVM improves oocyte maturation and the developmental potential of porcine embryos after PA.

Rhizobium leguminosarum Glutathione Peroxidase Is Essential for Oxidative Stress Resistance and Efficient Nodulation.

Glutathione (GSH) plays a key role in regulating the cellular Redox Homeostasis, and appears to be essential for initiation and development of root nodules. Glutathione peroxidase (Gpx) catalyzes the reduction of H2O2 and organic hydroperoxides by oxidation of GSH to oxidized GSH (GSSG), which in turn is reduced by glutathione reductase (GR). However, it has not been determined whether the Rhizobium leguminosarum Gpx or GR is required during symbiotic interactions with pea. To characterize the role of glutathione-dependent enzymes in the symbiotic process, single and double mutants were made in gpxA (encoding glutathione peroxidase) and gshR (encoding glutathione reductase) genes. All the mutations did not affect the rhizobial growth, but they increased the sensitivity of R. leguminosarum strains to H2O2. Mutant in GpxA had no effect on intracellular GSH levels, but can increase the expression of the catalase genes. The gshR mutant can induce the formation of normal nodules, while the gpxA single and double mutants exhibited a nodulation phenotype coupled to more than 50% reduction in the nitrogen fixation capacity, these defects in nodulation were characterized by the formation of ineffective nodules. In addition, the gpxA and gshR double mutant was severely impaired in rhizosphere colonization and competition. Quantitative proteomics using the TMT labeling method was applied to study the differential expression of proteins in bacteroids isolated from pea root nodules. A total of 27 differentially expressed proteins were identified in these root bacteroids including twenty down-regulated and seven up-regulated proteins. By sorting the down-regulated proteins, eight are transporter proteins, seven are dehydrogenase, deoxygenase, oxidase, and hydrolase. Moreover, three down-regulating proteins are directly involved in nodule process.

Comparison of Anti-Oxidative Effect of Human Adipose- and Amniotic Membrane-Derived Mesenchymal Stem Cell Conditioned Medium on Mouse Preimplantation Embryo Development.

Oxidative stress is a major cause of damage to the quantity and quality of embryos produced in vitro. Antioxidants are usually supplemented to protect embryos from the suboptimal in vitro culture (IVC) environment. Amniotic membrane-derived mesenchymal stem cells (AMSC) have emerged as a promising regenerative therapy, and their paracrine factors with anti-oxidative effects are present in AMSC conditioned medium (CM). We examined the anti-oxidative potential of human AMSC-CM treatment during IVC on mouse preimplantation embryo development and antioxidant gene expression in the forkhead box O (FoxO) pathway. AMSC-CM (10%) was optimal for overall preimplantation embryo developmental processes and upregulated the expression of FoxOs and their downstream antioxidants in blastocysts (BL). Subsequently, compared to adipose-derived mesenchymal stem cell (ASC)-CM, AMSC-CM enhanced antioxidant gene expression and intracellular GSH levels in the BL. Total antioxidant capacity and SOD activity were greater in AMSC-CM than in ASC-CM. Furthermore, SOD and catalase were more active in culture medium supplemented with AMSC-CM than in ASC-CM. Lastly, the anti-apoptotic effect of AMSC-CM was observed with the regulation of apoptosis-related genes and mitochondrial membrane potential in BL. In conclusion, the present study established AMSC-CM treatment at an optimal concentration as a novel antioxidant intervention for assisted reproduction.

The effect of copper supplementation on in vitro maturation of porcine cumulus-oocyte complexes and subsequent developmental competence after parthenogenetic activation

Copper (Cu) ions have redox activity and act as cofactors of enzymes related to respiration, radical detoxification, and iron metabolism. In this study, we aimed to examine the effects of copper (II) chloride dihydrate (CuCl2·2H2O) on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA). Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, cumulus expansion, the mRNA expression levels of various genes, and developmental competence were analyzed. During IVM, the maturation medium was supplemented with various concentrations of Cu (0, 0.7, 1.4, and 2.8 μg/mL). After 42 h of IVM, Cu supplementation significantly increased the number of oocytes in the metaphase II stage. Further, the 1.4 μg/mL Cu group showed significantly higher intracellular GSH levels than the control group. However, Cu supplementation increased intracellular ROS levels regardless of their concentration. Additionally, the mRNA levels of Has-2, the cumulus cell expansion-related gene, were higher in all the Cu-treated groups than in the control group. The cumulus cell expansion index was higher in the 0.7 and 1.4 μg/mL Cu groups than in the other groups. In the 0.7 μg/mL Cu group, the mRNA expression levels of PCNA, Zar1, and NPM2, which are related to developmental competence, were significantly higher than those in the control group. Moreover, increased levels of Sod1 transcript, correlated with the antioxidative response, were observed in the 0.7 and 1.4 μg/mL Cu groups. The apoptosis rate in Cu-treated cumulus cells and oocytes was decreased compared to that in the corresponding control groups. Upon evaluation of subsequent embryonic development after PA, the 0.7 μg/mL Cu group showed significantly improved cleavage and blastocyst formation rate compared to the control group. In conclusion, our results suggest that Cu supplementation at appropriate concentrations in IVM medium improves porcine oocyte maturation and the subsequent embryonic potential of PA embryos by reducing oxidative stress and apoptosis.

