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Vključevanje hidrolizirajočih taninov iz kostanja (Castanea satiava Mill.) kot dodatek v vodi za pitje ali v krmi lahko predstavlja alternativo uporabi nutritivnih antibiotikov. Uporaba živalskega celičnega modela predstavlja primerno orodje za določitev optimalnih koncentracij fitogenih snovi, ki se v prehrani živali lahko pojavljajo v vodotopni ali prašnati obliki. Absorpcija glukoze in transport skozi celice epitelija črevesja sta regulirana z glukoznimi prenašalci, ki pomembno vplivajo na razpoložljivo koncentracijo glukoze in posledično na oskrbljenost organizma z glukozo, na katero lahko imajo tanini pomemben učinek. Za ta namen smo v prvi fazi raziskave razvili in karakerizirali piščančji epitelijski celični model. Zatem smo na njem analizirali učinkovitost vodotopne oblike ekstrakta v različnih koncentracijah (0,025 %, 0,05 %, 0,1 %, in 0,2 %) in določili citotoksičnost, celično proliferacijo, presnovno aktivnost, nastajanje reaktivnih kisikovih snovi, znotrajcelični antioksidativni potencial, genotoksičnost in vpliv na celični cikel. V drugi fazi smo na že razvitem celičnem modelu prašiča (CLAB in PSI) testirali učinkovitost prašnatih oblik kostanjevega ekstrakta na gensko izraženost glukoznih prenašalcev (SGLT1 in GLUT2) in glukoznih prenašalcev tipa 4 (GLUT4) kot tudi biodostopnost glukoze in biodostopnost hidrolizabilnih taninov. V tretji fazi poskusa smo na piščancih v laboratorijskih pogojih z analizo proizvodnih parametrov preverili učinkovitost optimalnih koncentracij HT, ugotovljenih na piščančjem epitelijskem modelu in vivo. Koncentracije, uporabljene v testiranju, so pokazale značilno (P < 0,05) večji proliferativni učinek na CSIEC kot kontrolni medij (največja proliferacija pri 0,1 % VOTE, določeno z meritvami optične gostote). 0,2 % koncentracija VOTE je bila citotoksična, kar je povzročilo značilno večjo (P < 0,05) produkcijo dušikovega oksida in vodikovega peroksida, vendar brez kratkotrajne genotoksičnosti. Čeprav je povečana koncentracija VOTE povzročila upad presnovne aktivnosti celic (najnižja pri 0,1 % VOTE), je presnovna aktivnost ostala višja kot v kontrolnih pogojih. Antioksidativni potencial je bil 75 % boljši in značilno višji (P < 0,05) v izpostavljenih celicah z 0,1 % VOTE v primerjavi s kontrolo. Dodajanje ekstraktov, bogatih s HT, je značilno (P < 0,05) vplivalo na izražanje hišnih genov v celicah CLAB. Ugotovili smo, da dodajanje ekstraktov, bogatih s HT (Farmatan in Contan), v visokih koncentracijah (37 µg/mL) značilno (P < 0,05) vpliva na manjše izražanje hišnih genov RNK polimeraze II (POLR2A) v celicah CLAB. Poleg tega so pri koncentraciji 4 µg/mL različni HT ekstrakti povečali izraženost SGLT1. Koncentracija dodatka 4 µg/mL HT ekstrakta Tanex je značilno (P < 0,05) povečala izražanje GLUT2. Različni HT ekstrakti v koncentracijah 1 ali 4 µg/mL vplivajo na izražanje GLUT4. Analize biološke razpoložljivosti HT kažejo na značilno (P < 0,05) translokacijo galne kisline skozi 3D celični model v prvi uri po izpostavljenosti celic VOTE. Naši rezultati kažejo, da v vodi topni kostanjevi tanini ugodno vplivajo na črevesni epitelij, saj: i) spodbujajo proliferacijo enterocitov ii) povečajo antioksidativni potencial iii) nimajo genotoksičnega učinka in iv) ne vplivajo na celično presnovo. Dejstvo, da različni HT ekstrakti v koncentracijah 1 ali 4 µg/mL značilno (P < 0,05) povečajo izraženost SGLT1, GLUT2 in GLUT4 in imajo zato verjetno koristne prehranske učinke s povečanjem koncentracije glukoze v krvi v živali. Naši rezultati potrjujejo učinkovitost VOTE kot obetavnega kandidata za prehranski dodatek v komercialni proizvodnji. The cell model was used as a suitable tool to determine the optimum concentrations of hydrolyzable tannin-rich plant extracts that may occur in water-soluble or