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OBJECTIVE: This study evaluated the effect of sardine (SPH) and chickpea protein hydrolysates (CPH) on oxidant stress and inflammatory profile in cholesterol-fed rats. METHODS: The experiment was undertaken for thirty days on 18 cholesterol-fed Wistar rats (220±10 g) divided into three groups and receiving 1 g/kg of body weight either chickpea protein hydrolysate (CPH), sardine protein hydrolysate (SPH) or casein in water (CG). RESULTS: Compared to CG, SPH and CPH treatment reduced cholesterol, hydroperoxide and malondialdehyde contents in serum, lipoproteins, erythrocytes and aorta. These same treated groups showed also lower serum isoprostane levels. However, serum paraoxonase activity and HDL-antioxidant property were improved only by CPH compared to CG. SOD activity of aorta and erythrocytes was higher in CPH but in SPH group, SOD activity was lower in these tissues and remained unchanged in serum. Furthermore, CPH and SPH stimulated glutathione peroxidase and catalase activities of aorta and erythrocytes. In CPH group, nitric oxide levels of serum, erythrocytes and aorta were increased by respectively 1.4- to 1.8-fold compared to CG and SPH. In addition, among the three groups, CPH exhibited the best anti-inflammatory effect by lowering serum C reactive protein, uric acid and albumin concentrations. CONCLUSIONS: SPH and particularly CPH possess antioxidant and anti-inflammatory properties and could be useful as nutraceuticals for health improving and preventing numerous disorders such as cardiovascular diseases.

Squalene is a natural bioactive triterpene and an important intermediate in the biosynthesis of sterols. To assess the effect of this compound on the hepatic transcriptome, RNA-sequencing was carried out in two groups of male New Zealand rabbits fed either a diet enriched with 1% sunflower oil or the same diet with 0.5% squalene for 4 weeks. Hepatic lipids, lipid droplet area, squalene, and sterols were also monitored. The Squalene administration downregulated 9 transcripts and upregulated 13 transcripts. The gene ontology of transcripts fitted into the following main categories: transporter of proteins and sterols, lipid metabolism, lipogenesis, anti-inflammatory and anti-cancer properties. When the results were confirmed by RT-qPCR, rabbits receiving squalene displayed significant hepatic expression changes of LOC100344884 (PNPLA3), GCK, TFCP2L1, ASCL1, ACSS2, OST4, FAM91A1, MYH6, LRRC39, LOC108176846, GLT1D1 and TREH. A squalene-enriched diet increased hepatic levels of squalene, lanosterol, dihydrolanosterol, lathosterol, zymostenol and desmosterol. Strong correlations were found among specific sterols and some squalene-changed transcripts. Incubation of the murine AML12 hepatic cell line in the presence of lanosterol, dihydrolanosterol, zymostenol and desmosterol reproduced the observed changes in the expressions of Acss2, Fam91a1 and Pnpla3. In conclusion, these findings indicate that the squalene and post-squalene metabolites play important roles in hepatic transcriptional changes required to protect the liver against malfunction. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

Hepatic fat-specific protein 27 [cell death-inducing DNA fragmentation effector protein C (Cidec)/Fsp27] mRNA levels have been associated with hepatic lipid droplet extent under certain circumstances. To address its hepatic expression under different dietary conditions and in both sexes, apolipoprotein E (Apoe)-deficient mice were subjected to different experimental conditions for 11 wk to test the influence of cholesterol, Western diet, squalene, oleanolic acid, sex, and surgical castration on Cidec/Fsp27 mRNA expression. Dietary cholesterol increased hepatic Cidec/Fsp27β expression, an effect that was suppressed when cholesterol was combined with saturated fat as represented by Western diet feeding. Using the latter diet, neither oleanolic acid nor squalene modified its expression. Females showed lower levels of hepatic Cidec/Fsp27β expression than males when they were fed Western diets, a result that was translated into a lesser amount of CIDEC/FSP27 protein in lipid droplets and microsomes. This was also confirmed in low-density lipoprotein receptor (Ldlr)-deficient mice. Incubation with estradiol resulted in decreased Cidec/Fsp27β expression in AML12 cells. Whereas male surgical castration did not modify the expression, ovariectomized females did show increased levels compared with control females. Females also showed increased expression of peroxisome proliferator-activated receptor-γ coactivator 1-α (Pgc1a), suppressed by ovariectomy, and the values were significantly and inversely associated with those of Cidec/Fsp27β. When Pgc1a-deficient mice were used, the sex differences in Cidec/Fsp27β expression disappeared. Therefore, hepatic Cidec/Fsp27β expression has a complex regulation influenced by diet and sex hormonal milieu. The mRNA sex differences are controlled by Pgc1a., This research was supported by Ministerio de Economía y CompetitividadFondo Europeo de Desarrollo Regional (Grants SAF2015-63904-R, SAF2016-75441-R, and RTI2018-093864-B-I00), Fondo Social Europeo-Gobierno de Aragón (Grant B16_17R), and the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie Grant Agreement 721236-TREATMENT to M. Monsalve. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y Nutrición (CB06/03/1012) is an initiative of Instituto de Salud Carlos III. L. V. Herrera-Marcos and S. Sancho-Knapik were recipients of Fondo Social Europeo-Gobierno de Aragón and Fundación Cuenca-Villoro (BE 203/2009) fellowships, respectively.

