GPETAFLR, a peptide from Lupinus angustifolius L. prevents inflammation in microglial cells and confers neuroprotection in brain.

OBJECTIVES: Neuroinflammation is a complex inflammatory process in the central nervous system (CNS) where microglia may play a critical role. GPETAFLR is a peptide isolated from Lupinus angustifolius L. protein hydrolysates with functional activity in mononuclear phagocytes. However, it is unknown whether GPETAFLR has neuroprotective effects. METHODS: We analysed the potential anti-neuroinflammatory activity of GPETAFLR by using two different models of neuroinflammation: BV-2 microglial cells and mice with high-fat diet (HFD)-induced obesity. RESULTS: GPETAFLR hampered LPS-induced upregulation of pro-inflammatory and M1 marker genes in BV-2 cells. This effect was accompanied by an unchanged expression of anti-inflammatory IL-10 gene and by an increased expression of M2 marker genes. GPETAFLR also increased the transcriptional activity of M2 marker genes, while the microglia population remained unchanged in number and M1/M2 status in brain of mice with high-fat diet (HFD)-induced obesity. Furthermore, GPETAFLR counteracted HFD-induced downregulation of IL-10 and upregulation of pro-inflammatory markers in the mouse brain, both at gene and protein levels. DISCUSSION: This is the first report describing that a peptide from plant origin robustly restrained the pro-inflammatory activation of microglial cells in cultures and in brain. Our data suggest that GPETAFLR might be instrumental in maintaining CNS homeostasis by inhibiting neuroinflammation.

Nutraceutical Extract from Dulse (Palmaria palmata L.) Inhibits Primary Human Neutrophil Activation.

Palmaria palmata L. (Palmariaceae), commonly known as "dulse", is a red alga that grows on the northern coasts of the Atlantic and Pacific oceans, and is widely used as source of fiber and protein. Dulse is reported to contain anti-inflammatory and antioxidant compounds, albeit no study has investigated these effects in primary human neutrophils. Implication strategies to diminish neutrophil activation have the potential to prevent pathological states. We evaluated the ability of a phenolic dulse extract (DULEXT) to modulate the lipopolysaccharide (LPS)-mediated activation of primary human neutrophils. Intracellular reactive oxygen species (ROS) were measured by fluorescence analysis and nitric oxide (NO) production using the Griess reaction. Inflammatory enzymes and cytokines were detected by ELISA and RT-qPCR. The results show that DULEXT diminished the neutrophil activation related to the down-regulation of TLR4 mRNA expression, deceased gene expression and the LPS-induced release of the chemoattractant mediator IL-8 and the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α. ROS, NO, and myeloperoxidase (MPO) were also depressed. The data indicated that DULEXT has the potential to disrupt the activation of human primary neutrophils and the derived inflammatory and prooxidant conditions, and suggest a new role for Palmaria palmata L. in the regulation of the pathogenesis of health disorders in which neutrophils play a key role, including atherosclerosis.

Acyclic Diterpene Phytol from Hemp Seed Oil (Cannabis sativa L.) Exerts Anti-Inflammatory Activity on Primary Human Monocytes-Macrophages.

Seeds from non-drug varieties of hemp (Cannabis sativa L.) have been used for traditional medicine, food, and fiber production. Our study shows that phytol obtained from hemp seed oil (HSO) exerts anti-inflammatory activity in human monocyte-macrophages. Fresh human monocytes and human macrophages derived from circulating monocytes were used to evaluate both plasticity and anti-inflammatory effects of phytol from HSO at 10-100 mM using FACS analysis, ELISA, and RT-qPCR methods. The quantitative study of the acyclic alcohol fraction isolated from HSO shows that phytol is the most abundant component (167.59 ± 1.81 mg/Kg of HSO). Phytol was able to skew monocyte-macrophage plasticity toward the anti-inflammatory non-classical CD14+CD16++ monocyte phenotype and toward macrophage M2 (CD200Rhigh and MRC-1high), as well as to reduce the production of IL-1ß, IL-6, and TNF-α, diminishing the inflammatory competence of mature human macrophages after lipopolysaccharide (LPS) treatment. These findings point out for the first time the reprogramming and anti-inflammatory activity of phytol in human monocyte-macrophages. In addition, our study may help to understand the mechanisms by which phytol from HSO contributes to the constant and progressive plasticity of the human monocyte-macrophage linage.

