Prevented DNA Fragmentation Research Articles

MicroRNA-512-3p mediates Trypanosoma cruzi-induced apoptosis during ex vivo infection of human placental explants

IntroductionUpon infection, Trypanosoma cruzi, a protozoan parasite, crosses the placental barrier and causes congenital Chagas disease. Ex vivo infection of human placental explants (HPEs) with the parasite induces apoptotic cell death. This cellular process involves changes in gene expression, which are partially regulated by miRNAs. In this study, we investigated the role of miR-512-3p, a highly expressed miRNA in the placenta, in parasite-induced apoptosis. MethodsHPE cells were transfected with antagomirs or mimics of miR-512-3p and subsequently challenged with the parasite. The expression levels of miR-512-3p, caspase 3, caspase 8, and Livin were measured using RT-qPCR, and apoptotic cell death was analyzed based on caspase activity and DNA fragmentation assays. ResultsTargeted inhibition of miR-512-3p effectively prevented parasite-induced expression and enzymatic activity of caspase 3 and caspase 8. However, it did not completely prevent DNA fragmentation, indicating the involvement of other factors in this process. Furthermore, the findings suggest that Livin may be regulated by miR-512-3p. DiscussionOur findings suggest that miR-512-3p modulates parasite-induced apoptosis in the trophoblast. By understanding the mechanisms involved in this process, we can gain insights into the pathogenesis of congenital Chagas disease and develop targeted therapeutic strategies.

Curcumin modulation of L-dopa and rasagiline-induced neuroprotection in rotenone model of Parkinson's disease.

Parkinson's disease (PD) is one of the most incurable, chronic, and progressive neurodegenerative disorders Worldwide. Curcumin, a natural polyphenolic anti-oxidant compound, has a long history in traditional medicine. We investigate the effect of curcumin on brain oxidative stress, DNA fragmentation, and motor changes in rotenone-induced PD in mice. The possible modulation of the anti-parkinsonian action of drugs L-dopa and rasagiline by curcumin was also studied. Mice received rotenone 1.5 mg/kg and were treated with curcumin (150 mg/kg), L-dopa (25 mg/kg), rasagiline (1 mg/kg), L-dopa+curcumin, or rasagiline+curcumin. Striatal malondialdehyde, reduced glutathione, nitric oxide, tyrosine hydroxylase, and brain DNA fragmentations were measured. Histopathological examination of brain tissues was done. Motor coordination and behavioral tests such as wire-hanging, stair, and wood-waking tests were included. Rotenone caused elevation in brain malondialdehyde and nitric oxide contents, depletion of reduced glutathione accompanied by a reduction in rearing behavior, and impairment of motor activity in wire-hanging, stair, and wood-waking tests. Also, severe DNA fragmentation in the striatum, marked decrease of substantia nigra pigmented neurons, neuronal degeneration in the cerebral cortex and hippocampus, decreased glial fibrillary acidic protein reaction (GFAP) and glial cell size in the cerebral cortex were caused by rotenone. In rotenone-treated mice, brain oxidative stress was decreased by curcumin, L-dopa, rasagiline, curcumin+L-dopa, and curcumin+rasagiline. These treatments also prevented DNA fragmentation and markedly improved the motor and behavioral impairment caused by rotenone. Rotenone-induced histopathological changes were ameliorated by curcumin which had an additive effect to that of l-dopa or rasagiline. These data indicate that curcumin showed additive neuroprotective effects to L-dopa or rasagiline and ameliorated neurodegeneration, DNA fragmentation, and motor defects caused by rotenone in mice.

Short Communication: The use of lactose-astaxanthin to maintain the quality of green junglefowl frozen semen

