Reductase mRNA Levels Research Articles

Paternal exercise induces antioxidant defenses by α-Klotho/Keap1 pathways in the skeletal muscle of offspring exposed to a high fat-diet without changing telomere length

Although previous studies demonstrated that the ancestral lifestyle can enhance the metabolic health of offspring exposed to an obesogenic diet, the specific connections between these positive effects in redox state and telomere length are unknown. We investigated the impact of paternal resistance training (RT) on stress-responsive signaling and the pathways involved in telomere homeostasis in skeletal muscle. This investigation encompassed both the fathers and first-generation litter exposed to a long-term standard diet (24 weeks) and high fat diet (HFD). Wistar rats were randomized into sedentary or trained fathers (8 weeks of resistance training). The offspring were obtained by mating with sedentary females. Upon weaning, male offspring were divided into four groups: offspring of sedentary or trained fathers exposed to either a control diet or HFD. The gastrocnemius was prepared for reverse transcription-quantitative polymerase chain reaction, immunoblotting, ELISA, and electron paramagnetic resonance spectroscopy. RT upregulated shelterin mRNA levels and antioxidant protein, preserving muscle telomere in fathers. Conversely, HFD induced a disturbance in the redox balance, which may have contributed to the offspring telomere shortening from sedentary fathers. Preconceptional paternal RT downregulates Kelch-like ECH-associated protein 1 (Keap1) mRNA levels in the skeletal muscle of progeny exposed to HFD, driving an increase in Glutathione reductase mRNA levels, Sod1 and Catalase protein levels to mitigate ROS production. Also, paternal exercise upregulates α-Klotho protein levels, mediating antioxidative responses without altering shelterin mRNA levels and telomere length. We provide the first in-depth analysis that the offspring's redox state seems to be directly associated with the beneficial effects of paternal exercise.

Eugeniin improves cholesterol metabolism in HepG2 cells and Caco-2 cells.

Considering the absence of prior studies on the cholesterol metabolism-improving effects of eugeniin, the present investigation aimed to explore the potential impact of eugeniin on cholesterol metabolism. This study sought to elucidate the molecular mechanisms involved in this process using HepG2 and Caco-2 cells treated with 5 µm eugeniin. The intracellular cholesterol levels in HepG2 and Caco-2 cells were significantly decreased in the 24-h eugeniin-treated group. The protein and messenger ribonucleic acid (mRNA) levels of the low-density lipoprotein receptor (LDLR) were increased, while 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase protein and mRNA levels were decreased in HepG2 cells 6h of the eugeniin-treated group. Additionally, LDLR protein and mRNA levels were increased in HepG2 cells after 24h of eugeniin treatment. In Caco-2, the protein and mRNA levels of ATP-binding cassette transporter 1 were increased after 24h eugeniin treatment. This novel finding indicates that eugeniin improves cholesterol metabolism in human cell cultures.

Inhibition of Vascular Inflammation by Apolipoprotein A-IV.

BackgroundApolipoprotein (apo) A-IV, the third most abundant apolipoprotein in human high density lipoproteins (HDLs), inhibits intestinal and systemic inflammation. This study asks if apoA-IV also inhibits acute vascular inflammation.MethodsInflammation was induced in New Zealand White rabbits by placing a non-occlusive silastic collar around the common carotid artery. A single 1 mg/kg intravenous infusion of lipid-free apoA-IV or saline (control) was administered to the animals 24 h before collar insertion. The animals were euthanised 24 h post-collar insertion. Human coronary artery cells (HCAECs) were pre-incubated with reconstituted HDLs containing apoA-IV complexed with phosphatidylcholine, (A-IV)rHDLs, then activated by incubation with tumour necrosis factor (TNF)-α. Cell surface vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in the TNF-α-activated HCAECs was quantified by flow cytometry. VCAM-1, ICAM-1 and 3β-hydroxysteroid-Δ24 reductase (DHCR24) mRNA levels were quantified by real time PCR.ResultsApolipoprotein ApoA-IV treatment significantly decreased collar-induced endothelial expression of VCAM-1, ICAM-1 and neutrophil infiltration into the arterial intima by 67.6 ± 9.9% (p < 0.01), 75.4 ± 6.9% (p < 0.01) and 74.4 ± 8.5% (p < 0.05), respectively. It also increased endothelial expression of DHCR24 by 2.6-fold (p < 0.05). Pre-incubation of HCAECs with (A-IV)rHDLs prior to stimulation with TNF-α inhibited VCAM-1 and ICAM-1 protein levels by 62.2 ± 12.1% and 33.7 ± 5.7%, respectively. VCAM-1 and ICAM-1 mRNA levels were decreased by 55.8 ± 7.2% and 49.6 ± 7.9%, respectively, while DHCR24 mRNA expression increased by threefold. Transfection of HCAECs with DHCR24 siRNA attenuated the anti-inflammatory effects of (A-IV)rHDLs. Pre-incubation of TNF-α-activated HCAECs with (A-IV)rHDLs also inhibited nuclear translocation of the p65 subunit of nuclear factor-κB (NF-κB), and decreased IκBα phosphorylation.ConclusionThese results indicate that apoA-IV inhibits vascular inflammation in vitro and in vivo by inhibiting NF-κB activation in a DHCR24-dependent manner.

