Effect of long-term consumption of oxidized polyunsaturated fatty acids on atherosclerotic lesion in LDL receptor knockout mice / Efeito do consumo crônico de ácidos graxos poliinsaturados oxidados na aterosclerose em camundongos knockout para receptor de LDL

Polyunsaturated fatty acids (PUFA) are susceptible to enzymatic and non-enzymatic oxidation, leading to the production of secondary compounds that present different physiological effects. Among the PUFA, the products formed from Omega 6 (n-6 FA) and Omega 3 (n-3 FA) fatty acids oxidation can modulate inflammation, dyslipidemia and oxidative stress preventing or reducing the atherosclerosis progression. In fact, the effect of chronic intake of edible oils containing products of polyunsaturated fatty acids oxidation (POPs) on atherosclerosis is still controversial. In general POPs from n-6 FA have a more pro-inflammatory profile than POPs from n-3 FA. Thus, the objective of this study was to compare the chronic intake of partially oxidized n-6 FA and n-3 FA rich oils on atherosclerosis biomarkers. Initially, six edible oils containing a higher amount of n-6 and n-3 FA were submitted to oxidative conditions, simulating the steps of transport, storage and consume. It was observed that oxidative reaction started in all oils since the first step and at the moment of consumption, some oxidative chemical markers were out the legal range suggested by the Official Agencies. In addition, it was possible to identify the type of secondary product formed from each precursor oil, providing a better information for oils quality control. After this step, fish and soybean oils were chosen as n-3 FA and n-6 FA rich oils, respectively. Using LDLr(-/-) mice, the effect of three oxidative levels of soybean oil was evaluated after 24 weeks of supplementation. Animals fed with the oil with the highest level of oxidation (fried and reused oil) showed no body weight gain, suggesting that POPs from soybean oil at this level could promote a browning effect on white adipose tissue by increasing UCP-1 expression. This group also showed the highest concentration of lipoproteins in plasma. However, these metabolic differences did not accelerate atherosclerosis in the animals. Finally, the effect of POPs from n-3 FA and n-6 FA oxidation were compared also using LDLr(-/-) mice as model for experimental atherosclerosis. Some alterations observed after n-3 FA supplementation, such as the increase of liver weight, IL-6, SONPC, 8-HETE and 15-F2-Isop and the decrease of BAT and glucose, were reversed by their POPs. In addition, POPs from n-6 FA caused increased of LDL and 5-HETE. As observed in the previous study, these metabolic alterations were not enough to prevent or accelerate atherosclerosis, as measured by histological analysis of the lesion size in the aorta. These results suggest that although a significant amount of POPs are being consumed by diet, their metabolic effects did not influence atherosclerotic plaques in the animal model. However, besides lesion area in the aortas, new studies should also evaluate the plaques stability