Agro-food industries generate a huge amount of fruit and vegetable wastes. These residues are composed by structural parts (leaves, peels, pulps, seeds, roots and stems) after the extraction of their juices, and they are rich in bioactive compounds such polyphenols, protein, carbohydates, fibre, lignin, lipids and minerals. Therefore, these wastes constitute a source of bioactive compunds and their valorization leads to a circular economy in which industry and society benefit. Between the component of the residues, polyphenols compounds may contribute to prevent or treated cancer, neurodegenerative and cardiovascular diseases, among others. The aim of this work was the study of the potential therapeutic effect of fruit and vegetable extracts (peach, apple, cucumber and red pepper) obtained by ultrasounds, on different lines of cancer cells (colon, liver and breast) and on oxidative stress in a healthy colon. The results showed that extracts rich in polyphenols had an antiproliferativeeffect against tumor cells and their action was improved by encapsulation in maltodextrin. Encapsulation also increased the protection against oxidative stress in intestinal cells. This study opened the gate to use the extracts from fruit and vegetable wastes as pharmaceutical excipients, food additives, nutraceutical products or functional foods for therapeutic purposes and prevention of diseases related to oxidative stress., SOE1/P1/E0123 Interreg Sudoe REDVALUE, Technological Alliance to Complete the Forest and Agroindustrial Production Cycle. Authors also thank the Ministry of Economy and Innovation under Grant (SAF2016-75441-R); Aragón Regional Government (B16-20R, Fondos FEDER "Otra manera de hacer Europa") and CIBERobn under Grant (CB06/ 03/1012) of the Instituto de Salud Carlos III. Author Raquel Ibarz thanks the University of Lleida for the UdL-Impuls post-doctoral contract. In addition, this study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Pedro Santos is recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, info:eu-repo/semantics/publishedVersion

In this study, the total phenolic compounds content and profile, the nutritional value, the antioxidant and antiproliferative activities of avocado peel, seed coat, and seed extracts were characterized. Additionally, an in-silico analysis was performed to identify the phenolic compounds with the highest intestinal absorption and Caco-2 permeability. The avocado peel extract possessed the highest content of phenolic compounds (309.95 ± 25.33 mMol GA/100 g of extract) and the lowest effective concentration (EC50) against DPPH and ABTS radicals (72.64 ± 10.70 and 181.68 ± 18.47, respectively). On the other hand, the peel and seed coat extracts had the lowest energy densities (226.06 ± 0.06 kcal/100 g and 219.62 ± 0.49 kcal/100 g, respectively). Regarding the antiproliferative activity, the avocado peel extract (180 ± 40 µg/mL) showed the lowest inhibitory concentration (IC50), followed by the seed (200 ± 21 µg/mL) and seed coat (340 ± 32 µg/mL) extracts. The IC50 of the extracts induced apoptosis in Caco-2 cells at the early and late stages. According to the in-silico analysis, these results could be related to the higher Caco-2 permeability to hydroxysalidroside, salidroside, sakuranetin, and luteolin. Therefore, this study provides new insights regarding the potential use of these extracts as functional ingredients with antioxidant and antiproliferative properties and as medicinal agents in diseases related to oxidative stress such as cancer. This work was supported by the Spanish Ministry of Economy and Competitiveness and FEDER, UE (grants AGL2015-65975-R and RTI2018-094268-B-C21) and by grants from Ministry of Science and Technology (PID2019-104915RB-I00), CIBEROBN (CB06/03/1012), Gobierno de Aragón (B16-20R, Fondos FEDER “otra manera de hacer Europa”), SUDOE (Redvalue, SOE1/PI/E0123).

Squalene (SQ) is an intermediate hydrocarbon in the biosynthesis of phytosterols and terpenes in plants. It is widely used for applications such as skin moisturizers, vaccines, or in carriers for active lipophilic molecules. It has commonly been obtained from sharks, but restrictions on their use have created a need to find alternative sources. We present a review of studies concerning SQ in olive groves to characterize its content and to provide new aspects that may increase the circular economy of the olive tree. There is a large variation in SQ content in virgin olive oil due to cultivars and agronomic issues such as region, climate, types of soil, crop practices, and harvest date. Cultivars with the highest SQ content in their virgin olive oil were 'Nocellara de Belice', 'Drobnica', 'Souri', and 'Oblica'. An interaction between cultivar and aspects such as irrigation practices or agricultural season is frequently observed. Likewise, the production of high SQ content needs precise control of fruit maturation. Leaves represent an interesting source, if its extraction and yield compensate for the expenses of their disposal. Supercritical carbon dioxide extraction from olive oil deodorizer distillates offers an opportunity to obtain high-purity SQ from this derivative. Exploiting SQ obtained from olive groves for the pharmaceutical or cosmetic industries poses new challenges and opportunities to add value and recycle by-products. © 2019 Society of Chemical Industry.

The dynamic and complex interactions between enteric pathogens and the intestinal epithelium often lead to disturbances in the intestinal barrier, altered fluid, electrolyte, and nutrient transport and can produce an inflammatory response. Lipopolysaccharide (LPS) is a complex polymer forming part of the outer membrane of Gram-negative bacteria. On the other hand, squalene is a triterpene present in high levels in the extra-virgin olive oil that has beneficial effects against several diseases and it has also anti-oxidant and anti-inflammatory properties. The aim of this work was to study whether the squalene could eliminate the LPS effect on D-galactose intestinal absorption in rabbits and Caco-2 cells. The results have shown that squalene reduced the effects of LPS on sugar absorption. High LPS doses increased D-galactose uptake through via paracellular but also decreased the active sugar transport because the SGLT1 levels were diminished. However, the endotoxin effect on the paracellular way seemed to be more important than on the transcellular route. At the same time, an increased in RELM-β expression was observed. This event could be related to inflammation and cause a decrease in SGLT1 levels. In addition, MLCK protein is also increased by LPS which could lead to an increase in sugar transport through tight junctions. At low doses, the LPS could inhibit SGLT1 intrinsic activity. Bioinformatic studies by docking confirm the interaction between LPS-squalene as well as occur through MLCK and SGLT-1 proteins.

