Les derniers apports nutritionnels conseillés recommandent une consommation plus importante en acides gras polyinsaturés de la série n-3 que celle actuellement constatée dans l’alimentation française. Dans ce contexte, il convenait d’appréhender les facteurs susceptibles de moduler l’absorption et le devenir de leur chef de file, l’ALA, en faisant appel à deux approches, l’une in vivo (rat), l’autre in vitro (Caco-2). L’étude relative au devenir métabolique de l’ALA, selon sa forme physique et chimique de présentation, a été réalisée avec des lipides « modèles » (TAG structurés) ou naturels (huile de lin) riches en ALA, et selon différents systèmes lipidiques (huile en phase continue ou en émulsion de type huile dans eau et de composition en phospholipides variables). Les résultats obtenus in vivo et in vitro à l’égard de l’huile de lin (émulsionnée ou non) montrent que l’émulsification accélère non seulement le passage intestinal de l’ALA mais améliore également sa concentration lymphatique. L’étude cellulaire a par ailleurs démontré que la présence de lysophospholipides dans les micelles mixtes permet d’améliorer la sécrétion de l’ALA dans les lipoprotéines. D’autre part, le devenir métabolique de l’ALA dépend de sa régiolocalisation sur le triglycéride alimentaire. En effet, les résultats de l’étude faisant appel aux TAG structurés montrent que la position interne n’est que partiellement conservée dans la lymphe, suggérant qu’une hydrolyse des 2-MAG serait opérée par une MG lipase. En conséquence, l’ensemble des résultats obtenus lors de cette étude montre que l’absorption et le transport de l’ALA seraient uniquement modulés selon la forme physique de l’acide gras alors que son devenir et son utilisation métabolique dépendraient de sa régiolocalisation sur le TAG alimentaire. Ces deux facteurs réunis permettraient dés lors de prévenir l’ALA d’une β-oxydation précoce, en vue de favoriser son élongation en dérivés supérieurs dans les tissus cibles. / The last recommended nutrient intakes advise a higher consumption of n-3 polyunsaturated fatty acids series than currently found in French diet. In this context, it was appropriate to apprehend the factors that could modulate the absorption and fate of their leader, ALA, using two approaches, one in vivo (rat), the other one in vitro (Caco-2). The study on the metabolic fate of ALA, according to its physical and chemical submission form, was conducted with "models" lipids (structured TAG) or natural (flaxseed oil) rich in ALA, and according to different lipid systems (oil in continuous phase or in emulsion type oil in water and with variable phospholipid composition). Results obtained in vivo and in vitro for flaxseed oil (emulsion or not) show that emulsification enhances the recovery of ALA at the intestinal level but also improves its lymphatic concentration. The cell study also demonstrated that the presence of lysophospholipids in mixed micelles can improve the secretion of ALA in lipoproteins. On the other hand, the metabolic fate of ALA depends on its location on the glycerol backbone of the dietary triglyceride. The results of the study using structured TAG show that the internal position is partially preserved in lymph, suggesting that a hydrolysis of 2 - MAG by MG lipase could occur. Accordingly, all of the results obtained in this study shows that the absorption and transport of ALA would only be modulated according to the physical form of the fatty acid while its fate and its metabolic use would depend on its location on the dietary TAG. These two factors combined would then allow preventing the early β-oxidation of ALA in order to promote its elongation in higher derivatives in the target tissues.

Purpose: To 1) critically appraise available randomized controlled trials (RTCs) addressing the efficacy of long-chain ω-3 fatty acids as a secondary prevention agent of hypertriglyceridemia, and 2) make recommendations for clinical practice. Data Sources: All RCTs identified from several databases from 1993-2003 were reviewed by two independent reviewers who extracted data from each study and used the previously tested Boyack and Lookinland Methodological Quality Index (MQI) to determine study quality. Results: Ten studies reported long-chain ω-3 fatty acids to be effective in the treatment of hypertriglyceridemia. The average decrease in triglycerides (TG) was 29%, total cholesterol (TC) 11.6%, very low density lipoprotein (VLDL) 30.2%, and low-density lipoprotein (LDL) 32.5%. One study found LDLs to increase by 25%. The average increase in high-density lipoprotein (HDL) was 10%. The overall average MQI score was 36% (26%-54%). Many of the RCTs had serious shortcomings including short duration, lack of a power analysis, no intention to treat analysis, no report of blind assessment of outcome, and lack of dietary control as a confounding variable. Conclusions/Implications: Overall study methodology was weak. Although the evidence supporting the use of long-chain ω-3 fatty acids in the secondary prevention of hypertriglyceridemia is reasonably strong, until there are larger RCTs of stronger methodological quality, it is not recommended to treat hypertriglyceridemia with ω-3 fatty acid supplementation in lieu of lipid lowering medications.

