Induced Reactive Oxygen Species Generation Research Articles

Molecular insights of 2,6-dichlorobenzoquinone-induced cytotoxicity in zebrafish embryo: Activation of ROS-mediated cell cycle arrest and apoptosis.

2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), as an emerging disinfection by-product, has been frequently detected in waters, posing potential health risk on public health. Although some studies have pointed out that 2,6-DCBQ exposure can induce cytotoxicity, limited information is available for underlying mechanism for 2,6-DCBQ-induced cytotoxicity. To explore this mechanism, we assessed the levels of reactive oxygen species (ROS), acridine orange (AO) staining, and the mRNA transcriptions of genes (Chk2, Cdk2, Ccna, Ccnb and Ccne) involved in cell-cycle and genes (p53, bax, bcl-2 and caspase 3) involved in apoptosis in zebrafish embryo, after exposed to different concentrations (10, 30, 60, 90 and 120 μg/L) of 2,6-DCBQ for 72 h. Our results indicated that 2,6-DCBQ exposure induced ROS generation and cell apoptosis, and disturbed the mRNA transcription of genes related to cell cycle and apoptosis in zebrafish embryo. Moreover, we also found that 30 ~ 60 μg/L 2,6-DCBQ is the important transition from cell-cycle arrest to cell apoptosis. These results provided novel insight into 2,6-DCBQ-induced cytotoxicity.

Gallic acid suppresses the progression of triple-negative breast cancer HCC1806 cells via modulating PI3K/AKT/EGFR and MAPK signaling pathways.

Triple-negative breast cancer (TNBC) is a severe threat to women's health because of its aggressive nature, early age of onset, and high recurrence rate. Therefore, in this study, we aimed to evaluate the anti-tumor effects of Gallic acid (GA) on the TNBC HCC1806 cells in vitro. The cell proliferation was detected by MTT and plate clone formation assays, cell apoptosis, cell cycle, and mitochondrial membrane potential (MMP) were analyzed by flow cytometry and Hoechst 33258 staining assays, and the intracellular reactive oxygen species (ROS) accumulation were also investigated. Real-Time PCR and western blot were examined to explore the mechanism of action. The results indicated that GA suppressed HCC1806 cells proliferation and promoted HCC1806 cells apoptosis. Meanwhile, GA treatment changed the morphology of the HCC1806 cells. In addition, GA blocked the HCC1806 cells cycle in the S phase, and it induced cells apoptosis accompanied by ROS accumulation and MMP depolarization. Real-Time PCR results suggested that GA increased Bax, Caspase-3, Caspase-9, P53, JINK and P38 mRNA expression, and decreased Bcl-2, PI3K, AKT and EGFR mRNA expression. Western blotting results suggested that GA increased Bax, cleaved-Caspase-3, cleaved-Caspase-9, P53, P-ERK1/2, P-JNK, P-P38 proteins expression, and decreased Bcl-2, P-PI3K, P-AKT, P-EGFR proteins expression. Furthermore, molecular docking suggested that GA has the high affinity for PI3K, AKT, EGFR, ERK1/2, JNK, and P38. In conclusion, GA could suppress HCC1806 cells proliferation and promote HCC1806 cells apoptosis through the mitochondrial apoptosis pathway and induces ROS generation which further inhibits PI3K/AKT/EGFR and activates MAPK signaling pathways. Our study will provide some new references for using GA in the treatment of TNBC.

Induction of oxidative DNA damage, cell cycle arrest and p53 mediated apoptosis by calcium titanate nanoparticles in MCF-7 breast cancer cells