Background and aim: The type and amount of dietary protein has become a topic of re-newed interest in light of their involvement in metabolic diseases, atherosclerosis and thrombosis. However, little attention has been devoted to the effect of avian proteins despite their wide human consumption. The aim was to investigate the influence of chicken and turkey as sources of protein compared with that of soybean on atherosclerosis and fatty liver disease. Methods and results: To this purpose, male and female Apoe-deficient were fed purified Western diets differing in their protein sources for 12 weeks. After this period, blood, liver, aortic tree and heart base samples were taken for analyses of plasma lipids and atherosclerosis. Plasma triglycerides, non-esterified fatty acids, esterified cholesterol levels and radical oxygen species in lipoproteins changed depending on the diet and sex. Females consuming the turkey protein-containing diet showed decreased athero-sclerotic foci, as evidenced by the en face atherosclerosis analyses. The presence of macrophages and smooth muscle cells in plaques were not modified, and no changes were observed in hepatic lipid droplets in the studied groups either. Paraoxonase activity was higher in the group consuming turkey protein without sex differences, but only in females, it was significantly associated with aor-tic lesion areas. Conclusions: Compared to soybean protein, the consumption of avian proteins depending on sex resulted in similar or lower atherosclerosis development and comparable hepatic steatosis. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

To evaluate the effects of squalene, the main unsaponifiable component of virgin olive oil, on lipid metabolism, two groups of male New Zealand rabbits were fed a 1% sunflower oil-enriched regular diet or the same diet containing 0.5% squalene for 4 weeks. Plasma triglycerides, total- and HDL-cholesterol and their lipoproteins were assayed. Analyses of hepatic lipid droplets, triglycerides, total- and non-esterified cholesterol, squalene, protein and gene expression, and cholesterol precursors were carried out. In the jejunum, the squalene content and mRNA and protein APOB expressions were measured. Finally, we studied the effect of cholesterol precursors in AML12 cells. Squalene administration significantly increased plasma total cholesterol, mainly carried as non-esterified cholesterol in IDL and large LDL, and corresponded to an increased number of APOB100-containing particles without accumulation of triglycerides and decreased reactive oxygen species. Despite no significant changes in the APOB content in the jejunum, the latter displayed increased APOB mRNA and squalene levels. Increases in the amounts of non-esterified cholesterol, squalene, lanosterol, dihydrolanosterol, lathosterol, cholestanol, zymostenol, desmosterol and caspase 1 were also observed in the liver. Incubation of AML12 cells in the presence of lanosterol increased caspase 1. In conclusion, squalene administration in rabbits increases the number of modified APOB-containing lipoproteins, and hepatic cholesterol biosynthesis is linked to caspase 1 probably through lanosterol. © The Royal Society of Chemistry.

This article belongs to the Special Issue Anti-inflammatory and Antioxidant Properties of Plant Extracts., The application of plant extracts for therapeutic purposes has been used in traditional medicine because plants contain bioactive compounds with beneficial properties for health. Currently, the use of these compounds that are rich in polyphenols for the treatment and prevention of diseases such as cancer, diabetes, and cardiovascular diseases, many of them related to oxidative stress, is gaining certain relevance. Polyphenols have been shown to have antimutagenic, antioxidant, and anti-inflammatory properties. Therefore, the objective of the present work was to study the potential effect of grape stem extracts (GSE), rich in phenolic compounds, in the treatment of cancer, as well as their role in the prevention of this disease associated with its antioxidant power. For that purpose, three cancer lines (Caco-2, MCF-7, and MDA-MB-231) were used, and the results showed that grape stem extracts were capable of showing an antiproliferative effect in these cells through apoptosis cell death associated with a modification of the mitochondrial potential and reactive oxygen species (ROS) levels. Additionally, grape stem extracts showed an antioxidant effect on differentiated intestinal cells that could protect the intestine from diseases related to oxidative stress. Therefore, grape extracts contain bioactive principles with important biological properties and could be used as bio-functional food ingredients to prevent diseases or even to improve certain aspects of human health., This work was supported by grants from Ministerio de Economia y Competitividad, Gobierno de España (SAF2016-75441-R), CIBEROBN (CB06/03/1012), Gobierno de Aragón (B16-17R, Fondos FEDER “otra manera de hacer Europa”), SUDOE (Redvalue, SOE1/PI/E0123), Project ELENA (EFA 220/11 ELENA) and Gobierno de Navarra (Projects VARASVI PI022 and PI038).

Erythrodiol is a terpenic compound found in a large number of plants. To test the hypotheses that its long-term administration may influence hepatic transcriptome and this could be influenced by the presence of APOA1-containing high-density lipoproteins (HDL), Western diets containing 0.01% of erythrodiol (10 mg/kg dose) were provided to Apoe- and Apoa1-deficient mice. Hepatic RNA-sequencing was carried out in male Apoe-deficient mice fed purified Western diets differing in the erythrodiol content. The administration of this compound significantly up- regulated 68 and down-regulated 124 genes at the level of 2-fold change. These genes belonged to detoxification processes, protein metabolism and nucleic acid related metabolites. Gene expression changes of 21 selected transcripts were verified by RT-qPCR. Ccl19-ps2, Cyp2b10, Rbm14-rbm4, Sec61g, Tmem81, Prtn3, Amy2a5, Cyp2b9 and Mup1 showed significant changes by erythrodiol administration. When Cyp2b10, Dmbt1, Cyp2b13, Prtn3 and Cyp2b9 were analyzed in female Apoe-deficient mice, no change was observed. Likewise, no significant variation was observed in Apoa1- or in Apoe-deficient mice receiving doses ranging from 0.5 to 5 mg/kg erythrodiol. Our results give evidence that erythrodiol exerts a hepatic transcriptional role, but this is selective in terms of sex and requires a threshold dose. Furthermore, it requires an APOA1-containing HDL.