Diabetes mellitus tipo 2 (DM2) está associado a um estado de inflamação crônica e ativação do estresse do retículo endoplasmático (ERE). Nesse contexto, são necessários estudos para encontrar alternativas que melhorem o quadro inflamatório, como os ácidos graxos poli-insaturados ômega 3 (n-3 PUFA), um conhecido agente anti-inflamatório. Esse estudo teve por objetivo avaliar o efeito da suplementação do ácido alfa-linolênico (ALA, um n-3 PUFA) no estresse do retículo endoplasmático e no estado inflamatório no tecido adiposo subcutâneo abdominal (TASC) em pacientes com DM2. Foi conduzido um estudo duplo-cego, prospectivo, placebo-controlado. Vinte pacientes com DM2 foram randomizados para suplementação com 3g/dia de ALA ou placebo durante 60 dias. O tecido adiposo foi coletado através de punção aspirativa por agulha fina do abdome antes e após a suplementação e os genes e proteínas foram avaliados através de PCR em tempo real e western blot. Foi encontrada, após suplementação, uma redução da expressão gênica do XBP1 (20%), sXBP1 (70%) e aumento da expressão gênica do GRP78 (150%), confirmado na expressão proteica. Além disso, foi encontrado aumento da expressão gênica da adiponectina (90%) e redução da expressão gênica do IL-6 (80%) e IRS-1 (60%), sem correlação com a expressão proteica, no tempo pós-suplementação com ALA. Portanto, foi demonstrado que o ALA pode modular o ERE através da via da IRE1/XBP, levando ao aumento das chaperonas (BIP/GRP78), além de um efeito adicional na expressão gênica da adiponectina, IL-6 e IRS-1, o que pode demonstrar um potencial terapêutico do ALA em pacientes com DM2. / Type 2 diabetes mellitus (T2DM) is a state of chronic inflammation and activation of endoplasmic reticulum stress (ERS). In this context, studies are necessaries to find new possibilities to improve this inflammation such as the n-3 polyunsaturated fatty acid (n-3 PUFA) acting as an anti-inflammatory agent. In this study, we aimed to evaluate the effect of n-3 PUFA alpha-linolenic acid (ALA, a n-3 PUFA) supplementation in T2DM patients on the molecular expression of ERS genes in abdominal subcutaneous adipose tissue (SAT). We performed a placebo-controlled study, in a double-blind design with 20 T2DM patients, receiving, randomly, 3g/day of ALA or placebo for 60 days. The adipose tissue was collected by fine-needle aspiration in abdomen before and after the supplementation and the genes and proteins were evaluated by real-time PCR and western blot. It was seen, after the supplementation, a reduction in XBP1 (20%), sXBP1 (70%) and an increase in Grp78 (150%) gene expression, likewise same results in protein concentration. Furthermore, it was observed an increase in adiponectina (90%) gene expression and reduction in IL-6 (80%) and IRS-1 (60%) gene expression, with no correlation to protein expression after supplementation of ALA. Therefore, we have provided evidence that ALA may modulate ERS by the IRE1/XBP pathway leading to an increase in chaperones (BIP/GRP78), additionally its effect on adiponectina, IL-6 and IRS-1 gene expression can demonstrate a therapeutic potential in T2DM.