Antioxidant and Immunomodulatory Properties of Chia Protein Hydrolysates in Primary Human Monocyte-Macrophage Plasticity.

Chia (Salvia hispanica L.) seed has high potential in the development of functional food due to its protein content with a special amino acid profile. Among the hematopoietic-derived cells, monocytes are endowed with high plasticity, responsible for their pro- and anti-inflammatory function in M1 and M2 phenotype polarization, respectively. Indeed, monocytes are involved in several oxidative- and inflammatory-associated disorders such as cancer, obesity, and cardiovascular and neurodegenerative diseases. This study was designed to investigate the role of chia protein hydrolysates (CPHs) in primary human monocyte-macrophage plasticity response using biochemical, RT-qPCR, and ELISA assays. Our results showed that CPHs reduce ROS and nitrite output, as pro-inflammatory cytokine secretion, and enhance the expression and release of anti-inflammatory cytokines. In addition, CPHs reverse LPS-associated M1 polarization into M2. These findings open new opportunities for developing nutritional strategies with chia as a dietary source of biopeptides to prevent the development and progression of oxidative- and inflammatory-related diseases.

Kiwicha (Amaranthus caudatus L.) protein hydrolysates reduce intestinal inflammation by modulating the NLRP3 inflammasome pathway.

The increase in the world population along with new policies aimed at a more sustainable world has led to the need of searching for new food sources, which are environmentally friendly, implying healthy and nutritious diets. This study explored the biological activity of two kiwicha (Amaranthus caudatus L.) protein hydrolysates obtained with the aid of Bioprotease LA-660 regarding their anti-inflammatory response at the intestinal level, employing the CACO-2 cell line. The results obtained showed that the in vitro administration of these hydrolysates decreased the expression of proinflammatory cytokines, increased the expression of anti-inflammatory cytokines, and decreased the gene expression of the major components of inflammasomes in the intestinal CACO-2 cell model. To the best of our knowledge, this is the first study involving the evaluation of the anti-inflammatory activity of kiwicha hydrolysates at the intestinal level, employing the CACO-2 cell model and its ultrastructural characterization using scanning electron microscopy. We conclude that the Amaranthus caudatus hydrolysates are a valuable source of active peptides that take part as functional ingredients in food and nutraceutical preparations.

Obesity-Associated Metabolic Disturbances Reverse the Antioxidant and Anti-Inflammatory Properties of High-Density Lipoproteins in Microglial Cells.

High-density lipoproteins (HDLs) play an important role in reverse cholesterol transport and present antioxidant properties, among others. In the central nervous system (CNS), there are HDLs, where these lipoproteins could influence brain health. Owing to the new evidence of HDL functionality remodeling in obese patients, and the fact that obesity-associated metabolic disturbances is pro-inflammatory and pro-oxidant, the aim of this study was to investigate if HDL functions are depleted in obese patients and obesity-associated microenvironment. HDLs were isolated from normal-weight healthy (nwHDL) and obese men (obHDL). The oxHDL level was measured by malondialdehyde and 4-hydroxynoneal peroxided products. BV2 microglial cells were exposed to different concentrations of nwHDL and obHDL in different obesity-associated pro-inflammatory microenvironments. Our results showed that hyperleptinemia increased oxHDL levels. In addition, nwHDLs reduced pro-inflammatory cytokines' release and M1 marker gene expression in BV2 microglial cells. Nevertheless, both nwHDL co-administered with LPS+leptin and obHDL promoted BV2 microglial activation and a higher pro-inflammatory cytokine production, thus confirming that obesity-associated metabolic disturbances reverse the antioxidant and anti-inflammatory properties of HDLs in microglial cells.

Postprandial triglyceride-rich lipoproteins promote M1/M2 microglia polarization in a fatty-acid-dependent manner.