Scope To investigate the effects of squalene, the main hydrocarbon present in extra virgin olive oil, on liver transcriptome in different animal models and to test the influence of sex on this action and its relationship with hepatic lipids. Methods and results To this purpose, male C57BL/6J Apoe-deficient mice are fed a purified Western diet with or without squalene during 11 weeks and hepatic squalene content is assessed, so are hepatic lipids and lipid droplets. Hepatic transcriptomic changes are studied and confirmed by RT-qPCR. Dietary characteristics and influence of squalene doses are tested in Apoe-deficient on purified chow diets with or without squalene. These diets are also given to Apoa1 and wild-type mice on C57BL/6J background and to C57BL/6J xOla129 Apoe-deficient mice. Squalene supplementation increases its hepatic content without differences among sexes and hormonal status. The Cyp2b10 and Cyp2c55 gene expressions are significantly up-regulated by the squalene intake in all models, with independence of sex, sexual hormones, dietary fat content, genetic background and dose, and in Apoe-deficient mice consuming extra-virgin olive oil. Conclusion Hepatic squalene increases the expression of these cytochromes and their changes in virgin olive oil diets may be due to their squalene content.

Background and aims. The molecular mechanisms by which the liver develops steatotic disease still remain unclear. Previous studies using nutritional and genetic models of hepatic steatosis in mice showed that liver synaptotagmin 1 (Syt1) expression was associated with lipid droplet area. Hepatic Syt1 overexpression was used as a tool to explore its effect on hepatic and plasma lipids. Methods and results To find out a cause-effect, hepatic mouse Syt1 mRNA was cloned into a vector driving hepatocyte-specific expression and administered by hydrodynamic injection to male Apoe-deficient mice fed on a Western diet, the latter as a model of rapid spontaneous steatosis development. Hepatic microsomal, large vesicle, lysosomal and plasma membrane fractions were enriched in SYT1 protein following gene overexpression. In these conditions, very low density lipoprotein esterified cholesterol increased. Likewise, the transgene caused an alteration in lipid droplet surface and a positive correlation between Syt1 expression and hepatic total cholesterol content. A lipidomic approach evidenced a decrease in lysophosphatidylcholine, phosphatidylcholine and triglycerides in isolated plasma membrane fraction. Expressions of genes involved in biosynthesis of bile acids, fatty acid metabolism, lipoprotein dynamics and vesicular transport were modified by the increased SYT1 expression. Conclusions These results indicate that this protein is involved in hepatic management of lipids and in the regulation of genes involved in lipid metabolism.

Introduction. The hepatic steatosis of the nonalcoholic origin or NAFLD is increasing at present, particularly in Western countries, parallel to the increase in obesity, constituting one of the most prevalent hepatic processes in the Western society. Melatonin has been successfully tested in experimental models in mice as a drug capable of reversing steatosis. The effect of melatonin on fat metabolism can be summarized as a decrease in lipid peroxidation and a decrease in oxidative stress, biochemical phenomena intimately related to fat deposition in the hepatocyte. There are hardly any studies in large animals. Objective. In this study, we investigate the effects of melatonin administered orally at a dose of 10 mg/kg/day to reverse established hepatic steatosis induced by a special diet in a porcine animal model. Materials and Methods. We analyze the parameters of oxidative stress: malondialdehyde (MDA), 4-hydroxyalkenals (4-HDA), and carbonyls, degree of fat infiltration (analyzed by direct vision by a pathologist and by means of a computer program of image treatment), and serological parameters of lipid metabolism and hepatic damage. These parameters were analyzed in animals to which hepatic steatosis was induced by means of dietary modifications. Results. We have not been able to demonstrate globally a beneficial effect of melatonin in the improvement or reversal of liver steatosis once established, induced by diet in a porcine animal model. However, we have found several signs of improvement at the histological level, at the level of lipid metabolism, and at the level of oxidative stress parameters. We have verified in our study that, in the histological analysis of the liver sample by means of the program image treatment (free of subjectivity) of the animals that continue with the diet, those that consume melatonin do not increase steatosis as much as those that do not consume it significantly (p=0.002). Regarding the parameters of oxidative stress, MDA modifies in a significant manner within the group of animals that continue with the diet and take melatonin (p=0.004). As for lipid metabolism, animals that maintain the steatotic diet and take melatonin lower total and LDL cholesterol levels and increase HDL levels, although these results do not acquire statistical significance. Conclusions. In this study, it has not been possible to demonstrate a beneficial effect of melatonin in the improvement or reversal of liver steatosis once established and induced by diet in the porcine model. It is true that signs of improvement have been found at the histological level, at the level of lipid metabolism, and at the level of oxidative stress phenomena, when comparing animals with established steatosis that are treated with melatonin with those who do not take it. This work is the first study conducted in a large animal model in which the effect of melatonin is studied as a treatment in the reversal of established hepatic steatosis.

Given the alarming increase in colorectal cancer (CRC) worldwide, novel therapies are urgently needed. Plant-derived extracts have gained considerable interest in the last years due to their strong anticancer effect mediated by their unique bioactive compounds. Specifically, rosehips from Rosa canina have been successfully tested against several cancer models, including colon cancer. Moreover, gold derivatives are a promising alternative to the current platinum-based drugs commonly used in CRC chemotherapy due to their lack of affinity for DNA. Herein we have investigated the antitumor potential of a drug combination made of acidic polyphenols extracted from R. canina and the gold complex (Au(C&equiv, C-2-NC5H4) (PTA)) in Caco-2 cell line as a model of CRC. The combination triggered strong apoptosis mediated by a blockage of the autophagic flux, which might be a consequence of a reactive oxygen species (ROS) increase and mitochondrial dysfunctionality. Our results suggest that the clinical application of plant polyphenols might enhance the anticancer effect of metallodrugs and reduce drug exposure time and therefore its side effects.