BackgroundThe distinctive properties and high activity of calcium titanate nanoparticles (CaTiO3-NPs) increase their use in many products. However, the cytotoxic and genotoxic effects of CaTiO3-NPs in human cancer cell lines have not been well studied. Therefore, this study was conducted to explore CaTiO3-NPs induced cytotoxicity, genomic instability and apoptosis in human breast cancer (MCF-7) cells.MethodsSulforhodamine B (SRB) and the alkaline comet assays were done to study cell viability and DNA damage induction, respectively. Apoptosis induction and cell cycle distribution were analyzed using flow cytometry. The level of intracellular reactive oxygen species (ROS) was studied, and the expression levels of p53, Bax and Bcl2 genes were also measured.ResultsThe results of the Sulforhodamine B (SRB) cytotoxicity assay showed that viability of MCF-7 cells was not affected by CaTiO3-NPs treatment for 24 h, however, exposure to CaTiO3-NPs for 72 h caused concentrations dependent death of MCF-7 cells. Treatment with CaTiO3-NPs for 72 h caused marked increases in intracellular ROS level and induced DNA damage. Treatment of MCF-7 cells with CaTiO3-NPs also caused MCF-7 cell cycle arrest at the G0 and S phases and s triggered apoptosis of MCF-7 cells by causing simultaneous increases in the expression levels of apoptotic p53 and Bax genes and a decrease in the expression level of anti-apoptotic Bcl2 gene.ConclusionCollectively, it was concluded that CaTiO3-NPs cause time- and concentration-dependent cytotoxic effects in human MCF-7 cells through induction of ROS generation, genomic instability and apoptosis. Thus it is recommended that further in vitro and in vivo studies are therefore recommended to understand the cytotoxic and biological effects of CaTiO3-NPs.

Green and efficient synthesis of mesoporous sodalite assisted by persulfate or peroxymonosulfate

Mesoporous sodalite (SOD) zeolite was fabricated from industrial waste lithium silica fume (LSF) through the reactive oxygen species (ROS) method under ultraviolet (UV) irradiation without the template. Persulfate (PS) and peroxymonosulfate (PMS) under UV irradiation would further induce ROS generation and result in faster crystallization to obtain highly crystalline and abundant mesopores (mesopore volume, Vmes＞0.34 cm³/g) SOD within 2 h. The ROS produced in the synthesis system were detected by electron spin resonance (EPR), and the results revealed that ·OH, O2·−, 1O2, and SO4·− may be related to the accelerating process. Zeolite produced superior adsorptive capacity (112.4 mg/g) and reusability in copper ion (Cu(II)) adsorptive experiments. The abundance of mesoporous in zeolite facilitates the removal of Cu(II) by ion exchange and hydroxyl complexation. This work opens up a new route for green and fast synthesis of zeolite and highly efficient Cu(II) removal adsorbent.

A mitochondria-targeted supramolecular nanoplatform for peroxynitrite-potentiated oxidative therapy of orthotopic hepatoma

Oxidative therapy, which generates reactive oxygen species (ROS) via intracellular enzymatic reactions to achieve tumor ablation, is considered as an emerging approach to cancer treatment. Herein, nitric oxide (NO)-combined oxidative therapy is reported by integrating glutathione (GSH)-sensitive NO donor and pH-sensitive cinnamaldehyde (CA) prodrug into a mitochondria-targeted drug nanocarrier, which is prepared by the host-guest interaction between α-cyclodextrin (α-CD) and polyethylene glycol (PEG). After internalized by cancer cells, CA can be released in acidic endo/lysosome and finally induce ROS generation in mitochondria for oxidative therapy. At the same time, NO can be targeted delivered to mitochondria by a mitochondria-targeting strategy and then realize selective release of NO in mitochondria. NO can deplete intracellular predominant antioxidant GSH, which will enhance oxidative therapy of CA. Furthermore, peroxynitrite (ONOO−) with strong peroxidation and nitration capability can be produced in mitochondria by the reaction between NO and ROS for reactive nitrogen species (RNS)-mediated oxidative therapy. The generation of ONOO− in mitochondria is very effective in facilitating mitochondrial membrane permeabilization, which can cause mitochondrial dysfunction and finally induce mitochondrial apoptosis. The antitumor ability of mitochondria-targeted ONOO−-potentiated oxidative therapy is fully investigated on subcutaneous and orthotopic hepatoma model on nude mice. This innovative strategy for the selective generation of ONOO− in mitochondria may serve as an afflatus for future applications in cancer treatment.

Hibiscus sabdariffa Extract Protects HaCaT Cells against Phenanthrene-Induced Toxicity through the Regulation of Constitutive Androstane Receptor/Pregnane X Receptor Pathway.