Inhibiting M1 microglia phenotype while stimulating the M2 microglia has been suggested as a potential therapeutic approach for the treatment of neuroinflammatory diseases. Our hypothesis is that the type of dietary fatty acids (FAs) into human postprandial triglyceride-rich lipoproteins (TRLs) could modulate the plasticity of microglia. We isolated TRLs at the postprandial hypertriglyceridemic peak from blood samples of healthy volunteers after the ingestion of a meal rich in saturated FAs (SFAs), monounsaturated FAs (MUFAs) or MUFAs plus omega-3 long-chain polyunsaturated FAs. We observed that postprandial TRL-MUFAs enhance M2 microglia polarization, whereas postprandial TRL-SFAs made polarized microglia prone to an M1 phenotype. In addition, in contrast to dietary SFAs, dietary MUFAs primed for a reduced proinflammatory profile in the brain of mice fed with the different FA-enriched diets. Our study underlines a role of postprandial TRLs as a metabolic entity in regulating the plasticity of microglia and brings an understanding of the mechanisms by which dietary FAs are environmental factors fostering the innate immune responsiveness. These exciting findings open opportunities for developing nutraceutical strategies with olive oil as the principal source of MUFAs, notably oleic acid, to prevent development and progression of neuroinflammation-related diseases.

A lupine (Lupinus angustifolious L.) peptide prevents non-alcoholic fatty liver disease in high-fat-diet-induced obese mice.

Bioactive peptides are related to the prevention and treatment of many diseases. GPETAFLR is an octapeptide that has been isolated from lupine (Lupinus angustifolius L.) and shows anti-inflammatory properties. The aim of this study was to evaluate the potential activity of GPETAFLR to prevent non-alcoholic fatty liver disease (NAFLD) in high-fat-diet (HFD)-induced obese mice. C57BL/6J mice were fed a standard diet or HFD. Two of the groups fed the HFD diet were treated with GPETAFLR in drinking water at 0.5 mg kg-1 day-1 or 1 mg kg-1 day-1. To determine the ability of GPETAFLR to improve the onset and progression of non-alcoholic fatty liver disease, histological studies, hepatic enzyme profiles, inflammatory cytokine and lipid metabolism-related genes and proteins were analysed. Our results suggested that HFD-induced inflammatory metabolic disorders were alleviated by treatment with GPETAFLR. In conclusion, dietary lupine consumption can repair HFD-induced hepatic damage possibly via modifications of liver's lipid signalling pathways.

Hemp (Cannabis sativa L.) Protein Hydrolysates Promote Anti-Inflammatory Response in Primary Human Monocytes.

Hemp seeds have a wide variety of chemical compounds which present biological activity. Specifically, the focus on proteins and bioactive peptides are increasing as alternative sources of nutraceutical uses. In the literature, hemp protein products (HPPs) have reported antioxidant and anti-inflammatory properties. This study aimed to determine the inflammation-related modulatory effects of HPPs on lipopolysaccharide (LPS)-activated primary human monocytes. CD14+ cells were immunomagnetically isolated from buffy coats and the anti-inflammatory activity of hemp protein isolate (HPI) and hydrolysates (HPHs) was evaluated on LPS-stimulated human primary monocytes. The specific markers of inflammation, polarization, and chemoattraction were measured by RT-qPCR and ELISA assays. Our results showed that HPPs decreased the pro-inflammatory mediators (TNFα, IL-1ß, and IL-6) and increased the anti-inflammatory mediators (IL-10 and IL-4). In addition, M1 polarization marker gene expression (CCR7 and iNOS) was downregulated by HPPs and, M2 polarization marker gene expression (CD200R and MRC1) was upregulated. Finally, the mRNA expression of chemotaxis genes (CCR2 and CCL2) was downregulated by HPPs. In conclusion, this study suggests that HPPs may improve chronic inflammatory states and promote regenerative processes by reprogramming monocytes toward M2 polarization phenotype.

GPETAFLR, an octapeptide isolated from Lupinus angustifolius L. protein hydrolysate, promotes the skewing to the M2 phenotype in human primary monocytes.

The present study aimed to test the mechanisms by which GPETAFLR, released from the enzymatic hydrolysis of lupine protein, may modulate the inflammatory response and plasticity in human primary monocytes. Human circulating monocytes and mature macrophages were used to analyze the effects of GPETAFLR on plasticity and inflammatory response using biochemical, flow cytometry, quantitative real-time PCR, and ELISA assays. GPETAFLR skewed the monocyte plasticity towards the anti-inflammatory non-classical CD14+CD16++ monocyte subset and reduced the inflammatory competence of LPS-treated human monocytes diminishing IL-1ß, IL-6, and TNF-α and increasing IL-10 production and gene expression. Results showed that GPETAFLR decreased the frequency of the LPS-induced activated monocyte population (CD14++CD16-), diminished monocyte activation involved down-regulation of CCR2 mRNA expression and protein expression, and decreased gene expression of the LPS-induced chemoattractant mediator CCL2. Our findings imply a new understanding of the mechanisms by which GPETAFLR favor a continuous and gradual plasticity process in the human monocyte/macrophage system and offer novel benefits derived from the consumption of Lupinus angustifolius L. in the prevention of inflammatory-related diseases.