Oxidative stress response biomarker gene expression in Piaractus brachypomus (Characiformes: Serrasalmidae)

ABSTRACT Oxidative stress may occur in the fish brain under several conditions, including tissue reoxygenation during anaesthesia, injury and toxic exposure. An imbalance in oxidative homeostasis can lead to oxidative stress, triggering several adaptive mechanisms at both the genetic and protein levels. The aim of this study was to assess the transcription levels of five oxidative stress-related genes in the Colombian native fish red-bellied pacu, Piaractus brachypomus, under anaesthesia with menthol (50 mg/L) and eugenol (40 mg/L), as well as in models of acute brain injury and organophosphate toxicity. Relative gene expression was assessed in the brain, gill and liver tissues of fish under anaesthesia and in the brain tissues of fish in the brain injury and toxicity experiments. It was found that glutathione reductase (GSR) mRNA levels in the brain were significantly higher in the eugenol group than in the menthol group, ATP synthase subunit 6 (ATP6) transcription was downregulated after brain injury, and the GSR, ATP6 and superoxide dismutase 2 (SOD2) genes were upregulated in fish exposed to the organophosphate chlorpyrifos. This is the first evaluation of the relative gene expression of oxidative response biomarkers in P. brachypomus.

Bone marrow ribonucleotide reductase mRNA levels and methylation status as prognostic factors in patients with myelodysplastic syndrome treated with 5-Azacytidine

Ribonucleotide Reductase (RNR) is a two-subunit (RRM1, RRM2) enzyme, responsible for the conversion of ribonucleotides to deoxyribonucleotides required for DNA replication. To evaluate RNR as a biomarker of response to 5-azacytidine, we measured RNR mRNA levels by a quantitative real-time PCR in bone marrow samples of 98 patients with myelodysplastic syndrome (MDS) treated with 5-azacytidine with parallel quantification of the gene promoter’s methylation. Patients with low RRM1 levels had a high RRM1 methylation status (p = 0.005) and a better response to treatment with 5-azacytidine (p = 0.019). A next-generation sequencing for genes of interest in MDS was also carried out in a subset of 61 samples. Splicing factor mutations were correlated with lower RRM1 mRNA levels (p = 0.044). Our results suggest that the expression of RNR is correlated with clinical outcomes, thus its expression could be used as a prognostic factor for response to 5-azacytidine and a possible therapeutic target in MDS.

Beneficial effects of primidone in Niemann-Pick disease type C (NPC)-model cells and mice: Reduction of unesterified cholesterol levels in cells and extension of lifespan in mice

Niemann-Pick disease type C (NPC) is caused by a loss of function of either NPC1 or NPC2 protein, resulting in the accumulation of unesterified, free-cholesterol (free-C) in cells/tissues and thus leading to cell/tissue damage. In the brain of patients/animals with NPC, as a consequence of the accumulation of free-C in late endosomes/lysosomes (LE/LY) in cells, multiple lipids including complex sphingolipids are accumulated, and almost all patients/animals ultimately develop progressive/fatal neurodegeneration. Several reagents that are considered to act in the brain show beneficial effects on NPC-model animals. In the present study, we investigated the effects of antiepileptic drugs, such as primidone and valproic acid, on the accumulation of free-C in NPC1-null CHO cells and NPC1* fibroblasts, human fibroblasts established from a patient with NPC1 mutation. Like valproic acid, treatment with primidone reduced free-C levels in LE/LY in NPC1-null/mutant cells. Down-regulation of cholesterol ester levels in NPC1-null cells and up-regulation of HMG-CoA reductase and low-density lipoprotein receptor mRNA levels in NPC1* cells were partially recovered by primidone treatment. Thus, primidone was suggested to enhance free-C trafficking from LE/LY to endoplasmic reticulum in NPC1-null/mutant cells. In NPC1-null mice, oral application of primidone (100 mg/kg/day) extended lifespan by approximately 5 days, although the first days showing ataxia, a typical symptom of neuromotor dysfunction, were not affected. Our findings suggest the potential of primidone for the treatment of NPC.