Abstract. Bebas W, Agustina KK. 2022. Short Communication: The use of lactose-astaxanthin to maintain the quality of green junglefowl frozen semen.Biodiversitas 23: 5759-5764. The Green Junglefowl is Indonesian germplasm that is at risk of population decline. Therefore, efforts are needed to protect its existence and survival. Cryopreservation of spermatozoa in the form of frozen semen is required to preserve this germplasm. This research aims to determine the effect of lactose-astaxanthin in preventing DNA fragmentation and intact plasma membrane of Green Junglefowl spermatozoa during the freezing process. A completely randomized design with a 2x4 factorial pattern, including two treatment groups and four dimethylsulfoxide (DMSO) concentration factors, was used. The treatment groups are T1 and T2, consisting of egg yolk phosphate diluent and egg yolk phosphate diluent added with a combination of 0.6% lactose-0.004% astaxanthin, respectively. The four DMSO concentration factors were 4%, 6%, 8%, and 10% respectively. Subsequently, each treatment was repeated four times. The DNA fragmentation and intact plasma membrane of spermatozoa were observed, and the data obtained afterward were analyzed for variance, followed by the Ducan test. The results showed that the addition of 0.6% lactose-0.004% astaxanthin with 6% DMSO had a significantly better effect (P<0.05) on the quality of post-thawing spermatozoa with 19.75±0.96% DNA fragmentation and 53.75±3.86% intact plasma membranes. Therefore, it is recommended that a combination of 0.6% lactose-0.004% astaxanthin at 6% DMSO concentration should be added to Green Junglefowl semen during the freezing process to prevent DNA fragmentation and intact plasma membranes.

Nano-Curcumin Prevents Cardiac Injury, Oxidative Stress and Inflammation, and Modulates TLR4/NF-κB and MAPK Signaling in Copper Sulfate-Intoxicated Rats.

Copper (Cu) is essential for a plethora of biological processes; however, its high redox reactivity renders it potentially toxic. This study investigated the protective effect of curcumin (CUR) and nano-CUR (N-CUR) against Cu cardiotoxicity, emphasizing the role of oxidative stress, TLR4/NF-κB and mitogen-activated protein kinase (MAPK) signaling and cell death in rats. Rats received 100 mg/kg copper sulfate (CuSO4), a pesticide used for repelling pests, and were concurrently treated with CUR or N-CUR for 7 days. Cu caused cardiac injury manifested by elevated serum cardiac troponin I (cTnI), creatine kinase (CK)-MB, and lactate dehydrogenase (LDH), as well as histopathological alterations. Cardiac malondialdehyde (MDA), NF-κB p65, TNF-α, and IL-6 were increased, and reduced glutathione (GSH), superoxide dismutase (SOD) and catalase were decreased in Cu-treated rats. CUR and N-CUR prevented cardiac tissue injury, decreased serum cTnI, CK-MB, and LDH, and cardiac MDA, NF-κB p65, TNF-α, and IL-6, and enhanced cellular antioxidants. CUR and N-CUR downregulated TLR4 and AP-1, and decreased the phosphorylation levels of p38 MAPK, JNK, and ERK1/2. In addition, CUR and N-CUR increased cardiac Bcl-2 and BAG-1, decreased Bax and caspase-3, and prevented DNA fragmentation. In conclusion, N-CUR prevents Cu cardiotoxicity by attenuating oxidative injury, inflammatory response, and apoptosis, and modulating TLR4/NF-κB and MAPK signaling. The cardioprotective effect of N-CUR was more potent than the native form.

Nuciferine Attenuates Doxorubicin-Induced Cardiotoxicity: An In Vitro and In Vivo Study.

Chemotherapeutic drugs are a known factor that impairs the system of life due to their severe side effects. A more worrying fact is that the patients administered with doxorubicin fall under the risk of cardiotoxicity. The evolution of exploring plant-derived compounds is a possible way to combat health issues in therapeutic applications. Hence, this study focuses on the protective effect of plant-based compound nuciferine (NFN) against doxorubicin-induced cardiotoxicity in both in vitro and in vivo models. In this investigation, nuciferine significantly reduces DOX-mediated cardiotoxicity by mitigating reactive oxygen species, thereby preventing DNA fragmentation, regulating apoptosis genes and reducing the caspase 3/7 levels in vitro. Besides, nuciferine has shown significant protection against DOX-induced cardiac impairment and the upregulation of cardiogenic markers in vivo. The DOX-induced oxidative stress can be mitigated via enhancing the endogenous antioxidants, thereby controlling ROS-mediated apoptosis. In virtue of these potential features, nuciferine can be a budding candidate to address therapeutic needs.