Phenanthrene (Phe) exposure is associated with skin ageing, cardiotoxicity and developmental defects. Here, we investigated the mode of Phe toxicity in human keratinocytes (HaCaT cells) and the attenuation of toxicity on pre-treatment (6 h) with ethanol extract of Hibiscus sabdariffa calyxes (HS). Cell viability, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm) alteration, changes in the transcriptional activity of selected genes involved in phase I and II metabolism, antioxidant response and gluconeogenesis, western blot and docking studies were performed to determine the protective effect of HS against Phe. Phe (250 μM) induced cytotoxicity in HaCaT cells through AhR-independent, CAR/PXR/RXR-mediated activation of CYP1A1 and the subsequent alterations in phase I and II metabolism genes. Further, CYP1A1 activation by Phe induced ROS generation, reduced ΔΨm and modulated antioxidant response, phase II metabolism and gluconeogenesis-related gene expression. However, pre-treatment with HS extract restored the pathological changes observed upon Phe exposure through CYP1A1 inhibition. Docking studies showed the site-specific activation of PXR and CAR by Phe and inhibition of CYP1A1 and CYP3A4 by the bioactive compounds of HS similar to that of the positive controls tested. Our results conclude that HS extract can attenuate Phe-induced toxicity in HaCaT cells through CAR/PXR/RXR mediated inhibition of CYP1A1.

Ethalfluralin induces developmental toxicity in zebrafish via oxidative stress and inflammation

Ethalfluralin, of dinitroaniline herbicide family, is an effective weed controller. Following residue detection in herbicide-treated fields, ethalfluralin was reported to interfere with early stages of implantation in some vertebrate species. However, the role of ethalfluralin in the development of zebrafish embryos has not been elucidated yet. Therefore, in the present study, we investigated the morphological and physiological changes that occur in the embryonic development of zebrafish due to ethalfluralin exposure. Results indicated that ethalfluralin decreased survival rate along with reduction in the hatching ratio and heartbeat. It was observed to cause edema in the heart and yolk sac, and apoptosis in the anterior region of the developing zebrafish larvae; as visualized through acridine orange and TUNEL staining. In addition, ethalfluralin increased the expression of the apoptosis-associated genes including tp53, cyc1, casp8, casp9, and casp3. The Seahorse Mito Stress analysis revealed that ethalfluralin slightly reduced mitochondrial respiration in live zebrafish embryos. Reactive oxygen species (ROS) production was also observed to be elevated in zebrafish larvae in response to ethalfluralin. Treatment with ethalfluralin decreased blood vessel formation in brain and intestine in flk1 transgenic zebrafish embryos. The decrease in angiogenesis related gene expression was specifically observed in vegfc, flt1, and kdrl, and in the intestinal vasculature related genes apoa4a, aqp3, fabp2, and vil1. Moreover, an increase in inflammatory genes such as cox2a, cox2b, cxcl-c1c, il8, mcl1a, mcl1b, and nf-κb was observed using real-time PCR analysis. Collectively, these results indicate that oxidative stress generated by exposure to ethalfluralin induced ROS generation, apoptosis, inflammation and anti-angiogenic effects, and therefore, ethalfluralin may be toxic to the development of zebrafish embryos.

Immunosenescence in atherosclerosis: A role for chronic viral infections.

Immune system is a versatile and dynamic body organ which offers survival and endurance of human beings in their hostile living environment. However, similar to other cells, immune cells are hijacked by senescence. The ageing immune cells lose their beneficial functions but continue to produce inflammatory mediators which draw other immune and non-immune cells to the senescence loop. Immunosenescence has been shown to be associated with different pathological conditions and diseases, among which atherosclerosis has recently come to light. There are common drivers of both immunosenescence and atherosclerosis; e.g. inflammation, reactive oxygen species (ROS), chronic viral infections, genomic damage, oxidized-LDL, hypertension, cigarette smoke, hyperglycaemia, and mitochondrial failure. Chronic viral infections induce inflammaging, sustained cytokine signaling, ROS generation and DNA damage which are associated with atherogenesis. Accumulating evidence shows that several DNA and RNA viruses are stimulators of immunosenescence and atherosclerosis in an interrelated network. DNA viruses such as CMV, EBV and HBV upregulate p16, p21 and p53 senescence-associated molecules; induce inflammaging, metabolic reprogramming of infected cells, replicative senescence and telomere shortening. RNA viruses such as HCV and HIV induce ROS generation, DNA damage, induction of senescence-associated secretory phenotype (SASP), metabolic reprogramming of infected cells, G1 cell cycle arrest, telomere shortening, as well as epigenetic modifications of DNA and histones. The newly emerged SARS-CoV-2 virus is also a potent inducer of cytokine storm and SASP. The spike protein of SARS-CoV-2 promotes senescence phenotype in endothelial cells by augmenting p16, p21, senescence-associated β-galactosidase (SA-β-Gal) and adhesion molecules expression. The impact of SARS-CoV-2 mega-inflammation on atherogenesis, however, remains to be investigated. In this review we focus on the common processes in immunosenescence and atherogenesis caused by chronic viral infections and discuss the current knowledge on this topic.