Evaluation of the effect of silver and silver nanoparticles on the function of selenoproteins using an in-vitro model of the fish intestine: The cell line RTgutGC

Emerging research in mammalian cells suggests that ionic (AgNO3) and nano silver (AgNP) can disrupt the metabolism of selenium which plays a vital role in oxidative stress control. However, the effect of silver (Ag) on selenoprotein function in fish is poorly understood. Here we evaluate the effects of AgNO3 and citrate coated AgNP (cit-AgNP) on selenoprotein function and oxidative stress using a fish cell line derived from the rainbow trout (Oncorhynchus mykiss) intestine (RTgutGC). Cell viability was evaluated using a cytotoxicity assay which measures simultaneously metabolic activity, membrane integrity and lysosome integrity. Cells exposed to equimolar amounts of AgNO3 and cit-AgNP accumulated the same amount of silver intracellularly, however AgNO3 was more toxic than cit-AgNP. Selenoenzymes glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) mRNA levels and enzyme activity were measured. While mRNA levels remained unaffected by AgNO3 or cit-AgNP, the enzyme activity of GPx was inhibited by AgNO3 (1 µM) and cit-AgNP (5 µM) and TrxR activity was inhibited by AgNO3 (0.4 µM) and cit-AgNP (1, 5 µM). Moreover, cells exposed to 1 µM of AgNO3 and cit-AgNP showed an increase in metallothionein b (MTb) mRNA levels at 24 h of exposure, confirming the uptake of silver, but returned to control levels at 72 h suggesting silver scavenging by MTb. Oxidative stress was not observed at any of the doses of AgNO3 or cit-AgNP tested. Overall, this study shows that AgNO3 or cit-AgNP can inhibit the activity of selenoenzymes but do not induce oxidative stress in RTgutGC cells.

Plasma thymidylate synthase and dihydrofolate reductase mRNA levels as potential predictive biomarkers of pemetrexed sensitivity in patients with advanced non-small cell lung cancer.

BackgroundHigh levels of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) expression in tumour tissues are an indicator of ineffective responses to pemetrexed-based chemotherapy in various tumours, including non-small cell lung cancer (NSCLC). However, tumour tissues are highly heterogeneous, so a single biopsy may not reflect genetic alterations during disease progression. This study investigated the potential use of plasma TS and DHFR mRNA levels as biomarkers for predicting sensitivity to pemetrexed-based chemotherapy.MethodsPlasma samples were obtained from 245 patients with advanced NSCLC and 30 healthy donors. Total RNA was extracted from the plasma samples, and TS and DHFR mRNA levels were determined via real-time PCR. TS and DHFR mRNA levels between cancer patients and healthy controls were compared. The association between plasma TS and DHFR mRNA levels and tumour response to pemetrexed/cisplatin chemotherapy was analysed.ResultsThe plasma TS and DHFR mRNA levels decreased in patients with advanced NSCLC compared with healthy controls. Moreover, plasma TS and DHFR mRNA levels negatively correlated with tumour response to pemetrexed/cisplatin chemotherapy in patients with advanced NSCLC. Overall survival time was prolonged in patients with low TS mRNA expression compared with those with high TS mRNA expression, although the difference was not statistically significant.ConclusionsLow expression levels of plasma TS and DHFR mRNA confer increased tumour sensitivity to pemetrexed/cisplatin chemotherapy in patients with advanced NSCLC. The results suggested that plasma TS and DHFR mRNA levels are promising biomarkers for choosing patients who are likely to respond and benefit the most from pemetrexed-based chemotherapy.

Porphyromonas gingivalis infection alters Nrf2-phase II enzymes and nitric oxide in primary human aortic endothelial cells.