Betulinic acid purified from Alstonia boonei inhibits folate biosynthesis in malarial Plasmodium, enhances mitochondrial pore opening and F1F0 ATPase in mice

Activity-guided assay of antimalarial properties of Alstonia boonei led to the purification of betulinic acid from the chloroform fraction of the methanol stem bark extract. The structure and stereochemistry of betulinic acid from A. boonei was established by 1H, 13C, COSYHMBC, HSQC, LC-MS and DEPT techniques. This compound demonstrated antiplasmodial activities using chloroquine susceptible (NK 65) and resistant (ANKA) strains of Plasmodium berghei. Betulinic acid (BA) opens mitochondrial permeability transition pore (mPT), increased mitochondrial F1F0 ATPase activity, decreased lipid peroxidation and prevented DNA fragmentation. Molecular dynamics simulation studies against falcipain-2, dihydrofolate reductase, dihydropteroate synthase and mitochondrial F1F0 ATPase (F-2-BA, DHFR-BA, DHPS-BA and mATPase-BA, respectively) revealed that betulinic acid (BA) mechanistically disrupted the secondary structure of mATPase (mouse) with RMSD > 2 (RMSD=2.34 Å). Binding effects of BA was more prominent on DHFR as evidenced by high structural deviations (RMSD = 2.24 Å). However, Falcipain-2 had the least deviation (RMSD = 1.77 Å) correlative with the reduced activity of the compound. DHPS, on the other hand, had a mean RMSD value of 1.93 Å. The disruption of mATPase could explain the effect of the compound on the opening of mPT as experimentally reported.

The effects of luteinizing hormone as a suppression factor for apoptosis in bovine luteal cells in vitro.

The fate of the corpus luteum, a transient endocrine gland formed and degraded during an oestrous cycle, is decided by various physiological factors, such as luteinizing hormone (LH). As a stimulator of progesterone, LH is known to maintain corpus luteum functional and structural integrity by inhibiting apoptosis, a programmed cell death. Therefore, we aim to investigate its action during the mid-luteal phase hypothesized that LH suppresses the death mechanism of bovine luteal steroidogenic cells (LSC) by analysing the expression of proteins involved. Cultured bovine LSC obtained from corpus luteum were treated for 24hr with recombinant TNF and IFNG in the presence or absence of LH. The result showed that LH proved to have a protective effect by increased cell viability (p<.05) and prevented DNA fragmentation (p<.05), as demonstrated by the WST-1 colorimetric assay and TUNEL assay. Expression analysis of mRNA and protein levels showed that LH altered the expression ofBCL2 (p<.05), CASP3 (p<.05), FAS (p<.05),andBAX(p<.05) to support cell survival. In conclusion, our study suggests that LH prolongs the corpus luteum life span through the anti-apoptotic mechanism by increasing cell viability and suppressing apoptosis-related genes and protein expression.

Cytoprotective effect of ethyl acetate fraction from Ephedra fragilis on H2O2-induced oxidative damage in Tetrahymena pyriformis

The main purpose of the present study was to investigate the ability of ethyl acetate fraction (EAF) from Ephedra fragilis to function as a protective agent against hydrogen peroxide induced oxidative damage in Tetrahymena pyriformis. The cells were preincubated with EAF (50-200 μg/mL) or ascorbic acid (50 μg/mL) for 24 h, followed by incubation with 50% H2O2 inhibitory concentration for 48 h. Cell viability was assessed using trypan exclusion method. Cell morphology and mobility, antioxidant enzymes activities (catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR)), malondialdehyde (MDA) and protein carbonyl (PCO) levels, DNA fragmentation and metabolic enzymes activities (succinate dehydrogenase (SDH) and NADPH-cytochrome c reductase (NCCR)) were investigated. Our results indicate that, pretreatment of T. pyriformis cells with EAF improved the cell viability, restored normal cell mobility and morphology, decreased the levels of both MDA and PCO level, prevent DNA fragmentation and enhanced the activity of antioxidant (CAT, SOD and GR) and metabolic (SDH and NCCR) enzymes in H2O2 damaged cells. In conclusion, these results suggest for the first time that E. fragilis is a promising source of natural antioxidants, that could offer protection against oxidative stress and should be further exploited for its use in clinical medicine.

A REDESIGN OF THE PROTEX TO HARVEST FREE OXYGEN SPECIES APPEARS TO IMPROVE OVERALL SEMEN PARAMETERS

Previous research from this laboratory has demonstrated that semen quality, both physiological and biochemical, can be improved via a simple redesign of the semen collection cup. The redesign termed the Device for Improved Semen Collection (DISC – trade name ProteX), maintains a stable environment during and after collection, preventing cell shock and preserving cell function. However, the original design did nothing to prevent DNA fragmentation. Recently the system underwent a redesign incorporating a proprietary method to harvest free oxygen radicals, an identified cause of DNA fragmentation.

