H2O2 Cytotoxicity Research Articles

A Novel Benzothiazole-Based Fluorescent AIE Probe for the Detection of Hydrogen Peroxide in Living Cells

A benzothiazole-based derivative aggregation-induced emission (AIE) fluorescent ‘turn-on’ probe named 2-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)benzo[d]thiazole (probe BT-BO) was developed and synthesized successfully for detecting hydrogen peroxide (H2O2) in living cells. The synthesis method of probe BT-BO is facile. Probe BT-BO demonstrates a well-resolved emission peak at 604 nm and the ability to prevent the interference of reactive oxygen species (ROS), various metal ions and anion ions, and good sensitivity. Additionally, the probe boasts impressive pH range versatility, a fast response time to H2O2 and low cytotoxicity. Finally, probe BT-BO was applied successfully to image A549 and Hep G2 cells to monitor both exogenous and endogenous H2O2.

G protein-coupled estrogen receptor activates PI3K/AKT/mTOR signaling to suppress ferroptosis via SREBP1/SCD1-mediated lipogenesis.

Lung cancer is the leading cause of cancer-related death worldwide. The sex differences in the occurrence and fatality rates of non-small cell lung cancer (NSCLC), along with its association with estrogen dependence, suggest that estrogen receptors (ERs) contribute to the development of NSCLC. However, the influence of G protein-coupled estrogen receptor (GPER1) on NSCLC remains to be determined. Escape from ferroptosis is one of the hallmarks of tumor discovered in recent years. In this context, the present study evaluated whether GPER1 promotes NSCLC progression by preventing ferroptosis, and the underlying mechanism through which GPER1 protects against ferroptosis was also explored. The effects of GPER1 on the cytotoxicity of H2O2, the ferroptosis inducer RSL3, and Erastin were assessed using the CCK8 assay and plate cloning. Lipid peroxidation levels were measured based on the levels of MDA and BODIPY™581/591C11. GPER1 overexpression and knockdown were performed and G1 was used, and the expression of SCD1 and PI3K/AKT/mTOR signaling factors was measured. Immunofluorescence analysis and immunohistochemistry were performed on paired specimens to measure the correlation between the expression of GPER1 and SCD1 in NSCLC tissues. The effect of GPER1 on the cytotoxicity of cisplatin was measured in vitro using the CCK8 assay and in vivo using xenograft tumor models. GPER1 and G1 alleviated the cytotoxicity of H2O2, reduced sensitivity to RSL3, and impaired lipid peroxidation in NSCLC tissues. In addition, GPER1 and G1 promoted the protein and mRNA expression of SCD1 and the activation of PI3K/AKT/mTOR signaling. GPER1 and SCD1 expression were elevated and positively correlated in NSCLC tissues, and high GPER1 expression predicted a poor prognosis. GPER1 knockdown enhanced the antitumor activity of cisplatin in vitro and in vivo. GPER1 prevents ferroptosis in NSCLC by promoting the activation of PI3K/AKT/mTOR signaling, thereby inducing SCD1 expression. Therefore, treatments targeting GPER1 combined with cisplatin would exhibit better antitumor effects.

Effects of FGF2 Priming and Nrf2 Activation on the Antioxidant Activity of Several Human Dental Pulp Cell Clones Derived From Distinct Donors, and Therapeutic Effects of Transplantation on Rodents With Spinal Cord Injury.

In recent years, the interest in cell transplantation therapy using human dental pulp cells (DPCs) has been increasing. However, significant differences exist in the individual cellular characteristics of human DPC clones and in their therapeutic efficacy in rodent models of spinal cord injury (SCI); moreover, the cellular properties associated with their therapeutic efficacy for SCI remain unclear. Here, using DPC clones from seven different donors, we found that most of the clones were highly resistant to H2O2 cytotoxicity if, after transplantation, they significantly improved the locomotor function of rats with complete SCI. Therefore, we examined the effects of the basic fibroblast growth factor 2 (FGF2) and bardoxolone methyl (RTA402), which is a nuclear factor erythroid 2-related factor 2 (Nrf2) chemical activator, on the total antioxidant capacity (TAC) and the resistance to H2O2 cytotoxicity. FGF2 treatment enhanced the resistance of a subset of clones to H2O2 cytotoxicity. Regardless of FGF2 priming, RTA402 markedly enhanced the resistance of many DPC clones to H2O2 cytotoxicity, concomitant with the upregulation of heme oxygenase-1 (HO-1) and NAD(P)H-quinone dehydrogenase 1 (NQO1). With the exception of a subset of clones, the TAC was not increased by either FGF2 priming or RTA402 treatment alone, whereas it was significantly upregulated by both treatments in each clone, or among all seven DPC clones together. Thus, the TAC and resistance to H2O2 cytotoxicity were, to some extent, independently regulated and were strongly enhanced by both FGF2 priming and RTA402 treatment. Moreover, even a DPC clone that originally exhibited no therapeutic effect on SCI improved the locomotor function of mice with SCI after transplantation under both treatment regimens. Thus, combined with FGF2, RTA402 may increase the number of transplanted DPCs that migrate into and secrete neurotrophic factors at the lesion epicenter, where reactive oxygen species are produced at a high level.

