Melatonin Prevents Oxidative Stress and Hepatocyte Cell Death Induced by Experimental Cholestasis

The induction of oxidative stress precedes liver injury during experimental obstructive jaundice (OJ). In this sense, different evidences suggest that melatonin (MEL), as antioxidant, may be useful in the protection against apoptosis and necrosis during experimental cholestasis. In addition, we will also assess if MEL-dependent protection is related to a recovery of antioxidant status disturbances induced by OJ. Cholestasis was achieved by double ligature and sectioning of the principal bile duct. MEL was injected intraperitoneally (500?μg/kg/day). Lipid peroxidation was evaluated by the measurement of malondialdehyde (MDA) content in liver. Different parameters related to antioxidant status, such as reduced glutathione (GSH), glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD) were determined in liver. Liver injury was assessed by alanine aminotransferase (ALT) in serum, histological examination, DNA fragmentation and TUNEL assay. The activation of perisinusoidal stellate cells was evaluated by immunohistochemical measurement of α-smooth muscle actin in liver sections. The induction of OJ increased all the parameters related to apoptosis and necrosis in liver. The induction of liver injury was associated with stellate cell activation, as well as an increase in MDA (p<0.0001) and a reduction in GSH, GPx, catalase and SOD content (p<0.0001) in liver. MEL reduced hepatic apoptosis and necrosis (p<0.004) with a significant improvement in all oxidative stress markers. In conclusion, our results showed that MEL recovered the antioxidant status and reduced apoptosis and necrosis induced by experimental cholestasis.     

Novel Cell Preservation Technique to Extend Bovine In Vitro White Blood Cell Viability

Although cell-mediated immunity based diagnostics can be integral assays for early detection of various diseases of dairy cows, processing of blood samples for these tests is time-sensitive, often within 24 hours of collection, to maintain white blood cell viability. Therefore, to improve utility and practicality of such assays, the objective of this study was to assess the use of a novel white blood cell preservation technology in whole bovine blood. Blood samples from ten healthy cows were each divided into an unpreserved control sample and a test sample preserved with commercially-available cell transport medium. Samples were maintained at room temperature and stimulated with the mitogens pokeweed and concanavalinA, as well as with interleukin-12 p40. Stimulation was completed on days 1, 5, and 8 post-sampling. Viability of white blood cells was assessed through interferon gamma production determined with a commercial enzyme linked immunosorbent assay. In addition, mononuclear cell viability was assessed with propidium iodide flow cytometry. Greater interferon gamma production was observed on days 5 and 8 post-collection in preserved samples, with both pokeweed and concanavalinA stimulating positive interferon gamma production on day 5 post-collection. A greater proportion of the amount of interferon gamma produced on day 1 continued to be produced on days 5 and 8 post-collection with concanavalinA stimulation (with or without interleukin 12) as compared to pokeweed stimulation. Additionally, viable mononuclear cells were still present at eight days post-collection, with a higher mean proportion detected at days 5 and 8 in all stimulated preserved samples. This practical and simple method to extend in vitro white blood cell viability could benefit the efficient utilization of cell-based blood tests in ruminants.     

Tualang Honey Improves Human Corneal Epithelial Progenitor Cell Migration and Cellular Resistance to Oxidative Stress In Vitro

Stem cells with enhanced resistance to oxidative stress after in vitro expansion have been shown to have improved engraftment and regenerative capacities. Such cells can be generated by preconditioning them with exposure to an antioxidant. In this study we evaluated the effects of Tualang honey (TH), an antioxidant-containing honey, on human corneal epithelial progenitor (HCEP) cells in culture. Cytotoxicity, gene expression, migration, and cellular resistance to oxidative stress were evaluated. Immunofluorescence staining revealed that HCEP cells were holoclonal and expressed epithelial stem cell marker p63 without corneal cytokeratin 3. Cell viability remained unchanged after cells were cultured with 0.004, 0.04, and 0.4% TH in the medium, but it was significantly reduced when the concentration was increased to 3.33%. Cell migration, tested using scratch migration assay, was significantly enhanced when cells were cultured with TH at 0.04% and 0.4%. We also found that TH has hydrogen peroxide (H₂O₂) scavenging ability, although a trace level of H₂O₂ was detected in the honey in its native form. Preconditioning HCEP cells with 0.4% TH for 48 h showed better survival following H₂O₂-induced oxidative stress at 50 µM than untreated group, with a significantly lower number of dead cells (15.3±0.4%) were observed compared to the untreated population (20.5±0.9%, p<0.01). Both TH and ascorbic acid improved HCEP viability following induction of 100 µM H₂O₂, but the benefit was greater with TH treatment than with ascorbic acid. However, no significant advantage was demonstrated using 5-hydroxymethyl-2-furancarboxaldehyde, a compound that was found abundant in TH using GC/MS analysis. This suggests that the cellular anti-oxidative capacity in HCEP cells was augmented by native TH and was attributed to its antioxidant properties. In conclusion, TH possesses antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in HCEP cells in vitro.     