Os ácidos graxos poliinsaturados (PUFA) são suscetíveis à oxidação enzimática e não enzimática, levando à produção de compostos secundários que apresentam diferentes efeitos fisiológicos. Entre os PUFA, os produtos formados a partir da oxidação dos ácidos graxos ômega 6 (n-6 FA) e ômega 3 (n-3 FA) podem modular a inflamação, dislipidemia e estresse oxidativo, impedindo ou reduzindo a progressão da aterosclerose. De fato, o efeito da ingestão crônica de óleos contendo produtos da oxidação de ácidos graxos poliinsaturados (POPs) na aterosclerose ainda é controverso. Em geral, os POPs dos n-6 FA têm um perfil mais pró-inflamatório do que os POPs dos n-3 FA. Assim, o objetivo deste estudo foi comparar a ingestão crônica de POPs provenientes de óleos ricos em n-6 FA e n-3 FA em biomarcadores de aterosclerose. Inicialmente, seis óleos ricos em n-6 FA e n-3 FA foram submetidos a condições oxidativas, simulando as etapas de transporte, armazenamento e consumo. Observou-se que a reação oxidativa iniciou-se em todos os óleos desde a primeira etapa e, no momento do consumo, alguns marcadores oxidativos estavam fora da faixa legal sugerida pelas agências reguladoras. Além disso, foi possível identificar o tipo de produto secundário formado a partir de cada óleo precursor, fornecendo melhores informações para o controle de qualidade dos óleos. Após esta etapa, os óleos de peixe e soja foram escolhidos como óleos ricos em n-3 FA e n-6 FA, respectivamente. Utilizando camundongos LDLr(-/-), o efeito de três níveis oxidativos de óleo de soja foi avaliado após 24 semanas de suplementação. Os animais alimentados com o óleo com maior nível de oxidação (óleo frito e de reuso) não apresentaram ganho de peso corporal, sugerindo que os POPs do óleo de soja nesse nível de oxidação pudessem promover um efeito de Browning no tecido adiposo branco, aumentando a expressão de UCP-1. Este grupo também mostrou a maior concentração de lipoproteínas no plasma. No entanto, essas diferenças metabólicas não aceleraram a aterosclerose nos animais. Finalmente, o efeito de POPs da oxidação de óleos ricos em n-3 FA e n-6 FA foi comparado também usando camundongos LDLr(-/-), como modelo para aterosclerose experimental. Algumas alterações observadas após a suplementação com óleo de peixe fresco, como aumento do peso hepático, IL-6, SONPC, 8-HETE e 15-F2-IsoP e diminuição da BAT e glicose, foram revertidas por seus POPs. Além disso, os POPs do óleo de soja causaram aumento de LDL e 5-HETE. Como observado no estudo anterior, essas alterações metabólicas não foram suficientes para prevenir ou acelerar a aterosclerose, medida pela análise histológica do tamanho da lesão na aorta. Esses resultados sugerem que, embora uma quantidade significativa de POPs esteja sendo consumida pela dieta, seus efeitos metabólicos não influenciaram as placas ateroscleróticas no modelo animal. Porém, além da área de lesão nas aortas, novos estudos também devem avaliar a estabilidade das placas

ß-ionone inhibits nonalcoholic fatty liver disease and its association with hepatocarcinogenesis in male Wistar rats.

Among the primary neoplasias that affect the liver, hepatocellular carcinoma (HCC) is the most frequent and the third leading cause of death related to cancer. Several risk factors predispose individuals to HCC such as nonalcoholic fatty liver disease (NAFLD), whose incidence has significantly increased worldwide. ß-ionone (ßI) isoprenoid is a known chemopreventive of hepatocarcinogenesis. However, the effects of this compound on NAFLD isolated or in association with hepatocarcinogenesis have not yet been evaluated. A high-fat emulsion administered for 6 weeks resulted in NAFLD in male rats, and oral treatment with ßI during this period significantly attenuated its development. Moreover, the presence of NAFLD potentiated hepatocarcinogenesis induced by the resistant hepatocyte (RH) model in these animals by increasing the number and percentage of the liver section area occupied by placental glutathione S-transferase (GST-P)-positive persistent preneoplastic lesions (pPNLs), that are thought to evolve into HCC. This indicates that this NAFLD/RH protocol is suitable for studies of the influence of NAFLD on the HCC development. Therefore, here we also investigated the chemopreventive effect of ßI under these two associated conditions. In this context, ßI reduced the number and percentage of the liver section area occupied by pPNLs, as well as cell proliferation and the number of oval cells, which are considered potential targets for the development of HCC. Thus, ßI presents not only a promising inhibitory effect on NAFLD isolated but also chemopreventive activity when it is associated with hepatocarcinogenesis.

Protective effect of genetic deletion of pannexin1 in experimental mouse models of acute and chronic liver disease.

Pannexins are transmembrane proteins that form communication channels connecting the cytosol of an individual cell with its extracellular environment. A number of studies have documented the presence of pannexin1 in liver as well as its involvement in inflammatory responses. In this study, it was investigated whether pannexin1 plays a role in acute liver failure and non-alcoholic steatohepatitis, being prototypical acute and chronic liver pathologies, respectively, both featured by liver damage, oxidative stress and inflammation. To this end, wild-type and pannexin1-/- mice were overdosed with acetaminophen for 1, 6, 24 or 48h or were fed a choline-deficient high-fat diet for 8weeks. Evaluation of the effects of genetic pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, lipid accumulation, protein adduct formation, oxidative stress and inflammation. In parallel, in order to elucidate molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. The results of this study show that pannexin1-/- diseased mice present less liver damage and oxidative stress, while inflammation was only decreased in pannexin1-/- mice in which non-alcoholic steatohepatitis was induced. A multitude of genes related to inflammation, oxidative stress and xenobiotic metabolism were differentially modulated in both liver disease models in wild-type and in pannexin1-/- mice. Overall, the results of this study suggest that pannexin1 may play a role in the pathogenesis of liver disease.