An Exosome-Rich Conditioned Medium from Human Amniotic Membrane Stem Cells Facilitates Wound Healing via Increased Reepithelization, Collagen Synthesis, and Angiogenesis.

Tissue regeneration is an essential requirement for wound healing and recovery of organs' function. It has been demonstrated that wound healing can be facilitated by activating paracrine signaling mediated by exosomes secreted from stem cells, since exosomes deliver many functional molecules including growth factors (GFs) and neurotrophic factors (NFs) effective for tissue regeneration. In this study, an exosome-rich conditioned medium (ERCM) was collected from human amniotic membrane stem cells (AMSCs) by cultivating the cells under a low oxygen tension (2% O2 and 5% CO2). The contents of GFs and NFs including keratinocyte growth factor, epidermal growth factor, fibroblast growth factor 1, transforming growth factor-β, and vascular endothelial growth factor responsible for skin regeneration were much higher (10-30 folds) in the ERCM than in normal conditioned medium (NCM). In was found that CM-DiI-labeled exosomes readily entered keratinocytes and fibroblasts, and that ERCM not only facilitated the proliferation of keratinocytes in normal condition, but also protected against H2O2 cytotoxicity. In cell-migration assay, the scratch wound in keratinocyte culture dish was rapidly closed by treatment with ERCM. Such wound-healing effects of ERCM were confirmed in a rat whole skin-excision model: i.e., the wound closure was significantly accelerated, remaining minimal crusts, by topical application of ERCM solution (4 × 109 exosome particles/100 μL) at 4-day intervals. In the wounded skin, the deposition of collagens was enhanced by treatment with ERCM, which was supported by the increased production of collagen-1 and collagen-3. In addition, enhanced angiogenesis in ERCM-treated wounds was confirmed by increased von Willebrand factor (vWF)-positive endothelial cells. The results indicate that ERCM from AMSCs with high concentrations of GFs and NFs improves wound healing through tissue regeneration not only by facilitating keratinocyte proliferation for skin repair, but also activating fibroblasts for extracellular matrix production, in addition to the regulation of angiogenesis and scar tissue formation.

3D-Printed Microcubes for Catalase Drug Delivery.

Oxidative stress, i.e., excessive production of reactive oxygen species (ROS), plays an important role in the pathogenesis of inflammatory diseases such as cardiovascular diseases, cancer, and neurodegenerative diseases. Catalase, an antioxidant enzyme, has great therapeutic potential; however, its efficacy is limited by its delivery to target cells or tissues. In order to achieve efficient delivery, consistent drug distribution, and drug activity, small and uniformly sized drug delivery vehicles are needed. Here, three-dimensional (3D) microcubes were printed by Nanoscribe Photonic Professional GT2, a high-resolution 3D printer, and the characteristics of 3D-printed microcubes as drug delivery vehicles for the delivery of catalase were investigated. The size of the 3D-printed microcubes was 800 nm in length of a square and 600 nm in height, which is suitable for targeting macrophages passively. Microcubes were also tunable in shape and size, and high-resolution 3D printing could provide microparticles with little variation in shape and size. Catalase was loaded on 3D-printed microcubes by nonspecific adsorption, and catalase on 3D-printed microcubes (CAT-MC) retained 83.1 ± 1.3% activity of intact catalase. CAT-MC also saved macrophages, RAW 264.7, from the cytotoxicity of H2O2 by 86.4 ± 4.1%. As drug delivery vehicles, 3D-printed microparticles are very promising due to their small and uniform size, which provides consistent drug distribution and drug activity. Therefore, we anticipate numerous applications of 3D-printed microparticles for delivering therapeutic proteins.