Neuroprotective protein hydrolysates from hemp (Cannabis sativa L.) seeds.

Hemp (Cannabis sativa L.) seeds are well known for their potential use as a source of nutrients, fiber, and bioactive compounds. A hemp protein isolate, prepared from defatted hemp flour, was hydrolyzed by alcalase and flavourzyme under specific conditions. The resulting hydrolysates were evaluated for the selection of potentially bioactive hemp protein hydrolysates (HPHs) owing to their DPPH scavenging and ferric reducing antioxidant power activity. In vitro cell-free experiments led to the identification of two bioactive HPHs, HPH20A and HPH60A + 15AF, which were used at 50 and 100 µg mL-1 on BV-2 microglial cells in order to evaluate the anti-neuroinflammatory activities. Our results showed that HPH20A and HPH60A + 15AF down-regulated TNF-α, IL-1ß, and IL-6 mRNA transcriptional levels in LPS-stimulated BV-2 microglial cells. In addition, HPH20A and HPH60A + 15AF up-regulated the gene expression of anti-inflammatory cytokine IL-10. This study suggests for the first time that HPHs may improve the neuroinflammatory and inflammatory states, supporting the nutraceutical value of hemp seeds.

Minor compounds from virgin olive oil attenuate LPS-induced inflammation via visfatin-related gene modulation on primary human monocytes.

We have analyzed the effects of minor compounds found in the unsaponifiable fraction (UF) and in the phenolic fraction (PF) of virgin olive oil (VOO) on LPS-induced inflammatory response via visfatin modulation in human monocytes. For this purpose, monocytes were incubated with UF and PF at different concentrations and the pro-inflammatory stimulus LPS for 24 hr; squalene (SQ) and hydroxytyrosol (HTyr), the main components in UF and PF, respectively, were also used. The relative expression of both pro-inflammatory and anti-inflammatory genes, as well as other genes related to the NAD+-biosynthetic pathway was evaluated by RT-qPCR; and the secretion of some of these markers was assessed by ELISA procedures. We found that UF, SQ, PF, and HTyr prevented from LPS-induced dysfunctional gene expression and secretion via visfatin-related gene modulation in human monocytes. These findings unveil a potential beneficial role for minor compounds of VOO in the prevention of inflammatory-disorders. PRACTICAL APPLICATION: In this project, potential health benefits of VOO micronutrients (unsaponifiable and phenolic compounds) were confirmed through anti-inflammatory assays. Our results reveal new interesting researching goals concerning nutrition by considering the role of bioactive VOO compounds in the prevention and progress of diseases related to inflammation.

GPETAFLR, a biopeptide from Lupinus angustifolius L., protects against oxidative and inflammatory damage in retinal pigment epithelium cells.

GPETAFLR, an octapeptide released from the enzymatic hydrolysis of lupine (Lupinus angustifolius L.) protein, has demonstrated anti-inflammatory effect in myeloid lineage. This work aims to evaluate in retinal pigment epithelium (RPE) cells the protective role of GPETAFLR on both oxidative and inflammatory markers known to be involved in age-related macular degeneration (AMD). In comparison with stimulated control cells, GPETAFLR increased glutathione production and diminished the secretion and gene expression of VEFG, IL-1ß, IL-6, IFNγ, and TNF-α, as well as reactive oxygen species, and nitrite output. Our findings reveal that GPETAFLR, a novel plant peptide, is able to protect against RPE oxidative stress and inflammation. Taken together, these results strongly support innovative nutritional strategies considering Lupinus angustifolius L. as source of proteins to prevent the onset and progression of AMD. PRACTICAL APPLICATIONS: We reveal a novel nutraceutical impact of GPETAFLR peptide in human RPE cells to prevent oxidative and inflammatory mediators. Our results support that the intake of Lupine angustifolius L., proposed to be a reservoir of GPETAFLR, could lessen the functional decay of RPE cells, leading therefore to a slowdown of the progress of AMD during age. Not only this work, but also future simple clinical studies should raise new nutritional strategies focused on understanding the etiological role of the foods, nutrition, and metabolism in the pathogenesis of ocular disorders.