Royal jelly alleviates the detrimental effects of aging on immune functions by enhancing the in vitro cellular proliferation, cytokines, and nitric oxide release in aged human PBMCS

Aging strongly delays the immunity. Our research aims to assess the in vitro effects of royal jelly (RJ) on the immune function of aged PBMCs. PBMCs were obtained from 10 healthy aged and young donors by the gradient density centrifugation method and further cultured in RPMI-1640 medium supplemented with or without RJ in the presence of Con A. Cell proliferation was assessed by MTT assay along with the measurement of interleukins, Nitric oxide (NO), Glutathione (GSH), and Malondialdehydes (MDA). Our results showed that RJ improved PBMCs proliferation significantly in the elderly subjects, accompanied by the increase in NO (p=.001) and the release of IL-2, IL-4, and IL-6 cytokines. RJ also increased the intracellular GSH (p= .001) and MDA (p=.001) levels in aged PBMCs. In young subjects, RJ enhanced PBMCs proliferation potency, IL-4, IL-6, GSH, and intracellular MDA levels but with a concomitant decrease in NO and IL-2 cytokine secretion as compared with non RJ-treated cells. In conclusion, RJ restored functions of the aged PBMCs as well as the young control subjects, indicating a beneficial effect on immune status during the aging process. PRACTICAL APPLICATIONS: Royal jelly is a well-known edible dietary compound, used traditionally to treat many diseases throughout the world. Since antiquity, it was shown to have medicinal importance. The immuno-enhancing potential of this food was largely and scientifically established by the lipid and protein fractions. The present study illustrates the anti-aging and stimulatory effects of the fresh RJ whole extract, from local Algerian honey bee: Apis mellifera intermissa, on the immunity of aged men. This study provides the experimental evidence supporting anti-immunosenesence effects of royal jelly. RJ supplementation can be used in the old age management and human age-related complications, especially, associated with the weaknesses of the immune response.

Superoxide produced by mitochondrial complex III plays a pivotal role in the execution of ferroptosis induced by cysteine starvation

Ferroptosis is a type of iron-dependent, non-apoptotic cell death, which is typically induced by cysteine starvation or by the inhibition of glutathione peroxidase 4 (GPX4) activity with the accompanying elevation in lipid peroxidation product levels. Despite the central role of mitochondria in oxidative metabolism and hence, as main sources of superoxide, the issue of whether mitochondrial superoxide participates in the execution of ferroptosis remains unclear. To gain additional insights into this issue, we employed suppressors of the site IQ electron leak (S1QEL) and suppressors of the site IIIQo electron leak (S3QEL), small molecules that suppress mitochondrial superoxide production from complex I and III, respectively. The findings indicate that S3QEL, but not S1QEL, significantly protected mouse hepatoma Hepa 1–6 cells from lipid peroxidation and the subsequent ferroptosis induced by cysteine (Cys) starvation (cystine deprivation from culture media or xCT inhibition by erastin). The intracellular levels of Cys and GSH remained low irrespective of life or death. Moreover, S3QEL also suppressed ferroptosis in xCT-knockout mouse-derived embryonic fibroblasts, which usually die under conventional cultivating conditions due to the absence of intracellular Cys and GSH. Although it has been reported that erastin induces the hyperpolarization of the mitochondrial membrane potential, no correlation was observed between hyperpolarization and cell death in xCT-knockout cells. Collectively, these results indicate that superoxide production from complex III plays a pivotal role in the ferroptosis that is induced by Cys starvation, suggesting that protecting mitochondria is a promising therapeutic strategy for the treatment of multiple diseases featuring ferroptosis.