Periodontal disease (PD) is known to be associated with endothelial dysfunction in patients with coronary artery and/or cardiovascular disease. In our study, we sought to explore the virulence of P. gingivalis (Pg) affecting glycogen synthase kinase 3 beta (GSK-3β)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/tetrahydrobiopterin (BH4 )/ nitric oxide synthase (NOS) expression in primary human aortic endothelial cells (pHAECs). pHAECs were infected for 48 hours with Pg in vitro using the Human oxygen-Bacteria anaerobic coculture technique. Cell viability was determined, and target gene expression changes were evaluated by quantitative real-time polymerase chain reaction at the end of each incubation period. Pg impaired pHAEC viability 24 hours post-infection. Pg infection reduced mRNA expression levels of endothelial NOS (eNOS), Nrf2, and Phase II enzymes (heme oxygenase-1, catalase, superoxide dismutase-1) in a time-dependent manner. Significant (P<0.05) increase in the inflammatory markers (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α) were observed in the medium as well as in the infected cells. Interestingly, inducible NOS mRNA levels showed a significant (P<0.05) increase at 12 hours and 24 hours and were reduced at later time points. BH4 (cofactor of eNOS) biosynthesis enzyme dihydrofolate reductase (DHFR, salvage pathway) mRNA levels showed a significant (P<0.05) decrease, while mRNA levels of GSK-3β were elevated. These results suggest that periodontal bacterial infection may cause significant changes in the endothelial GSK-3β/BH4 /eNOS/Nrf2 pathways, which may lead to impaired vascular relaxation. Greater understanding of the factors that adversely affect endothelial cell function could contribute to the development of new therapeutic compounds to treat PD-induced vascular diseases.

Abstract 1899: Extracellular lipid starvation modulates the effects of BRAF inhibitors in melanoma

Abstract Melanoma is the most aggressive of all skin cancers, being the leading cause of death for cutaneous malignancy, with elevated metastatic spread and drug resistance. Because the deregulation of components of lipid metabolism may contribute to melanoma cell resistance, the aim of this study was to examine the dependence of melanoma cell growth on extracellular lipids, the associated lipid metabolism gene expression profiles and lipid impact on sensitivity to BRAF inhibitors. We applied preclinical pharmacology approaches including western blot analyses, growth inhibition assays quantitative Real-time PCR (qRT-PCR) in melanoma cell lines including variants with acquired resistance to BRAF inhibitors. Two matched pairs of sensitive and PLX4032-resistant cell lines (LM16 and LM36) were used. Both pairs carried V600E BRAF gene mutation and BRAF gene amplification, and LM36R displayed mutant NRAS. When compared to the parental sensitive cells, LM16R cells showed increased activation of MAPK-ERK and PI3K-Akt-mTOR pathways, while LM36R cells displayed increased activation of IGF1R and LKB1. Under standard culture conditions, when compared to the parental sensitive cells, both the resistant variants showed reduced expression of DHCR24 (24-Dehydrocholesterol Reductase), and LM16R showed reduced protein level of the lipogenic enzyme FASN (Fatty Acid Synthase) while LM36R cells displayed similar FASN protein levels. When grown in lipid-free medium, LM16 and LM16R cells increased the expression of both FASN and DHCR24 proteins. These changes were particularly evident for DHCR24 in LM16R cells after 48 h of growth in the absence of lipids. Lipid starvation markedly reduced cell growth of LM16-LM16R as well as LM36-LM36R pairs over time, albeit with a lower impact at higher cell density. Growth inhibition assays indicated that all the cells cultured in lipid-free medium were more sensitive to PLX4032 than those cultured in standard medium. An analysis of the expression of lipid metabolism genes by qRT-PCR showed that after 48 hours under lipid-free culture conditions the levels of FASN were slightly increased in LM16 and LM16R cells but not in the LM36 pair. Conversely, the mRNA levels of DHCR24 were enhanced upon lipid starvation only in LM16 and LM16R cell lines. Besides, under these conditions, HMGCR (3-Hydroxy-3-Methylglutaryl-CoA Reductase) mRNA levels increased in LM16, LM16R and LM36R cell lines. ACSS2 (Acetyl-coenzyme A synthetase 2) was increased particularly in the LM16 cells. These findings suggest that lipid starvation redirects lipid pathways toward cholesterol synthesis (acetate-cholesterol) by increasing ACSS2, HMGCR as well as DHCR24 levels. Thus, extracellular lipid availability may influence tumor cell response to treatment, a finding to be considered in the frame of a personalized therapy tailored to the specific characteristics of each patient. Citation Format: Giovanni L. Beretta, Elisabetta Vergani, Cristina Corno, Stella Tinelli, Monica Rodolfo, Laura Gatti, Paola Perego. Extracellular lipid starvation modulates the effects of BRAF inhibitors in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1899.

