Eicosapentaenoic (EPA) and Docosahexaenoic (DHA) Acid Differentially Modulate Rat Neutrophil Function In Vitro

Fish oils are used as therapeutic agents in chronic inflammatory diseases. The omega-3 fatty acids (FA) found in these oils are mainly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. The anti-inflammatory properties of fish oils are attributed to both omega-3 fatty acids. However, it is unknown whether such effects are due to either EPA or DHA. In this study, the effects of EPA and DHA on rat neutrophil function in vitro were compared. Both EPA and DHA increased the production of H₂O₂ when cells were stimulated or not with lipopolysaccharides (LPS). However, EPA was more potent than DHA in triggering an increase in superoxide release by cells in the basal condition or when stimulated with phorbol myristate acetate (PMA) or zymosan. Only DHA increased the phagocytic capacity and fungicidal activity of neutrophils. Both FA increased the release of tumor necrosis factor-α (TNF-α) in nonstimulated cells, but only EPA increased the production of cytokine-inducing neutrophil chemoattractant-2 (CINC-2) in the absence or presence of LPS, whereas production of interleukin-1 beta (IL-1β) was only increased by DHA in the presence of LPS. In addition, there was no alteration in the production of nitric oxide. In conclusion, we show herein that EPA and DHA can differently modulate aspects of the neutrophil response, which may be relevant for the development of therapies rich in one or other FA depending on the effect required.     

Oxidative Properties of Polystyrene Nanoparticles with Different Diameters in Human Peripheral Blood Mononuclear Cells (In Vitro Study)

With the ongoing commercialization, human exposure to plastic nanoparticles will dramatically increase, and evaluation of their potential toxicity is essential. There is an ongoing discussion on the human health effects induced by plastic particles. For this reason, in our work, we assessed the effect of polystyrene nanoparticles (PS-NPs) of various diameters (29, 44 and 72 nm) on selected parameters of oxidative stress and the viability of human peripheral blood mononuclear cells (PBMCs) in the in vitro system. Cells were incubated with PS-NPs for 24 h in the concentration range of 0.001 to 100 µg/mL and then labeled: formation of reactive oxygen species (ROS) (including hydroxyl radical), protein and lipid oxidation and cell viability. We showed that PS-NPs disturbed the redox balance in PBMCs. They increased ROS levels and induced lipid and protein oxidation, and, finally, the tested nanoparticles induced a decrease in PBMCs viability. The earliest changes in the PBMCs were observed in cells incubated with the smallest PS-NPs, at a concentration of 0.01 μg/mL. A comparison of the action of the studied nanoparticles showed that PS-NPs (29 nm) exhibited a stronger oxidative potential in PBMCs. We concluded that the toxicity and oxidative properties of the PS-NPs examined depended to significant degree on their diameter.     

The Relationship between the Refractive Index of Fish Oils and their Content of Eicosapentaenoic Acid (EPA), Docosahexaenoic Acid (DHA) and Total Polyunsaturated Fatty Acids (PUFA)

Highly significant correlations were found to exist between the refractive index of fish oils and their content of eicosapentaenoic acid, the sum of eicosapentaenoic and docosahexaenoic acid and the total polyunsaturated fatty acids. The refractive index of a fish oil can be used therefore as an indicator of the content of the abovementioned fatty acids.     

Indigo Pulverata Levis (Chung-Dae,Persicaria tinctoria) Alleviates Atopic Dermatitis-like Inflammatory Responses In Vivo and In Vitro