Polyprodrug with glutathione depletion and cascade drug activation for multi-drug resistance reversal

High intracellular glutathione (GSH) levels play an important role in multidrug resistance (MDR) in cancer cells. It remains challenging to develop a drug delivery system that is simultaneously capable of GSH depletion and drug activation for multidrug resistance reversal. Herein, we designed a polyprodrug (denoted as PSSD) based on poly(disulfide) conjugated with doxorubicin (DOX) on the polymer side chains that exhibits GSH depletion and cascade DOX activation for drug resistance reversal. The poly(disulfide) backbone with a high disulfide density depletes intracellular antioxidant GSH via the disulfide-thiol exchange reaction to disrupt intracellular redox homeostasis in cells. Simultaneously, DOX can be activated through a cascade reaction, and degradation of the poly(disulfide) backbone further facilitates its drug release. Therefore, poly(disulfide) can be used as a GSH scavenger to reverse MDR as well as a prodrug backbone to target high intracellular GSH levels in cancer cells, providing a general strategy for drug resistance reversal.

Inhibitory effects of Panax ginseng glycoproteins in models of doxorubicin-induced cardiac toxicity in vivo and in vitro.

Doxorubicin (DOX) is an effective antineoplastic drug; however, its clinical application is limited owing to the side effect of fatal heart dysfunction on its use. Panax ginseng glycoproteins have antioxidant, antiapoptotic, and anti-inflammatory properties. Thus, the aim of this study was to investigate the effects and possible action mechanisms of P. ginseng glycoproteins against DOX-induced cardiotoxicity. To this end, we used an in vitro model of DOX-treated H9C2 cells and an in vivo model of DOX-treated rats. We found that P. ginseng glycoproteins markedly increased H9C2 cell viability, decreased creatine kinase and lactate dehydrogenase levels, and improved histopathological and electrocardiogram changes in rats, protecting them from DOX-induced cardiotoxicity. Furthermore, P. ginseng glycoproteins significantly inhibited myocardial oxidative insult through adjusting the intracellular ROS, MDA, SOD, and GSH levels in vitro and in vivo. In conclusion, our data suggest that P. ginseng glycoproteins alleviated DOX-induced myocardial oxidative stress-related cardiotoxicity. This natural product could be developed as a new candidate for alleviating DOX-induced cardiotoxicity.

Tumor cell-activated "Sustainable ROS Generator" with homogeneous intratumoral distribution property for improved anti-tumor therapy.

Photodynamic therapy (PDT) holds a number of advantages for tumor therapy. However, its therapeutic efficiency is limited by non-sustainable reactive oxygen species (ROS) generation and heterogeneous distribution of photosensitizer (PS) in tumor. Herein, a “Sustainable ROS Generator” (SRG) is developed for efficient antitumor therapy.Methods: SRG was prepared by encapsulating small-sized Mn3O4-Ce6 nanoparticles (MC) into dendritic mesoporous silica nanoparticles (DMSNs) and then enveloped with hyaluronic acid (HA). Due to the high concentration of HAase in tumor tissue, the small-sized MC could be released from DMSNs and homogeneously distributed in whole tumor. Then, the released MC would be uptaken by tumor cells and degraded by high levels of intracellular glutathione (GSH), disrupting intracellular redox homeostasis. More importantly, the released Ce6 could efficiently generate singlet oxygen (1O2) under laser irradiation until the tissue oxygen was exhausted, and the manganese ion (Mn2+) generated by degraded MC would then convert the low toxic by-product (H2O2) of PDT to the most harmful ROS (·OH) for sustainable and recyclable ROS generation.Results: MC could be homogeneously distributed in whole tumor and significantly reduced the level of intracellular GSH. At 2 h after PDT, obvious intracellular ROS production was still observed. Moreover, during oxygen recovery in tumor tissue, ·OH could be continuously produced, and the nanosystem could induce 82% of cell death comparing with 30% of cell death induced by free Ce6. For in vivo PDT, SRG achieved a complete inhibition on tumor growth.Conclusion: Based on these findings, we conclude that the designed SRG could induce sustainable ROS generation, homogeneous intratumoral distribution and intracellular redox homeostasis disruption, presenting an efficient strategy for enhanced ROS-mediated anti-tumor therapy.

Alpha-linolenic acid enhances maturation and developmental competence via regulation of glutathione, cAMP and fatty acid accumulation during in vitro maturation of porcine oocytes

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

Effect of Rosmarinic Acid and Ionizing Radiation on Glutathione in Melanoma B16F10 Cells: A Translational Opportunity.