Murine Adipose Tissue-Derived Stromal Cell Apoptosis and Susceptibility to Oxidative Stress In Vitro Are Regulated by Genetic Background

Adipose tissue-derived stromal cells (ADSCs) are of interest for regenerative medicine as they are isolated easily and can differentiate into multiple cell lineages. Studies of their in vitro proliferation, survival, and differentiation are common; however, genetic effects on these phenotypes remain unknown. To test if these phenotypes are genetically regulated, ADSCs were isolated from three genetically diverse inbred mouse strains- C57BL/6J (B6), BALB/cByJ (BALB), and DBA/2J (D2)- in which genetic regulation of hematopoietic stem function is well known. ADSCs from all three strains differentiated into osteogenic and chondrogenic lineages in vitro. ADSCs from BALB grew least well in vitro, probably due to apoptotic cell death after several days in culture. BALB ADSCs were also the most susceptible to the free radical inducers menadione and H₂O₂. ADSCs from the three possible F1 hybrids were employed to further define genetic regulation of ADSC phenotypes. D2, but not B6, alleles stimulated ADSC expansion in BALB cells. In contrast, B6, but not D2, alleles rescued BALB H₂O₂ resistance. We conclude that low oxidative stress resistance does not limit BALB ADSC growth in vitro, as these phenotypes are genetically regulated independently. In addition, ADSCs from these strains are an appropriate model system to investigate genetic regulation of ADSC apoptosis and stress resistance in future studies. Such investigations are essential to optimize cell expansion and differentiation and thus, potential for regenerative medicine.     

Production of Iridoids and Phenolics by Transformed Harpagophytum procumbens Root Cultures

Harpagophytum procumbens (Devil's claw) is an important medicinal plant, which tubers are used for the treatment of different inflammatory diseases. In this study, the time courses of growth of Devil's claw hairy roots and accumulation of intracellular total iridoids and phenolics were investigated during cultivation under submerged conditions. After 21 days of growth in liquid hormone‐free MS medium, a growth index of 81.3 was achieved and the accumulated biomass reached 0.58 g/flask. It was found that the time courses of total iridoids and phenolics production showed almost the same patterns with a maximum on day 21 from the beginning of cultivation (15.93 mg harpagoside equivalents/L and 261 mg gallic acid equivalents/L, respectively). The methanolic extracts possessed relatively high DPPH‐radical scavenging properties (IC₅₀ 4.08 mg/mL), while for the culture media no antiradical activity was detected. According to our best knowledge, the present report is the first one dealing with the investigation of transformed root cultures from Harpagophytum procumbens plants.     

In Vitro Electrochemical Corrosion and Cell Viability Studies on Nickel-Free Stainless Steel Orthopedic Implants

The corrosion and cell viability behaviors of nanostructured, nickel-free stainless steel implants were studied and compared with AISI 316L. The electrochemical studies were conducted by potentiodynamic polarization and electrochemical impedance spectroscopic measurements in a simulated body fluid. Cytocompatibility was also evaluated by the adhesion behavior of adult human stem cells on the surface of the samples. According to the results, the electrochemical behavior is affected by a compromise among the specimen''s structural characteristics, comprising composition, density, and grain size. The cell viability is interpreted by considering the results of the electrochemical impedance spectroscopic experiments.     

