@misc{Zając-Grabiec_Anna_Absorption_2022, author={Zając-Grabiec, Anna}, address={Kraków}, howpublished={online}, year={2022}, school={Wydział Farmaceutyczny}, language={pol; eng}, abstract={Inflammation is the defense response of tissues to damage. Acute inflammation can take a chronic form and result in many diseases. Research on inflammatory mediators has identified the anti-inflammatory potential of polyunsaturated fatty acids (PUFAs) and their derivatives. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) belong to the n-3 PUFAs. They are precursors of anti-inflammatory and pro-resolving lipid mediators. Supplementation with EPA or DHA may reduce the risk of chronic inflammatory diseases. Changing the composition of the phospholipids, the organization of membrane microdomains, metabolism of lipids, and modifications of signaling pathways in pathological states are becoming new pharmacological targets. Adipose tissue is one of the leading centers of energy metabolism regulation. Fatty acid receptors play an essential role in glucose metabolism. They regulate both glucose uptake in adipose tissue by influencing the expression of GLUT-4 glucose transporters, insulin receptor expression, and the adipogenesis process. Among others, adipogenesis disorders caused by inflammation in the adipose tissue led to insulin resistance, lipotoxicity, and steatosis. Factors influencing the development of obesity also include changes in the composition of the microflora, increased levels of lipopolysaccharide (LPS), leading to an increase in intestinal permeability and}, abstract={chronic, low-grade inflammation, and an increase in energy intake while reducing energy expenditure. Therefore, this dissertation used the in vitro model of adipocytes (differentiated 3T3-L1 cells) and enterocytes (Caco-2 cells). Fully differentiated 3T3-L1 adipocytes have the most morphological and biochemical features typical of adipose tissue cells in vivo. Due to its morphological and functional analogy, the Caco-2 lineage is considered the in vivo counterpart of enterocytes of the small intestine. Therefore, it is a suitable model for bioavailability studies. This study aimed to determine the effect of n-3 fatty acids, EPA, and DHA on 3T3-L1 fibroblasts and Caco-2 intestinal epithelial cells, activated by inflammatory factors, and thus targeted programming of fatty acid metabolism in inflammatory conditions. No cytotoxic effects or apoptosis was observed in 3T3-L1 cells before and after differentiation into adipocytes incubated with EPA or DHA, metformin (MET), and lovastatin (LOVA) in Caco-2 cells after supplementation with EPA and DHA and activation with inflammatory factors. Murine embryonic 3T3-L1 fibroblasts were differentiated into adipocytes. Cells were incubated with 50 μmol EPA for 48 hours and then activated with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). Cycloxygenase-2 (prostaglandin E2 synthase, Ptgs2, COX-2), cytosolic prostaglandin E2 synt}, abstract={hase (cPGES), fatty acid-binding protein 4 (FABP4), toll-like receptor 4 (TLR4), glucose type 4 receptor (GLUT-4) and the cannabinoid2 receptor (CB2) were determined by Western blotting. Gene expression of phospholipase A2 (Pla2g4a) and prostaglandin E2 synthase (Ptgs2) was analyzed by real-time qPCR. After the activation of EPA and IF, a significant decrease in COX-2, cPGES, and TLR4 proteins were observed. Incubation of cells with EPA and IF resulted in repression of Ptgs2 and increased expression of the Pla2g4a gene. A significant increase in CB2 protein in adipocytes incubated simultaneously with EPA and IF was noted. The obtained results indicate the anti-inflammatory properties of EPA. The activation of the GLUT4 receptor by EPA suggests a unique role for this fatty acid in regulating adipocyte metabolism and preventing insulin resistance. In another experiment, pre-adipocytes and adipocytes were supplemented with 40 μmol EPA, 40 μmol DHA, and 1 nmol of Resolvin D1 (RvD1) and activated with 1 μmol of benzo (a) pyrene (BaP). Identification and quantification of the isoprostanes 8-iso-prostaglandin F2α (8-isoPGF2α), 8-isoPGF3α, prostaglandin F2α (PGF2α), and PGF3α were performed using ultra-high-performance liquid chromatography UHPLC UltiMate 3000 RS system coupled with a mass spectroscope and a transfer time analyzer. The highest amounts of 8-isoPF2α, 8-isoPGF3α, and PGF}, abstract={2α were detected in BaP-activated cells. The addition of EPA, DHA, and RvD1 to 3T3-L1 cells, both before and after differentiation, significantly reduced the number of isoprostanes, which indicates the anti-inflammatory and antioxidant properties of n-3 fatty acids and their derivatives. The study confirming the properties of fatty acids was also described in another experiment, where adipocytes were incubated with 25 μmol of EPA or DHA and 10 μmol of metformin (MET), and 2.5 μmol of lovastatin (LOVA). 3T3-L1 cells were activated with tumor necrosis factor TNF-α. Next, the levels of cyclooxygenase 2 (COX-2) and glucose transporter (GLUT-4) proteins were examined by the Western Blot technique. The highest level of COX-2 was demonstrated in TNF-α activated adipocytes, while in 3T3-L1 cells, the level of GLUT-4 protein was the lowest after activation with the inflammatory factor. The addition of metformin significantly increased the level of GLUT-4. Incubation of cells with fatty acids and LOVA also increased GLUT4 levels, indicating the tested compounds' synergistic role in regulating adipocyte metabolism and preventing diabetes. In adipocytes incubated with TNF-α, the Ptgs2 and Pla2g4a gene was overexpressed, while for the Pparg gene, a statistically significant reduction was noted. Pparg overexpression was observed in cells incubated with MET, LOVA, EPA, and DHA. The obtaine}, abstract={d results suggest that EPA and DHA have anti-inflammatory and anti-inflammatory properties. Caco-2 human intestinal epithelial cells were cultured in parallel. In Caco-2 cells supplemented with EPA and activated with LPS, the level of pro-inflammatory proteins was significantly decreased, suggesting the anti-inflammatory properties of eicosapentaenoic acid. Supplementation of cells with 10 μmol EPA and 25 μmol EPA despite LPS activation resulted in a significant reduction of COX-2, cPGES, and AHR levels. After LPS activation, the highest concentration of IL-6 was recorded compared to the control. The addition of EPA significantly lowered the level of IL-6 in Caco-2 cells. The highest level of cyclooxygenase 2, cPGES, and FP receptor was observed in enterocytes incubated with toxin A. Supplementation with docosahexaenoic acid at 10 μmol, and 50 μmol significantly lowered the level of the proteins mentioned above, despite the presence of the inflammatory factor. A lower gene expression of cyclooxygenase isoforms was demonstrated in Caco-2 cells activated with toxin A and incubated with DHA. A significant increase in the expression of phospholipase A2 was also shown in the DHA + TOXA groups, compared to the results obtained for TOXA alone. The above results may indicate the reduction of toxin A-induced inflammation in enterocytes by docosahexaenoic acid. Experimental results demon}, abstract={strate the anti-inflammatory and antioxidant properties of n-3 fatty acids. They may contribute to developing new pharmacological strategies for treating obesity, insulin resistance, and intestinal inflammation. Fatty acids and their metabolites as ligands for PPAR transcription factors can be used as nutraceuticals to regulate the immune response.}, title={Absorption and metabolism of n-3 fatty acid in inflammation in vitro models}, type={Praca doktorska}, keywords={adipocytes, enterocytes, n-3 fatty acids, inflammation, pro-inflammatory genes and proteins expression}, }