To explain a paradoxical radiosensitizing effect of rosmarinic acid (RA) on the melanoma B16F10 cells, we analyzed the glutathione (GSH) intracellular production on this cell (traditionally considered radioresistant) in comparison with human prostate epithelial cells (PNT2) (considered to be radiosensitive). In PNT2 cells, the administration of RA increased the total GSH content during the first 3 h (p < 0.01) as well as increased the GSH/oxidized glutathione (GSSG) ratio in all irradiated cultures during all periods studied (1h and 3h) (p < 0.001), portraying an increase in the radioprotective capacity. However, in B16F10 cells, administration of RA had no effect on the total intracellular GSH levels, decreasing the GSH/GSSG ratio (p < 0.01); in addition, it caused a significant reduction in the GSH/GSSG ratio in irradiated cells (p < 0.001), an expression of radioinduced cell damage. In B16F10 cells, the administration of RA possibly activates the metabolic pathway of eumelanin synthesis that would consume intracellular GSH, thereby reducing its possible use as a protector against oxidative stress. The administration of this type of substance during radiotherapy could potentially protect healthy cells for which RA is a powerful radioprotector, and at the same time, cause significant damage to melanoma cells for which it could act as a radiosensitive agent.

Asiatic acid protects oocytes against in vitro aging-induced deterioration and improves subsequent embryonic development in pigs.

As a pentacyclic triterpene in Centella asiatica, asiatic acid (AA) is a powerful antioxidant with many bioactivities. In the present research, we investigated whether AA has the potential to rescue the decrease in porcine oocyte quality that occurs during in vitro aging (IVA). Mature porcine oocytes were collected and then continuously cultured for an additional 24 h or 48 h with or without AA in maturation medium as an IVA model. The results revealed that AA supplementation reduced the percentage of abnormal aged porcine oocytes during IVA. Furthermore, AA supplementation effectively maintained aged porcine oocyte developmental competence, both parthenogenetic activation and in vitro fertilization. The number of sperm that bound to the zona pellucida on aged porcine oocytes was higher in the AA-supplemented group than in the non-supplemented group. Moreover, AA supplementation not only blocked IVA-induced oxidative stress but also maintained intracellular GSH levels and reduced the percentage of early apoptosis aged porcine oocytes. Mitochondrial functions were disordered during the IVA process. The intracellular ATP levels and mitochondrial membrane potential in aged porcine oocytes were dramatically increased by AA supplementation. Therefore, AA has beneficial effects on porcine oocyte quality and developmental potential maintenance during IVA.

Designed Spider Silk-Based Drug Carrier for Redox- or pH-Triggered Drug Release.

Targeted drug delivery and controlled drug release can be obtained using specifically designed polymers as carriers. Due to their biocompatibility and biodegradability and especially the lack of an immune response, materials made of spider silk proteins are promising candidates for use in such applications. Particles made of recombinant spider silk proteins have previously been shown to be suitable drug and gene carriers as they could readily be loaded with various drug substances or biologicals, and subsequent release was observed over a defined period of time. However, the respective substances were bound non-covalently via hydrophobic or charge-charge interactions, and hence, the release of loaded substances could not be spatio-temporally controlled. Here, we present a setup of chemically modified recombinant spider silk protein eADF4 and variants thereof, combining their well-established biocompatible properties with covalent drug binding and triggered release upon changes in the pH or redox state, respectively. The usefulness of the spider silk platform technology was shown with model substances and cytostatic drugs bound to spider silk particles or films via a pH-labile hydrazine linker as one option, and the drugs could be released from the spider silk carriers upon acidification of the environment as seen, e.g., in tumorous tissues or endo/lysosomes. Sulfhydryl-bearing spider silk variants allowed model substance release if exposed to intracellular GSH (glutathione) levels as a second coupling option. The combination of non-immunogenic, nontoxic spider silk materials as drug carriers with precisely triggerable release chemistry presents a platform technology for a wide range of applications.