Alginate-Based Oral Delivery Systems to Enhance Protection, Release, and Absorption of Catalase.

Oxidative stress, overproduction of reactive oxygen species (ROS), plays an important role in the development of inflammatory bowel diseases. Catalase has great therapeutic potential by scavenging hydrogen peroxide, one of the ROSs produced in cellular metabolisms. However, in vivo application to scavenge ROS is currently limited especially in oral administrations. Here, we introduced an alginate-based oral drug delivery system that effectively protected catalase from the simulated harsh conditions of the gastrointestinal (GI) tract, released it in the small intestine mimicked condition, and enhanced its absorption via M cells, highly specialized epithelium cells in the small intestine. First of all, catalase was encapsulated in alginate-based microparticles with different amounts of polygalacturonic acid or pectin, which achieved an encapsulation efficiency of more than 90%. It was further shown that catalase was released from alginate-based microparticles in a pH-dependent manner. Results indicated that alginate-polygalacturonic acid microparticles (60 wt % Alg:40 wt % Gal) released 79.5 ± 2.4% of encapsulated catalase at pH 9.1 in 3 h, while they only released 9.2 ± 1.5% of encapsulated catalase at pH 2.0. Even when catalase was encapsulated in microparticles (60 wt % Alg:40 wt % Gal) and exposed to pH 2.0 followed by pH 9.1, it still retained 81.0 ± 11.3% enzyme activity compared to that in microparticles prior to the pH treatment. We then investigated the efficiency of RGD conjugation to catalase on the catalase uptake by M-like cells, the coculturing of human epithelial colorectal adenocarcinoma; Caco-2 cells and B lymphocyte; Raji cells. RGD-catalase protected M-cells more efficiently from the cytotoxicity of H2O2, a typical ROS. RGD conjugation to catalase enhanced the uptake by M-cells with 87.6 ± 0.8% RGD-catalase, whereas 11.5 ± 9.2% of RGD-free catalase passed across M-cells. From the results of protection, release, and absorption of model therapeutic proteins from the harsh pH conditions, alginate-based oral drug delivery systems will have numerous applications for the controlled release of drugs that are easily degradable in the GI tract.

Daurisoline attenuates H2O2-induced chondrocyte autophagy by activating the PI3K/Akt/mTOR signaling pathway

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degeneration and intra-articular inflammation. Daurisoline (DAS) is an isoquinoline alkaloid isolated from Rhizoma Menispermi, whose antitumor and anti-inflammatory pharmacological effects have been demonstrated, but the effects of DAS on OA have rarely been researched. In this study, we aimed to explore the potential role of DAS in OA and its partial mechanism.Materials and methodsThe cytotoxicity of H2O2 and DAS toward chondrocytes was detected by the Cell Counting Kit-8 assay. Safranin O staining was used to detect chondrocyte phenotype changes. Cell apoptosis was measured by both flow cytometry and quantitative analysis of the protein levels of the apoptosis-related factors Bax, Bcl-2 and cleaved caspase-3 by western blot. Western blotting and immunofluorescence were used to assess the expression of the autophagy-related proteins LC3, Beclin-1 and p62. In addition, key signal pathway targets and matrix-degrading indicators were measured by western blot.ResultsOur results indicated that H2O2 induced human chondrocyte apoptosis and activated autophagy in a dose-dependent manner. DAS treatment dose-dependently reversed the expression of apoptosis-related proteins (Bax, Bcl-2 and cleaved caspase3) and the apoptosis rate induced by H2O2. Western blot and immunofluorescence analyses showed that DAS decreased the H2O2-induced upregulation of the autophagy marker Beclin-1 and the LC3 II/LC3 I ratio and upregulated the p62 protein level. Mechanistically, DAS inhibited autophagy through the activation of the classical PI3K/AKT/mTOR signaling pathway and protected chondrocytes from apoptosis. In addition, DAS alleviated the H2O2-induced degradation of type II collagen and the high expression of matrix metalloproteinase 3 (MMP3) and MMP13.ConclusionOur research demonstrated that DAS alleviated chondrocyte autophagy caused by H2O2 through activation of the PI3K/AKT/mTOR signaling pathway and protected chondrocytes from apoptosis and matrix degradation. In conclusion, these findings suggest that DAS may serve as a promising therapeutic strategy for OA.