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-β1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-β1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target, This work was supported by the Government of Aragón (DGA-METIC–consortium; B04_17R), FP7-PEOPLE-MC-CIG to RK, FIS-CB06/07/1036 to PG and RK, ERC-2014-StG-638284, DPI2016-75458-R, T39_17R to EP; FIS-PI16/02112 to ALGO; NHLBI-R01-HL080173, NCRRP20-RR018751 to HM. JLG received a DGA-PhD scholarship (C072/2014). BMB was supported by the NCATS (UL1 TR001860 and linked award TL1 TR001861)

Squalene is the main unsaponifiable component of virgin olive oil, the main source of dietary fat in Mediterranean diet, traditionally associated with a less frequency of cardiovascular diseases. In this study, two experimental approaches were used. In the first, New Zealand rabbits fed for 4 weeks with a chow diet enriched in 1% sunflower oil for the control group, and in 1% of sunflower oil and 0.5% squalene for the squalene group. In the second, APOE KO mice received either Western diet or Western diet enriched in 0.5% squalene for 11 weeks. In both studies, liver samples were obtained and analyzed for their squalene content by gas chromatography-mass spectrometry. Hepatic distribution of squalene was also characterized in isolated subcellular organelles. Our results show that dietary squalene accumulates in the liver and a differential distribution according to studied model. In this regard, rabbits accumulated in cytoplasm within small size vesicles, whose size was not big enough to be considered lipid droplets, rough endoplasmic reticulum, and nuclear and plasma membranes. On the contrary, mice accumulated in large lipid droplets, and smooth reticulum fractions in addition to nuclear and plasma membranes. These results show that the squalene cellular localization may change according to experimental setting and be a starting point to characterize the mechanisms involved in the protective action of dietary squalene in several pathologies.

Purpose The purpose of this study was to determine the effects of chickpea (Cicer arietinum) protein hydrolysates prepared at two degrees of hydrolysis (DH) on lipoprotein profile and on oxidant status in cholesterol-fed rats. Design/methodology/approach Eighteen male Wistar rats (220 ± 10 g) were divided into three groups and fed for 30 days a diet containing 20 per cent casein supplemented with 1 per cent cholesterol and 0.5 per cent cholic acid. During the experimentation, the first and the second groups received daily by gavage 250 mg of chickpea protein hydrolysates/rat at DH = 8 per cent (CPH8) and DH = 17 per cent (CPH17), respectively. The third group, named control group (CG), received water under the same conditions. Findings Serum total cholesterol concentrations were reduced in CPH8 (p < 0.0073) and CPH17 (p < 0.0004) groups versus CG. This reduction corresponded to a lower very-low-density lipoprotein (VLDL)-cholesterol (p < 0,0019). CPH17 reduced low-density lipoprotein- and high-density lipoprotein (HDL)-cholesterol (p < 0.0001) but increased apolipoprotein A4 (p < 0.002) concentrations and lecithin-cholesterol acyltransferase activity (p < 0.0001). APOA1 remained unchanged in the treated groups. Liver total and esterified cholesterol contents were twofold lower in both treated groups versus CG. CPH8 increased triacylglycerols and phospholipids (p < 0.0001) contents, while CPH17 decreased those of unesterified cholesterol (p < 0.0016). Compared with CG, CPH8 and CPH17 reduced serum (p < 0.0001) and lipoprotein hydroperoxides by stimulating paraoxonase activity (p < 0.0001). However, only CPH17 treatment reduced serum, VLDL- and HDL-malondialdehyde contents and improved glutathione peroxidase activity (p < 0.061). Originality/value Thus, chickpea protein hydrolysates and especially hydrolysed at DH = 17 per cent may have a great potential for use as a nutraceutical to reduce hypercholesterolaemia and, by consequence, oxidative stress. Therefore, the degree of enzymatic hydrolysis has a significant influence on the production of potent bioactive peptides.

[Objective]: Map3k8 (Cot/Tpl2) activates the MKK1/2-ERK1/2, MAPK pathway downstream from interleukin-1R, tumor necrosis factor-αR, NOD-2R (nucleotide-binding oligomerization domain-like 2R), adiponectinR, and Toll-like receptors. Map3k8 plays a key role in innate and adaptive immunity and influences inflammatory processes by modulating the functions of different cell types. However, its role in atherogenesis remains unknown. In this study, we analyzed the role of this kinase in this pathology. [Approach and Results]: We show here that Map3k8 deficiency results in smaller numbers of Ly6C high CD11c low and Ly6C low CD11c high monocytes in ApoE - /- mice fed a high-fat diet (HFD). Map3k8 ApoE monocytes displayed high rates of apoptosis and reduced amounts of Nr4a1, a transcription factor known to modulate apoptosis in Ly6C low CD11c high monocytes. Map3k8 ApoE splenocytes and macrophages showed irregular patterns of cytokine and chemokine expression. Map3k8 deficiency altered cell adhesion and migration in vivo and decreased CCR2 expression, a determinant chemokine receptor for monocyte mobilization, on circulating Ly6C high CD11c low monocytes. Map3k8 ApoE mice fed an HFD showed decreased cellular infiltration in the atherosclerotic plaque, with low lipid content. Lesions had similar size after Map3k8 +/+ ApoE bone marrow transplant into Map3k8 ApoE and Map3k8 +/+ ApoE mice fed an HFD, whereas smaller plaques were observed after the transplantation of bone marrow lacking both ApoE and Map3k8. [Conclusions]: Map3k8 decreases apoptosis of monocytes and enhances CCR2 expression on Ly6C high CD11c low monocytes of ApoE mice fed an HFD. These findings explain the smaller aortic lesions in ApoE mice with Map3k8 ApoE bone marrow cells fed an HFD, supporting further studies of Map3k8 as an antiatherosclerotic target.