Lipid metabolism and plasma metabolomics of juvenile largemouth bass Micropterus salmoides were affected by dietary oxidized fish oil

During the storage, fish oil is susceptible to peroxidation to form toxic lipid derivatives, dietary intake of oxidized fish oil would induce oxidative stress and inflammation in animals, but it was not clear about the relation between oxidized fish oil intake, lipid metabolism and oxidative stress. Four isonitrogenous and isolipidic diets containing fresh fish oil (peroxide value, POV: 7.2 meq kg−1, diet FR) and oxidized fish oil (POV: 155, 275 and 564 meq kg−1, diet OX155, OX275 and OX564, respectively) were formulated to investigate the effects of dietary oxidized fish oil on growth performance, plasma metabolomics and lipid metabolism of juvenile largemouth bass, Micropterus salmoides. After the 12-week feeding trial, fish fed the OX155 obtained significantly higher (P < .05) weight gain than fish fed FR because of their higher feed intake. Malonaldehyde (MDA) levels were increased in plasma and liver of largemouth bass fed OX564 diet than those fed FR. Untargeted metabolomics ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) was performed to invest the differences of plasma metabolites in fish fed different oxidative degrees of fish oil. Lipid metabolism, especially phospholipids and sterol metabolism in largemouth bass were significantly affected by oxidized fish oil. Dietary oxidized fish oil decreased membrane phospholipids unsaturated degree and increased plasma lysophospholipid contents in largemouth bass. Several bile acids were decreased, while many cholesterols and vitamin D3 related metabolites were increased with the increasing oxidative degree of fish oil. Lipoprotein transport related genes and lipid oxidation related genes mRNA levels were increased in fish fed highly oxidized fish oil. Cholesterol synthesis related gene 7-dehydrocholesterol reductase (DHCR7) mRNA level was increased with the increasing oxidative degree of fish oil. Bile acid metabolism related gene 12a-hydroxylase (CYP8B1) was higher in fish fed OX275 diet. The increased vitamin D3 related metabolites in fish fed oxidized oil may associate with the decreased mRNA levels of RXRα and 24 - hydroxylase (CYP24A1). In conclusion, dietary oxidized fish oil may affect the health of fish through decrease the unsaturated degree of phospholipids and fatty acids, increase the contents of oxidized cholesterol and lysophospholipid in plasma, and repress the bile acid synthesis.

Annexin-1 Mimetic Peptide Ac2-26 Suppresses Inflammatory Mediators in LPS-Induced Astrocytes and Ameliorates Pain Hypersensitivity in a Rat Model of Inflammatory Pain.

Ac2-26, a mimetic peptide of Annexin-A1, plays a vital role in the anti-inflammatory response mediated by astrocytes. In this study, we aimed to explore the underlying mechanisms of Ac2-26-mediated anti-inflammatory effect. Specifically, we investigated the inhibitory effects of Ac2-26 on lipopolysaccharide (LPS)-induced astrocyte migration and on pro-inflammatory cytokines and chemokines expressions, as well as one glutathione (GSH) reductase mRNA and total intracellular GSH levels in LPS-induced astrocytes. Additionally, we investigated whether mitogen-activated protein kinases (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathway were involved in this process. Finally, we evaluated the analgesic effect of Ac2-26 in complete Freund's adjuvant (CFA)-induced inflammatory pain model. Our results demonstrated that Ac2-26 inhibited LPS-induced astrocytes migration, reduced the production of pro-inflammatory mediators [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1α)] and upregulated GSH reductase mRNA and GSH levels in LPS-induced astrocytes in vitro. This process was mediated through the p38, JNK-MAPK signaling pathway, but not dependent on the NF-κB pathway. Furthermore, the p38 and JNK inhibitors mimicked the effects of Ac2-26, whereas a p38 and JNK activator anisomycin partially reversed its function. Finally, Ac2-26 treatment reduced CFA-induced activation of astrocytes and production of inflammatory mediators in the spinal cord. These results suggest that Ac2-26 attenuates pain by inhibiting astrocyte activation and the production of inflammatory mediators; thus, this work presents Ac2-26 as a potential drug to treat neuropathic pain.

