Evaluación del efecto protector de un extracto enzimático de salvado de arroz frente al estado inflamatorio presente en el proceso aterosclerótico

Resumen

TITLE: “Evaluation of the protective effect of a rice bran enzymatic extract against the inflammatory state present in the atherosclerotic process” INTRODUCTION Rice bran is a phytochemical-rich layer that surrounds the endosperm of whole grain brown rice. It is produced as a by-product of white rice milling and discarded or designated to animal feeding due to its tendency to rancid reactions that impede its safe consumption. However, there is accumulating evidence that consumption of stabilized rice bran promotes health benefits in humans due to the multifactorial activities of the minor constituents. gamma-oryzanol, the main bioactive molecule in the unsaponificable fraction of rice bran, accounts for well recognized lipid lowering and antioxidant activities. Gamma-oryzanol is cleaved into ferulic acid and sterols by intestinal lipase enzymes. Sterols in the intestine promote cholesterol elimination by competing with cholesterol absorption, while ferulic acid is absorbed and inhibits hepatic cholesterol synthesis by HMG-CoA-reductase. Moreover, ferulic acid has been found to reduce oxidative stress by inducing a more favourable balance between the expression of enzymes related to oxidative stress in favour of the antioxidant defences. In addition to the lipid lowering and antioxidant activities, rice bran is known by its anti-inflammatory actions through reduction of NF-κβ activation. Moreover, rice bran has an important antidiabetic activity related to the normalization of liver enzymes implicated in the metabolism and synthesis of glucose, and to increased secretion of insulin by the pancreas. Prior works of our group showed improvement of several markers of metabolic syndrome and obesity of animals whose diet was supplemented with a rice bran enzymatic extract (RBEE). The enzymatic extraction is carried out at controlled pH and temperature to preserve the activity of the phytochemicals, and allows the stabilization of the original raw material by inactivation of endogenous lipases. The final product is a water-soluble syrup with higher biological quality compared to raw rice bran. There is a net increase in the bioactive components, mainly of gamma-oryzanol and tocotrienols, and the fragmentation of the proteins into peptides of low molecular weight allows the solubilisation of the hydrophobic components by interaction. RBEE supplements to high fat diet improved total cholesterol and HDL cholesterol in Wistar rats. Moreover, RBEE-supplemented diet was able to attenuate hypertension insulin resistance and to improve serum lipids in obese Zucker rats. Moreover, vascular function of the aorta and mesenteric arteries of Zucker rats was improved by increasing the expression of eNOS and calcium activated potassium channels and decreasing vascular oxidative stress and inflammation. Moreover, RBEE supplements attenuated structural changes of adipose tissue and normalized the expression of pro-inflammatory markers such as TNF-alpha, IL-6, IL-1β and iNOS and proinflammatory macrophage polarization of Zucker rats and diet induced obesity-mice. These promising protective cardiometabolic activities derived from diet supplementation with RBEE are of great interest for atherogenesis prevention. Atherosclerosis is the underlying cause for 80% of the deaths caused by cardiovascular diseases. Atherosclerosis is characterized by a low-grade systemic inflammatory and prooxidante state, causing the narrowing of the arteries caused by the accumulation of cholesterol, collagen, vascular smooth muscular cells and other cell types in the vessel wall. These plaques can eventually break causing thrombosis that could block blood flow to tissues, precipitating ischemic events. The process is trigged by small endothelial damages that cause a change in the endothelium, incrementing its permeability to cholesterol and immune cells. Cumulative LDL cholesterol in the arterial wall and its oxidative modifications stimulates monocyte infiltration and foam cell growth by cholesterol phagocytosis. The process is enhanced by oxidative stress and inflammation, which are the underlying cause of other pathologies that come along and potentiate atherosclerosis. Endothelial dysfunction is a predictive factor for atherosclerosis development. Hypercholesterolemia induces the activation of NADPHox reducing the amount of NO available for vasorelaxation, and reduces the activity of eNOS. In further stages of the progression of atherosclerosis, vascular remodelling due to plaque growth, VSMC proliferation and collagen deposition can alter vascular function, both in conductance as well as resistance vessels. Another consequence of hypercholesterolemia is the development of non-alcoholic fatty liver, which can promote