Given the alarming increase in colorectal cancer (CRC) worldwide, novel therapies are urgently needed. Plant-derived extracts have gained considerable interest in the last years due to their strong anticancer effect mediated by their unique bioactive compounds. Specifically, rosehips from Rosa canina have been successfully tested against several cancer models, including colon cancer. Moreover, gold derivatives are a promising alternative to the current platinum-based drugs commonly used in CRC chemotherapy due to their lack of affinity for DNA. Herein we have investigated the antitumor potential of a drug combination made of acidic polyphenols extracted from R. canina and the gold complex (Au(C≡C-2-NC5H4) (PTA)) in Caco-2 cell line as a model of CRC. The combination triggered strong apoptosis mediated by a blockage of the autophagic flux, which might be a consequence of a reactive oxygen species (ROS) increase and mitochondrial dysfunctionality. Our results suggest that the clinical application of plant polyphenols might enhance the anticancer effect of metallodrugs and reduce drug exposure time and therefore its side effects.

Hypertensive cardiac hypertrophy (HCH) is a common cause of heart failure (HF), a major public health problem worldwide. However, the molecular bases of HCH have not been completely elucidated. Neuron-derived orphan receptor-1 (NOR-1) is a nuclear receptor whose role in cardiac remodelling is poorly understood. The aim of the present study was to generate a transgenic mouse over-expressing NOR-1 in the heart (TgNOR-1) and assess the impact of this gain-of-function on HCH. The CAG promoter-driven transgenesis led to viable animals that over-expressed NOR-1 in the heart, mainly in cardiomyocytes and also in cardiofibroblasts. Cardiomyocytes from TgNOR-1 exhibited an enhanced cell surface area and myosin heavy chain 7 (Myh7)/Myh6 expression ratio, and increased cell shortening elicited by electric field stimulation. TgNOR-1 cardiofibroblasts expressed higher levels of myofibroblast markers than wild-type (WT) cells (α 1 skeletal muscle actin (Acta1), transgelin (Sm22α)) and were more prone to synthesise collagen and migrate. TgNOR-1 mice experienced an age-associated remodelling of the left ventricle (LV). Angiotensin II (AngII) induced the cardiac expression of NOR-1, and NOR-1 transgenesis exacerbated AngII-induced cardiac hypertrophy and fibrosis. This effect was associated with the up-regulation of hypertrophic (brain natriuretic peptide (Bnp), Acta1 and Myh7) and fibrotic markers (collagen type I α 1 chain (Col1a1), Pai-1 and lysyl oxidase-like 2 (Loxl2)). NOR-1 transgenesis up-regulated two key genes involved in cardiac hypertrophy (Myh7, encoding for β-myosin heavy chain (β-MHC)) and fibrosis (Loxl2, encoding for the extracellular matrix (ECM) modifying enzyme, Loxl2). Interestigly, in transient transfection assays, NOR-1 drove the transcription of Myh7 and Loxl2 promoters. Our findings suggest that NOR-1 is involved in the transcriptional programme leading to HCH.

The Mediterranean diet is considered as the foremost dietary regimen and its adoption is associated with the prevention of degenerative diseases and an extended longevity. The preeminent features of the Mediterranean diet have been agreed upon and the consumption of olive oil stands out as the most peculiar one. Indeed, the use of olive oil as the nearly exclusive dietary fat is what mostly characterizes the Mediterranean area. Plenty of epidemiological studies have correlated that the consumption of olive oil was associated with better overall health. Indeed, extra virgin olive oil contains (poly)phenolic compounds that are being actively investigated for their purported biological and pharma-nutritional properties. On 18 and 19 May 2018, several experts convened in Jaen (Spain) to discuss the most recent research on the benefits of olive oil and its components. We reported a summary of that meeting (reviewing several topics related to olive oil, not limited to health) and concluded that substantial evidence is accruing to support the widespread opinion that extra virgin olive oil should, indeed, be the fat of choice when it comes to human health and sustainable agronomy., The meeting-III International Conference on Virgin Olive Oil and Health (O_LIVE 2018)-was supported by the Diputación de Jaén (Spain)., http://creativecommons.org/licenses/by/4.0/

Hydroxytyrosol (HT) is involved in healthful activities and is beneficial to lipid metabolism. Many investigations focused on finding tissue-specific targets of HT through the use of different omics approaches such as transcriptomics and proteomics. However, it is not clear which (if any) of the potential molecular targets of HT reported in different studies are concurrently affected in various tissues. Following the bioinformatic analyses of publicly available data from a selection of in vivo studies involving HT-supplementation, we selected differentially expressed lipid metabolism-related genes and proteins common to more than one study, for validation in rodent liver samples from the entire selection. Four miRNAs (miR-802-5p, miR-423-3p, miR-30a-5p, and miR-146b-5p) responded to HT supplementation. Of note, miR-802-5p was commonly regulated in the liver and intestine. Our premise was that, in an organ crucial for lipid metabolism such as the liver, consistent modulation should be found for a specific target of HT even if different doses and duration of HT supplementation were used in vivo. Even though our results show inconsistency regarding differentially expressed lipid metabolism-related genes and proteins across studies, we found Fgf21 and Rora as potential novel targets of HT. Omics approaches should be fine-tuned to better exploit the available databases., This research was funded by grants from the Spanish “Agencia Estatal de Investigación” and the European FEDER Funds to AD and RMH (AGL2016-78922-R); the Ministerio de Economía y Competitividad–Fondo Europeo de Desarrollo Regional (SAF2016-75441-R) and the Fondo Social Europeo–Gobierno de Aragón (B16_17R) to MAN and JO; the Fundación Ramón Areces (CIVP18A3888) to AD, JG-Z, JTC, MCC, FV and RMH; the CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERObn) to JAM, MPP, JO and MAN; and POR FESR 3S4H to FV. CIBERObn. is an initiative of the ISCIII, Spain. MCLH, LdP and MB R-R were recipients of contracts from the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid, Fondo Social Europeo, and the Iniciativa de Empleo Juvenil YEI (PEJD-2016/BIO-2781, PEJD-2017-PRE/BIO-5100, and PEJD-2018-POST/BIO 8933), respectively.