Diabetes mellitus tipo 2 (DM2) está associado a um estado de inflamação crônica e ativação do estresse do retículo endoplasmático (ERE). Nesse contexto, são necessários estudos para encontrar alternativas que melhorem o quadro inflamatório, como os ácidos graxos poli-insaturados ômega 3 (n-3 PUFA), um conhecido agente anti-inflamatório. Esse estudo teve por objetivo avaliar o efeito da suplementação do ácido alfa-linolênico (ALA, um n-3 PUFA) no estresse do retículo endoplasmático e no estado inflamatório no tecido adiposo subcutâneo abdominal (TASC) em pacientes com DM2. Foi conduzido um estudo duplo-cego, prospectivo, placebo-controlado. Vinte pacientes com DM2 foram randomizados para suplementação com 3g/dia de ALA ou placebo durante 60 dias. O tecido adiposo foi coletado através de punção aspirativa por agulha fina do abdome antes e após a suplementação e os genes e proteínas foram avaliados através de PCR em tempo real e western blot. Foi encontrada, após suplementação, uma redução da expressão gênica do XBP1 (20%), sXBP1 (70%) e aumento da expressão gênica do GRP78 (150%), confirmado na expressão proteica. Além disso, foi encontrado aumento da expressão gênica da adiponectina (90%) e redução da expressão gênica do IL-6 (80%) e IRS-1 (60%), sem correlação com a expressão proteica, no tempo pós-suplementação com ALA. Portanto, foi demonstrado que o ALA pode modular o ERE através da via da IRE1/XBP, levando ao aumento das chaperonas (BIP/GRP78), além de um efeito adicional na expressão gênica da adiponectina, IL-6 e IRS-1, o que pode demonstrar um potencial terapêutico do ALA em pacientes com DM2. / Type 2 diabetes mellitus (T2DM) is a state of chronic inflammation and activation of endoplasmic reticulum stress (ERS). In this context, studies are necessaries to find new possibilities to improve this inflammation such as the n-3 polyunsaturated fatty acid (n-3 PUFA) acting as an anti-inflammatory agent. In this study, we aimed to evaluate the effect of n-3 PUFA alpha-linolenic acid (ALA, a n-3 PUFA) supplementation in T2DM patients on the molecular expression of ERS genes in abdominal subcutaneous adipose tissue (SAT). We performed a placebo-controlled study, in a double-blind design with 20 T2DM patients, receiving, randomly, 3g/day of ALA or placebo for 60 days. The adipose tissue was collected by fine-needle aspiration in abdomen before and after the supplementation and the genes and proteins were evaluated by real-time PCR and western blot. It was seen, after the supplementation, a reduction in XBP1 (20%), sXBP1 (70%) and an increase in Grp78 (150%) gene expression, likewise same results in protein concentration. Furthermore, it was observed an increase in adiponectina (90%) gene expression and reduction in IL-6 (80%) and IRS-1 (60%) gene expression, with no correlation to protein expression after supplementation of ALA. Therefore, we have provided evidence that ALA may modulate ERS by the IRE1/XBP pathway leading to an increase in chaperones (BIP/GRP78), additionally its effect on adiponectina, IL-6 and IRS-1 gene expression can demonstrate a therapeutic potential in T2DM.

Background Iron deficiency (ID) is the world‟s most prevalent micronutrient deficiency and predominantly affects developing countries, also South Africa. In areas with low fish consumption and high n-6 PUFA vegetable oil intake, there is a risk for having inadequate n-3 PUFA status. Both iron and n-3 PUFA play important roles in the immune response, and supplementation is a strategy to alleviate deficiencies. However, little is known about potential interactive effects between concurrent iron and n-3 PUFA supplementation on the immune system. This is also important in the context that iron supplementation may be unsafe and may increase morbidity and mortality. Aim The overall aim of this thesis was to assess the effects of iron and docosahexaenoic (DHA)/eicosapentaenoic acid (EPA) supplementation, alone and in combination, on the immune system of ID children. More specifically, these effects were investigated on the occurrence and duration of illness and school-absenteeism due to illness, peripheral blood mononuclear cell (PBMC), red blood cell (RBC) and plasma total phospholipid fatty acid composition, iron status, fatty acid-derived immune modulators and targeted PBMC gene expression. Furthermore, association of PBMC, RBC and plasma total phospholipid fatty acid composition with allergic disease, were also examined. Design In a 2-by-2 factorial, randomised, double-blind, placebo-controlled trial, South African children (n = 321, aged 6–11 y) were randomly assigned to receive oral supplements of either 1) iron (50 mg as ferrous sulphate) plus placebo; 2) DHA/EPA (420/80 mg) plus placebo; 3) iron plus DHA/EPA (420/80 mg); or 4) placebo plus placebo for 8.5 mo, four times per week. Absenteeism and illness symptoms were recorded and biochemical parameters for compliance as well as parameters fundamental to immune function were assessed at baseline and endpoint. Furthermore, in a cross-sectional design, associations of allergic disease with baseline fatty acid composition of PBMC, RBC and plasma were examined. Results The combination of iron and DHA/EPA significantly attenuated respiratory illness caused by iron supplementation. DHA/EPA supplementation alone improved respiratory symptoms at school, but increased headache-related absenteeism. DHA/EPA and iron supplementation individually tended to increase and decrease anti-inflammatory DHA and EPA-derived mediators, respectively. Furthermore the anti-inflammatory DHA-derived immune mediator, 17HDHA was higher in the DHA/EPA plus placebo and iron plus DHA/EPA groups than in the iron plus placebo group. Also, the pro-inflammatory arachidonic acid (AA)-derived modulators (5- and 15-hydroxyeicosapentaenoic acid) were significantly lower in the iron plus DHA/EPA group compared to the placebo plus placebo groups. In the study population, 27.2% of the children had allergic disease and AA in PBMC phospholipids was significantly lower in the allergic children than in the non-allergic children. In RBC phospholipids dihomo-gamma-linolenic acid (DGLA) and the ratio of DGLA: linoleic acid (LA) correlated negatively and the n-6:n-3 PUFA ratio positively with total immunoglobulin E (tIgE). Furthermore, trans-C18:1n-9, tended to be higher in the allergic group. Conclusion DHA/EPA prevented respiratory illness caused by iron supplementation and although DHA/EPA on its own reduced respiratory morbidity when the children were present at school, surprisingly it increased the likelihood of being absent with headache and fever. The biochemical findings compliment the clinical results and support previous observations about DHA/EPA supplementation to reduce inflammation, but add to the current knowledge base that a relatively high oral dose of non-haem iron modulates circulating lipid-derived immune modulators and related gene expression. Furthermore, when supplementing with iron and DHA/EPA combined, in this ID population with low fish intake, the anti-inflammatory effect of DHA/EPA is maintained concurrently with attenuation of respiratory morbidity. This finding support the notion that excess iron (probably as non-transferrin bound iron) becomes available for pathogens and is probably why we found that iron increased respiratory infectious morbidity. The improved clinical outcome with combined supplementation seems to be related to increased lipid-mediator synthesis gene expression and the availability of DHA/EPA, leading to a more pro-resolving profile and enhanced immune competence. Overall these results give better insight into immune function and infectious morbidity in relation to n-3 PUFA and iron status and treatment, as well as the possible association of fatty acid status with allergic disease in young South-African school children. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

Background Iron deficiency (ID) is the world‟s most prevalent micronutrient deficiency and predominantly affects developing countries, also South Africa. In areas with low fish consumption and high n-6 PUFA vegetable oil intake, there is a risk for having inadequate n-3 PUFA status. Both iron and n-3 PUFA play important roles in the immune response, and supplementation is a strategy to alleviate deficiencies. However, little is known about potential interactive effects between concurrent iron and n-3 PUFA supplementation on the immune system. This is also important in the context that iron supplementation may be unsafe and may increase morbidity and mortality. Aim The overall aim of this thesis was to assess the effects of iron and docosahexaenoic (DHA)/eicosapentaenoic acid (EPA) supplementation, alone and in combination, on the immune system of ID children. More specifically, these effects were investigated on the occurrence and duration of illness and school-absenteeism due to illness, peripheral blood mononuclear cell (PBMC), red blood cell (RBC) and plasma total phospholipid fatty acid composition, iron status, fatty acid-derived immune modulators and targeted PBMC gene expression. Furthermore, association of PBMC, RBC and plasma total phospholipid fatty acid composition with allergic disease, were also examined. Design In a 2-by-2 factorial, randomised, double-blind, placebo-controlled