Cuprous Oxide Nanoparticles Decorated Fabric Materials with Anti-biofilm Properties.

Considering the global spread of bacterial infections, the development of anti-biofilm surfaces with high antimicrobial activities is highly desired. This work unraveled a simple, sonochemical method for coating Cu2O nanoparticles (NPs) on three different flexible substrates: polyester (PE), nylon 2 (N2), and polyethylene (PEL). The introduction of Cu2O NPs on these substrates enhanced their surface hydrophobicity, induced ROS generation, and completely inhibited the growth of sensitive (Escherichia coli and Staphyloccocus aureus) and drug-resistant (MDR E. coli and MRSA) planktonic and biofilm. The experimental results confirmed that Cu2O-PE exhibited complete biofilm mass reduction ability for all four strains, whereas Cu2O-N2 showed more than 99% biomass inhibition against both drug-resistant and sensitive pathogens in 6 h. Moreover, Cu2O-PEL also indicated a 99.95, 97.73, 98.00, and 99.20% biomass reduction of MRSA, MDR E. coli, E. coli, and S. aureus, respectively. All substrates were investigated for time-dependent inhibitions, and the associated biofilm mass and log reduction were evaluated. The mechanisms of Cu2O NP action against the mature biofilms include the generation of reactive oxygen species (ROS) as well as electrostatic interaction between Cu2O NPs and bacterial membranes. The current study could pave the way for the commercialization of sonochemically coated Cu2O NP flexible substrates for the prevention of microbial contamination in hospitals and industrial environments.

Anticancer activity of a novel naringin‒dextrin nanoformula: Preparation, characterization, and in vitro induction of apoptosis in human hepatocellular carcinoma cells by inducing ROS generation, DNA fragmentation, and cell cycle arrest

Dextrin is a polysaccharide but is small and of low complexity. Naringin is a flavonoid widely known for its pharmacological properties. This study aimed to create a naringin–dextrin nanoformula (NDN) to improve the effects of free naringin against human hepatocellular carcinoma cell lines (HepG2). Transmission electron microscopy, particle size distribution, entrapment efficiency, X-ray diffraction, and Fourier transform infrared spectroscopy were used to characterize NDN. The findings showed that the biological activity of naringin and NDN may be enhanced by inducing reactive oxygen species (ROS) generation and anti-inflammatory reactions mediated by decreased expressions of nuclear factor kappa B and interleukin-8. These changes induced apoptosis via a decrease in the expression levels of B-cell lymphoma-2 and an increase in the expression levels of Bcl-2-associated X protein, caspase-3, caspase-9, p53, and programmed cell death 5. Moreover, the results revealed a decrease in the expressions of isoleucine–glutamine motif-containing GTPase-activating protein 1, isoleucine–glutamine motif-containing GTPase-activating protein 3, and Ras signaling and an increase in the expression of isoleucine–glutamine motif-containing GTPase-activating protein 2. Thus, NDN improved naringin's therapeutic action against HepG2 by activating anti-inflammatory responses and inducing apoptosis via ROS generation, DNA fragmentation, and cell cycle arrest.

ALL-TRANS RETINOIC ACID ALLEVIATES ARSENIC-INDUCED ENDOCRINE DISRUPTION IN SWISS ALBINO MICE

Exposure to endocrine-disrupting chemicals (EDCs), such as arsenic, leads to severe health problems and surreptitiously accentuates stressful conditions in humans. Additionally, arsenic-induced endocrine stress leads to severe disturbances in glucose metabolism, mostly by disrupting the structure-function adroitness of the adrenal, thyroid and pancreas. Mechanistically, elevated levels of reactive oxygen species (ROS) generated by arsenic accentuate perturbation of cellular redox balance and eventually cell death. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, is known for its anti-oxidant properties. Therefore, ATRA was used as a protection against arsenic-induced deteriorations of physiological conditions in mice. The present study reveals arsenic induced ROS generation in the adrenal and thyroid glands, accompanied by a decline in the activities of ROS scavenging enzymes, leading to disruption of the architecture of the tissues and induction of apoptosis therein, culminating in an imbalance in the hormonal secretions from the respective tissues. Structural damage to the pancreas accompanied with severe imbalances in glucose metabolism and associated biochemical parameters like glucose, pancreatic amylase and liver glycogen were also noted. Treatment with ATRA could efciently reverse the deleterious effects induced by arsenic. Hence, ATRA can be used as an efcient nutraceutical which can lead to attenuation of endocrine stress induced by arsenic.