Simultaneous Manipulation and Imaging of Chemogenetically Induced Hydrogen Peroxide in Hardly Transfectable Endothelial Cells

Hydrogen peroxide (H2O2) is a critical signaling molecule in vascular cells, which controls signaling events, yet it can cause pathological oxidative stress in excess. The lack of suitable tools undermined experimental approaches to study the role of oxidative eu- and distress in cellular ultra-locales. This study exploits a yeast-derived D-amino acid oxidase (mDAAO) as a chemogenetic tool to induce, visualize and test the cytotoxicity of H2O2 in hardly transfectable endothelial cells. Due to the poor transfectability of endothelial cells, lentiviral vectors have been used to generate cell lines stably expressing mDAAOs. mDAAOs are substrate-based chemogenetic enzymes that convert D-amino acids to their corresponding alpha-keto acids and generate H2O2 as a byproduct, which can be visualized with a novel ultrasensitive, and ratiometric H2O2 biosensor termed HyPer7. This study tested the suitability of two different D-amino acids, including D-alanine and D-methionine, to induce oxidative stress in endothelial cells. Live-cell imaging experiments unveiled that 10 mM D-methionine generated significantly higher and faster H2O2 signals than D-alanine. However, both D-amino acids induced comparable levels of cell death documented by a colorimetric cell metabolic activity assay (MTT). This study provides a guide for manipulating and monitoring the cytotoxic effect of H2O2 in endothelial cells.

Effect of Spiny-Cheek Crayfish (Faxonius limosus) on H2O2-Induced Oxidative Stress in Normal Fibroblast Cells

Faxonius limosus (spiny-cheek) crayfish is an invasive species that is widespread in Europe. The aim of the study was to evaluate the possibility of using extracts from this crustacean as a source of natural antioxidants. For this purpose, selected bioactive compounds (total phenols, glutathione, vitamins A, E, D, β-carotene and amino acids) were characterised and the antioxidant activity of the extract was assessed in vitro and in relation to HFFF-2 fibroblast cells, in which oxidative stress was caused by the additive hydrogen peroxide (H2O2). The extract abolished the cytotoxicity of H2O2, decreased reactive oxygen species (ROS) secretion, increased cell viability and decreased the expression of caspase-3. The results suggest that F. limosus extract is a promising raw material with antioxidant properties.

Manganese oxide increases bleaching efficacy and reduces the cytotoxicity of a 10% hydrogen peroxide bleaching gel

The study aims to assess the effects of a 10% H2O2 bleaching gel with different MnO2 concentrations on the bleaching efficacy (BE), degradation kinetics (DK) of H2O2, and trans-amelodentinal cytotoxicity (TC). Standardized bovine enamel/dentin disks (n = 96) were placed in artificial pulp chambers, and the bleaching gels were applied for 45min. Thus, the following groups were established: (G1) no treatment (negative control/NC); (G2) 35% H2O2 (positive control/PC); (G3) 10% H2O2; (G4) 10% H2O2 + 2mg/mL MnO2; (G5) 10% H2O2 + 6mg/mL MnO2; and (G6) 10% H2O2 + 10mg/mL MnO2. After analyzing bleaching efficacy (ΔE00 and ΔWI), the degradation kinetics of H2O2 and trans-amelodentinal cytotoxicity were determined (n = 8, ANOVA/Tukey; p < 0.05). G6 presented BE (ΔE00 and ΔWI) statistically similar to G2, which represented conventional in-office bleaching (p = 0.6795; p > 0.9999). A significant reduction in the diffusion of H2O2 occurred in G3, G4, G5, and G6 compared to G2 (p < 0.0001). The highest DK of H2O2 occurred in G6 (p < 0.0001), which had the lowest TC in comparison with all other bleached groups (p ≤ 0.0186). The addition of 10mg/mL of MnO2 in a 10% H2O2 bleaching gel potentiates the degradation of this reactive molecule, which increases the BE of the product and decreases TC. Replacing a 35% H2O2 gel commonly used for conventional in-office dental bleaching by a 10% H2O2 gel containing 10mg/mL of MnO2 reduces the cytotoxicity of this professional therapy, maintaining its excellent esthetic efficacy.