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with a type 2 T helper cell (Th2) immune response. The Indigo Pulverata Levis extract (CHD) is used in traditional Southeast Asian medicine; however, its beneficial effects on AD remain uninvestigated. Therefore, we investigated the therapeutic effects of CHD in 2,4-dinitrochlorobenzene (DNCB)-induced BALB/c mice and tumor necrosis factor (TNF)-α- and interferon gamma (IFN)-γ-stimulated HaCaT cells. We evaluated immune cell infiltration, skin thickness, and the serum IgE and TNF-α levels in DNCB-induced AD mice. Moreover, we measured the expression levels of pro-inflammatory cytokines, mitogen-activated protein kinase (MAPK), and the nuclear factor-kappa B (NF-κB) in the mice dorsal skin. We also studied the effect of CHD on the translocation of NF-κB p65 and inflammatory chemokines in HaCaT cells. Our in vivo results revealed that CHD reduced the dermis and epidermis thicknesses and inhibited immune cell infiltration. Furthermore, it suppressed the proinflammatory cytokine expression and MAPK and NF-κB phosphorylations in the skin tissue and decreased serum IgE and TNF-α levels. In vitro results indicated that CHD downregulated inflammatory chemokines and blocked NF-κB p65 translocation. Thus, we deduced that CHD is a potential drug candidate for AD treatment.     

Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells

Therapeutic oligonucleotides have achieved great clinical interest since their approval as drug agents by regulatory agencies but their access and distribution in blood cells are not completely known. We evaluated by flow cytometry the ability of short fluorescent scramble oligonucleotides (ON*) to access human peripheral blood mononuclear cells (PBMC) after incubating with ON* during 1 h and 7 days of culture follow-up ‘in vitro’. Blood samples were treated with chemically modified oligonucleotides (phosphorothioate backbone and 2′ O-Me ends) to resist nuclease digestion under culture conditions. The ON* internalization was determined after discarding the membrane-associated fluorescence by trypan blue quenching. Whereas the oligonucleotide accessed neutrophils and monocytes rapidly, achieving their maximum in 1 h and 24 h, respectively, lymphocytes required 7 days to achieve the maximum (80% of cells) transfection. The ON*ability to access lymphocyte types (T, B, and NK) and T cell subtypes (CD4+, CD8+, and CD4-CD8-) were similar, with T cells being more accessible. Regulatory CD4+ and CD8+ T cells were classified in low and high Foxp3 expressers, whose expression proved not to alter the ON* internalization during the first hour, achieving 53% of CD4+Foxp3+ and 40% of CD8+Foxp3+ cells. Our results contribute to understanding and improving the management of therapeutic ONs.     

Influence of Soybean Oil Blending with Polylactic Acid (PLA) Films: In Vitro and In Vivo Evaluation

Due to the great interest in oil‐based polymers, which are prepared from renewable resources, different forms and amounts of soybean oil‐based PLA films were prepared and evaluated for their potential usage as a medical biomaterial. Soybean oil, epoxidized soybean oil and auto‐oxidized soybean oil were blended with PLA and PLA/oil films with appropriate oil amounts [2, 7, 14 and 20% (w/w)] were obtained by solvent casting. Thermal stability and plasticization effect were determined by adjusting oil amounts and type. Epoxidized soybean oil blended films showed the smallest increase in elongation breaks (13–20%) and the highest decrease in thermal decomposition temperatures (364–327 °C) compared to other oil blended films. In vitro quantitative and qualitative cytotoxicity results showed no reactivity (grade 0) for the L929 cells treated with 14% (w/w) oil blended PLA films. In vivo irritation and implantation tests concluded that 14% (w/w) oil blended PLA films were non‐irritant. No erythema, no oedema reactions, no traumatic necrosis and foreign debris were observed. Thus, along with superior biocompatibility, PLA/oil films can replace petroleum‐based products for several biomedical uses.     

Ascorbic Acid Supplementation Improves Skeletal Muscle Growth in Pacu (Piaractus mesopotamicus) Juveniles: In Vivo and In Vitro Studies

In fish, fasting leads to loss of muscle mass. This condition triggers oxidative stress, and therefore, antioxidants can be an alternative to muscle recovery. We investigated the effects of antioxidant ascorbic acid (AA) on the morphology, antioxidant enzyme activity, and gene expression in the skeletal muscle of pacu (Piaractus mesopotamicus) following fasting, using in vitro and in vivo strategies. Isolated muscle cells of the pacu were subjected to 72 h of nutrient restriction, followed by 24 h of incubation with nutrients or nutrients and AA (200 µM). Fish were fasted for 15 days, followed by 6 h and 15 and 30 days of refeeding with 100, 200, and 400 mg/kg of AA supplementation. AA addition increased cell diameter and the expression of anabolic and cell proliferation genes in vitro. In vivo, 400 mg/kg of AA increased anabolic and proliferative genes expression at 6 h of refeeding, the fiber diameter and the expression of genes related to cell proliferation at 15 days, and the expression of catabolic and oxidative metabolism genes at 30 days. Catalase activity remained low in the higher supplementation group. In conclusion, AA directly affected the isolated muscle cells, and the higher AA supplementation positively influenced muscle growth after fasting.     