Critical role of glutamine metabolism in cardiomyocytes under oxidative stress

BackgroundMetabolic remodeling in cardiomyocytes is deeply associated with the pathogenesis of heart failure (HF). Glutaminolysis is an anaplerotic pathway that incorporates α-ketoglutarate (αKG) derived from glutamine into the tricarboxylic acid (TCA) cycle. It is well known that cancer cells depend on glutamine for their increased energy demand and proliferation; however, the physiological roles of glutamine metabolism in failing hearts remain unclear. ObjectiveTo investigate the regulatory mechanisms and biological effects of glutamine metabolism in oxidative stress-induced failing myocardium. Methods and resultsThe intracellular levels of glutamine, glutamate, and αKG were significantly decreased by H2O2 stimulation in rat neonatal cardiomyocytes (RNCMs). To better understand the metabolic flux in failing myocardium, we performed a stable isotope tracing study and found that glutaminolysis was upregulated in RNCMs under oxidative stress. Consistent with this, the enzymatic activity of glutaminase (Gls), which converts glutamine to glutamate, was augmented in RNCMs treated with H2O2. These findings suggest that glutamine anaplerosis is enhanced in cardiomyocytes under oxidative stress to compensate for the reduction of αKG. Furthermore, the inhibition of Gls reduced cardiac cell viability, ATP production, and glutathione (GSH) synthesis in RNCMs with H2O2 stimulation. Finally, we evaluated the effects of αKG on failing myocardium and observed that dimethyl α-ketoglutarate (DMKG) suppressed oxidative stress-induced cell death likely due to the enhancement of intracellular ATP and GSH levels. ConclusionOur study demonstrates that under oxidative stress, glutaminolysis is upregulated to compensate for the loss of αKG and its replenishment into the TCA cycle, thereby exerting cardioprotective effects by maintaining ATP and GSH levels. Modulation of glutamine metabolism in failing hearts might provide a new therapeutic strategy for HF.

Cinnamoyl-N-Acylhydrazone-Donepezil Hybrids: Synthesis and Evaluation of Novel Multifunctional Ligands Against Neurodegenerative Diseases.

A new series of ten multifunctional Cinnamoyl-N-acylhydrazone-donepezil hybrids was synthesized and evaluated as multifunctional ligands against neurodegenerative diseases. The molecular hybridization approach was based on the combination of 1-benzyl-4-piperidine fragment from the anti-Alzheimer AChE inhibitor donepezil (1) and the cinnamoyl subunit from curcumin (2), a natural product with remarkable antioxidant, neuroprotective and anti-inflammatory properties, using a N-acylhydrazone fragment as a spacer subunit. Compounds 4a and 4d showed moderate inhibitory activity towards AChE with IC50 values of 13.04 and 9.1µM, respectively. In addition, compound 4a and 4d showed a similar predicted binding mode to that observed for donepezil in the molecular docking studies. On the other hand, compounds 4a and 4c exhibited significant radical scavenging activity, showing the best effects on the DPPH test and also exhibited a significant protective neuronal cell viability exposed to t-BuOOH and against 6-OHDA insult to prevent the oxidative stress in Parkinson's disease. Similarly, compound 4c was capable to prevent the ROS formation, with indirect antioxidant activity increasing intracellular GSH levels and the ability to counteract the neurotoxicity induced by both OAβ1-42 and 3-NP. In addition, ADMET in silico prediction indicated that both compounds 4a and 4c did not show relevant toxic effects. Due to their above-mentioned biological properties, compounds 4a and 4c could be explored as lead compounds in search of more effective and low toxic small molecules with multiple neuroprotective effects for neurodegenerative diseases.

Biodegradable Nanoplatforms with the Multiple Modulation of Tumor Microenvironment for Enhanced Sonodynamic Therapy

The specific microenvironment of solid tumors, which is characterized by hypoxia, overexpression of glutathione (GSH) and high accumulation of anti-inflammatory tumor associated macrophages (TAMs), limits the efficiency of sonodynamic therapy (SDT). Herein, a multifunctional nanoplatform was engineered to modulate the tumor microenvironment for highly efficient SDT. In this system, sonosensitizers and catalase were encapsulated in disulfide-bridged mesoporous organosilicon nanoparticles with high loading, which protected the activity of catalase and ensure the stability of sonosensitizers and enzyme. Subsequently hyaluronic acid was grafted onto the nanoplatform to reeducate TAMs and induce the secretion of exogenous hydrogen peroxide. Due to the good protection of enzyme, the catalase within the nanoplatform efficiently produced the mount of O 2 through decomposing the H 2 O 2 in tumor tissues, which remarkably alleviated tumor hypoxia. Furthermore, degradation of the nanoparticles was observed in response to GSH, which effectively decreased the intracellular GSH level, further favoring SDT-triggered anticancer effect. Based on the multiple adjustments to tumor microenvironment, our nanoplatform displayed extraordinary sonodynamic therapeutic effect with low systemic toxicity.