Abstract 1779: Inability to regulate ROS content confers sensitivity to ferroptosis in breast cancer cells resistant to T-DM1

Abstract Clinical success of Trastuzumab-emtansine (T-DM1, Kadcyla) lead its extended approval as a post-surgical adjuvant treatment in early stage HER-2-positive breast cancer. However, intrinsic and acquired resistance remains a major limitation to its clinical efficacy. Adipocytes were shown to exhibit a pro-survival cross talk with breast cancer cells, limiting their sensitivity to T-DM1 and raising the question of metabolic alterations involved in this resistance phenotype. To explore the role of tumor cell metabolic plasticity in T-DM1 resistance, xenografts of the human HER-2 positive breast cancer model BT-474 were exposed in vivo to increasing doses of T-DM1. BT-474 WT and R tumors were characterized by RNAseq and primary explants of in vivo tumors were cultured and analyzed in vitro. Although HER-2 levels were found to be strongly reduced in resistant cells, no increase of its pro-survival downstream partners were observed at the transcriptomic level. Conversely, PIK3CA and KRAS were respectively 2.5- and 2-times downregulated in BT-474 R according to RNAseq data, suggesting the implementation of compensatory pathways for cell survival and proliferation. Tumors resistant to T-DM1 exhibited alterations in lipid metabolism, including an increase in lipid droplet content observed by ORO staining, upregulation of fatty acid metabolism pathways, and lipid transporter FABP4 overexpression. Cells resistant to T-DM1 survived to 2-deoxyglucose treatment (100 mM) as well as glucose deprivation in vitro while these conditions were cytotoxic to BT-474 WT cells. In addition, BT-474 R cells were resistant to palmitate exposure, suggesting energy production is monitored by an increased lipid import and metabolism. Ferroptotic cell death and associated lipid peroxidation have recently been associated with alterations in lipid metabolism. H2D-CFDA staining revealed a 2-fold increase in ROS content in R cells at the basal state compared to WT cells. In vitro, BT-474 R cells were highly sensitive to ROS inducers such as H2O2 and bleomycin when compared to WT cells (IC50s were 7- and 30-fold higher in WT compared to R cells for H202 and bleomycin, respectively). This unusually strong cytotoxicity of H2O2 was found to be correlated with a significant increase of ROS content within the resistant cells compared to wild type cells after H2O2 treatment. Additionally, inhibitors of ferroptosis such as GPX4, and the antiporter system xct were found to be downregulated in resistant cells, while the GPX4 inhibitor RSL3 was 5-fold more cytotoxic in cells resistant to T-DM1 than in WT cells. T-DM1 -resistant cells thus demonstrate increased fatty acid uptake, decreased ability to eliminate ROS and enhanced sensitivity to ferroptosis inducers. Altogether, these results suggest that ferroptosis could constitute a promising target for T-DM1-resistant tumors. Citation Format: Louise Conilh, Juliette Sauveur, Sabine Beaumel, Claire Scheffges, Estelle Nunez, David Cheillan, Charles Dumontet. Inability to regulate ROS content confers sensitivity to ferroptosis in breast cancer cells resistant to T-DM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1779.

Antidepressant-like effects of a chlorogenic acid- and cynarine-enriched fraction from Dittrichia viscosa root extract

Dittrichia viscosa is a perennial Mediterranean plant used in traditional medicine for “calming purposes”, pointing at a possible antidepressant activity of the plant. We conducted chromatographic and bioassay-guided fractionation of D. viscosa root extract to isolate a specific fraction (fraction “K”) with antidepressant-like characteristics in vivo and strong antioxidant properties in vitro. A single dose of “K” reduced immobility time in the forced swim test with a mouse model possessing a depressive-like phenotype. Neurochemical profiling for 5-hydroxytryptamine (5-HT) and its primary metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in prefrontal cortex and hippocampus of “K”-treated mice showed reduction in 5-HIAA, indicative of either serotonin uptake transporter or monoamine oxidase-A inhibition, as well as slight increases in 5-HT content. These neurochemical alterations, as well as the behavioral changes observed, were comparable to the effects of paroxetine. “K” also protected PC12 cells in a H2O2 cytotoxicity assay, thus demonstrating antioxidant properties, yet paroxetine augmented oxidative damage and cell death. Identification of the main compounds in “K” by high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) indicated that chlorogenic acid and cynarine comprised 87% of the total components. D. viscosa root extract appears to produce antidepressant and cytoprotective effects and may serve as an attractive alternative to standard therapies for depression.