atherosclerosis through liberation of proinflammatory cytokines. These prooxidant and proinflammatory stimuli increase vascular apoptosis causing a high cellular turnover that will lead to early cellular senescence, another cause of atherosclerosis promotion. However, macrophage apoptosis could be beneficial to prevent foam cell formation as long as apoptotic bodies are removed and not accumulated in the plaque core. Cellular apoptosis is also induced by dysfunctional mitochondria. Mitochondria are both, source and target of oxidative stress. Reactive oxygen species are key mediators of signalling pathways underlying vascular inflammation in atherosclerosis progression. High oxidative environment causes the modification of mitochondrial DNA leading to dysfunctional proteins and uncoupled mitochondria, which will produce vast amounts of superoxide promoting atherogenesis. Antioxidant-rich foods, such as rice bran can help to abrogate oxidative stress and inflammation. However, to understand the underlying mechanisms and the biological relevance, it is of great importance to investigate the absorption and metabolism of the active molecules. Although gamma-oryzanol is the major phytochemical in rice bran, its appearance in serum was never detected due to its low water solubility, the size of the molecule and more importantly, to pancreatic esterase activity in the intestine which leads to the release of ferulic acid. Diet is the first basic prevention measure as well as the first approach to hyperlipidaemia. Rice bran, rice bran oil and its main bioactive molecules have shown ability to reduce plasma cholesterol and the complications derived from hypercholesterolemia in several clinical studies. Therefore, rice bran and its technological- and qualitatively-improved presentation, the RBEE, appear as promising functional food ingredients for the prevention and treatment of atherosclerosis and atherosclerosis-related disorders. OBJECTIVES Given the biological potential of rice bran for the prevention of cardiovascular diseases and the technological improvement and in the concentration of the bioactive ingredients introduced by the enzymatic extraction, the general objective of this thesis was the study of the effects of diet supplementation with the RBEE on the progression of atherosclerosis in a genetic model of hypercholesterolemia and atherosclerotic plaque development. The specific objectives for this thesis were the following: - To carry out a review of the cardiometabolic and vascular effects of rice bran known to date, as well as its derived preparations and main bioactive molecules. - To study the effect of RBEE on atherogenesis. - To study the hypocholesterolemic mechanism of RBEE. - To study the effects of RBEE consumption on the mechanisms related to the atherosclerotic process: oxidative stress and inflammation. - To study the effects of dietary supplementation with RBEE on endothelial dysfunction in the aorta and resistance arteries. - To study the effects of RBEE consumption on the appearance of functional alterations resulting from the atherosclerotic process, such as steatosis, mitochondrial dysfunction, cellular senescence and apoptosis. - To study the bioavailability of one of the major bioactive molecules of RBEE, ferulic acid. - To identify the bioactive molecules present in the RBEE that are responsible for the observed effects. - To study the antioxidant effects derived from the consumption of ferulic acid in human polymorphonuclear cells. CHAPTER I "Contribution of ferulic acid, gamma-oryzanol and tocotrienols to the cardiometabolic protective effects of rice bran" Rice bran is an excellent nutritional source of bioactive compounds, including phytochemicals such as ferulic acid, gamma-oryzanol, phytosterols and tocols. These bioactive molecules have shown cardiometabolic protection, such as anti-diabetic and anti-hypertensive effects, but more importantly, lipid lowering effects due to cholesterol synthesis downregulation and increased faecal excretion. Moreover, rice bran phytochemicals have been described to mitigate oxidative stress by increasing antioxidant enzymes and by reducing oxygen radical production, and to possess anti-inflammatory activity due to downregulation of NF-κβ activation and reduction of pro-inflammatory cytokines production. This review aims to update and summarize clinical and animal studies, describing the multifactorial activities of rice bran and the individual contribution of its main bioactive compounds, namely, gamma-oryzanol, ferulic acid, phytosterols, triterpenic alcohols and tocotrienols. Technological factors affecting biological activities of the different rice bran-derived preparations are also discussed. CHAPTER II “Rice bran enzymatic extract reduces atherosclerotic plaque development and steatosis in high fat fed ApoE-/- mice.” Here, we aimed to identify RBEE hypolipidemic mechanism and to study the effects of RBEE on the progression of atherosclerosis disease and linked vascular dysfunction and liver steatosis in ApoE-/- mice fed low (LFD) or high (HFD) fat diets. ApoE-/- mice were fed LFD (13% kcal) or HFD (42% kcal) supplemented or not with 1 or 5% RBEE (w/w) for 23 weeks. Then, serum, aorta, liver and faeces were collected and flash frozen for further analysis. RBEE supplementation of HFD improved serum values by augmenting HDL-C and preventing total cholesterol and AST increase. HMG-CoA reductase activity was attenuated (1% and 5% RBEE) and cholesterol excretion increased (5% RBEE). 