trial, South African children (n = 321, aged 6–11 y) were randomly assigned to receive oral supplements of either 1) iron (50 mg as ferrous sulphate) plus placebo; 2) DHA/EPA (420/80 mg) plus placebo; 3) iron plus DHA/EPA (420/80 mg); or 4) placebo plus placebo for 8.5 mo, four times per week. Absenteeism and illness symptoms were recorded and biochemical parameters for compliance as well as parameters fundamental to immune function were assessed at baseline and endpoint. Furthermore, in a cross-sectional design, associations of allergic disease with baseline fatty acid composition of PBMC, RBC and plasma were examined. Results The combination of iron and DHA/EPA significantly attenuated respiratory illness caused by iron supplementation. DHA/EPA supplementation alone improved respiratory symptoms at school, but increased headache-related absenteeism. DHA/EPA and iron supplementation individually tended to increase and decrease anti-inflammatory DHA and EPA-derived mediators, respectively. Furthermore the anti-inflammatory DHA-derived immune mediator, 17HDHA was higher in the DHA/EPA plus placebo and iron plus DHA/EPA groups than in the iron plus placebo group. Also, the pro-inflammatory arachidonic acid (AA)-derived modulators (5- and 15-hydroxyeicosapentaenoic acid) were significantly lower in the iron plus DHA/EPA group compared to the placebo plus placebo groups. In the study population, 27.2% of the children had allergic disease and AA in PBMC phospholipids was significantly lower in the allergic children than in the non-allergic children. In RBC phospholipids dihomo-gamma-linolenic acid (DGLA) and the ratio of DGLA: linoleic acid (LA) correlated negatively and the n-6:n-3 PUFA ratio positively with total immunoglobulin E (tIgE). Furthermore, trans-C18:1n-9, tended to be higher in the allergic group. Conclusion DHA/EPA prevented respiratory illness caused by iron supplementation and although DHA/EPA on its own reduced respiratory morbidity when the children were present at school, surprisingly it increased the likelihood of being absent with headache and fever. The biochemical findings compliment the clinical results and support previous observations about DHA/EPA supplementation to reduce inflammation, but add to the current knowledge base that a relatively high oral dose of non-haem iron modulates circulating lipid-derived immune modulators and related gene expression. Furthermore, when supplementing with iron and DHA/EPA combined, in this ID population with low fish intake, the anti-inflammatory effect of DHA/EPA is maintained concurrently with attenuation of respiratory morbidity. This finding support the notion that excess iron (probably as non-transferrin bound iron) becomes available for pathogens and is probably why we found that iron increased respiratory infectious morbidity. The improved clinical outcome with combined supplementation seems to be related to increased lipid-mediator synthesis gene expression and the availability of DHA/EPA, leading to a more pro-resolving profile and enhanced immune competence. Overall these results give better insight into immune function and infectious morbidity in relation to n-3 PUFA and iron status and treatment, as well as the possible association of fatty acid status with allergic disease in young South-African school children. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

INTRODUÇÃO: A esteato-hepatite não alcóolica (NASH) é considerada problema de saúde pública, dada sua crescente incidência e seu possível papel na carcinogênese hepato-celular. Terapias atuais envolvem alterações de dieta e estilo de vida, mas têm seu resultado prejudicado pela baixa aderência dos pacientes. Abordagens farmacológicas ainda são precárias. Uma grande dificuldade no manejo de NASH reside no limitado entendimento de sua fisiopatologia, que parece envolver complexas alterações metabólicas e inflamatórias. Ácidos graxos poli-insaturados ômega-3 (AGPIs n-3) são reconhecidos por suas propriedades moduladoras do metabolismo lipídico e da inflamação, e estão diminuídos em pacientes com NASH. O uso clínico de AGPIs n-3 tem mostrado benefício no controle da esteatose e na produção de marcadores da resposta metabólica e inflamatória em NASH, embora com algumas observações contraditórias. A compreensão de mecanismos moleculares modulados por AGPIs n-3 em NASH podem ser úteis para identificar alvos moleculares que auxiliem no desenho de intervenção farmacológica efetiva. Nesse sentido, ciências ômicas são particularmente úteis para a compreensão de mecanismos moleculares com alto valor translacional para a prática clínica e podem contribuir para a