Cell Proliferation and Oxidative Stress

Antioxidants such as mannitol, butylated hydroxytoluene and a-tocopherol enhance the growth of pol-yoma virus transformed and non-transformed BHK-21 cells. In the case of mannitol this is observed even in the absence of added calf serum. In part these effects may operate to protect cellular growth control mechanisms. On the other hand oxidants such as H₂O₂ and t-butyl hydroperoxide can inhibit growth and overall cellular protein synthesis, through mechanisms that are likely to involve radicals. In the case of H₂O₂, the inhibitory effects can nevertheless be reduced by 'prestressing' the cells with mild heat or with H₂O₂ itself.

Paradoxically very low concentrations (10^?8 M) of H₂ 0₂ or t-butyl hydroperoxide can actually stimulate cell growth, even in the absence of serum. These stimulatory effects however do not appear to involve radicals as they are enhanced by inclusion of mannitol or DMSO in the medium.     

Celastrol Prevents Atherosclerosis via Inhibiting LOX-1 and Oxidative Stress

Celastrol is a triterpenoid compound extracted from the Chinese herb Tripterygium wilfordii Hook F. Previous research has revealed its anti-oxidant, anti-inflammatory, anti-cancer and immunosuppressive properties. Here, we investigated whether celastrol inhibits oxidized low-density lipoprotein (oxLDL) induced oxidative stress in RAW 264.7 cells. In addition, the effect of celastrol on atherosclerosis in vivo was assessed in apolipoprotein E knockout (apoE^−/−) mouse fed a high-fat/high-cholesterol diet (HFC). We found that celastrol significantly attenuated oxLDL-induced excessive expression of lectin-like oxidized low density lipoprotein receptor-1(LOX-1) and generation of reactive oxygen species (ROS) in cultured RAW264.7 macrophages. Celastrol also decreased IκB phosphorylation and degradation and reduced production of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor (TNF)-α and IL-6. Celastrol reduced atherosclerotic plaque size in apoE^−/− mice. The expression of LOX-1 within the atherosclerotic lesions and generation of superoxide in mouse aorta were also significantly reduced by celastrol while the lipid profile was not improved. In conclusion, our results show that celastrol inhibits atherosclerotic plaque developing in apoE^−/− mice via inhibiting LOX-1 and oxidative stress.     

Oxidative Stress and DNA Lesions: The Role of 8-Oxoguanine Lesions in Trypanosoma cruzi Cell Viability

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.     

Oxidative Stress and Tumour Cell Proliferation

The effects of oxidant stress were studied in immortalised hamster (BHK-21) and rat (208F) cell lines before and after transformation to the malignant state with polyoma virus, or activated H-ras, respectively. Whilst intracellular superoxide production was detectable in both transformed and immortalised cells the rate was somewhat higher in the transformed cells which have lower levels of superoxide dismutase. Because growth of transformed cells was particularly depressed in the presence of MTT, a tetrazolium compound reduced by superoxide, the possible role of active oxygen species in the promotion of cell growth was examined. Low levels of hydrogen peroxide were stimulatory towards both immortalised and transformed cells. In the case of H-ras transformed rat cells, paraquat was also stimulatory provided serum was present in the growth medium. In the absence of serum, paraquat was notably inhibitory but inhibition could be alleviated by addition of low concentrations of α-tocopherol (10^?8 M) to the serum-depleted medium.

Although depletion of serum from the growth medium also leads to lower cell proliferation, subsequent experiments showed that a-tocopherol addition to serum-free medium was sufficient to restimulate growth. In the case of transformed cells, yields of cells were even greater than that encountered in the presence of 10% serum. Thus whilst certain active oxygen species (e. g. hydrogen peroxide) may have a role in promoting the growth of transformed and immortalised cells the necessity for antioxidant protection is important.     

Efficacy and tolerance of Harpagophytum procumbens versus diacerhein in treatment of osteoarthritis.