MicroRNA-21 guide and passenger strand regulation of adenylosuccinate lyase-mediated purine metabolism promotes transition to an EGFR-TKI-tolerant persister state

In EGFR-mutant lung cancer, drug-tolerant persister cells (DTPCs) show prolonged survival when receiving EGFR tyrosine kinase inhibitor (TKI) treatments. They are a likely source of drug resistance, but little is known about how these cells tolerate drugs. Ribonucleic acids (RNAs) molecules control cell growth and stress responses. Nucleic acid metabolism provides metabolites, such as purines, supporting RNA synthesis and downstream functions. Recently, noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), have received attention due to their capacity to repress gene expression via inhibitory binding to downstream messenger RNAs (mRNAs). Here, our study links miRNA expression to purine metabolism and drug tolerance. MiR-21-5p (guide strand) is a commonly upregulated miRNA in disease states, including cancer and drug resistance. However, the expression and function of miR-21-3p (passenger strand) are not well understood. We found that upregulation of miR-21-5p and miR-21-3p tune purine metabolism leading to increased drug tolerance. Metabolomics data demonstrated that purine metabolism was the top pathway in the DTPCs compared with the parental cells. The changes in purine metabolites in the DTPCs were partially rescued by targeting miR-21. Analysis of protein levels in the DTPCs showed that reduced expression of adenylosuccinate lyase (ADSL) was reversed after the miR-21 knockdown. ADSL is an essential enzyme in the de novo purine biosynthesis pathway by converting succino-5-aminoimidazole-4-carboxamide riboside (succino-AICAR or SAICAR) to AICAR (or acadesine) as well as adenylosuccinate to adenosine monophosphate (AMP). In the DTPCs, miR-21-5p and miR-21-3p repress ADSL expression. The levels of top decreased metabolite in the DTPCs, AICAR was reversed when miR-21 was blocked. AICAR induced oxidative stress, evidenced by increased reactive oxygen species (ROS) and reduced expression of nuclear factor erythroid-2-related factor 2 (NRF2). Concurrently, miR-21 knockdown induced ROS generation. Therapeutically, a combination of AICAR and osimertinib increased ROS levels and decreased osimertinib-induced NRF2 expression. In a MIR21 knockout mouse model, MIR21 loss-of-function led to increased purine metabolites but reduced ROS scavenging capacity in lung tissues in physiological conditions. Our data has established a link between ncRNAs, purine metabolism, and the redox imbalance pathway. This discovery will increase knowledge of the complexity of the regulatory RNA network and potentially enable novel therapeutic options for drug-resistant patients.

Miltirone Attenuates Reactive Oxygen Species-Dependent Neuronal Apoptosis in MPP+-Induced Cell Model of Parkinson's Disease Through Regulating the PI3K/Akt Pathway.

Miltirone is a phenanthrene-quinone derived from Salvia miltiorrhiza Bunge with anti-inflammatory and anti-oxidant effects. Our study aimed to explore the protective effect of miltirone on 1-methyl-4-phenylpyridinium (MPP+)-induced cell model of Parkinson's disease (PD). PharmMapper database was employed to predict the targets of miltirone. PD-related genes were identified using GeneCards database. The overlapping genes between miltirone and PD were screened out using Venn diagram. KEGG analysis was performed using DAVID and KOBAS databases. Cell viability, reactive oxygen species (ROS) generation, apoptosis, and caspase-3 activity were detected by CCK-8 assay, a ROS assay kit, TUNEL, and caspase-3 activity assay, respectively. Effect of miltirone on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway was explored by western blot analysis. A total of 214 targets of miltirone and 372 targets related to PD were attained, including 29 overlapping targets. KEGG analysis demonstrated that the 29 overlapping targets were both significantly enriched in the PI3K/Akt pathway. MPP+ stimulation reduced the cell viability in SH-SY5Y cells and neuronal primary cultures derived from human brain. Miltirone or N-acetylcysteine (NAC) attenuated MPP+-induced reduction in cell viability, ROS production, SOD activity reduction, apoptosis, and increase of caspase-3 activity. Additionally, miltirone recuperated MPP+-induced inactivation of the PI3K/Akt pathway. Moreover, treatment with LY294002, an inhibitor of the PI3K/Akt pathway, reversed the inhibitory effect of miltirone on MPP+-induced ROS generation and apoptosis in SH-SY5Y cells and neuronal primary cultures. In conclusion, miltirone attenuated ROS-dependent apoptosis in MPP+-induced cellular model of PD through activating the PI3K/Akt pathway.