Protective effect of l-carnitine-loaded solid lipid nanoparticles against H2O2-induced genotoxicity and apoptosis

L-carnitine (LC) is a highly water-soluble compound involved in the β-oxidation of lipids and transportation of long-chain fatty acids across the membrane of mitochondria. However, the higher hydrophilicity of LC limits its free diffusion across the bilayer lipid membrane of intestinal epithelium in oral administration, decreasing oral bioavailability. Drug delivery with nanoparticles enhances cargo bioavailability and cellular uptake and improves therapeutic outcomes while decreasing unwanted side effects. Here, we proposed solid lipid nanoparticles (SLNs) as a hydrophobic carrier for LC delivery, aiming at increasing LC bioavailability and its protective role against intracellular oxidative stress damages. The LC-SLNs were prepared using the hot homogenization technique, and different physicochemical properties were investigated. The inhibition of H2O2-induced ROS generation in human umbilical vein endothelial cells (HUVECs) with plain LC and LC-SLNs was investigated. Moreover, various in vitro experiments were performed to assess whether LC-SLNs can protect HUVECs from H2O2-induced genotoxicity and apoptosis. The monodispersed and spherical blank SLNs and LC-SLNs were 104 ± 1.8 and 128 ± 1.5 nm, respectively with a drug loading (DL) of 11.49 ± 0.78 mg/mL and acceptable encapsulation efficiency (EE%) (69.09 ± 1.12) of LC-SLNs. The formulation process did not affect the antioxidant properties of LC. MTT assay and comet assay demonstrated that the LC-SLNs decreased cytotoxicity and genotoxicity of H2O2, respectively on HUVECs. Besides, LC-SLNs more inhibited ROS generation, along with apoptotic events in H2O2-treated HUVECs compared to the plain LC. Altogether, our findings affirmed the protective effects of LC-SLNs against H2O2-induced genotoxicity and apoptosis in HUVECs. In conclusion, LC-SLN formulation is a promising drug delivery system to overcome the bioavailability issue of hydrophilic LC, enhancing the antioxidant and biological properties of the plain LC.

DK-MGAR101, an extract of adventitious roots of mountain ginseng, improves blood circulation by inhibiting endothelial cell injury, platelet aggregation, and thrombus formation

BackgroundSince ginsenosides exert an anti-thrombotic activity, blood flow-improving effects of DK-MGAR101, an extract of mountain ginseng adventitious roots (MGAR) containing various ginsenosides, were investigated in comparison with an extract of Korean Red Ginseng (ERG). MethodsIn Sprague-Dawley rats orally administered with DK-MGAR101 or ERG, oxidative carotid arterial thrombosis was induced with FeCl3 (35%), and their blood flow and occlusion time were measured. To elucidate underlying mechanisms, the cytoprotective activities on rat aortic endothelial cells (RAOECs) exposed to hydrogen peroxide (H2O2) were confirmed. In addition, the inhibitory activities of DK-MGAR101 and ERG on agonist-induced platelet aggregation, thromboxane B2 production, and ATP granule release from stimulated platelets as well as blood coagulation were analyzed. ResultsDK-MGAR101 containing high concentrations of Rb1, Rg1, Rg3, Rg5, and Rk1 ginsenosides (55.07 mg/g) was more effective than ERG (ginsenosides 8.45 mg/g) in protecting RAOECs against H2O2 cytotoxicity. DK-MGAR101 was superior to ERG not only in suppressing platelet aggregation, thromboxane B2 production, and granule release, but also in delaying blood coagulation, FeCl3-induced arterial occlusion, and thrombus formation. ConclusionsThe results indicate that DK-MGAR101 prevents blood vessel occlusion by suppressing platelet aggregation, thrombosis, and blood coagulation, in addition to endothelial cell injury.

ACTH(6-9)PGP Peptide Protects SH-SY5Y Cells from H2O2, tert-Butyl Hydroperoxide, and Cyanide Cytotoxicity via Stimulation of Proliferation and Induction of Prosurvival-Related Genes.