Antileukemic Activity of 8-Chloro-Adenosine (8-Cl-Ado) Is Mediated By Mir-155 Degradation and ErbB3 Binding Protein (Ebp1)-Dependent p53 Activation: A Novel Therapeutic Approach for FLT3-ITD Acute Myeloid Leukemia (AML)

AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362]. As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values <1 at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90)]. In an orthotopic mouse model where FLT3-ITD-positive MV4-11 cells were xenografted, mice treated with 75 mg 8-Cl-Ado/kg/day plus quizartinib (0.5 mg/kg/day) survived significantly longer than mice treated with the individual agents or vehicle alone (median survival 54.2 days vs. 27.3 days for controls, p=0.016).Taken together, these results support 8-Cl-Ado as a promising novel agent via a unique mechanism of action for FLT3-ITD AML, mediated by miR-155 degradation. 8-Cl-Ado also synergizes with TKIs both in vitro and in vivo thereby representing a potentially novel clinical approach for FLT3-ITD AML. A single-agent phase I/II clinical trial in relapsed/refractory AML is already ongoing at our institution. [Display omitted] DisclosuresWennerberg:Novartis: Research Funding. Gandhi:Pharmacyclics: Research Funding.

Photobiomodulation prevents DNA fragmentation of alveolar epithelial cells and alters the mRNA levels of caspase 3 and Bcl-2 genes in acute lung injury

Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are defined as pulmonary inflammation that could occur from sepsis and lead to pulmonary permeability and alveolar edema making them life-threatening diseases. Photobiomodulation (PBM) properties have been widely described in the literature in several inflammatory diseases; although the mechanisms of action are not always clear, this could be a possible treatment for ARDS/ALI. Thus, the aim of this study was to evaluate the mRNA levels from caspase-3 and BCL-2 genes and DNA fragmentation in lung tissue from Wistar rats affected by ALI and subjected to photobiomodulation by exposure to a low power infrared laser (808 nm; 100 mW; 3.571 W cm-2; four points per lung). Adult male Wistar rats were randomized into 6 groups (n = 5, for each group): control, PBM10 (10 J cm-2, 2 J and 2 seconds), PBM20 (20 J cm-2, 5 J and 5 seconds), ALI, ALI + PBM10 and ALI + PBM20. ALI was induced by intraperitoneal Escherichia coli lipopolysaccharide injection. Lung samples were collected and divided for mRNA expression of caspase-3 and Bcl-2 and DNA fragmentation quantifications. Data show that caspase-3 mRNA levels are reduced and Bcl-2 mRNA levels increased in ALI after low power infrared laser exposure when compared to the non-exposed ALI group. DNA fragmentation increased in inflammatory infiltrate cells and reduced in alveolar cells. Our research shows that photobiomodulation can alter relative mRNA levels in genes involved in the apoptotic process and DNA fragmentation in inflammatory and alveolar cells after lipopolysaccharide-induced acute lung injury. Also, inflammatory cell apoptosis is part of the photobiomodulation effects induced by exposure to a low power infrared laser.

Zebra blenny protein hydrolysates as a source of bioactive peptides with prevention effect against oxidative dysfunctions and DNA damage in heart tissues of rats fed a cholesterol-rich diet

High-cholesterol diet (HCD) is suspected to produce in excess free radicals having adverse effects on human health and causing atherosclerosis damage in heart tissues. In our study, the effects of zebra blenny protein hydrolysates (ZBPHs) were investigated on cardiac oxidant/antioxidant status as well as DNA damage and histopathological disorders in rats, fed with a hypercholesterolemic diet. The molecular weight distribution of the hydrolysates was determined by size exclusion chromatography, which analyzed a representative hydrolysate type with a weight range of 3–20kDa. ZBPHs effectively protected heart genomic DNA against oxidative damage induced by Fenton's reagent. HCD promoted oxidative stress with a rise in serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities and thiobarbituric acid reactive substance (TBARS), advanced oxidation protein product (AOPP) and hydrogen peroxide (H2O2) levels in heart tissues. An increase in glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) activities as well as a fall in ATPase activities and glutathione (GSH) level was also noted in heart of hypercholesterolemic rats. Treatment with ZBPHs ameliorated the biochemical parameters cited above. In addition, pre-treatment with ZBPHs prevented DNA fragmentation. The histopathological findings confirmed the biochemical results and the potential antioxidant activities of ZBPHs which can help the cure and management of cardiovascular diseases induced by high-cholesterol levels.