Retroconversion of Docosapentaenoic Acid (n-6): an Alternative Pathway for Biosynthesis of Arachidonic Acid in Daphnia magna

The aim of this study was to assess metabolic pathways for arachidonic acid (20:4n-6) biosynthesis in Daphnia magna. Neonates of D. magna were maintained on [¹³C] enriched Scenedesmus obliquus and supplemented with liposomes that contained separate treatments of unlabeled docosapentaenoic acid (22:5n-6), 20:4n-6, linoleic acid (18:2n-6) or oleic acid (18:1n-9). Daphnia in the control treatment, without any supplementary fatty acids (FA) containing only trace amounts of 20:4n-6 (~0.3 % of all FA). As expected, the highest proportion of 20:4n-6 (~6.3 %) was detected in Daphnia that received liposomes supplemented with this FA. Higher availability of 18:2n-6 in the diet increased the proportion of 18:2n-6 in Daphnia, but the proportion of 20:4n-6 was not affected. Daphnia supplemented with 22:5n-6 contained ~3.5 % 20:4n-6 in the lipids and FA specific stable isotope analyses validated that the increase in the proportion of 20:4n-6 was due to retroconversion of unlabeled 22:5n-6. These results suggest that chain shortening of 22:5n-6 is a more efficient pathway to synthesize 20:4n-6 in D. magna than elongation and desaturation of 18:2n-6. These results may at least partially explain the discrepancies noticed between phytoplankton FA composition and the expected FA composition in freshwater cladocerans. Finally, retroconversion of dietary 22:5n-6 to 20:4n-6 indicates Daphnia efficiently retain long chain n-6 FA in lake food webs, which might be important for the nutritional ecology of fish.     

Blood–Nerve Barrier (BNB) Pathology in Diabetic Peripheral Neuropathy and In Vitro Human BNB Model

In diabetic peripheral neuropathy (DPN), metabolic disorder by hyperglycemia progresses in peripheral nerves. In addition to the direct damage to peripheral neural axons, the homeostatic mechanism of peripheral nerves is disrupted by dysfunction of the blood–nerve barrier (BNB) and Schwann cells. The disruption of the BNB, which is a crucial factor in DPN development and exacerbation, causes axonal degeneration via various pathways. Although many reports revealed that hyperglycemia and other important factors, such as dyslipidemia-induced dysfunction of Schwann cells, contributed to DPN, the molecular mechanisms underlying BNB disruption have not been sufficiently elucidated, mainly because of the lack of in vitro studies owing to difficulties in establishing human cell lines from vascular endothelial cells and pericytes that form the BNB. We have developed, for the first time, temperature-sensitive immortalized cell lines of vascular endothelial cells and pericytes originating from the BNB of human sciatic nerves, and we have elucidated the disruption to the BNB mainly in response to advanced glycation end products in DPN. Recently, we succeeded in developing an in vitro BNB model to reflect the anatomical characteristics of the BNB using cell sheet engineering, and we established immortalized cell lines originating from the human BNB. In this article, we review the pathologic evidence of the pathology of DPN in terms of BNB disruption, and we introduce the current in vitro BNB models.     