Hyperthermia increases HSP production in human PDMCs by stimulating ROS formation, p38 MAPK and Akt signaling, and increasing HSF1 activity

BackgroundHuman placenta-derived multipotent cells (hPDMCs) are isolated from a source uncomplicated by ethical issues and are ideal for therapeutic applications because of their capacity for multilineage differentiation and proven immunosuppressive properties. It is known that heat shock preconditioning induces the upregulation of heat shock proteins (HSPs), which enhance survival and engraftment of embryonic stem cells (ESCs) during transplantation in live animal models, although whether heat shock preconditioning has the same effects in hPDMCs is unclear.MethodsThe hPDMCs were isolated from placenta of healthy donors. The cells were treated with heat shock (43 °C, 15 min), followed by evaluation of cell viability. Furthermore, the HSPs expression was assessed by Western blot, qPCR. The reactive oxygen species (ROS) production and signal pathway activation were determined by flow cytometry and Western blot, respectively. The regulatory pathways involved in HSPs expression were examined by pretreatment with chemical inhibitors, and siRNAs of MAPK, Akt, and heat shock factor 1 (HSF1), followed by determination of HSPs expression.ResultsThis study demonstrates that heat shock treatment induced ROS generation and HPSs expression in hPDMCs. Heat shock stimulation also increased p38 MAPK and Akt phosphorylation. These effects were reduced by inhibitors of ROS, p38 MAPK and Akt. Moreover, we found that heat shock treatment enhanced nuclear translocation of the HSF1 in hPDMCs, representing activation of HSF1. Pretreatment of hPDMCs with ROS scavengers, SB203580 and Akt inhibitors also reduced the translocation of HSF1 induced by heat shock.ConclusionsOur data indicate that heat shock acts via ROS to activate p38 MAPK and Akt signaling, which subsequently activates HSF1, leading to HSP activation and contributing to the protective role of hPDMCs.

Effects of arbutin on glucose uptake by glucose transporter 4 (GLUT4) and its cytoprotective properties in L6 skeletal muscle cell line.

It has been well known that oxidative stress and increased intracellular reactive oxygen species (ROS) have a pivotal role in disrupting the insulin signaling pathways leading to cellular insulin resistance. In this study, we evaluated arbutin's effects on glucose uptake by GLUT4 and cytoprotective properties in the L6 skeletal muscle cell line. The effect of arbutin and tertiary butyl hydrogen peroxide(t-BHP) on glucose uptake in cultured L6 cells was investigated by flow cytometry. We also evaluated gene expression levels of GLUT1 and GLUT4 in the L6 cells by quantitative real-time polymerase chain reaction analysis. The results from the study demonstrated that the optimum ROS generation occurred 3 h after 100 µM t-BHP treatment and pretreatment with arbutin (500 and 1000 µM) significantly inhibited the t-BHP induced ROS generation (p < .05). Our result indicated that 3 h pretreatment of L6 cells with 1000 μM of arbutin before 50 μM t-BHP significantly increased glucose uptake than the 50 μM t-BHP alone group (p < .05). Our findings may suggest that an increase in the uptake of 2-NBDG by L6 cells with arbutin pretreatment can be associated with increased expression of GLUT4 and GLUT1 under oxidative stress.

NADPH Oxidase 1 Mediates Acute Blood Pressure Response to Angiotensin II by Contributing to Calcium Influx in Vascular Smooth Muscle Cells.