identificação desses alvos. OBJETIVO: O presente estudo avaliou a resposta proteômica hepática e lipidômica plasmática de pacientes com NASH perante o tratamento com AGPIs n-3. MÉTODO: As avaliações proteômicas e lipidômicas foram desenvolvidas por espectometria de massas e/ou cromatografia gasosa em amostras de biópsias hepáticas e plasma coletadas de pacientes envolvidos em estudo preliminar, realizado no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. O referido estudo envolveu pacientes adultos, de ambos os sexos e com diagnóstico de NASH tratados diariamente, durante 6 meses, com 3 cápsulas contendo mistura de óleo de linhaça e óleo de peixe [0,315 g AGPIs: sendo 0,065 g de ácido eicosapentaenoico (EPA), 0,050 g de docosahexaenoico (DHA) e 0,2 g alfa linolênico (ALA) por cápsula]. Pacientes, após o tratamento com AGPIs n-3, que apresentaram altas concentrações plasmáticas de ALA e/ou DHA e/ou baixas de ácido araquidônico (AA) mostraram melhora parcial das alterações de histologia hepática. No presente estudo, avaliamos as vias proteômicas e marcadores lipidômicos resultantes do tratamento com AGPIs n-3. Isto foi feito por meio da comparação, antes (grupo AT) e depois do tratamento (grupo DT), de pools de tecido hepático (análise por interactoma) e amostras de plasma (OPLS-DA). RESULTADOS: Foram identificadas proteínas hepáticas, exclusivamente e/ou alteradamente expressas, no grupo DT, relacionadas com vias de matriz celular, metabolismo lipídico, de estresse oxidativo, e de retículo endoplasmático e respiração celular. Com excessão da via de matriz celular, a análise do interactoma revelou alteração funcional significativa das vias moduladas por essas proteínas. Em conjunto, essas alterações foram sugestivas de diminuição de lipotoxicidade, estresse oxidativo e respiração anaeróbia, e aumento de respiração aeróbia após tratamento com AGPIs n-3. Estas modificações são marcadores potenciais de melhora de função de retículo endoplasmático e mitocondrial. Em adição, após o tratamento com AGPIs n-3, o perfil lipidômico plasmático mostrou-se alterado com significativo aumento de glicerofosfolípides, ALA e EPA, e diminuição de ácido araquidônico (n-6) e da razão AGPIs n-6/n-3. Estes dados são concordantes com potencial melhora das funções de retículo endoplasmático e mitocondriais. CONCLUSÃO: O tratamento com AGPIs n-3 em pacientes com NASH influenciou favoravelmente o perfil proteômico hepático e lipidômico sistêmico. Em conjunto, essas alterações sugerem melhora da função de retículo endoplasmático e mitocondrial, com potencial impacto na homeostase celular, por meio da modulação de diferentes vias biológicas / INTRODUCTION: Non-alcoholic steatohepatitis (NASH) is considered a public health problem, given its increasing incidence and its possible role in hepatocellular carcinogenesis. Current therapies involve diet and lifestyle changes, but its applicability suffers from low patients adherence. Pharmacological approaches are still missing. A main difficulty in the NASH management lies in the limited understanding of its pathophysiology, which seems to involve complex metabolic and inflammatory disturbances. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are recognized for its modulatory properties on lipid metabolism and inflammation and are decreased in patients with NASH. The clinical use of these PUFAs has shown benefit in controlling steatosis and the production of metabolic and inflammatory response markers in NASH, despite some conflicting reports. Understanding mechanisms modulated by n-3 PUFAs in NASH may be useful for identifying molecular targets that could assist in the design of effective pharmacologic interventions. In this sense, omics sciences are particularly useful for understanding molecular mechanisms with high translational value to clinical practice and may contribute to the identification of these targets. AIM: This study evaluated the liver proteomic and plasma lipidomics responses of patients with NASH towards treatment with n-3 PUFAs. METHODS: The proteomic and lipidomic evaluations were studied by mass spectrometry and / or gas chromatography in samples from liver biopsies and plasma collected from patients enrolled in a preliminary clinical trial of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. This study involved adult patients of both sexes diagnosed with NASH treated daily for 6 months, with 3 capsules containing a mixture of linseed and fish oils [0.315 g PUFAs: 0.065 g eicosapentaenoic acid (EPA) , 0.050 g docosahexaenoic (DHA) and 0.2 g alpha linolenic acid (ALA) per capsule]. Patients, after treatment with n-3 PUFAs, with higher