Bone Marrow Ribonucleotide Reductase mRNA Levels and Methylation Status As a Prognostic Factor in Patients with Myelodysplastic Syndrome Treated with 5-Azacytidine

Bone Marrow Ribonucleotide Reductase mRNA Levels and Methylation Status As a Prognostic Factor in Patients with Myelodysplastic Syndrome Treated with 5-Azacytidine

Differential effects of red yeast rice, Berberis aristata and Morus alba extracts on PCSK9 and LDL uptake

Background and aimsThe novel nutraceutical combination containing red yeast rice (monacolin K 3.3 mg), Berberis aristata cortex extract (Berberine 531.25 mg) and Morus alba leaves extract (1-deoxynojirimycin 4 mg) is effective in the management of elevated plasma low-density lipoprotein cholesterol (LDL-C) levels. The aim of the present study was to investigate the effects of the three components on proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of LDL receptor (LDLR) expression, in hepatocyte cell lines and to compare their effects on LDL cellular uptake. Methods and resultsHepG2 and Huh7 cells were incubated with B. aristata cortex extract (BCE), red yeast rice (RYR) and M. alba leaves extract (MLE) alone or in combination for 24 h. RYR (50 μg/mL) increased PCSK9 protein expression (Western blot analysis and ELISA), PCSK9 mRNA (qPCR) and its promoter activity (luciferase reporter assay). BCE (40 μg/mL) reduced instead PCSK9 expression, mRNA levels and promoter activity. MLE determined a concentration-dependent reduction of PCSK9 at the mRNA and protein levels, with a maximal reduction at 1 mg/mL, without significant changes of PCSK9 promoter activity. MLE also downregulated the expression of 3-hydroxy-3-methyl-3-glutaryl coenzyme A reductase and fatty acid synthase mRNA levels. The combination of RYR, BCE and MLE reduced the PCSK9 mRNA and protein levels, as well as the promoter activity. Finally, the single components and their combination induced LDL receptor and LDL uptake by the hepatocytes. ConclusionThe positive effect of MLE on PCSK9 supports the rationale of using the nutraceutical combination of RYR, BCE and MLE to control hyperlipidemic conditions.

Curcumin and its synthetic analogue dimethoxycurcumin differentially modulates antioxidant status of normal human peripheral blood mononuclear cells

Curcumin is a polyphenol derived from the herb Curcuma longa, which has been extensively studied in terms of its antitumour, antioxidant, and chemopreventive activity as well as various other effects. In the present work we compared curcumin with its synthetic analogue dimethoxycurcumin (dimc) in terms of its antioxidant enzyme-modulating effects in human peripheral blood mononuclear cells (PBMC). We found that these compounds modulate antioxidant enzymes differentially. Both curcumin and dimethoxycurcumin effected a decrease in lipid peroxidation status in PBMC, however, curcumin had better activity in this regard. An increase in the activity of catalase was seen in the case of curcumin-treated PBMC, whereas dimc increased catalase activity significantly to almost twofold level. Real time-polymerase chain reaction (RT-PCR) analysis revealed significant up-regulation of catalase at mRNA level post treatment with curcumin as well as dimc, however, dimc had better activity in this regard. Glutathione reductase (GR) activity and reduced glutathione levels increased in the case of peripheral blood mononuclear cells (PBMC) treated with curcumin, however, the trend was reversed with dimethoxycurcumin where, both glutathione reductase activity and reduced glutathione levels were significantly reduced. RT-PCR analysis of glutathione reductase mRNA levels showed decrease in mRNA levels post treatment with dimethoxycurcumin (dimc) further corroborating GR enzyme assay results, however, we could not obtain significant result post curcumin treatment. NFkB reporter assay and western blot analysis of nuclear as well as cytosolic fractions of NFkB revealed that curcumin inhibits NFkB activation whereas inhibition was much less with dimc. It has been reported that curcumin and dimc exerts differential cytotoxicity in normal and tumour cells and the reason for this had been attributed to the differential uptake of these compounds by normal cells and tumour cells. Based on our results we propose that differential modulation of antioxidant enzymes via NFkB pathway could be the reason behind differential cytotoxicity of dimc as well as curcumin in normal cells and tumour cells in addition to differential uptake of these compounds as reported previously.

Roles of spermine in modulating the antioxidant status and Nrf2 signalling molecules expression in the thymus and spleen of suckling piglets-new insight.