Reactive oxygen species (ROS) and calcium ions (Ca2+) are among the major effectors of Ang II (angiotensin II) in vascular smooth muscle cells. ROS are related to Ca2+ signaling or contraction induced by Ang II, but little is known about their detailed functions. Here, NOX (NADPH oxidase), a major ROS source responsive to Ang II, was investigated regarding its contribution to Ca2+ signaling. Vascular smooth muscle cells were primary cultured from rat aorta. Ca2+ and ROS were monitored mainly using fura-2 and HyPer family probes' respectively. Signals activating NOX were examined with relevant pharmacological inhibitors and genetic manipulation techniques. Ang II-induced ROS generation was found to be biphasic: the first phase of ROS production, which was mainly mediated by NOX1, was small and transient, preceding a rise in Ca2+, and the second phase of ROS generation, mediated by NOX1 and NOX4, was slow but sizeable, continuing over tens of minutes. NOX1-derived superoxide in the first phase is required for Ca2+ influx through nonselective cation channels. AT1R (Ang II type 1 receptor)-Gβγ-PI3Kγ (phosphoinositide 3-kinase γ) signaling pathway was responsible for the rapid activation of NOX1 in the first phase, while in the second phase, NOX1 was further activated by a separate AT1R-Gαq/11-PLC (phospholipase C)-PKCβ (protein kinase C β) signaling axis. Consistent with these observations, aortas from NOX1-knockout mice exhibited reduced contractility in response to Ang II, and thus the acute pressor response to Ang II was also attenuated in NOX1-knockout mice. NOX1 mediates Ca2+ signal generation and thereby contributes to vascular contraction and blood pressure elevation by Ang II.

Pretreatment of hydroethanolic extract of Dillenia indica L. attenuates oleic acid induced NAFLD in HepG2 cells via modulating SIRT-1/p-LKB-1/AMPK, HMGCR & PPAR-α signaling pathways

Ethnopharmacological relevanceDillenia indica L. is an edible plant from the Dilleniaceae family present in the forest of India and other Asian countries. Different parts of this plant are being used in the traditional system of medicines for various diseases like diabetes, indigestion, asthma, jaundice, and rheumatic pain by various rural communities. This plant is very common among Khamptis traditional healers, the rural community of the Dhemaji district of Assam, ethnic communities of Dibru-Saikhowa Biosphere Reserve of Northeast, India for various medicinal uses. It is observed as a ‘vat’ suppressant and ‘pitta’ boosting medicine in Ayurveda. Aim of the studyThe aim of this research was to evaluate the effect of hydroethanolic extract of Dillenia indica leaf (DI-HET) against non-alcoholic fatty liver disease (NAFLD) as it is reported effective against jaundice in traditional medicine. We are also planning to see the various molecular mechanisms responsible for its effect if it is efficacious. Study design/methodAn in vitro model for NAFLD was employed in this study. For this HepG2 cells were incubated with 100 μM of oleic acid (OA) for 24 h. For evaluation of the effect of DI-HET, the extracts (5 or 10 μg/mL) were pretreated to the OA group. Fenofibrate was the positive control. Various parameters relevant to lipogenesis and β-oxidation of fatty acids like intracellular lipid accumulation, reactive oxygen species (ROS), mitochondrial stress, and key proteins were studied. ResultsDI-HET significantly reduced the intracellular lipid accumulation in OA treated cells. And also substantially decreased the expression of lipogenic proteins and increased β-oxidation in the OA group. OA induced ROS generation was found to reduce with DI-HET treatment. Western blot analysis showed that the expression of LXR-α, SREBP-1C, SREBP-2, HMGCR, FAS, CD-36, and ACOX-1 were downregulated while that of SIRT-1, p-LKB-, p-AMPK, p-ACC, CPT-1, and PPAR-α upregulated in DI-HET treatment. LCMS/MS analysis showed the presence of polyphenols like naringenin, catechin, epicatechin, shikimic acid, syringic acid, vanillic acid, and kaempferol. ConclusionThese results suggest that DI-HET is effective against NAFLD by activation of the SIRT-1/p-LKB-1/AMPK signaling pathway via polyphenols present in the extract.

TSLP regulates mitochondrial ROS-induced mitophagy via histone modification in human monocytes