dusty forms in animal nutrition. Feed and water incorporated powdered hydrolysable tannins from chestnuts, representing a valuable alternative strategy to antibiotics in animal nutrition. Glucose uptake and transportation across intestinal epithelial cells is mediated by glucose transporters and significantly affects postprandial glucose levels and consequently the energy supplies in an organism. For this purpose, in the first phase of the study, a chicken epithelial cell model was developed, where the expression of epithelial features was first checked. The efficacy of the water-soluble form of the extract at various concentrations (0.025%, 0.05%, 0.1%, and 0.2%) was analysed and determined for cytotoxicity, cell proliferation, metabolic activity, the production of reactive oxygen species, intracellular antioxidative potential, genotoxicity, and their influences on the cell cycle. In the second phase, the efficacy of chestnut extract powders on the gene expression of sodium glucose transporters (SGLT1 and GLUT2) and glucose transporter type 4 (GLUT4) was tested, as well as the bioavailability of glucose and wood extracts constituents itself on a previously developed pig model (CLAB and PSI). In the third phase of the experiment, the performance of the optimal concentrations determined on the chicken epithelial model was tested in chickens under laboratory conditions by means of a productivity analysis. The tested concentrations showed a significantly (P < 0.05) greater proliferative effect on CSIEC than the control medium (maximum proliferation at 0.1% WSTE as determined by optical density measurements). The 0.2% concentration of WST was cytotoxic, causing a significantly higher (P < 0.05) nitric oxide and hydrogen peroxide production but with no short-term genotoxicity. Although increasing concentrations caused a decrease in the metabolism of the challenged cells (lowest at 0.1% WST), the metabolic activity remained higher than that in control cells. The antioxidant potential was 75% better and significantly (P < 0.05) higher in the 0.1% WST cultured cells compared to the control. The addition of wood extracts had a notable effect on the expression of target genes in CLAB cells. Here, the demonstration that the addition of HT-rich extracts of Farmatan and Contan at high concentrations (37 µg/mL) significantly (P < 0.05) downregulated the expression of the housekeeping gene RNA polymerase II (POLR2A) in CLAB cells, with a median Ct values of 3.72 and 1.46, respectively has been shown. Additionally, at the concentration of 4 µg/mL, different HT extracts upregulated the expression of SGLT1. The treatment with 4 µg/mL HT Tanex significantly (P < 0.05) upregulated GLUT2 expression. Different HT extracts at concentrations of 1 or 4 µg/mL upregulated the expression of GLUT4. The HT bioavailability analyses indicate significant (P < 0.05) gallic acid translocation through the 3D cell model within the first hour after compound exposure. In conclusion, cultured CSIECs are a useful tool in basic and clinical research for the study of intestinal physiology, as they retain physiological and biochemical properties and epithelial morphology close to tissue origin and in many ways reflect the in vivo state. Our results indicate that water-soluble chestnut tannins have a beneficial effect on intestinal epithelia, as they: i) stimulate proliferation of enterocytes ii) increasing antioxidative potential iii) have no genotoxic effect and iv) do not affect cellular metabolism. The fact that different HT extracts at concentrations of 1 or 4 µg/mL significantly (P < 0.05) upregulated the expression of SGLT1, GLUT2 and GLUT4 indicate that HT may act as SGLT1 and GLUT2 upregulators and thus may have beneficial nutritional effects by increasing the blood glucose supply in the animal. Our r |
