The Mechanism Of SGLT1 Regulating P-38MAPK/AKt2 Signal Pathway On NHE3 Translocation In TGEV Infection

Transmissible gastroenteritis(TGE)is a highly contact enteric infectious disease caused by transmissible gastroenteritis virus(TGEV),which of main clinical symptoms are severe diarrhea,vomiting and dehydration.TGEV mainly infect piglets within 2 weeks of age,and the mortality rate is close to 100%.It is a fatal epidemic disease of early death and growth retardation in piglets of all pig-raising countries in the world.In recent years,with the increasing of epidemic of TGE,it has caused serious economic losses to the pig industry.The vaccines are available for preventing TGEV infection,however the pathogenesis of the virus has not been elucidated,it is therefore difficult to control the occurrence of the disease.TGEV mainly infect the small intestine through the digestive tract,and replicate in small intestine epithelial cells,which can destroy the disorder of sodium ion transport in the epithelial cells,leading to an increase in intestinal osmotic pressure and causing diarrhea.It is known that the membrane transporter NHE3(Na~+/H~+exchanger 3)mediate Na~+/H~+exchange in intestinal cells,which is closely related to the development of diarrhea.Under physiological conditions,SGLT1 can regulate the translocation of NHE3 and affect the absorption of Na~+.However,it is unknow that whether SGLT1 still regulate NHE3translocation after TGEV infect pIECs.Therefore,In this study,the jejunal epithelium of porcine IPEC-J2 was used as an infection model,and the expression of SGLT1 was inhibited by constructing lentiviral interference vector and inhibitor Phlorizin,respectively,investigating the effect and molecular mechanism of TGEV infection on NHE3 translocation,and provide a new theoretical basis for revealing the pathogenesis of TGEV.The contents are as follows:1.The expression of SGLT1,NHE3 and activity of Na~+/H~+exchange after TGEV infectionAfter TGEV infect IPEC-J2,the protein expression of SGLT1 and NHE3 were detected by Western blot,and using the real-time quantitative RT-qPCR to detect the relative mRNA levels of SGLT1 and NHE3.The content of NHE3 on the apical surface of IPEC-J2 cells was determined by cell-surface biotinylation,and Na~+/H~+exchange activity was measured fluorometrically using BCECF-AM.Results showed that TGEV infection can up-regulate the protein expression and the mRNA level of SGLT1,and the total protein expression of NHE3was not significantly change,whereas the protein expression of NHE3 was down-regulated on the plasma membrane,as well as the decreased of the activity of NHE3.These results revealed that TGEV infection can down-regulate the amount of NHE3 on the plasma membrane,which in turn reduces the activity of NHE3.2.The mechanism of SGLT1 regulate NHE3 translocation under physiological conditions(1)According to the GenBank accession(NM-001012297)from NCBI for SGLT1 CDS sequences,four siRNA sequences were designed,then constructing the lentiviral vector piLenti-siRNA-GFP,and infect IPEC-J2 cells to identify the optimal MOI of lentivirus infection.Exploring the concentration of the resistant drug puromycin to screen for stable expression of interfering cell lines simultaneously.After screening for the best interference fragment,Western-blot method was used to detect the phosphorylation of key proteins MAPKAPK-2 and EZRIN in p-38MAPK/AKt2 signaling pathway.The surface NHE3 was detected by biotinylation method and applying Flame Atomic Absorption Spectrometry(FAAS)to detect the concentration of Na~+between the inside and outside of cells.Results showed that the MOI value of lentivirus was 5,and the optimal drug concentration of puromycin puromycin was 0.8?g/mL.piLenti-siRNAb-GFP has the best interference effect,with the inhibition rate of SGLT1 was more than 60%.Silencing of SGLT1 using piLenti-siRNAb-GFP can significantly up-regulate the expression of p-MAPKAPK-2 and p-EZRIN,as well as the expression of NHE3 on the plasma membrane was significantly up-regulated.These above results indicate that inhibition of SGLT1 can promote the translocation of NHE3 through regulating p-38MAPK/AKt2 pathway.(2)MTT assay showed that the optimal drug concentration of inhibitor Phlorizin was 200?M.The expression of p-MAPKAPK-2,p-EZRIN and surface NHE3 were significantly up-regulated after inhibiting the expression of SGLT1.Similarly,the activity of NHE3 was also significantly enhanced.The results suggesting that inhibition of SGLT1 promote NHE3translocation and enhanced NHE3 exchange activity.3.Regulation of NHE3 translocation by SGLT1-mediated p-38MAPK/AKt2 signaling pathway after TGEV infectionThe expression of SGLT1 was significantly reduced by lentiviral vector and inhibitor Phlorizin,respectively,then inoculation with TGEV.Western-blot and biotin methods were used to detect the phosphorylation of key proteins MAPKAPK-2 and EZRIN in the signaling pathway and the surface NHE3,simultaneously.Applying Flame Atomic Absorption Spectrometry(FAAS)method to detect the concentration of Na~+in and out of the cell.The activity of NHE3 was detected by pH fluorescent probe.After interfering with the expression SGLT1 and cells infect TGEV,the phosphorylation levels of MAPKAPK-2 and EZRIN were significantly up-regulated,and the expression of NHE3 on the cell membrane was significantly increased.It was also found that intracellular Na~+concentration increased significantly,while the extracellular concentration Na~+significantly decreased.After inhibiting the expression of SGLT1 and cells infected TGEV,the expression of p-MAPKAPK-2 was no significant change,however,the expression of p-EZRIN was significantly up-regulated,also showed a significant increase in the surface protein NHE3 and the activity.These results indicated that inhibition of SGLT1 expression can promote NHE3translocation and enhance Na~+uptake under TGEV infection,suggesting that SGLT1 is involved in the regulation of NHE3 translocation and sodium hydrogen activity,which laying a new theoretical foundation for revealing the pathogenesis of TGEV.