槲皮素对猪肠上皮细胞利用蛋白质的作用机制

本试验旨在研究槲皮素促进猪肠上皮细胞利用蛋白质的作用及机制。猪肠上皮细胞孵育48 h后试验组分别用含0.1、0.2、0.4、0.8和1.6 mg/L槲皮素的二甲基亚砜(DMSO)溶液处理72 h,对照组采用0.2%DMSO处理。采用二喹啉甲酸(BCA)测定受试细胞中蛋白质的含量;采用实时荧光定量PCR(RT-qPCR)法测定氨基酸和小肽转运载体以及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关基因的mRNA相对表达量;采用Western blot法测定mTOR信号通路相关基因的蛋白表达。结果表明:与对照组相比,1)0.4和0.8 mg/L槲皮素均极显著增加猪肠上皮细胞中蛋白质的含量(P<0.01)。2)1.6 mg/L槲皮素极显著提高猪肠上皮细胞中兴奋性氨基酸转运载体1(EAAC1)、谷氨酰胺载体2(ASCT2)、氨基酸转运载体A2(ATA2)、L型氨基酸转运载体2(LAT2)、阳离子氨基酸转运载体1(CAT1)、b 0,+系统氨基酸转运载体(rBAT)、y+L系统氨基酸转运载体1(y+LAT1)、y+L系统氨基酸转运载体2(y+LAT2)和寡肽转运载体1(PepT1)mRNA相对表达量(P<0.01)。3)0.4 mg/L槲皮素极显著降低猪肠上皮细胞中结节性硬化复合物1(TSC1)mRNA相对表达量(P<0.01);0.8 mg/L槲皮素极显著增加mTOR和核糖体蛋白S6(RPS6)mRNA相对表达量并极显著降低TSC1 mRNA相对表达量(P<0.01);1.6 mg/L槲皮素极显著增加mTOR、真核起始因子4E结合蛋白1(4E-BP1)、真核细胞翻译起始因子4E(eIF4E)、真核细胞翻译起始因子4B(eIF4B)、真核细胞翻译起始因子4A(eIF4A)和RPS6 mRNA相对表达量(P<0.01)。4)0.1和1.6 mg/L槲皮素极显著提高猪肠上皮细胞中mTOR、eIF4E和eIF4A蛋白表达量并极显著降低4E-BP1蛋白表达量(P<0.01)。由此可见,槲皮素可通过调控氨基酸转运载体、小肽转运载体及mTOR信号通路相关基因的表达来促进猪肠上皮细胞对蛋白质的利用。

Mechanism of Quercetin on Protein Availability in Porcine Intestinal Epithelial Cells

This experiment was conducted to investigate the mechanism of quercetin promoting protein availability in porcine intestinal epithelial cells.The cells in experimental groups were treated with dimethyl sulfoxide(DMSO)solution containing quercetin at different supplemental levels of 0.1,0.2,0.4,0.8 and 1.6 mg/L for 72 h after incubated for 48 h,and the cells in control group were treated with 0.2%DMSO solution.Thereafter,the protein content in cells was determined by bicinchoninic acid(BCA)method.The effects of quercetin on mRNA relative expression levels of mammalian target of rapamycin(mTOR)signaling pathway,amino acids and small peptide transporters using RT-qPCR.The effects of quercetin on protein expression levels of genes related to mTOR signaling pathway using Western blot.The results showed as follows:compared with control group,1)the content of protein in porcine intestinal epithelial cells was significantly increased by adding 0.4 and 0.8 mg/L quercetin for 72 h(P<0.01);2)1.6 mg/L quercetin significantly increased mRNA relative expression levels of excitatory amino acid carrier 1(EAAC1),alanine-serine-cysteine transporter 2(ASCT2),amino acid transporter A2(ATA2),L-type amino acid transporter 2(LAT2),cation amino acid transporter 1(CAT1),related to b 0,+amino acid transporter(rBAT),y+L amino acid transporter 1(y+LAT1),y+L amino acid transporter 2(y+LAT2)and oligopeptide transporter 1(PepT1)in porcine intestinal epithelial cells(P<0.01);3)0.4 mg/L quercetin significantly decreased mRNA relative expression level of tuberous sclerosis complex 1(TSC1)in porcine intestinal epithelial cells(P<0.01);0.8 mg/L quercetin significantly increased mRNA relative expression levels of mTOR and ribosomal protein S6(RPS6)(P<0.01),whereas significantly decreased that of TSC1(P<0.01);1.6 mg/L quercetin significantly increased mRNA relative expression levels mTOR,eukaryotic initiation factor 4E-binding protein 1(4E-BP1),eukaryotic translation initiation factor 4E(eIF4E),eukaryotic translation initiation factor 4B(eIF4B),eukaryotic translation initiation factor 4A(eIF4A)and ribosomal protein S6(RPS6)(P<0.01);4)0.1 and 1.6 mg/L quercetin significantly increased protein expression levels of mTOR,eIF4E and eIF4A in porcine intestinal epithelial cells(P<0.01),whereas significantly decreased that of 4E-BP1(P<0.01).In conclusion,quercetin at appropriate dose increase the utilization of protein in porcine intestinal epithelial cells by regulating gene expression of amino acid transporters,small peptide transporters and genes related to mTOR signaling pathway.