Protective effect of a polysaccharide from Anoectochilus roxburghii against carbon tetrachloride-induced acute liver injury in mice

Ethnopharmacological relevanceAnoectochilus roxburghii (Wall.) Lindl. is traditionally used for the treatment of various types of chronic and acute hepatitis in China. Considering that Anoectochilus roxburghii polysaccharide (ARPT) is the main constituent of Anoectochilus roxburghii, the present study was designed to investigate the hepatoprotective effect of ARPT and its possible mechanism in carbon tetrachloride (CCl4)-induced mice. Material and methodsThe hepatoprotective activity of ARPT (150, 300 and 500mg/kg) were investigated on CCl4-induced acute liver damage in mice. The activities of alanine transaminase (ALT), aspartate transaminase (AST) were determined in serum. The hepatic levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured in liver homogenates. The levels of cytochrome P450 sub family 2E1 (CYP2E1), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), KC (Murine IL-8 ortholog), transforming growth factor-beta1 (TGF-β1), Bcl-2 and Bax were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of CYP2E1, nuclear factor-kappa B (NF-κB) p65 and caspase-3 were evaluated by western blot assays. The hepatic levels of TNF-α, IL-6, MIP-2 and TGF-β1 were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, histopathological observation and terminal-deoxynucleoitidyl transferase mediated nick end labeling assay (TUNEL) were carried out on the separated livers of mice. ResultsARPT significantly decreased serum ALT and AST activities, hepatic MDA level, and markedly enhanced antioxidant enzyme (SOD, CAT and GSH-Px) activities and GSH level in hepatic tissue, in a dose-dependent manner, when compared to the model group. Histopathological observation revealed the hepatoprotective effect of ARPT against the damage. Furthermore, ARPT remarkably inhibited CYP2E1 mRNA expression, decreased NF-κB p65 expression and therefore to prevent the secretion of pro-inflammatory cytokines (TNF-α and IL-6) and chemokines (MCP-1, MIP-2 and KC), suppressed TGF-β1 expression and hepatocytes apoptosis. Moreover, ARPT could prevent DNA fragmentation based on TUNEL assay results. ConclusionThese findings suggested that ARPT possessed hepatoprotective effect against CCl4-induced hepatotoxicity in mice and the action might in part be through reducing oxidative stress, inflammation and apoptosis.

Melatonin diminishes oxidative damage in sperm cells, improving assisted reproductive techniques.

Sperm preparation procedures are a potential generator of oxidative stress-induced DNA damage, which leads to a dramatic drop in fertility. An increasing number of studies suggest that melatonin reduces the oxidative stress induced by manipulation. However, very little is known about the preservative role of melatonin in sperm preparation medium during assisted reproduction procedures. For this aim to be achieved, semen was divided into two fractions and preincubated with and without 1 mM melatonin. Afterwards, both fractions were divided into two subfractions to perform swim-up in the presence and absence of 1 mM melatonin. Labeling with anti-CD46 and antiactive caspase-3 allowed the monitoring of acrosome reaction and apoptosis by flow cytometry. Sperm DNA fragmentation and compaction were analyzed through propidium iodide staining. The normozoospermic and oligozoospermic samples that were preincubated with melatonin underwent a significant increase in the ratio of adequate spermatozoa and a reduction of caspase-3 activation. Additionally, preincubation with melatonin enhanced the migration of sperm cells with compacted DNA in oligozoospermic samples (P < 0.05) and prevented DNA fragmentation in normozoospermic samples (P < 0.05). In light of the current results, the cytoprotective capacity and innocuousness of melatonin make it a great candidate to be applied in assisted reproduction techniques in order to prevent triaogenic oxidative damage.

Cytolethal Distending Toxin From Campylobacter jejuni Requires the Cytoskeleton for Toxic Activity.

BackgroundCampylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus.ObjectivesThe aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus.MethodsCampylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA).ResultsThe results obtained showed that the eight strains of C. jejuni, including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated.ConclusionsThis work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA, cdtB and cdtC genes.

Silver Nanoparticle Based Codelivery of Oseltamivir to Inhibit the Activity of the H1N1 Influenza Virus through ROS-Mediated Signaling Pathways.

As the therapeutic agent for antiviral applications, the clinical use of oseltamivir is limited with the appearance of drug-resistant viruses. It is important to explore novel anti-influenza drugs. The antiviral activity of silver nanoparticles (AgNPs) has attracted increasing attention in recent years and was a possibility to be employed as a biomedical intervention. Herein, we describe the synthesis of surface decoration of AgNPs by using oseltamivir (OTV) with antiviral properties and inhibition of drug resistance. Compared to silver and oseltamivir, oseltamivir-modified AgNPs (Ag@OTV) have remarkable inhibition against H1N1 infection, and less toxicity was found for MDCK cells by controlled-potential electrolysis (CPE), MTT, and transmission electron microscopy (TEM). Furthermore, Ag@OTV inhibited the activity of neuraminidase (NA) and hemagglutinin (HA) and then prevented the attachment of the H1N1 influenza virus to host cells. The investigations of the mechanism revealed that Ag@OTV could block H1N1 from infecting MDCK cells and prevent DNA fragmentation, chromatin condensation, and the activity of caspase-3. Ag@OTV remarkably inhibited the accumulation of reactive oxygen species (ROS) by the H1N1 virus and activation of AKT and p53 phosphorylation. Silver nanoparticle based codelivery of oseltamivir inhibits the activity of the H1N1 influenza virus through ROS-mediated signaling pathways. These findings demonstrate that Ag@OTV is a novel promising efficient virucide for H1N1.

The carbonic anhydrase inhibitor methazolamide prevents amyloid beta-induced mitochondrial dysfunction and caspase activation protecting neuronal and glial cells in vitro and in the mouse brain

Mitochondrial dysfunction has been recognized as an early event in Alzheimer's disease (AD) pathology, preceding and inducing neurodegeneration and memory loss. The presence of cytochrome c (CytC) released from the mitochondria into the cytoplasm is often detected after acute or chronic neurodegenerative insults, including AD. The carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) was identified among a library of drugs as an inhibitor of CytC release and proved to be neuroprotective in Huntington's disease and stroke models. Here, using neuronal and glial cell cultures, in addition to an acute model of amyloid beta (Aβ) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aβ, we analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer's amyloid. MTZ prevented DNA fragmentation, CytC release and activation of caspase 9 and caspase 3 induced by Aβ in neuronal and glial cells in culture through the inhibition of mitochondrial hydrogen peroxide production. Moreover, intraperitoneal administration of MTZ prevented neurodegeneration induced by intra-hippocampal Aβ injection in the mouse brain and was effective at reducing caspase 3 activation in neurons and microglia in the area surrounding the injection site. Our results, delineating the molecular mechanism of action of MTZ against Aβ-mediated mitochondrial dysfunction and caspase activation, and demonstrating its efficiency in a model of acute amyloid-mediated toxicity, provide the first combined in vitro and in vivo evidence supporting the potential of a new therapy employing FDA-approved CAIs in AD.

Dioscin attenuates hepatic ischemia-reperfusion injury in rats through inhibition of oxidative-nitrative stress, inflammation and apoptosis.

Dioscin shows potent effects against liver damage in our previous studies; however, the action of it on hepatic ischemia-reperfusion (I/R) injury is still unknown. In the present article, the effects and possible mechanisms of dioscin against hepatic I/R injury were investigated. Seventy percent partial hepatic warm ischemia was induced in Wistar rats for 60 min followed by succedent reperfusion. In the prophylactic test, dioscin was administered intragastrically to the rats at doses of 20, 40, and 60 mg/kg once daily for seven consecutive days before I/R. In the therapeutic test, the rats received dioscin intragastrically at a dose of 60 mg/kg once 2 hr before I/R. We found that dioscin significantly decreased serum alanine aminotransferase and aspartate aminotransferase activities, increased survival rate of rats, and improved I/R-induced hepatocyte abnormality. In addition, dioscin obviously increased the levels of SOD, CAT, GSH-Px, GSH, decreased the levels of MDA, TNOS, iNOS, NO, and prevented DNA fragmentation caused by I/R injury. Further research indicated that dioscin markedly decreased the gene expressions of interleukin-1β, interleukin-6, tumor necrosis factor-α, intercellular adhesion molecule-1, MIP-1α, MIP-2, Fas, FasL, decreased the protein expressions of NF-κB, AP-1, COX-2, HMGB-1, CYP2E1, Bak, caspase-3, p53, PARP, Caspase-9, decreased the levels of JNK, ERK and p38 MAPKs phosphorylation, and upregulated the levels of Bcl-2 and Bcl-x. Our results suggest that dioscin has potent actions against hepatic I/R injury through suppression of inflammation, oxidative-nitrative stress, and apoptosis, which should be developed as a new drug to treat hepatic I/R injury in the future.

