Effect Of Iron On Manganese Absorption And The Mechanisms In Caco-2 Cells And Broilers

Two experiments were conducted to study the effect of iron(Fe) on manganese(Mn) absorption and the possible mechanisms in Caco-2 cells and broilers.The first experiment included 2 trials.Trial 1 was conducted to investigate the effect of cell density on differentiation and depolarization of Caco-2 cell monolayers and determine the optimal time for absorption and transport experiment. In conventional culture condition, the Caco-2 cells were inoculated at a density of 1×105(high, H), 5×104(medium, M) or 3×104(low, L) cells/cm2 on six-well transwell plates and cultured for 29 days. The transepithelial electrical resistance(TEER), alkaline phosphatase activities of apical and basolateral side, and diffusion of phenol red were measured to evaluate the integrity and permeability of cell monolayers. The results showed that TEER value exceeded 600 Ω/cm2 at d 12, 15, and 18, and decreased at d 27, 29, and 29 when the cells were inoculated at a density of H, M or L, respectively. The ratios of alkaline phosphatase activities of apical to basolateral side were more than 10 at d 15, 18, and 21 when the cells were inoculated at a density of H, M or L, respectively. The diffusions of phenol red from the apical to basolateral side were lower than 0.5% at d 9, 12 and 15 when the cells were inoculated at a density of H, M or L, respectively. The results suggested that the integrity and permeability of cell monolayers were satisfactory for absorption and transport experiment from d 15, 18 and 21 to 27 when the cells were inoculated at a density of H, M or L, respectively.Trial 2 was conducted to investigate the effect of Fe on Mn absorption and the mechanism in caco-2 cell monolayers. There were 5 groups in this trial as normal serum-containing medium(control), serum-containing medium supplemented with 100 or 200μmol/L of Fe, serum-free medium supplemented with 100 or 200μmol/L of Fe. Each group contained 6 replicates. Iron was supplemented as Fe(NTA)4 into the media from d 18 to 23 after seeding, and then all groups were incubated with 800μmol/L of Mn at 37℃, 50r/min for 120 min. Cells, solutions in the upper and lower chambers were collected for the analyses of Mn, Fe and expressions of divalent metaltransporter1(dmt1),ferroportin1(fpn1)andduodenalcytochromeb(dcytb)gene.theresultsshowedthat,comparedwiththecontrol,thefecontentsinserum-freefe-supplementedgroupswereincreased(p<0.09),butthefecontentsinserum-containingfe-supplementedgroupswerenotaffected(p>0.31).theuptake(p<0.03)andabsorption(p<0.02)ofmnwerelowerinserum-freefe-supplementedgroupsandserum-containingfe-supplementedwith200μmol/lgroupwhencellsweretreatedwithfefor120 h.themrnalevelsofdmt1(p<0.01)andfpn1(p<0.05)inserum-freefe-supplementedgroupsweredecreasedwhencellsweretreatedwithfeforeither72or120 h.themrnalevelsofdcytbinserum-freefe-supplementedgroupswerenotaffected(p>0.14).however,themrnalevelsofdmt1(p>0.38)andfpn1(p>0.43)inserum-containingfe-supplementedgroupswerenotaffectedwhencellsweretreatedwithfefor72 h.whencellsweretreatedwithserum-containingfe-supplementedfor120 h,themrnalevelsofdcytbweredecreased,aswellasthemrnalevelsofdmt1(p<0.01)inserum-containingfe-supplementedwith200μmol/lgroup.thefpn1proteinlevelswerenotaffected(p>0.89)inallfe-supplementedgroupsateither72or120 h.thedmt1proteinlevelsinserum-freefe-supplementedgroupsweredecreased(p<0.08),butthedmt1proteinlevelsinserum-containingfe-supplementedgroupswerenotaffected(p>0.44).theresultssuggestedthatcellularfedecreasetheuptakeandabsortionofmnthroughthedown-regulationsofdmt1andfpn1geneexpressionincaco-2cells.thesecondexperimentwasconductedtostudytheeffectofdietaryfeontheabsorptionofmnandthemechanismsinbroilers.atotalof3361-dross-308malechicksweredividedinto4groupswith6replicatesand14 chickseachreplicatecages,andfedthebasaldiet(control,addedfe0mg/kg,containingfe77.7mg/kg)orbasaldietsupplementedwith100,250,or500mgfe/kgfor28 days.blood,liver,heart,pancreas,duodenalmucosaandlefttibiawerecollectedfortheanalysesofmineralcontent,andthemrnalevelsofdmt1andfpn1induodenalmucosaond7,14,21,and28,respectively.theligatedduodenalsegmentsinthegroupsofdietsadded-fewith100and500mg/kgwereperfusedwithbuffercontaining8.74mmolmn/l.theresultsshowedthatdietaryfeleveldidnotaffect(p>0.16)growthperformanceexceptadg(p<0.10)duringd1~14andadfi(p<0.03)duringd1~7indietadded-fewith500mg/kggroup.plasmatotalironbindingcapacity,hemoglobinconcentration,andhematocritwerenotaffect(p>0.16)bydietaryfelevel.plasmafecontentandtransferrinsaturationincreased(p<0.01)asdietaryfelevelincreased.ironcontensinheartatd7and14,liver,duodenum,pancreasandtibiaashinadded-fegroupsincreased(p<0.02)exceptforheartfecontents(p>0.23)atd21and28.themncontentsofliverandheartwerenotaffected(p>0.13)bydietaryfelevel,however,mncontentsofduodenum,pancreasandtibiaashinadded-fegroupsdecreased(p<0.01)asdietaryfelevelincreased.zinccontentsinpancreasatd8(p<0.01)weredecreasedbydietaryaddedfe.contentsofznandcuintheanalyzedorganswerenotaffected(p>0.13).themrnalevelsofdmt1andfpn1induodenumdecreased(p<0.10)asdietaryfelevelincreased,respectively.themnabsorptioninligatedduodenumfromadded-fe500mg/kggroupwaslower(p<0.04)thanthatfromadded-fe100mg/kggroup.theresultssuggestedthatdietaryfedecreasetheabsorptionofmnthroughthedown-regulationsofdmt1andfpn1 geneexpressioninbroilers.insummary,theresultsfrominvitroandinvivoexperimentsshowedthatthemnabsorptionsweredecreasedbyfethroughthedown-regulationsofdmt1andfpn1 geneexpression,whichprovidedanovelinsightintotheinteractionbetweenfeandmn.