concentrations of ALA and DHA and lower arachidonic acid (AA) showed improvement of liver histology alterations. In the present study we evaluated the proteomics pathways and lipidomics markers resulted from treatment with PUFAs n-3. This was performed by comparing, before (BT group) and after (AT group) treatment, liver tissue pools (analysis interactome) and plasma samples (OPLS-DA). RESULTS: It was identified, in a way exclusive and altered, the expressed liver proteins in AT group, related to pathways of cellular matrix, lipid metabolism, oxidative and endoplasmic reticulum stress and cellular respiration. With the exception of cell matrix, the analysis of the interactome revealed substantial functional alterations of the pathways modulated by these proteins. Together, these changes were suggestive of decreased lipotoxicity, oxidative stress and anaerobic respiration and increased aerobic respiration following treatment with PUFAs n-3. These modifications are potential markers of endoplasmic reticulum and mitochondrial functions improvement. In addition, after treatment with n-3 PUFAs, the lipidomics profile was modified, with significant increase in glycerophospholipids, ALA and EPA and decrease of arachidonic acid (AA) and n-6/n-3 AGPIs ratio. These findings are concordant with potential improvement of reticulum endoplasmic and mitochondrial functions. CONCLUSION: In patients with NASH the treatment with n-3 PUFAs favorably influenced hepatic proteomic and systemic lipidomics profiles. Together, these changes suggest improved endoplasmic reticulum and mitochondrial functions, with potential impact on cellular homeostasis through the modulation of different biological pathways

INTRODUÇÃO: A esteato-hepatite não alcóolica (NASH) é considerada problema de saúde pública, dada sua crescente incidência e seu possível papel na carcinogênese hepato-celular. Terapias atuais envolvem alterações de dieta e estilo de vida, mas têm seu resultado prejudicado pela baixa aderência dos pacientes. Abordagens farmacológicas ainda são precárias. Uma grande dificuldade no manejo de NASH reside no limitado entendimento de sua fisiopatologia, que parece envolver complexas alterações metabólicas e inflamatórias. Ácidos graxos poli-insaturados ômega-3 (AGPIs n-3) são reconhecidos por suas propriedades moduladoras do metabolismo lipídico e da inflamação, e estão diminuídos em pacientes com NASH. O uso clínico de AGPIs n-3 tem mostrado benefício no controle da esteatose e na produção de marcadores da resposta metabólica e inflamatória em NASH, embora com algumas observações contraditórias. A compreensão de mecanismos moleculares modulados por AGPIs n-3 em NASH podem ser úteis para identificar alvos moleculares que auxiliem no desenho de intervenção farmacológica efetiva. Nesse sentido, ciências ômicas são particularmente úteis para a compreensão de mecanismos moleculares com alto valor translacional para a prática clínica e podem contribuir para a identificação desses alvos. OBJETIVO: O presente estudo avaliou a resposta proteômica hepática e lipidômica plasmática de pacientes com NASH perante o tratamento com AGPIs n-3. MÉTODO: As avaliações proteômicas e lipidômicas foram desenvolvidas por espectometria de massas e/ou cromatografia gasosa em amostras de biópsias hepáticas e plasma coletadas de pacientes envolvidos em estudo preliminar, realizado no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. O referido estudo envolveu pacientes adultos, de ambos os sexos e com diagnóstico de NASH tratados diariamente, durante 6 meses, com 3 cápsulas contendo mistura de óleo de linhaça e óleo de peixe [0,315 g AGPIs: sendo 0,065 g de ácido eicosapentaenoico (EPA), 0,050 g de docosahexaenoico (DHA) e 0,2 g alfa linolênico (ALA) por cápsula]. Pacientes, após o tratamento com AGPIs n-3, que apresentaram altas concentrações plasmáticas de ALA e/ou DHA e/ou baixas de ácido araquidônico (AA) mostraram melhora parcial das alterações de histologia hepática. No presente estudo, avaliamos as vias proteômicas e marcadores lipidômicos resultantes do tratamento com AGPIs n-3. Isto foi feito por meio da comparação, antes (grupo AT) e depois do tratamento (grupo DT), de pools de tecido hepático (análise por interactoma) e amostras de plasma (OPLS-DA). RESULTADOS: Foram identificadas proteínas hepáticas, exclusivamente e/ou alteradamente expressas, no grupo DT, relacionadas com vias de matriz celular, metabolismo lipídico, de estresse oxidativo, e de retículo endoplasmático e respiração celular. Com excessão da via de matriz celular, a análise do interactoma revelou alteração funcional significativa das vias moduladas por essas proteínas. Em