In a double-blind, randomized, multicentre clinical study, the efficacy and tolerance of a herbal medicine product, Harpadol (6 capsules/day, each containing 435 mg of powdered cryoground powder Harpagophytum procumbens), was compared with diacerhein 100 mg/day in the treatment, for 4 months, of 122 patients suffering from osteoarthritis of the knee and hip. Assessments of pain and functional disability were made on a 10 cm horizontal visual analogue scale; severity of osteoarthritis was evaluated by Lequesne's index. Spontaneous pain showed a significant improvement during the course of the study and there was no difference in the efficacy of the two treatments. Similarly, there was a progressive and significant reduction in the Lequesne functional index and no statistical difference was found between Harpadol and diacerhein. At completion of the study, patients taking Harpadol were using significantly less NSAIDs and antalgic drugs. The frequency of adverse events was significantly lower in the Harpadol group. The most frequent event reported was diarrhea, occurring in 8.1% and 26.7% of Harpadol and diacerhein patients respectively. The global tolerance assessment by patients at the end of treatment favoured Harpadol. The results of this study demonstrate that Harpadol is comparable in efficacy and superior in safety to diacerhein.     

Melatonin Prevents Oxidative Stress in Ovariectomized Rats Treated with Aluminium

This study is designed to determine the simultaneous effect of aluminium (Al) and melatonin (Mel) treatment in intact and ovariectomized (Ovx) female rats on oxidative stress and their inter-organ relationship in the kidney and liver. Al-treated rats received an intra-peritoneal injection of solution of aluminium lactate (0.575?mg Al/100?g of body weight, three times a week), during 12?weeks. Mel groups received intra-peritoneal injections of melatonin at a dose of 10?mg/kg/day, 5?days/week, during 12?weeks. The results of this study showed that Al treatment in female rats modifies homeostasis of glutathione and the antioxidant capacity of the rat liver and kidney. The alteration of glutathione homeostasis and oxidative status was not associated with an increased lipid peroxidation in both organs with the exception of the increase observed in the liver of Ovx rats. Al also induced modifications in the activity of some enzymes related to the glutathione cycle: GSH-Px in the liver and kidney and glutathione reductase only in the kidney. Al exposure decreased CAT activity in both the kidney and liver of intact and Ovx groups. The administration of Mel in the intact and castrated females treated with Al seems to reduce oxidative changes in the liver and kidney of intact and Ovx rats.     

Impact of Genetic Reduction of NMNAT2 on Chemotherapy-Induced Losses in Cell Viability In Vitro and Peripheral Neuropathy In Vivo

Nicotinamide mononucleotide adenylyl transferases (NMNATs) are essential neuronal maintenance factors postulated to preserve neuronal function and protect against axonal degeneration in various neurodegenerative disease states. We used in vitro and in vivo approaches to assess the impact of NMNAT2 reduction on cellular and physiological functions induced by treatment with a vinca alkaloid (vincristine) and a taxane-based (paclitaxel) chemotherapeutic agent. NMNAT2 null (NMNAT2-/-) mutant mice die at birth and cannot be used to probe functions of NMNAT2 in adult animals. Nonetheless, primary cortical cultures derived from NMNAT2-/- embryos showed reduced cell viability in response to either vincristine or paclitaxel treatment whereas those derived from NMNAT2 heterozygous (NMNAT2+/-) mice were preferentially sensitive to vincristine-induced degeneration. Adult NMNAT2+/- mice, which survive to adulthood, exhibited a 50% reduction of NMNAT2 protein levels in dorsal root ganglia relative to wildtype (WT) mice with no change in levels of other NMNAT isoforms (NMNAT1 or NMNAT3), NMNAT enzyme activity (i.e. NAD/NADH levels) or microtubule associated protein-2 (MAP2) or neurofilament protein levels. We therefore compared the impact of NMNAT2 knockdown on the development and maintenance of chemotherapy-induced peripheral neuropathy induced by vincristine and paclitaxel treatment using NMNAT2+/- and WT mice. NMNAT2+/- did not differ from WT mice in either the development or maintenance of either mechanical or cold allodynia induced by either vincristine or paclitaxel treatment. Intradermal injection of capsaicin, the pungent ingredient in hot chili peppers, produced equivalent hypersensitivity in NMNAT2+/- and WT mice receiving vehicle in lieu of paclitaxel. Capsaicin-evoked hypersensitivity was enhanced by prior paclitaxel treatment but did not differ in either NMNAT2+/- or WT mice. Thus, capsaicin failed to unmask differences in nociceptive behaviors in either paclitaxel-treated or paclitaxel-untreated NMNAT2+/- and WT mice. Moreover, no differences in motor behavior were detected between genotypes in the rotarod test. Our studies do not preclude the possibility that complete knockout of NMNAT2 in a conditional knockout animal could unmask a role for NMNAT2 in protection against detrimental effects of chemotherapeutic treatment.     