In Vitro and in Vivo Models of Non-Alcoholic Fatty Liver Disease (NAFLD)

By now, non-alcoholic fatty liver disease (NAFLD) is considered to be among the most common liver diseases world-wide. NAFLD encompasses a broad spectrum of pathological conditions ranging from simple steatosis to steatohepatitis, fibrosis and finally even cirrhosis; however, only a minority of patients progress to end-stages of the disease, and the course of the disease progression to the later stages seems to be slow, developing progressively over several years. Key risk factors including overweight, insulin resistance, a sedentary life-style and an altered dietary pattern, as well as genetic factors and disturbances of the intestinal barrier function have been identified in recent years. Despite intense research efforts that lead to the identification of these risk factors, knowledge about disease initiation and molecular mechanisms involved in progression is still limited. This review summarizes diet-induced and genetic animal models, as well as cell culture models commonly used in recent years to add to the understanding of the mechanisms involved in NAFLD, also referring to their advantages and disadvantages.     

In Vitro and in Vivo Anti-Hyperglycemic Effects of Omija (Schizandra chinensis) Fruit

The entrocytes of the small intestine can only absorb monosaccharides such as glucose and fructose from our diet. The intestinal absorption of dietary carbohydrates such as maltose and sucrose is carried out by a group of α-glucosidases. Inhibition of these enzymes can significantly decrease the postprandial increase of blood glucose level after a mixed carbohydrate diet. Therefore, the inhibitory activity of Omija (Schizandra chinensis) extract against rat intestinal α-glucosidase and porcine pancreatic α-amylase were investigated in vitro and in vivo. The in vitro inhibitory activities of water extract of Omija pulp/skin (OPE) on α-glucosidase and α-amylase were potent when compared to Omija seeds extract (OSE). The postprandial blood glucose lowering effect of Omija extracts was compared to a known type 2 diabetes drug (Acarbose), a strong α-glucosidase inhibitor in the Sprague-Dawley (SD) rat model. In rats fed on sucrose, OPE significantly reduced the blood glucose increase after sucrose loading. Furthermore, the oxygen radical absorbance capacity (ORAC) of OSE and OPE was evaluated. OPE had higher peroxyl radical absorbing activity than OSE. These results suggest that Omija, which has high ORAC value with α-glucosidase inhibitory activity and blood glucose lowering effect, could be physiologically useful for treatment of diabetes, although clinical trials are needed.     

Mitogen Kinase Kinase (MKK7) Controls Cytokine Production In Vitro and In Vivo in Mice

Mitogen kinase kinase 4 (MKK4) and mitogen kinase kinase 7 (MKK7) are members of the MAP2K family that can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, while MKK7 has been reported to activate only JNK in response to different stimuli. The stimuli, as well as the cell type determine which MAP2K member will mediate a given response. In various cell types, MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have also implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK4 and MKK7 contribute to innate immune responses in macrophages or during inflammation in vivo. To address this question and to elucidate the role of MKK4 and MKK7 in macrophage and in vivo, we developed MKK4- and MKK7-deficient mouse models with tamoxifen-inducible Rosa26 Cre^ERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for lipopolysaccharide (LPS)-induced cytokine production, M1 polarization, and migration, which appear to be a major contributor to the inflammatory response in vivo. Conversely, MKK4 plays a significant, but minor role in cytokine production in vivo.     

Chebulagic Acid,a Hydrolyzable Tannin,Exhibited Antiviral Activity in Vitro and in Vivo against Human Enterovirus 71

Human enterovirus 71 is one of the major causative agents of hand, foot and mouth disease in children under six years of age. Presently, no vaccines or antiviral drugs have been clinically available to employ against EV71. In this study, we demonstrate that treatment with chebulagic acid reduced the viral cytopathic effect on rhabdomyosarcoma cells with an IC₅₀ of 12.5 μg/mL. The utilization of the chebulagic acid treatment on mice challenged with a lethal dose of enterovirus 71 was able to efficiently reduce mortality and relieve clinical symptoms through the inhibition of viral replication. Chebulagic acid may represent a potential therapeutic agent to control infections to enterovirus 71.     

