ACE2 Mediates The Proliferation Of Pig Intestinal Epithelial Cells And The Related Pathway Of Tryptophan Transport

Angiotensin-converting enzyme 2(ACE2)is a new member of the renin-angiotensin system(RAS)and is widely distributed in the intestine.Recent studies have found that ACE2 synergizes with intestinal amino acid transport in both rats and mice.Tryptophan is a major limiting amino acid in corn-soybean meal diets,although it has a small amount but plays an important metabolic role.At present,there are few studies on the nutrient regulation mechanisms of tryptophan uptake in pigs,Therefore,in this study,intestinal epithelial cell IPEC-J2 was used as the research object to deeply analyze the ACE2-mediated pig intestinal epithelial cell proliferation and tryptophan transport related pathways.The test was divided into two parts:the first part was treated with different tryptophan concentrations(0.044 mmol/L(ie control),0.1 mmol/L,0.2 mmol/L,0.4mmol/L,0.8 mmol/L,1.6 mmol/L).Cells,cell number and cell viability were measured by cell counting method and Cell Counting Kit-8 method.The expression levels of ACE2and B~0AT1 genes and proteins were detected by RT-PCR and Western blotting.The concentration of free tryptophan in the culture supernatant was determined by HPLC.In the second part,In the second part,the changes of B~0AT1,AhR,ERK1/2,mTOR and S6K1,4EBP1 Gene and Protein were measured by RT-PCR and Western blotting using ACE2gene overexpression and ACE2 gene RNA interference.The concentration of free amino acids in the culture supernatant was measured by HPLC.The effect of ACE2 on tryptophan transport in IPEC-J2 cells was analyzed.The results were as follows:(1)After the treatment of IPEC-J2 cell 48h with different concentrations of tryptophan,the cell morphology was changed:the cells in the control group,the 0.1 mmol/L L-Trp group and the 0.2 mmol/L L-Trp treatment group were normal,the cells of the 0.4 mmol/L L-Trp treatment group began to die,and the cells in the 0.8 and 1.6 mmol/L L-Trp treatment group died out in large numbers;cell number and cell viability aspects:Compared with the control group,the 0.1-and 0.2-mmol/L L-Trp treatment groups had no significant differences(P>0.05),and 0.4,0.8,and 1.6 mmol/L L-Trp treatment groups had significant differences(P<0.05).(2)As the concentration of tryptophan in the medium increases,the concentration of tryptophan that enters the cells also increases.Increased tryptophan concentration in the cells will increase the expression of ACE2 mRNA and protein,and will also increase the mRNA abundance and protein expression of the ne utral amino acid transporter B~0AT1when the concentration of tryptophan in the cell culture medium reaches 0.4 mmol/L.The relative abundances of ACE2 mRNA and B~0AT1 mRNA were significantly higher than those of other groups(P<0.05),but the expression levels of ACE2 and B~0AT1 protein were inconsistent,when the concentration of tryptophan in the cell medium reached 0.4 mmol/L,the expression of ACE2 protein was highest.The B~0AT1 protein expression level was highest in the 0.2 mmol/L-Trp treatment group.(3)For the first time,a porcine eukaryotic GFP expression vector pEGFP-C3/ACE2was successfully constructed.(4)ACE2 overexpression can significantly increase the expression of B~0AT1 gene and protein and can significantly up-regulate the expression of key proteins in mTOR signaling pathway;promote cell proliferation;significantly increase the relative abundance and protein expression of AhR mRNA;significantly down-regulate ERK1/2 protein phosphorylation(P<0.05).Increased expression of ACE2 increases transport of tryptophan,serine,glycine,glutamate,glutamine,aspartate,cysteine,methionine,valine,and leucine.Most of them are neutral amino acids.(5)ACE2 RNA interference can significantly reduce B~0AT1 gene and protein expression and can significantly down-regulate the expression of key proteins in mTOR signaling pathway;significantly reduce the relative abundance and protein expression of AhR mRNA;significantly up-regulate ERK1/2 protein phosphorylation(P<0.05).Decreased expression of ACE2 reduces tryptophan,glycine,histidine,and glutamate transport.In summary,porcine ACE2 co-regulates tryptophan transport in intestinal epithelial cell IPEC-J2 via B~0AT1 and activates the mTOR signaling pathway and its downstream proteins S6K1 and 4EBP1 phosphorylation.At the same time,it was found that porcine ACE2 regulates the transport of tryptophan in IPEC-J2 of intestinal epithelial cells and regulates the expression of AhR transcription factor and the phosphorylation of ERK1/2protein.