This study aimed to investigate the effects of spermine and extended spermine administration on the antioxidant status and expression of NF-E2-related factor 2 (Nrf2) signalling molecules in the thymus and spleen in suckling piglets. One half of eighty 12-day-old suckling piglets obtained sufficient nutrient intake supplemented with spermine (0.4mmol/kg body weight), and another half received restricted nutrient intake supplemented with physiological saline in equal doses once a day for 7hr or 3, 6 or 9days in pairs. Spermine supplementation and its extended duration significantly decreased malondialdehyde (MDA) and protein carbonyl (PC) contents (p<.05), but markedly improved antisuperoxide anion (ASA), antihydroxyl radical (AHR), catalase (CAT), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities (p<.05) and glutathione (GSH) content (p<.05) in the thymus and spleen. Additionally, real-timePCR analysis showed that spermine administration and extended spermine intake reduced Kelch-like ECH-associated protein 1 (Keap1) gene transcription and enhanced SOD1, GPx1, CAT, glutathione reductase (GR) and Nrf2 mRNA levels of the thymus and spleen (p<.05), and also improved GST gene expression in the thymus (p<.05). Notably, the spermine-supplemented time for the optimal effects of suckling piglet was determined to be 6days. Collectively, the current study suggested that spermine supplementation and extended spermine administration could protect the health of the thymus and spleen from early weaning by enhancing the antioxidant status and regulating the expression of antioxidant-related signalling molecules.

Effect of media composition on bioavailability and toxicity of silver and silver nanoparticles in fish intestinal cells (RTgutGC)

To understand conditions affecting bioavailability and toxicity of citrate-coated silver nanoparticles (cit-AgNP) and dissolved silver at the luminal enterocyte interface, we exposed rainbow trout (Oncorhynchus mykiss) gut cells (RTgutGC) in media of contrasting composition: two amino acid-containing media, one of which was supplemented with proteins, as can be expected during digestion; and two protein and amino acid-free media contrasting low and high chloride content, as can be expected in the lumen of fish adapting to freshwater or seawater, respectively. Dose–response curves were generated measuring cell metabolic activity, membrane and lysosome integrity over a period of 72 hours. Then, nontoxic doses were applied and total silver accumulation, metallothionein and glutathione reductase mRNA levels were determined. The presence of proteins stabilized cit-AgNP keeping them in suspension. Conversely, in protein-free media, cit-AgNP agglomerated and settled, resulting in higher cellular accumulation of silver and toxicity. Chloride concentrations in exposure media modulated the toxicity of AgNO3 but not of cit-AgNP. Moreover, while amino acid-containing media are protective against AgNO3, likely due to the formation of thiolate complexes, they are only partially protective against cit-AgNP. Viability assays indicated that lysosomes are targets of cit-AgNP, supporting the hypothesis that cit-AgNP exert toxicity intracellularly. Metallothionein, a sensor of metal bioavailability, was induced by cit-AgNP in high chloride medium but not in low chloride medium, indicating that chloride might have a role in mobilizing silver from intercellular vesicles. Overall, this study shows that AgNP bioavailability and toxicity in the intestine is linked to its luminal content.

Radix Puerariae lobatae (Gegen) suppresses the anticoagulation effect of warfarin: a pharmacokinetic and pharmacodynamics study.

BackgroundRadix Salvia miltiorrhiza (Danshen) and Radix Puerariae lobatae (Gegen) are used in Traditional Chinese Medicine to treat cardiovascular diseases. However, adverse herb-drug interactions were observed between warfarin and herbal remedies containing Danshen and Gegen. This study aims to investigate the pharmacokinetic and pharmacodynamic interactions between warfarin and the different components found in Danshen and Gegen.MethodsSixty Sprague–Dawley rats were used to investigate the effects of warfarin (0.2 mg/kg), Danshen (240 or 480 mg/kg) and Gegen (240 or 480 mg/kg) both in isolation and combination. The rats in the warfarin and Danshen/Gegen combination groups were given an oral dose of Danshen or Gegen 2 h after being given an oral dose of warfarin. After five consecutive days of treatment, the pharmacokinetic interactions between Danshen/Gegen and warfarin were investigated by simultaneously monitoring and comparing the cytochrome P450 (CYP) activities, mRNA and protein expression levels in the livers of the rats from the different treatment groups. The pharmacodynamic interactions were evaluated by monitoring and comparing the vitamin K epoxide reductase (VKOR) activities, mRNA and protein expression levels in the livers of rats from the different groups, as well as the thrombomodulin (TM) activities, mRNA and protein in the lungs of these animals. The rat plasma soluble thrombomodulin concentrations of the different treatment groups were also evaluated. Microsomes incubation, Real Time-Polymerase Chain Reaction and Western blot was applied respectively to study the activity, mRNA expression and protein expression of CYP, VKOR and TM.ResultsThe activities and expression levels of the CYP and VKOR enzymes in the warfarin-Gegen combination groups increased by nearly 30 % (P = 0.02) compared with the warfarin-alone group, whereas those of TM decreased by almost 25 % (P = 0.02). The administration of Danshen did not lead to any changes in the activities or the expression levels of the CYP, VKOR or TM enzymes compared with those of the control group. Gegen induced several warfarin-metabolizing CYP enzymes and neutralized the effects of warfarin towards VKOR and TM.ConclusionGegen, rather than Danshen at the same tested dosage, offsets the anticoagulant effects of warfarin by accelerating the phase I liver metabolism of warfarin, as well as increasing the activity, mRNA and protein expression of VKOR while decreasing those of TM.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0078-9) contains supplementary material, which is available to authorized users.

