Research On NHE3 Dynamic Transport Mechanism Regulated By EGFR/ERK Pathway In IPEC-J2 Cells With TGEV Infection

Transmissible gastroenteritis（TGE）caused by the transmissible gastroenteritis virus（TGEV）belongs to Coronaviridae is a highly contagious disease of digestive tract,which is mainly characterized by vomiting and severe diarrhea in piglets.Suckling piglets within 2 weeks of age are the most susceptible and the mortality rate can reach high up to 100%,which brings terrible losses to the healthy development of worldwide swine industry.Transport function of electrolytes in epithelial cells lining the intestines can occur with obstacles after TGEV infection,resulted in change of osmotic pressure in the lumen,with diarrhea and dehydration.Currently there is no demonstrably right answer about molecule pathogenic mechanism of diarrhea caused by TGEV in piglets.Intestinal diarrhea is closely related to Na⁺transport function of Na⁺/H⁺exchanger protein NHE3 in brush border membrane of small intestine epithelial cells.The early studies of research group have shown that epidermal growth factor receptor（EGFR）may participate in regulation of NHE3 expression in the brush border of intestinal epithelial cells.It was also known that EGFR was conducive to viral invasion of host cells due to its character of TGEV receptor.There is a unresolved question that NHE3 activity whether can be regulated through alteration of EGFR expression to affect Na⁺absorption in intestinal epithelial cells after TGEV infection.To prove above assumptions intestinal epithelial cells model was established by this study to accomplish detection of NHE3 expression and Na⁺concentration in epithelial cells after TGEV infection.Then use the EGFR inhibitor（AG1478）to inhibit EGFR so as to check expression and phosphorylation levels of critical proteins in EGFR/ERK pathway.NHE3 dynamic changes in intestinal epithelial cell membrane can be evaluated by methods of fluorescence recovery after photobleaching（FRAP）and confocal laser scanning microscope（CLSM）to analyse mechanism that EGFR regulate NHE3 activity,providing a theoretical basis to clarify pathogenic mechanism of diarrhea piglets caused by TGEV.Main contents and results are as follows:1.The impact on activity and expression of NHE3 after TGEV infection in porcine jejunum intestinal cells（IPEC-J2）（1）Use TGEV Miller（MOI=0.1）to infect IPEC-J2 cells applying Flame Atomic Absorption Spectrometry（FAAS）to detect Na⁺concentration variation in intra-and extracellular cells at different time,results showed that extracellular Na⁺concentration has an increasing trend,but intracellular Na⁺concentration firstly increased and then decreased,IPEC-J2 cells will eventually lead to weak intracellular Na⁺absorption function and NHE3 activity after TGEV infection.NHE3 mRNA and protein levels in intestinal cells were evaluated by Quantitative Real-time PCR（qRT-PCR）.NHE3 mRNA and protein relative expression in IPEC-J2 cells both up-regulated obviously.It indicated that decrease of Na⁺absorptive function and NHE3 levels in IPEC-J2 cells were caused by TGEV infection,having an effect on NHE3 activity and expression.2.Study on mechanism of NHE3 expression regulated by EGFR in IPEC-J2 with TGEV infection（1）Reseach found that EGFR phosphorylation increased in IPEC-J2 cells with TGEV infection were detected by Western-blot.Cytotoxicity of AG1478 to cells was measured by MTT,results showed that maximum non-toxic dose of AG1478 treated in IPEC-J2 was 30μM.（2）After respective treatment of AG1478 and DMSO to IPEC-J2 cells,continued to culture with TGEV.The proliferation ability of TGEV in cells was determined by TCID₅₀.Na⁺concentration of IPEC-J2 cells was evaluated by flame atomic absorption method.The results showed that the virus titer of the DMSO group decreased 32.5%compared with the mock group,and virus titer of AG1478 group reduced 93.3%lower than DMSO group.On the basis,after inhibition to EGFR,continued to culture with TGEV.Results indicated that phosphorylation levels of EGFR and ERK for TGEV group had increased matched to blank control,NHE3 expression significantly decreased（p<0.01）.The phosphorylation levels of EGFR and ERK was inhibited in AG1478 group infected with TGEV,meanwhile NHE3 expression increased markedly（0.01<p<0.05）.Results indicated that the inhibition of EGFR on IPEC-J2 cells could avoid proliferation of TGEV in cells and enhance Na⁺absorptive function in IPEC-J2 cells with TGEV infection.,increasing activity and expression of NHE3.3.Study on the dynamic transport in membranes of IPEC-J2 cells through EGFR after TGEV infectionAccording to GenBank accession（XM₀21077062.1）from NCBI for NHE3CDS sequences,NHE3 gene segments were created synthetically and inserted into eukaryotic expression vectors pEGFP-N3 to construct recombinant fluorescence expression vector pEGFP-NHE3,then transfected into IPEC-J2 cells to observe stable cycles of expression.When pEGFP-NHE3 was transfected into IPEC-J2 cells,AG1478 treated group was infected with TGEV,at the same time,using the only pEGFP-NHE3 transfected group as the parallel control group also could be infected mwith TGEV.Fluorescence Recovery After Photobleaching（FRAP）was used to detect NHE3 recovery rate and mobile fraction（M_f）in order to identify NHE3mobility.NHE3 mobility in TGEV-infected group was significantly slower than non-infected group（p<0.01）,TGEV group after AG1478 treatment was higher than alone TGEV-infected group（0.01<p<0.05）.Results indicated that,the stable expression of recombinant vector pEGFP-NHE3 in IPEC-J2 cells was up to about 5days,NHE3 mobility in membrane of TGEV-infected cells could be enhanced after inhibition to EGFR.