Molecular Mechanism Of Tight Junction Regulated By Leucine In Porcine Intestinal

The intact Intestinal mucosal barrier of the body is a strong guarantee to prevent harmful substances such as bacteria and endotoxins from entering the internal environment due to translocation,and the mechanical barrier is the most important.The tight junction is the crucial structure of mechanical barrier,and its damage can increase permeability and cause intestinal diseases.Leucine,an essential amino acid,has been shown to have effects on tight junction of intestine,but the molecular mechanism is unclear.Our study aims to reveal effect of L-Leucine on tight junction and its molecular mechanism further.Intestinal porcine epithelial cells(IPEC-J2)cultured in vitro were used as an experimental model in our study.Firstly,we found that leucine decreased epithelial cell permeability by detecting transepithelial electrical resistance(TEER)after cells were treated with leucine(0?0.1?0.5?2?and 4 mM)and discovered 0.5 mM Leu remarkably induced the expression of claudins through RT-qPCR analysis.To further explore the molecular mechanism of expression of claudins caused by leucine,cells were pretreated with rapamycin and qPCR results showed that expression of claudins induced by Leu was repressed.Later,we constructed sestrin2/mTORC1 knockdown cell line and qPCR results showed that mTORC1 was involved in the upregulation of claudin-3?-5?and-8 promoted by Leu,in addition,expression of claudin-3 was inhibited by sestrin2.Meanwhile,we discovered that expression of tight junctionrelated genes was suppressed when cells were pre-treated with BAY11-7082(an inhibitor of IKK),and transcription factor NF-?B was also involved in the upregulation of claudin-3 and claudin-8 via experiment in RelA knockdown cells.Last,western blotting presented that leucine can induce NF-?B phosphorylation,which can be prevented by rapamycin,and all of these indicated that Leu induced NF-?B phosphorylation through mTORC1.In summary,leucine might regulate the expression of tight junctionrelated genes through sestrin2-mTORC1-NF-?B signaling pathway in IPEC-J2 cells,thereby effecting permeability and improving intestinal barrier.