5% RBEE diet supplementation reduced plaque development regardless of the diet. In HFD-fed mice, both doses of RBEE reduced lipid deposition and macrophage infiltration in the aortic sinus and downregulated ICAM-1 and VCAM-1 expression. None of these effects were observed in mice fed LFD. Liver steatosis was reduced by RBEE supplementation of LFD (1% RBEE) and HFD (1 and 5% RBEE) and nuclear PPAR-α expression upregulated in HDF 5% RBEE group. Regular consumption of RBEE-supplemented HFD reduced plaque development and liver steatosis by decreasing inflammation and hyperlipidemia through a HMG-CoA reductase activity and lipid excretion related mechanism. CHAPTER III “Diet supplementation with rice bran enzymatic extract restores endothelial impairment and wall remodelling of ApoE-/- mice microvessels” Small mesenteric artery resistance and functionality are key factors for the maintenance of blood homeostasis. We attained to evaluate the effects of a rice bran enzymatic extract (RBEE) on structural, mechanic and myogenic alterations and endothelial dysfunction secondary to atherosclerosis disease. Seven week-old ApoE-/- mice were fed on standard (ST) or high fat (HF) diets supplemented or not with 1 or 5 % RBEE (w/w) for 23 weeks. Wild-type C57BL/6J mice fed on ST diet served as controls. Small mesenteric arteries were mounted in a pressure myograph in order to evaluate structural, mechanical and myogenic properties. Vascular reactivity was assessed in the presence of different combinations of inhibitors: L-NAME, indometacin, apamin and charybdotoxin. ApoE-/- mice fed on ST and HF diets showed different structural and mechanical alterations, alleviated by RBEE supplementation of ST and HF diets. C57BL/6J was characterized by increased expression of IKCa (199.3%, p=0.023) and SKCa (133.2%, p=0.026), resulting in higher EDHF participation (p=0.0001). However, NO release was more relevant to ApoE-/- mice vasodilatation. HF diet reduced the amount of NO released due to 2-fold increase of eNOS phosphorylation in the inhibitory residue Thr495 (p=0.034), which was fully counteracted by RBEE supplementation (p=0.028), restoring ACh-induced vasodilatation (p=0.00006). Dihydroethidium fluorescence of superoxide and picrosirius red staining of collagen were reduced by RBEE supplementation of HF diet by 76.91% (p=0.022) and 65.87% (p=0.030), respectively. RBEE supplemented diet reduced vessel remodelling and oxidative stress. Moreover, RBEE supplemented diet increased NO release by downregulating p-eNOSThr495, thus, protecting the endothelial function. CHAPTER IV “Atherosclerosis-related inflammation and oxidative stress are improved by Rice Bran Enzymatic Extract” The objective of this work was to test the antioxidant and anti-inflammatory potential of Rice Bran Enzymatic Extract diet supplementation in atherosclerotic mice. ApoE-/- mice were fed high fat diet supplemented with 1 or 5% rice bran enzymatic extract (RBEE). In parallel, 100 ng/ml-LPS induced human monocytes were treated with 20 µg/ml RBEE, ferulic acid (FA) or gamma-oryzanol (g-Ory). RBEE diet supplements reduced NF-κβ activation, TNF-α, COX-2 and iNOS expression and serum NO-derived metabolites. Additionally, NADPH oxidase subunits were downregulated, resulting in lower superoxide production, as evidenced by lower dihydroethidium fluorescence and oxLDL deposition in the aorta. RBEE, FA and g-Ory reduced pro-inflammatory monocyte (CD14++ CD16+) phenotype and increased non-classical monocytes (CD14- CD16++). A shift towards M2 polarized macrophages was observed, leading to reduced IL-6 and TNF-α mRNA expression. RBEE chronic consumption ameliorates atherosclerosis-related oxidative stress and inflammation showing its potential as nutritional supplement. FA moiety of g-Ory was identified as the main responsible for the actions observed. CHAPTER V “Food supplementation with rice bran enzymatic extract prevents vascular apoptosis and atherogenesis in ApoE-/- mice” Here we tested the hypothesis that rice bran enzymatic extract (RBEE) impacts on apoptosis, telomere length and atherogenesis in mice. Seven weeks-old male ApoE-/- mice were fed high fat diet (HFD) or isocaloric HFD supplemented with 5% (w/w) RBEE for 23 weeks. RBEE treatment reduced total