| To investigate the effect of quercetin on protein availability in porcine intestinal epithelial cells,experiments were conducted by adding 0.2%dimethyl sulphoxide and different concentrations of quercetin in cells;cell proliferation,protein content,mRNA expression of amino acid transporters and small peptide transporter(PepT1),mammalian target of rapamycin signaling pathway were determined via MTT,CCK8,BCA,qRT-PCR and Western blot.Results are as follows:(1)Cell proliferation could be promoted by 0.001~1 mg/L quercetin compared to control,especially 1 mg/L.On this basis,the half-effective concentration of quercetin was 0.50,1.10,2.08,3.52,4.17 and 5.43 mg/L during 24~144 h,respectively.(2)There was no significant difference in protein content of quercetin treatments(P>0.05)at 24 h;protein content of 0.4,0.8,1.6 mg/L quercetin group was increased significantly(P<0.05)at 48 h;protein content of 0.4 mg/L quercetin treatment was increased significantly(P<0.05)at72h using on BCA method.(3)The expression of intracellular transporters was detected by RT-PCR.Compared with control,1.6 mg/L quercetin significantly increased mRNA expression of ASCT2,ATA2,rBAT,PepT1 at 24 h(P<0.01);significantly increased mRNA expression of rBAT,y~+LAT1,y~+LAT2(P<0.01)and PepT1(P<0.05)at 48 h;significantly increased mRNA expression of EAAC1,ASCT2,ATA2,LAT2,CAT-1,rBAT,y~+LAT1 transporters(P<0.01)and PepT1 at 72 h(P<0.05).Results indicated that 1.6 mg/L quercetin significantly promoted expression of amino acid transporters and PepT1.(4)Detection of intracellular signaling pathways revealed by fluorescence quantitative analysis when compared with control 1.6 mg/L quercetin significantly increased mRNA expression of tuberous sclerosis complex 1(P<0.05),significantly increased mRNA expression of mammalian target of rapamycin(mTOR),eukaryotic initiation factor 4E-binding protein 1(4E-BP1),eukaryotic initiation factor 4B(eIF4B),eukaryotic initiation factor 4A(eIF4A),ribosomal protein S6(RPS6)at 24 h(P<0.01),respectively;significantly increased mRNA expression of eIF4E(P<0.05),4E-BP1 and ribisomal S6 kinase 1(P<0.01)at 48 h;significantly increased mRNA expression of mTOR,4E-BP1,eukaryotic initiation factor 4E(eIF4E),eIF4B,eIF4A and RPS6(P<0.01)at 72 h.(5)Compared with control,1.6 mg/L quercetin significantly increased protein expression of mTOR,eIF4A,eIF4E,and significantly decreased protein expression of 4E-BP1(P<0.01),as detected using Western blot.In conclusion,1.6 mg/L quercetin promoted proliferation of porcine intestinal epithelial cells and increased intracellular protein content.The specific effect was accomplished probably via promoting expression of amino acid and small peptide transporters,and activating mTOR signaling pathway. |
