Study On The Mechanism Of AAAOPs’ Absorption And Induction Of Oxidative Stress In Infant Formula Milk Powder Based On Caco-2 Cell Model

Infant formula is the best substitute for breast milk and contains essential nutrients for the growth and development of infants and young children.However,heat treatment and other factors during production and processing can lead to oxidative modification of proteins in infant formula,which can affect the functional structure and nutritional value of proteins.It has been shown that the aromatic amino acid oxidation products(AAAOPs)in infant formulae(especially protein hydrolyzed formulae)are high and can be partially transported and absorbed by Caco-2 monolayer cell membranes,but the mechanism of absorption and transport is not clear.At the same time,dietary intake of oxidized proteins and amino acids can reduce the antioxidant defense capacity of the body,exacerbate oxidative stress in the body,and endanger the health of the body,but the potential molecular mechanism of oxidative stress damage induced by AAAOPs in cells has not been elucidated.Therefore,in this study,we analyzed the oxidation level of proteins and the content of AAAOPs in two types of milk powder(4 types each):regular infant formula and protein-hydrolyzed infant formula,mainly including meta-Tyrosine(m-Tyr),Kynurenine(Kyn),3-hydroxykynurenine(3-hydroxylkynurenine),and 3-Tyr.Hydroxykynurenine(3OHKyn)and di-Tyrosine(di-Tyr).We also explored the uptake and transport mechanism of AAAOPs using Caco-2 monolayer cell membrane model;on this basis,we investigated the molecular mechanism of oxidative stress damage induced by AAAOPs in Caco-2 cells.The main research contents and results are as follows:(1)The average content of protein carbonyl group in protein hydrolyzed milk powder(2.76 nmol/mg protein)was higher than that in normal infant formula(2.40 nmol/mg protein),and the average content of sulfhydryl group(10.34 nmol/mg protein)was lower than that in normal infant formula(13.38 nmol/mg protein),and the protein The average content of3OHKyn and di-Tyr in protein-hydrolyzed milk powder was significantly higher than that in normal infant formula(p<0.05),while the average content of m-Tyr was not significantly different(p>0.05)and Kyn was not detected(below the detection limit of 10 nmol/L by LC/MS).-PAGE protein staining experiments showed that the molecular weight of proteins in infant formula was concentrated in the range of 13 k Da～70 k Da,and the molecular weight of proteins in hydrolyzed formula was<10 k Da,and there was protein oxidative aggregation caused by disulfide bond cross-linking in infant formula.(2)The transmembrane electrical resistance(TEER)of Caco-2 cell monolayers reached about 400Ω·cm~2 at 21 d,and the alkaline phosphatase activity on the apical side(AP)was2.83 times higher than that on the basolateral side(BL)(p<0.05),indicating that Caco-2 cells had The results of the uptake assay showed that the m-AP was more than 2.83 times more active than the basolateral side(BL).The results of the uptake assay showed that the uptake rates of m-Tyr and Kyn in the Caco-2 cell model were 1.01±0.20%and 0.81±0.05%,respectively,and there was no significant difference in the uptake rates of the two substances(p<0.05);the uptake rates of 3OHkyn and di-Tyr(3.35±0.33%and 3.09±0.44%,respectively)were significantly higher than that of m-Tyr and Kyn(p<0.05).In combination with Caco-2cell uptake and transport pathway related promoters and inhibitors,it was found that m-Tyr,Kyn and 3OHKyn were mainly taken up and transported through the amino acid transport carrier pathway,while di-Tyr was taken up and transported by Caco-2 cells depending on Pep T1 carrier protein.(3)When Caco-2 cells were exposed to AAAOPs for 24 h,cellular metabolic activity was inhibited,while cellular metabolism and proliferation were stimulated to some extent after 48h incubation(1μmol/L和10μmol/L);AAAOPs induced an increase in intracellular reactive oxygen species(ROS)content in a concentration-dependent manner;AAAOPs inhibited intracellular superoxide AAAOPs inhibited the expression of superoxide dismutase(SOD),catalase(CAT),and reduced glutathione(GSH),resulting in an increase in malondialdehyde(MDA)and lactate dehydrogenase(LDH)release.The RT-PCR results showed that incubation of Caco-2 cells with AAAOPs(except di-Tyr)for 48 h resulted in differential down-regulation of Nrf2 gene expression,and also caused differential increases in the expression levels of the downstream related antioxidant enzymes heme oxygenase 1(HO-1),glutamate cysteine ligase(GCLM)and NADP(H)quinone oxidoreductase 1(NQO1).The Western-blot experiment showed that the expression levels of Nrf2 protein in the AAAOPs-treated group decreased compared with the control group,which was consistent with the RT-PCR results,however,the expression levels of the related antioxidant enzymes HO-1,NQO1and GCLM differed from the RT-PCR results.