Lactobacillus plantarum 299v Prevents Caspase-Dependent Apoptosis In Vitro

Selective microbes used as probiotics can enhance epithelial cell protection. We have previously shown that a Lactobacillus plantarum strain 299v (Lp299v) has the ability to induce mucin genes. In the current study, we utilized a cytokine model of inflammation in cell culture to study the modulation of apoptosis by this probiotic. HT-29 cells were pre-incubated with the Lp299v or L. plantarum strain adh- (Lpadh-), a non-adherent derivative of Lp299v. Cells were challenged with a mixture of cytokines (TNF-α, IFN-γ, and IL-1a) to imitate conditions of inflammation. To assess for cell death, we evaluated TUNEL, multi-caspase, and caspase-3 and caspase-7 activity assays. There was a marked decrease in apoptosis as measured by TUNEL⁺ cells in samples pre-treated with Lp299v (18.7 ± 4.1%, p < 0.01) and Lpadh- (16.6 ± 3.2%, p < 0.05) prior to cytokine exposure when compared to cells (43.6 ± 6.2%) exposed to the cytokine mixture. Lp299v pre-incubation with HT-29 cells reduced caspase⁺ cells in the multi-caspase activity assay (3.6 ± 0.6%, p < 0.05) compared to cells exposed to cytokines (68.9 ± 5.1%) whereas Lpadh- did not (46.8 ± 17.5%, p > 0.05). Similarly, caspase-3, caspase-7 activity was also reduced by Lp299v. Selected probiotics may confer an exogenous protective effect at the mucosal–luminal interface for intestinal epithelial cells via alteration of caspase-dependent apoptotic pathways.     

Harpagophytum Procumbens Ethyl Acetate Fraction Reduces Fluphenazine-Induced Vacuous Chewing Movements and Oxidative Stress in Rat Brain

Neurochemical Research - Long-term treatment with fluphenazine is associated with manifestation of extrapyramidal side effects, such as tardive dyskinesia. The molecular mechanisms related to the...     

Arginase 2 Deficiency Prevents Oxidative Stress and Limits Hyperoxia-Induced Retinal Vascular Degeneration

Background

Hyperoxia exposure of premature infants causes obliteration of the immature retinal microvessels, leading to a condition of proliferative vitreoretinal neovascularization termed retinopathy of prematurity (ROP). Previous work has demonstrated that the hyperoxia-induced vascular injury is mediated by dysfunction of endothelial nitric oxide synthase resulting in peroxynitrite formation. This study was undertaken to determine the involvement of the ureahydrolase enzyme arginase in this pathology.

Methods and Findings

Studies were performed using hyperoxia-treated bovine retinal endothelial cells (BRE) and mice with oxygen-induced retinopathy (OIR) as experimental models of ROP. Treatment with the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) prevented hyperoxia-induced apoptosis of BRE cells and reduced vaso-obliteration in the OIR model. Furthermore, deletion of the arginase 2 gene protected against hyperoxia-induced vaso-obliteration, enhanced physiological vascular repair, and reduced retinal neovascularization in the OIR model. Additional deletion of one copy of arginase 1 did not improve the vascular pathology. Analyses of peroxynitrite by quantitation of its biomarker nitrotyrosine, superoxide by dihydroethidium imaging and NO formation by diaminofluoroscein imaging showed that the protective actions of arginase 2 deletion were associated with blockade of superoxide and peroxynitrite formation and normalization of NOS activity.

Conclusions

Our data demonstrate the involvement of arginase activity and arginase 2 expression in hyperoxia-induced vascular injury. Arginase 2 deletion prevents hyperoxia-induced retinal vascular injury by preventing NOS uncoupling resulting in decreased reactive oxygen species formation and increased nitric oxide bioavailability.