Stabilized melanocortin analog peptide ACTH(6–9)PGP (HFRWPGP) possesses a wide range of neuroprotective activities. However, its mechanism of action remains poorly understood. In this paper, we present a study of the proproliferative and cytoprotective activity of the adrenocorticotropic hormone fragment 6–9 (HFRW) linked with the peptide prolyine–glycyl–proline on the SH-SY5Y cells in the model of oxidative stress-related toxicity. The peptide dose-dependently protected cells from H2O2, tert-butyl hydroperoxide, and KCN and demonstrated proproliferative activity. The mechanism of its action was the modulation of proliferation-related NF-κB genes and stimulation of prosurvival NRF2-gene-related pathway, as well as a decrease in apoptosis.

Vinpocetine-based therapy is an attractive strategy against oxidative stress-induced hepatotoxicity in vitro by targeting Nrf2/HO-1 pathway.

Vinpocetine (Vin), a synthetic-derivative of Vincamine, monoterpenoid indole alkaloid, has been reported to have various medicinal benefits. The purpose of our study was to investigate the pivotal role of “nuclear factor erythroid 2-related factor-2” (Nrf2)-mediated antioxidant protection of Vin against H2O2 and paracetamol (APAP)-induced liver toxicity. For this purpose, a normal human hepatic cell line (L02 cells) was incubated with cytotoxic concentrations of H2O2 or APAP in the presence or absence of Vin. To evaluate the responses, MTS Cell Viability assay, immunoblotting, biochemical assays, and molecular docking approach were used. Viability analysis showed that treatment of L02 cells with Vin prevented the cytotoxicity induced by H2O2 and APAP. It was evidenced by the fact that Vin dumped H2O2- and APAP-cytotoxicity and reactive oxygen species (ROS) generation. The immunoblotting analysis shows that Vin increased Nrf2 expression along with the expression of target protein, heme oxygenase-1 (HO-1), and increased intracellular glutathione (GSH) level. Interestingly, we found that Vin could protect the protein expression-level of Nrf2, which indicated the prospective interaction between Vin and Keap1 protein. Additionally, molecular docking-study revealed that Vin competed with Nrf2 for Keap1-binding site, with hydrogen and stearic interactions. Collectively, Vin effectively protects against H2O2 and APAP-induced cytotoxicity via executing Nrf2-mediated restoration of antioxidative/oxidative balance. Meanwhile, Vin interrupts protein-protein interaction between Nrf2 and Keap1, which might also contribute to decrease Nrf2 degradation and stabilize protein expression. Thus, Vin-based adjuvant therapy may represent a smart drug regimen to mitigate drug-induced oxidative stress and liver injuries.

Protective effect of Phyllanthus niruri leaf extracts on Saccharomyces cerevisiae cells subjected to oxidative stress

Phyllanthus niruri has been widely used to treat a number of ailments in traditional medicine. The pharmacological and preventive potential of Phyllanthus niruri, such as in urolithiasis, hyperglycemia, hypertension, pain and mild cases of malaria, are well known. The present investigation was designed to study the protective effects of aqueous, methanol and chloroform extract of Phyllanthus niruri against oxidative stress and cytotoxicity induced by hydrogen peroxide (H2O2) in Saccharomyces cerevisiae cells. Cytotoxicity of leaf extracts and H2O2 was identified by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sulforhodamine B assay (SRB) and neutral red uptake (NRU) assays. All the three extracts showed protection against H2O2 induced toxicity and reduced the loss of cell viability proving the cytoprotective effect of the extracts against H2O2 induced toxicity in Saccharomyces cerevisiae cells. The aqueous extract rendered maximum protection when compared to that of methanolic and chloroform extract.

Protective effect of vanillic acid in hydrogen peroxide-induced oxidative stress in D.Mel-2 cell line.

The overproduction of reactive oxygen species (ROS) causes oxidative stress, such as Hydrogen peroxide (H2O2). Acute oxidative stress is one of the main reasons for cell death. In this study, the antioxidant properties of vanillic acid- a polyphenolic compound was evaluated. Therefore, this study aims to check the effectiveness of vanillic acid in H2O2-induced oxidative stress in D. Mel-2 cell line. The efficacy was determined by biochemical tests to check the ROS production. The cytotoxicity of H2O2 and vanillic acid was checked by MTT assay. The DNA fragmentation was visualized by gel electrophoresis. Protein biomarkers of oxidative stress were analyzed by western blotting. The results depict a promising antioxidant effect of vanillic acid. The IC50 value of vanillic acid and H2O2 was found 250 μg/ml and 125 μg/ml, respectively. The catalase activity, SOF, GPx, and PC was seen less in H2O2 treated group compared with the control and vanillic acid treated group. However, the TBRAS activity was hight in H2O2 treated group. The effect of H2O2 on DNA fragmentation was high as compared with vanillic acid-treated cells. The protein expression of Hsp70, IL-6 and iNOS was seen significant in a vanillic acid-treated group as compared with H2O2 treated group. These results reinforce that at low concentration, vanillic acid could be used as an antioxidant agent in the food and pharmaceutical industries.