Aims: The North-to-South gradient in cardiovascular disease in Europe could be partially explained by the low prevalence of lipid-rich atheroma plaques (responsible for most culprit lesions) in Southern Europe. Consumption of certain foods delays atherosclerosis progression. In a mouse model of accelerated atherosclerosis we investigated whether the inclusion of walnuts within an atherogenic diet stabilizes advanced atheroma plaque.Methods: Apolipoprotein E-deficient mice (male, 10-week old) were randomized to receive either a control diet (9.6% of energy as fat, n=14), a palm oil-based high-fat diet (40% of energy as fat, n=15) or an isocaloric diet which fat was partially supplied by walnuts, in a dose equivalent to 30 g/d in humans (n=14). All diets contained 0.2% cholesterol. After 15 weeks of intervention, we evaluated changes in aortic atherosclerotic lesions (size and composition) and expression levels of markers for inflammation and oxidative stress. Results: There were no among-group differences in atherosclerotic size and extension, or oxidative stress. Compared to control diet, palm oil-diet induced plaque instability (higher lipid and necrosis, and lower collagen-to-lipid ratio). Walnut inclusion attenuated these features and decreased macrophage infiltration. Palm oil-based diet increased expression of chemokines, cytokines, inflammasome, and M1-macrophage marker compared to control diet. Such response was not observed in walnut group. Findings for walnut group could be explained by the differential aortic activation of NFkB (down-regulated) and Nrf2 (up-regulated). Conclusions: Isocaloric inclusion of walnuts in an unhealthy high-fat diet stabilizes advanced atheroma plaque. This contributes novel mechanistic evidence for the cardiovascular benefits of walnuts.

The application of plant extracts for therapeutic purposes has been used in traditional medicine since the plants are a source of a great variety of chemical compounds that possess biological activity. Actually, the effect of these extracts on diseases such as cancer is being widely studied. Colorectal adenocarcinoma is one of the main causes of cancer related to death and the second most prevalent carcinoma in Western countries. The aim of this work is to study the possible effect of two fenugreek (Trigonella foenum graecum) protein hydrolysates on treatment and progression of colorectal cancer. Fenugreek proteins from seeds were hydrolysed by using two enzymes separately, which are named Purafect and Esperase, and were then tested on differentiated and undifferentiated human colonic adenocarcinoma Caco2/TC7 cells. Both hydrolysates did not affect the growth of differentiated cells, while they caused a decrease in undifferentiated cell proliferation by early apoptosis and cell cycle arrest in phase G1. This was triggered by a mitochondrial membrane permeabilization, cytochrome C release to cytoplasm, and caspase-3 activation. In addition, the hydrolysates of fenugreek proteins displayed antioxidant activity since they reduce the intracellular levels of ROS. These findings suggest that fenugreek protein hydrolysates could be used as nutraceutical molecules in colorectal cancer treatment.

OBJECTIVES: The presence of lipid-rich, unstable atheroma plaques in the vascular tree is the harbinger of cardiovascular events. There is a low prevalence of unstable atheroma plaques in Mediterranean countries. This might explain in part the lower rates of cardiovascular disease in Southern Europe compared to Northern Europe and US. Consumption of certain foods paradigmatic of the Mediterranean Diet delays atherosclerosis progression. In a mouse model of accelerated atherosclerosis, we investigated whether the inclusion of walnuts within an atherogenic diet stabilizes advanced atheroma plaque. METHODS: Apolipoprotein E-deficient mice (male, 10-week old) were randomized to receive either regular chow (9.6% of energy as fat, n = 14); a palm oil-based high-fat diet (43% of energy as fat, n = 15); or an isocaloric high-fat diet in which palm oil was partially replaced by walnuts in a dose equivalent to 30 g/d in humans (n = 14). All diets contained 0.2% cholesterol. After 15 weeks of intervention, we evaluated changes in aortic atherosclerotic lesions (size and composition) and markers for inflammation, oxidative stress, necrosis and autophagy. RESULTS: There were no among-group differences in atherosclerotic size and extension, or oxidative stress. Compared to control diet, palm oil-diet induced plaque instability (higher lipid and necrosis, and lower collagen-to-lipid ratio). Walnut inclusion attenuated these features and decreased macrophage infiltration. Palm oil-based diet increased expression of chemokines, cytokines, inflammasome, and M1-macrophage marker compared to control diet. Such response was not observed in walnut group. Findings for walnut group could be explained by the differential aortic activation of NF-kB (down-regulated) and Nrf2 and autophagy (up-regulated). CONCLUSIONS: Isocaloric inclusion of walnuts within an unhealthy high-fat diet stabilizes advanced atheroma plaque. This contributes novel mechanistic evidence for the cardiovascular benefits of sustained walnut consumption. FUNDING SOURCES: Instituto de Salud Carlos III, Spain; Fondo de Investigación Sanitaria–Fondo Europeo de Desarrollo Regional, Spain. California Walnut Commission.

Squalene is a triterpenic compound found in a large number of plants and other sources with a long tradition of research since it was first reported in 1926. Herein a systematic review of studies concerning squalene published in the last 8 years is presented. These studies have provided further support for its antioxidant, anti-inflammatory, and anti-atherosclerotic properties in vivo and in vitro. Moreover, an antineoplastic effect in nutrigenetic-type treatments, which depends on the failing metabolic pathway of tumors, has also been reported. The bioavailability of squalene in cell cultures, animal models, and in humans has been well established, and further progress has been made in regard to the intracellular transport of this lipophilic molecule. Squalene accumulates in the liver and decreases hepatic cholesterol and triglycerides, with these actions being exerted via a complex network of changes in gene expression at both transcriptional and post-transcriptional levels. Its presence in different biological fluids has also been studied. The combination of squalene with other bioactive compounds has been shown to enhance its pleiotropic properties and might lead to the formulation of functional foods and nutraceuticals to control oxidative stress and, therefore, numerous age-related diseases in human and veterinary medicine.