Recurrent Herpes Simplex Virus Type 1 (HSV-1) Infection Modulates Neuronal Aging Marks in In Vitro and In Vivo Models

Herpes simplex virus 1 (HSV-1) is a widespread neurotropic virus establishing a life-long latent infection in neurons with periodic reactivations. Recent studies linked HSV-1 to neurodegenerative processes related to age-related disorders such as Alzheimer’s disease. Here, we explored whether recurrent HSV-1 infection might accelerate aging in neurons, focusing on peculiar marks of aged cells, such as the increase in histone H4 lysine (K) 16 acetylation (ac) (H4K16ac); the decrease of H3K56ac, and the modified expression of Sin3/HDAC1 and HIRA proteins. By exploiting both in vitro and in vivo models of recurrent HSV-1 infection, we found a significant increase in H4K16ac, Sin3, and HDAC1 levels, suggesting that the neuronal response to virus latency and reactivation includes the upregulation of these aging markers. On the contrary, we found a significant decrease in H3K56ac that was specifically linked to viral reactivation and apparently not related to aging-related markers. A complex modulation of HIRA expression and localization was found in the brain from HSV-1 infected mice suggesting a specific role of this protein in viral latency and reactivation. Overall, our results pointed out novel molecular mechanisms through which recurrent HSV-1 infection may affect neuronal aging, likely contributing to neurodegeneration.     

Products of Bisphenol A Degradation Induce Cytotoxicity in Human Erythrocytes (In Vitro)

The aim of this work has been to study the possible degradation path of BPA under the Fenton reaction, namely to determine the energetically favorable intermediate products and to compare the cytotoxicity of BPA and its intermediate products of degradation. The DFT calculations of the Gibbs free energy at M06-2X/6-311G(d,p) level of theory showed that the formation of hydroquinone was the most energetically favorable path in a water environment. To explore the cytotoxicity the erythrocytes were incubated with BPA and three intermediate products of its degradation, i.e., phenol, hydroquinone and 4-isopropylphenol, in the concentrations 5–200 μg/mL, for 1, 4 and 24 h. BPA induced the strongest hemolytic changes in erythrocytes, followed by hydroquinone, phenol and 4-isopropylphenol. In the presence of hydroquinone, the highest level of RONS was observed, whereas BPA had the weakest effect on RONS generation. In addition, hydroquinone decreased the level of GSH the most. Generally, our results suggest that a preferable BPA degradation path under a Fenton reaction should be controlled in order to avoid the formation of hydroquinone. This is applicable to the degradation of BPA during waste water treatment and during chemical degradation in sea water.     

Estradiol Favors the Formation of Eicosapentaenoic Acid (20:5n-3) and n-3 Docosapentaenoic Acid (22:5n-3) from Alpha-Linolenic Acid (18:3n-3) in SH-SY5Y Neuroblastoma Cells

Whether neurosteroids regulate the synthesis of long chain polyunsaturated fatty acids in brain cells is unknown. We examined the influence of 17-β-estradiol (E2) on the capacity of SH-SY5Y cells supplemented with α-linolenic acid (ALA), to produce eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Cells were incubated for 24 or 72 h with ALA added alone or in combination with E2 (ALA + E2). Fatty acids were analyzed by gas chromatography of ethanolamine glycerophospholipids (EtnGpl) and phosphatidylcholine (PtdCho). Incubation for 24 h with ALA alone increased EPA and DPA in EtnGpl, by 330 and 430% compared to controls (P < 0.001) and DHA by only 10% (P < 0.05). Although DHA increased by 30% (P < 0.001) in ALA + E2-treated cells, the difference between the ALA and ALA + E2 treatments were not significant after 24 h (Anova-1, Fisher’s test). After 72 h, EPA, DPA and DHA further increased in EtnGpl and PtdCho of cells supplemented with ALA or ALA + E2. Incubation for 72 h with ALA + E2 specifically increased EPA (+34% in EtnGpl, P < 0.001) and DPA (+15%, P < 0.001) compared to ALA alone. Thus, SH-SY5Y cells produced membrane EPA, DPA and DHA from supplemental ALA. The formation of DHA was limited, even in the presence of E2. E2 significantly favored EPA and DPA production in cells grown for 72 h. Enhanced synthesis of ALA-elongation products in neuroblastoma cells treated with E2 supports the hypothesis that neurosteroids could modulate the metabolism of PUFA.