Phillyrin Mitigates Apoptosis and Oxidative Stress in Hydrogen Peroxide-Treated RPE Cells through Activation of the Nrf2 Signaling Pathway.

Oxidative stress-induced dysfunction or apoptosis in retinal pigment epithelial (RPE) cells is an important cause of dry age-related macular degeneration (AMD). Although phillyrin has been shown to exert significant antioxidant effects, the underlying mechanism of action remains unclear. The purpose of this study was to investigate the protective effect of phillyrin on hydrogen peroxide- (H2O2-) induced oxidative stress damage in RPE cells and the potential mechanism involved. It was found that phillyrin significantly protected RPE cells from H2O2 cytotoxicity. Furthermore, phillyrin alleviated oxidative stress-induced apoptosis via inhibition of endogenous and exogenous apoptotic pathways. Compared with the H2O2-treated group, the expressions of cleaved caspase-3, cleaved caspase-9, cleaved polymerase (PARP), death receptor Fas, and cleaved caspase-8, as well as Bax/Bcl-2 ratio were decreased in RPE cells after the phillyrin intervention. In addition, phillyrin reversed the oxidative stress-induced reductions in superoxide dismutase (SOD) and glutathione (GSH) levels and annulled the elevations in reactive oxygen species (ROS) and malondialdehyde (MDA), thereby restoring oxidant-antioxidant homeostasis. Phillyrin treatment upregulated the expressions of cyclin E, cyclin-dependent kinase 2 (CDK2), and cyclin A and downregulated the expressions of p21 and p-p53, thereby reversing the G0/G1 cell cycle arrest in H2O2-treated RPE cells. Pretreatment with phillyrin also increased the expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2), total Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductases-1 (NQO-1) in RPE cells and inhibited the formation of Kelch-like ECH-associated protein 1 (Keap1)/Nrf2 protein complex. Thus, phillyrin effectively protected RPE cells from oxidative stress through activation of the Nrf2 signaling pathway and inhibition of the mitochondria-dependent apoptosis pathway.

Biosynthesis of Folates by Streptococcus thermophilus and Lactobacillus delbruekii ssp. bulgaricus

The folate synthesizing ability of yogurt bacteria, including Streptococcus thermophilus MC and ATCC 19258 and Lactobacillus delbrueckii ssp. bulgaricus 448 and 449, were evaluated. The major effort of this study focused on increasing the folate contents of yogurt bacteria fermented milk by adding lactose or calcium chloride. The addition of 2% lactose in reconstituted non-fat dry milk increased folate synthesis. Although folates increased only about 3 to 7% for yogurt cultures incubated for 6 hr, folates increased 12 to 198% for yogurt bacteria grown for 18 hr. The folate content decreased for reconstituted milk fermented with S. thermophilus MC or L. bulgaricus 448 or 449 when 0.02% calcium chloride was added. The folate level increased only for reconstituted milk fermented with S. thennophilus ATCC 19258 when calcium chloride was added. The addition of 2% lactose increased the cell counts for yogurt bacteria grown for 6 and 18 hr. On the other hand, the results of the cell counts from the addition of 0.02% calcium chloride were not consistent. However, the cell counts increased for both L. bulgaricus strains. The folate levels and cell counts during refrigerated storage at 4°C were determined for reconstituted non-fat dry milk fermented with these bacteria. Folate levels decreased about 9 to 28% at the end of the second week. The viable yogurt bacterial counts remained stable for the 2-week shelf life for all four strains tested in this study. To determine the effect of folate on protecting cells from the cytotoxicity of oxidant H2O2, Intestine 407 was used in this study for evaluation. The cell viability of Intestine 407 was increased due to the inhibition of oxidant H2O2 cytotoxicity by folate and Intestine 407 cells demonstrated higher viability when treated with higher concentration of folate.