cholesterol (744±63 vs 947±66 mg/dL) and triglycerides (97±16 vs 155±20 mg/dL) and augmented HDL-cholesterol (69±8 vs 35±8 mg/dL). RBEE attenuated macrophage infiltration by 56.69±4.65% and plaque development (7,737±836 vs 12,040±1,001 μm2) in the aortic sinus. In the aorta, RBEE treatment reduced expression of the apoptosis markers p16, p53 and bax/bcl2-ratio. RBEE prevented apoptosis of aortic endothelial cells (2.81±0.71 to 1.14±0.35 apoptotic nuclei/ring for ApoE-/- HFD and ApoE-/- HFD 5% RBEE, respectively). In contrast, MNC of RBEE-fed mice exhibited enhanced apoptosis marker expression with increased p53 and bax/bcl-2 protein levels. Compared to WT, ApoE-/- mice on HFD were characterized by significant telomere shortening in aorta (11±2%) and MNC (73±7%), which was reduced by supplementation with RBEE (aorta: 40±7%; MNC: 105±10%). Expression of telomere repeat-binding factor 2 was increased in RBEE fed mice. Long-term food supplementation with RBEE lowers cholesterol and prevents atherosclerotic plaque development in ApoE-/- mice. Differential regulation of vascular and MNC apoptosis and senescence were identified as potential mechanisms. CHAPTER VI “Bioavailability of the ferulic acid-derived phenolic compounds of a rice bran enzymatic extract. Activity against the superoxide production” The aims of this work were to describe the oral bioavailability and metabolic pathways of the ferulic acid-derived phenolic compounds contained in a rice bran enzymatic extract (RBEE), and to determine its activity upon NADPH oxidase activity. Wistar rats were administrated 10 g RBEE per kg and sacrificed at different times over a period of 24 h to obtain plasma. Urine and faeces were collected for 48 h. The phenolic metabolites were determined by liquid chromatography (UHPLC-MS/MS), and plasma pharmacokinetic parameters were calculated. In parallel, aortic rings were incubated in the plasma of rats sacrificed 30 min after water or RBEE gavage, or in HEPES-KHS in the presence of RBEE, ferulic acid or gamma-oryzanol, respectively. Endothelin-1-induced superoxide production was recorded by lucigenin enhanced luminescence. Twenty-five ferulic acid metabolites were found in the plasma showing a biphasic behaviour. Most of the urine metabolites were the same as those found in the plasma while in the faeces, colonic metabolism led to the simple phenol compounds. Finally, superoxide production was abrogated by phenolic compounds-enriched plasma and by RBEE and ferulic acid showing the biological potential of RBEE as a nutraceutical ingredient due to the benefits derived from NADPH oxidase inhibition. CHAPTER VII “Ferulic acid, a bioactive component of rice bran, improves oxidative stress and mitochondrial biogenesis and dynamics in mice and in human mononuclear cells” The objective of this work was to characterize the molecular effects of rice bran and its components on vascular oxidative stress and mitochondrial dysfunction during atherogenesis in mice and in humans. ApoE-/- mice were fed a high-fat, high-cholesterol diet (HFD) or HFD supplemented with 5% rice bran enzymatic extract (RBEE) for 21 weeks. RBEE prevented development of atherosclerotic plaques and oxidative stress in mouse aorta, as exemplified by increased GSH/GSSG ratio, reduced peroxiredoxin-SO3 and reduced lipid peroxidation. RBEE prevented the HFD-mediated downregulation of mRNA expression of markers of mitochondrial biogenesis (Pgc-1α, Pgc-1β, Nrf-1) and dynamics (Mfn1, Mfn2, Fis1, Beclin-1). Moreover, RBEE increased p-AMPK leading to increased deacetylation of PGC-1α. Analysis of the bioactive components of RBEE in bovine aortic endothelial cells identified ferulic acid as the component responsible for the observed effects. Ferulic acid metabolites such as sulfonates and glucuronides significantly increased in murine liver and kidney after treatment. To confirm these findings in humans, the kinetics of ferulic acid and its metabolites were determined in healthy volunteers who consumed 500 mg ferulic acid/day for 15 days. Ferulic acid intake reduced NADPH oxidase activity, superoxide release, apoptosis and necrosis and increased PGC-1α and MFN1 mRNA expression in human peripheral blood mononuclear cells. Moreover, differentiation and proliferation of endothelial progenitor cells were improved. Ferulic acid was identified as a major active component of rice bran, which improved mitochondrial biogenesis and dynamics and reduced oxidative stress in mouse aorta and human mononuclear cells. CONCLUSION The results obtained in this thesis suggest that the supplementation of a high fat and high cholesterol diet with RBEE improves the pro-inflammatory and prooxidant state present in atherosclerosis, preventing its progress and other alterations associated with atherosclerosis: vascular dysfunction and remodelling, steatosis, apoptosis, cellular senescence and mitochondrial dysfunction.