哺乳动物雷帕霉素靶蛋白信号通路调控猪肠上皮细胞精氨酸转运的机制  

Regulatory Mechanism of Mammalian Target of Rapamycin Signaling Pathway on Arginine Transport in Porcine Intestinal Epithelial Cells

作　　者：肖昊 赵一[1] 王丽 谭碧娥 XIAO Hao;ZHAO Yi;WANG Li;TAN Bi’e(State Key Laboratory of Livestock and Poultry Breeding,Key Laboratory of Animal Nutrition and Feed Science in South China Ministry of Agriculture,Maoming Branch,Guangdong Laboratory for Lingnan Modern Agriculture,Guangdong Key Laboratory of Animal Breeding and Nutrition,Institute of Animal Science,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;Observation and Experiment Station of Animal Nutrition and Feed Science in South-Central China,Ministry of Agriculture,Hunan Provincial Engineering Research Center for Healthy Livestock and Poultry Production,Key Laboratory of Agro-Ecological Processes in Subtropical Region,Institute of Subtropical Agriculture,Chinese Academy of Sciences,Changsha 410125,China)

机构地区：[1]广东省农业科学院动物科学研究所,农业部华南动物营养与饲料重点实验室,畜禽育种国家重点实验室,岭南现代农业科学与技术广东省实验室茂名分中心,广东省畜禽育种与营养研究重点实验室,广州510640 [2]中国科学院亚热带农业生态研究所,中国科学院亚热带农业生态过程重点实验室,湖南省动物营养生理与代谢过程湖南省重点实验室,长沙410125

出　　处：《动物营养学报》2021年第5期2896-2906,共11页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金(31672433);科技创新战略专项资金(高水平农科院建设)——优秀博士(R2017YJ-YB1004)及青年副研究员(R2018PY-QF001);国家生猪产业技术体系建设专项(CARS-35);广东省现代农业产业技术体系生猪创新团队项目(2019KJ126)。

摘　　要：本研究旨在基于哺乳动物雷帕霉素靶蛋白(mTOR)信号通路探讨猪肠上皮细胞增殖和精氨酸(Arg)转运的调控机制。在含100或350μmol/L Arg的培养基中添加(10 nmol/L)或不添加(0 nmol/L)雷帕霉素(Rap),培养猪肠上皮细胞(IPEC-J2细胞)3 d后,对细胞活力、细胞周期以及Arg转运、增殖和凋亡相关通路基因和蛋白表达进行检测。结果表明:1)添加Rap极显著降低了G2期和S期细胞数量(P<0.01),而提高Arg浓度有效缓解了Rap对细胞增殖的抑制作用;Rap通过激活磷脂酰肌醇-3-羟激酶(PI3K)-蛋白激酶(Akt)-B细胞淋巴瘤2(Bcl2)信号通路抑制细胞凋亡。2)添加Rap抑制mTOR信号通路后极显著提高了Arg摄取率(P<0.01),极显著提高了100μmol/L Arg培养下细胞中阳离子氨基酸转运载体2(CAT2)的mRNA和蛋白表达量(P<0.01);进一步试验证明蛋白激酶Cα(PKCα)-细胞外信号调节激酶(Erk)/cFos-CAT2信号通路可能是Rap促进CAT2表达,进而提高Arg摄取的重要通路。综上可知,Rap应激下猪肠上皮细胞增殖被抑制,提高Arg浓度能有效缓解Rap对细胞增殖的抑制作用;Rap通过调控PKCα-Erk/cFos-CAT2信号通路促进猪肠上皮细胞对Arg的摄取,且提高Arg浓度可促进细胞对Arg的摄取。结果提示,mTOR信号通路在调控猪肠上皮细胞Arg利用过程中发挥重要作用。The purpose of this study was to investigate the mechanism of mammalian target of rapamycin(mTOR)signaling pathway regulating the proliferation of porcine intestinal epithelial cells and arginine(Arg)transport.Porcine intestinal epithelial cells(IPEC-J2 cells)were cultured in Dulbecco’s modified Eagle’shigh glucose Ham(DMEM-H)medium containing 100 or 350μmol/L Arg,0 or 10 nmol/L rapamycin(Rap)for 3 days,and then collected for the determination of cell proliferation,cell cycle,and the expression of related genes and protein of Arg transport,proliferation and apoptosis pathways.The results showed as follows:1)inhibition of mTOR signaling pathway by adding Rap could significantly reduce the cell number of G2-phase and S-phase(P<0.01),while elevating the Arg concentration could alleviate the inhibitory effect of Rap on cell proliferation.Rap reduced the cell apoptosis by activating the phosphatidylinositol-3-hydroxykinase(PI3K)-protein kinase(Akt)-B-cell lymphoma/leukemia-2(Bcl2)signaling pathway.2)Inhibition of mTOR signaling pathway by adding Rap could significantly increase the Arg uptake rate(P<0.01),and the mRNA and protein expression levels of cationic amino acid transporter 2(CAT2)were significantly promoted after added Rap into the medium which containing 100μmol/L Arg(P<0.01).It is further demonstrated that Rap might promote CAT2 expression through the protein kinase Cα(PKCα)-extracellular signal-regulated kinase(Erk)/cFos-CAT2 signaling pathway,thereby promoting Arg uptake.It is concluded that inhibition of mTOR signaling pathway by Rap can inhibit the proliferation of porcine intestinal epithelial cells,while increasing Arg concentration can effectively alleviate the inhibitory effects of Rap;Rap promotes Arg uptake by regulating PKCα-Erk/cFos-CAT2 signaling pathway in porcine intestinal epithelial cells,and increasing Arg concentration can promote the Arg uptake of cells.The results suggest that mTOR signaling pathway plays an important role in the utilization of Arg in porcine intestinal epithel

关 键 词：MTOR信号通路 猪肠上皮细胞 精氨酸摄取 PKCα-Erk/cFos-CAT2信号通路 

分 类 号：S811[农业科学—畜牧学]