Lipid Lowering Effects of Hydroalcoholic Extract of Anethum graveolens L. and Dill Tablet in High Cholesterol Fed Hamsters

Objective. This study was aimed to determine the effect of Anethum graveolens extract and Anethum graveolens (dill) tablet on lipid profile, liver enzymes, and gene expression and enzymatic activity of HMG-CoA reductase in high cholesterol fed hamsters. Materials and Methods. Golden Syrian male hamsters (130 ± 10 g) were randomly divided into 6 groups (n = 6) and received daily the following: group 1 received chow + 2% cholesterol + 0.5% cholic acid (HCD), groups 2 and 3 received HCD diet plus 100 and 200 mg/kg hydroalcoholic extract of dill, respectively, and groups 4 and 5 received HCD diet plus 100 and 200 mg/kg dill tablet, respectively. Group 6 received only chow. After 1 month feeding serum biochemical factors were determined. HMG-CoA reductase mRNA level was measured (real-time PCR) and its activity was determined spectrophotometrically. Results. Compared with hypercholesterolemic group 1, lipid profile, blood glucose, and liver enzymes significantly decreased in all dill tablet or dill extract treated groups (p < 0.05). The changes in HMG-CoA reductase gene expression level and enzyme activity significantly reduced in animals that received 200 mg/kg of extract or tablet. Conclusion. Dill extract and dill tablet showed potential hypocholesterolemic properties in hamsters by inhibition of HMG-CoA reductase activity.

Dietary alpha-linolenic acid/linoleic acid ratios modulate intestinal immunity, tight junctions, anti-oxidant status and mRNA levels of NF-κB p65, MLCK and Nrf2 in juvenile grass carp (Ctenopharyngodon idella).

This study was conducted to investigate the effects of dietary alpha-linolenic acid/linoleic acid (ALA/LNA) ratios on the immune response, tight junctions, antioxidant status and immune-related signaling molecules mRNA levels in the intestine of juvenile grass carp (Ctenopharyngodon idella). A total of 1260 juvenile grass carp with an average initial weight of 8.78 ± 0.03 g were fed diets with different ALA/LNA ratios (0.01, 0.34, 0.68, 1.03, 1.41, 1.76 and 2.15) for 60 days. Results indicated that ALA/LNA ratio of 1.03 significantly increased acid phosphatase, lysozyme activities and complement C3 contents, promoted interleukin 10, transforming growth factor β1 and κB inhibitor α mRNA abundance, whereas suppressed pro-inflammatory cytokines (interleukin 1β, interleukin 8, tumor necrosis factor α and interferon γ2) and signal molecules (IκB kinase β, IκB kinase γ and nuclear factor κB p65) mRNA levels in the intestine (P < 0.05), suggesting that optimal dietary ALA/LNA ratio improved intestinal immune response of juvenile fish. Additionally, ALA/LNA ratio of 1.03 significantly promoted Claudin-3, Claudin-b, Claudin-c, Occludin and ZO-1 gene transcription, whereas reduced Claudin-15a and myosin light-chain kinase mRNA levels in the intestine, suggesting that appropriate dietary ALA/LNA ratio strengthened tight junctions in the intestine of juvenile fish. Meanwhile, ALA/LNA ratio of 1.03 noticeably elevated glutathione contents, copper/zinc superoxide dismutase, glutathione peroxidase, glutathione S-transferase and glutathione reductase activities and mRNA levels, as well as signaling molecule nuclear factor erythoid 2-related factor 2 gene transcriptional abundance in the intestine, suggesting that proper ratio of dietary ALA/LNA ameliorate the intestinal antioxidant status of juvenile fish. Based on the quadratic regression analysis of the complement C3 content in the distal intestine and malondialdehyde content in the whole intestine, optimal ALA/LNA ratio for maximum growth of juvenile grass carp (8.78-72.00 g) were estimated to be 1.13 and 1.12, respectively.