Purpose This paper aims to investigate the in vivo hypocholesterolemic property of fenugreek proteins (FP), Purafect-fenugreek protein hydrolysate (PFPH) and Esperase-fenugreek protein hydrolysate (EFPH) on high cholesterol (HC)-fed rats. Design/methodology/approach Rats were randomized into five groups: four were fed for four weeks a hypercholesterolemic diet and the tested products were given by gavage. The fifth group was taken as control (C) receiving the same diet without cholesterol. Findings Results showed that the elevated aspartate aminotransferase activity in HC group plasma was significantly corrected by FP and EFPH administration (−33 per cent; p = 0.0003). HC liver lipids and total cholesterol (TC) contents were not markedly affected by FP and EFPH. However, liver triglycerides (TG) contents trended to decrease in FP rats vs HC (p = 0.07), while, the TG decrease was significant in groups fed the proteins hydrolysates (p = 0.02). On the other hand, serum TC and TG decreased by 53 per cent (p = 0.0003) and 20 per cent (p = 0.04), respectively, in FP treated rats compared to HC group. This decrease was associated with a high fecal cholesterol excretion (2.5-fold higher in FP vs HC; p = 0.0001). Likewise, EFPH-treated rats exhibited lower TC compared to HC rats (p = 0.004). The very low density lipoprotins was the main affected fraction in these two groups, while there were no significant difference in apolipoproteins (Apo) B, A-I and A-IV contents between the different groups, except in FP group, where Apo A-I and A-IV decreased by 26 and 17 per cent, respectively, compared to C rats (p = 0.02). The high density lipoproteins (HDL) of rats treated with proteins hydrolysates showed a better antioxidant property compared to those of HC rats, which was accompanied with an increase in paraoxonase activity when compared to HC group. Originality/value Unlike PFPH which had almost no effect, FPs and EFPH could constitute a nutraceutical ingredient in cardiovascular disease management.

Purpose– The purpose of this study was to evaluate the antioxidant and hypocholesterolemic activities of sardine and bogue protein hydrolysates in cholesterol-fed rats.Design/methodology/approach– In total, 18 male Wistar rats (220 ± 10 g) fed 20 per cent casein, 1 per cent cholesterol and 0.5 per cent cholic acid were divided into three groups and received a daily gavage of 250 mg of sardine (SPH) or bogue (BPH) protein hydrolysates for 30 days. The third group, named control group (CG), received in the same conditions water. Lipoproteins were fractionated by size-exclusion fast protein liquid chromatography, and serum lipids, apolipoproteins and lipoproteins were assayed.Findings– In SPH and BPH groups, serum total cholesterol concentrations were −66 per cent lower than in CG. This corresponded to the decreased very low-density lipoprotein-C in the former groups. Moreover, BPH treatment reduced low-density lipoprotein-C compared with CG and SPH groups. Compared with CG, serum phospholipids were reduced by SPH and BPH. Furthermore, BPH increased significantly APOA4 and sphingomyelin but lowered phosphatidylcholine. In the latter group, serum lecithin cholesterol acyltransferase activity was +23 per cent higher, but with SPH, this activity was −35 per cent reduced compared with CG. Apolipoprotein A-I contents were similar in the three groups. Compared with CG, hydroperoxide and lipid peroxidation contents in serum and lipoprotein fractions were reduced by SPH and BPH. Compared with CG, serum superoxide dismutase and glutathione peroxidase activities were increased in the treated groups, particularly in the BPH group.Originality/value– These results suggest that sardine protein hydrolysates and particularly those of bogue could be a very useful natural compound to prevent hypercholesterolemia by both improving the lipid profile and modulating oxidative stress in cholesterol-fed rats.

The Mediterranean diet has been proven to be highly effective in the prevention of cardiovascular diseases. Paraoxonase 1 (PON1) has been implicated in the development of those conditions, especially atherosclerosis. The present work describes a systematic review of current evidence supporting the influence of Mediterranean diet and its constituents on this enzyme. Despite the differential response of some genetic polymorphisms, the Mediterranean diet has been shown to exert a protective action on this enzyme. Extra virgin olive oil, the main source of fat, has been particularly effective in increasing PON1 activity, an action that could be due to low saturated fatty acid intake, oleic acid enrichment of phospholipids present in high-density lipoproteins that favor the activity, and increasing hepatic PON1 mRNA and protein expressions induced by minor components present in this oil. Other Mediterranean diet constituents, such as nuts, fruits and vegetables, have been effective in modulating the activity of the enzyme, pomegranate and its compounds being the best characterized items. Ongoing research on compounds isolated from all these natural products, mainly phenolic compounds and carotenoids, indicates that some of them are particularly effective, and this may enhance the use of nutraceuticals and functional foods capable of potentiating PON1 activity.

The potential use of cholesterol esterases was tested to avoid alkaline hydrolysis for cleavage of plasma esterified oxysterols. The enzymatic hydrolysis was optimized by testing two sources of enzyme— Pseudomonas and bovine pancreas, presence of surfactants, incubation time and amount of enzyme. Free forms of 4β-, 7-, 24-, 25- and 27-hydroxycholesterol (HC) as well 7-ketocholesterol (7-KC) were analyzed by liquid chromatography and mass-spectrometry using the deuterated internal standard, 25-HC(d6). Enzymatic hydrolysis was more effective using the Pseudomonas enzyme and in presence of surfactants. Compared to alkaline hydrolysis, it generated a cleaner chromatographic baseline and better recovery of the internal standard. Oxysterols were assayed with detection limits between 7 and 31 pg/mL. Interassay coefficients of variation were lower than 10% and extraction recovery efficiencies, higher than 90%. The procedure was used to characterize plasma levels of Cyp7b1- deficient rat, where it showed increased plasma levels of 7, 24 and 25-HC. Due to the low volume of sample required, it may be used in other animal models, particularly rodents, as well as in pediatric samples where sample amount is always a problem. Thus, the proposed new method offers mild enzymatic processing that greatly facilitates oxysterol determinations to delineate their role in physiopathology.