Inhibition of connexin hemichannels alleviates non-alcoholic steatohepatitis in mice.

While gap junctions mediate intercellular communication and support liver homeostasis, connexin hemichannels are preferentially opened by pathological stimuli, including inflammation and oxidative stress. The latter are essential features of non-alcoholic steatohepatitis. In this study, it was investigated whether connexin32 and connexin43 hemichannels play a role in non-alcoholic steatohepatitis. Mice were fed a choline-deficient high-fat diet or normal diet for 8 weeks. Thereafter, TAT-Gap24 or TAT-Gap19, specific inhibitors of hemichannels composed of connexin32 and connexin43, respectively, were administered for 2 weeks. Subsequently, histopathological examination was carried out and various indicators of inflammation, liver damage and oxidative stress were tested. In addition, whole transcriptome microarray analysis of liver tissue was performed. Channel specificity of TAT-Gap24 and TAT-Gap19 was examined in vitro by fluorescence recovery after photobleaching analysis and measurement of extracellular release of adenosine triphosphate. TAT-Gap24 and TAT-Gap19 were shown to be hemichannel-specific in cultured primary hepatocytes. Diet-fed animals treated with TAT-Gap24 or TAT-Gap19 displayed decreased amounts of liver lipids and inflammatory markers, and augmented levels of superoxide dismutase, which was supported by the microarray results. These findings show the involvement of connexin32 and connexin43 hemichannels in non-alcoholic steatohepatitis and, simultaneously, suggest a role as potential drug targets in non-alcoholic steatohepatitis.

Connexin32 deficiency is associated with liver injury, inflammation and oxidative stress in experimental non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis is a highly prevalent liver pathology featured by hepatocellular fat deposition and inflammation. Connexin32, which is the major building block of hepatocellular gap junctions, has a protective role in hepatocarcinogenesis and is downregulated in chronic liver diseases. However, the role of connexin32 in non-alcoholic steatohepatitis remains unclear. Connexin32-/- mice and their wild-type littermates were fed a choline-deficient high-fat diet. The manifestation of non-alcoholic steatohepatitis was evaluated based on a battery of clinically relevant read-outs, including histopathological examination, diverse indicators of inflammation and liver damage, in-depth lipid analysis, assessment of oxidative stress, insulin and glucose tolerance, liver regeneration and lipid-related biomarkers. Overall, more pronounced liver damage, inflammation and oxidative stress were observed in connexin32-/- mice compared to wild-type animals. No differences were found in insulin and glucose tolerance measurements and liver regeneration. However, two lipid-related genes, srebf1 and fabp3, were upregulated in Cx32-/- mice in comparison with wild-type animals. These findings suggest that connexin32-based signalling is not directly involved in steatosis as such, but rather in the sequelae of this process, which underlie progression of non-alcoholic steatohepatitis.

Protection of echium oil by microencapsulation with phenolic compounds.

The consumption of omega-3 enables the reduction of cardiovascular disease risk; however they are unstable. The aim of this work was to encapsulate echium oil (Echium plantagineum L.), a rich source of omega-3 fatty acids, with phenolic compounds (sinapic acid and rutin) by double emulsion followed by complex coacervation or by complex coacervation with sinapic acid in the capsule wall. Analyses of morphology, particle size, circularity, water activity, moisture, Fourier transform infrared spectroscopy, thermogravimetry, process yield, accelerated oxidation and identification and quantification of fatty acids present in the encapsulated oil were performed. Samples presented values of encapsulation process yield of phenolics and oil in the range of 39-80% and 73-99%, respectively. Moreover, all samples protected the oil against oxidation, obtaining induction time (accelerated oxidation) of 5h for pure oil and values in the range from 10 to 18h for samples. Thus, better protection to the oil was possible with sinapic acid applied in the capsule wall, which enhances its protection against lipid oxidation.