conjunto, essas alterações foram sugestivas de diminuição de lipotoxicidade, estresse oxidativo e respiração anaeróbia, e aumento de respiração aeróbia após tratamento com AGPIs n-3. Estas modificações são marcadores potenciais de melhora de função de retículo endoplasmático e mitocondrial. Em adição, após o tratamento com AGPIs n-3, o perfil lipidômico plasmático mostrou-se alterado com significativo aumento de glicerofosfolípides, ALA e EPA, e diminuição de ácido araquidônico (n-6) e da razão AGPIs n-6/n-3. Estes dados são concordantes com potencial melhora das funções de retículo endoplasmático e mitocondriais. CONCLUSÃO: O tratamento com AGPIs n-3 em pacientes com NASH influenciou favoravelmente o perfil proteômico hepático e lipidômico sistêmico. Em conjunto, essas alterações sugerem melhora da função de retículo endoplasmático e mitocondrial, com potencial impacto na homeostase celular, por meio da modulação de diferentes vias biológicas / INTRODUCTION: Non-alcoholic steatohepatitis (NASH) is considered a public health problem, given its increasing incidence and its possible role in hepatocellular carcinogenesis. Current therapies involve diet and lifestyle changes, but its applicability suffers from low patients adherence. Pharmacological approaches are still missing. A main difficulty in the NASH management lies in the limited understanding of its pathophysiology, which seems to involve complex metabolic and inflammatory disturbances. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are recognized for its modulatory properties on lipid metabolism and inflammation and are decreased in patients with NASH. The clinical use of these PUFAs has shown benefit in controlling steatosis and the production of metabolic and inflammatory response markers in NASH, despite some conflicting reports. Understanding mechanisms modulated by n-3 PUFAs in NASH may be useful for identifying molecular targets that could assist in the design of effective pharmacologic interventions. In this sense, omics sciences are particularly useful for understanding molecular mechanisms with high translational value to clinical practice and may contribute to the identification of these targets. AIM: This study evaluated the liver proteomic and plasma lipidomics responses of patients with NASH towards treatment with n-3 PUFAs. METHODS: The proteomic and lipidomic evaluations were studied by mass spectrometry and / or gas chromatography in samples from liver biopsies and plasma collected from patients enrolled in a preliminary clinical trial of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. This study involved adult patients of both sexes diagnosed with NASH treated daily for 6 months, with 3 capsules containing a mixture of linseed and fish oils [0.315 g PUFAs: 0.065 g eicosapentaenoic acid (EPA) , 0.050 g docosahexaenoic (DHA) and 0.2 g alpha linolenic acid (ALA) per capsule]. Patients, after treatment with n-3 PUFAs, with higher concentrations of ALA and DHA and lower arachidonic acid (AA) showed improvement of liver histology alterations. In the present study we evaluated the proteomics pathways and lipidomics markers resulted from treatment with PUFAs n-3. This was performed by comparing, before (BT group) and after (AT group) treatment, liver tissue pools (analysis interactome) and plasma samples (OPLS-DA). RESULTS: It was identified, in a way exclusive and altered, the expressed liver proteins in AT group, related to pathways of cellular matrix, lipid metabolism, oxidative and endoplasmic reticulum stress and cellular respiration. With the exception of cell matrix, the analysis of the interactome revealed substantial functional alterations of the pathways modulated by these proteins. Together, these changes were suggestive of decreased lipotoxicity, oxidative stress and anaerobic respiration and increased aerobic respiration following treatment with PUFAs n-3. These modifications are potential markers of endoplasmic reticulum and mitochondrial functions improvement. In addition, after treatment with n-3 PUFAs, the lipidomics profile was modified, with significant increase in glycerophospholipids, ALA and EPA and decrease of arachidonic acid (AA) and n-6/n-3 AGPIs ratio. These findings are concordant with potential improvement of reticulum endoplasmic and mitochondrial functions. CONCLUSION: In patients with NASH the treatment with n-3 PUFAs favorably influenced hepatic proteomic and systemic lipidomics profiles. Together, these changes suggest improved endoplasmic reticulum and mitochondrial functions, with potential impact on cellular homeostasis through the modulation of different biological pathways