BackgroundThymic stromal lymphopoietin (TSLP) is a Th2-like cytokine involved in asthma pathogenesis. Excessive reactive oxygen species (ROS) production can lead to airway inflammation, hyperresponsiveness and remodeling. Mitophagy, followed by ROS production, is the selective degradation of mitochondria by autophagy and often occurs in defective mitochondria. In the present study, we aimed to examine the effects of TSLP on ROS production and mitophagy in human monocytes and to investigate the underlying mechanisms, including epigenetic regulation.ResultsTSLP induced ROS generation, and the effects were reversed by the antioxidant N-acetylcysteine (NAC) in THP-1 cells. Transmission electron microscopy images showed donut-shaped mitochondria that lost the cristae ultrastructure after TSLP stimulation. A decrease in mitochondrial membrane potential, decreased MTCO2 expression, and increased mitochondrial DNA release after TSLP stimulation were found. TSLP enhanced mitochondrial complex I and complex II/III activity and increased mitochondrial copy numbers and the expression of the complex II SHDA gene. TSLP-induced SHDA expression was inhibited by the histone acetyltransferase inhibitor anacardic acid (AA) and the histone methyltransferase inhibitor methylthioadenosine (MTA), and chromatin immunoprecipitation assays revealed that TSLP enhanced H3 acetylation, H4 acetylation, and H3K4 and H3K36 trimethylation in the SHDA promoter. Confocal laser microscopy showed that TSLP treatment increased the signals of the mitophagy-related proteins PINK1, LC3, phospho-parkin and phospho-ubiquitin, and pretreatment with AA and MTA reduced TSLP-induced PINK1 and LC3 accumulation in mitochondria. Western blot analysis showed that TSLP significantly increased phosphor-AMPK signal intensity, and the effects were inhibited by the antioxidant NAC. The increased signal intensities of the mitophagy-related proteins PINK1, Parkin and LC3 I/II were decreased by dorsomorphin, an AMPK inhibitor. TSLP decreased M1-related cytokine CXCL-10 production and increased M2-related cytokine CCL-1 and CCL-22 production, which was suppressed by the mitophagy inhibitor Mdivi-1 and PINK1 gene knockdown.ConclusionsEpithelial-derived TSLP regulates ROS production and mitophagy through AMPK activation and histone modification and alters M1/M2 chemokine expression in human monocytes.Graphical

Hydrogen-Rich Water Prevents Dehydration-Induced Cellular Oxidative Stress and Cell Death in Human Skin Keratinocytes

Hypohydration is linked to increased risk of a variety of diseases and can be life-threatening, especially in elderly populations. Dehydration induces cellular damage partially through the production of reactive oxygen species (ROS) in cells, tissues and organs. Hydrogen molecules are known to convert ROS to harmless water. Therefore, theoretically hydrogen-rich water (HW) might eliminate dehydration-induced ROS and reverse its harmful effects in cells. In this in vitro study, we demonstrated that air-drying for 5 min could induce ROS generation in both nucleus and cytoplasm of human keratinocytes HaCaT as quantified by CellROX® Green/Orange reagents (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.), respectively. Conversely, when the air-drying time was increased to 10 and 20 min, HaCaT cells lost the ability to produce ROS. Scanning electron microscopic (SEM) images showed that 10 min air-drying could induce severe membrane damage in HaCaT cells. PrestoBlue assay showed that, when HaCaT cells were air-dried for 20 min, cell viability was decreased to 27.6% of the control cells 48 h later. However, once HaCaT cells were pretreated with HW-prepared media, dehydration-induced intracellular ROS, cell membrane damage and cell death were significantly reduced as compared with double distilled water (DDW) under the same conditions. In conclusion, our data suggested that HW can decrease dehydration-induced harmful effects in human cells partially through its antioxidant capacity.

Levistolide A Inhibits PEDV Replication via Inducing ROS Generation.

Porcine epidemic diarrhea virus (PEDV) variant strains adversely affect the production of pigs globally. Vaccines derived from PEDV traditional strains impart less protection against the variant strains. Moreover, sequence diversity among different PEDV variant strains is also complicated. This necessitates developing alternative antiviral strategies for defending against PEDV. This study explored a natural product, Levistolide A (LA), to possess antiviral activity against PEDV. LA was found to suppress PEDV replication in a dose-dependent manner. And the inhibitory effect of LA against PEDV was maintained in the course of time. In terms of viral RNA and protein production, LA also showed a strong inhibitory effect. In addition, LA was indicated to inhibit PEDV from attaching to the cellular membrane or penetrating the cells. Further study revealed that LA can induce the generation of reactive oxygen species (ROS), and the corresponding inhibitor, NAC, was found to antagonize the effect of LA on inhibiting PEDV replication. This illustrated that the LA-induced ROS generation played an important role in its anti-PEDV activity. LA was also identified to stimulate ER stress, which is an important consequence of ROS production and was proven to be able to inhibit PEDV replication. To conclude, this study revealed that LA can inhibit PEDV replication via inducing ROS generation.