The protective role of ellagitannins flavonoids pretreatment against N-nitrosodiethylamine induced-hepatocellular carcinoma

Ellagitannins are esters of glucose with hexahydroxydiphenic acid; when hydrolyzed, they yield ellagic acid (EA), the dilactone of hexahydroxydiphenic acid. EA has been receiving the most attention, because it has potent antioxidant activity, radical scavenging capacity, chemopreventive and antiapoptotic properties. Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of liver, and accounts for as many as one million deaths worldwide in a year. The aim of the present study was to evaluate the antioxidant and chemopreventive efficiency of ellagic acid against N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in rats. Rats were classified into four groups as follows: normal control group, group injected i.p. with a single dose (200mg/kgb.wt.) of NDEA, third group daily administered orally EA with a dose of 50mg/kgb.wt. for 7days before and 14days after NDEA administration, and fourth group received a similar dose of EA for 21days after the dose of NDEA administration. The model of NDEA-injected hepatocellular carcinomic (HCC) rats elicited significant declines in liver antioxidant enzyme activities; glutathione peroxidase (GPX), gamma glutamyl transferase (γ-GT) and glutathione-S-transferase (GST), with a reduction in reduced glutathione (GSH) and serum total protein with concomitant significant elevations in tumor markers arginase and α-l-fucosidase, and liver enzymes; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and glutathione-S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PD), direct and total bilirubin. The oral administration of EA as a protective agent, produced significant increases in tested antioxidant enzyme activities and serum total protein concomitant with significant decreases in the levels of tumor markers arginase and α-l-fucosidase as well as liver enzymes, direct and total bilirubin. Similarly, the oral administration of EA, as a curative agent produced similar changes to those when EA was used as a protective agent, but to a lesser extent. In addition, it was noted that HCC rats exhibited a degree of DNA fragmentation; however, EA administration partially inhibited the DNA fragmentation. Therefore, EA has the ability to scavenge free radicals, prevent DNA fragmentation, reduce liver injury and protect against oxidative stress.

Silver nanoparticles induce p53-mediated apoptosis in human bronchial epithelial (BEAS-2B) cells.

Deregulated apoptosis has been associated with many lung diseases. Although many studies have reported the apoptotic effects exhibited by silver nanoparticles (Ag-NPs) in various circumstances, the apoptosis mechanism of Ag-NPs is unclear. We investigated oxidative stress and apoptosis in human normal bronchial epithelial (BEAS-2B) cells to elucidate the role of p53 in apoptosis by Ag-NPs. First, dispersion and stability of Ag-NPs improved usingbronchialepithelial cell growth mediumwith 5% fetal bovine serum. Then, we observed oxidative stress in BEAS-2B cells exposed to Ag-NPs. Second, we carried out a cell death assay to measure DNA fragmentation as a biomarker of apoptosis. BEAS-2B cells were treated with p53-specific short interfering RNA (siRNA) or p53 inhibitor (pifithrin-α) to investigate whether p53 is involved in apoptosis by Ag-NPs. As a result, Ag-NPs significantly enhanced DNA fragmentation dose-dependently and treatment with p53 siRNA or pifithrin-α prevented DNA fragmentation. We also found that apoptosis-related genes (caspase-3, Bax, and Bcl-2) were regulated by Ag-NPs, which was detected by mRNA and protein levels. These results suggest that Ag-NPs induced p53-mediated apoptosis in BEAS-2B cells. Our findings may contribute to understanding the potential roles of Ag-NPs in pulmonary disease.