CD44 Mediated TGEV Regulation Of NHE3 Induced Na~+ Imbalance In Piglets And Screening Of Activators

Transmissible gastroenteritis（TGE）is an acute,highly contagious intestinal disease caused by Transmissible gastroenteritis virus（TGEV）in piglets with severe diarrhoea,vomiting and dehydration of piglets as clinical symptoms,piglets under 2 weeks of age are most susceptible to infection,and mortality can reach up to 100%.It seriously affects the production and development of the pig industry and causes serious losses to the pig industry.Physiologically,water is mostly absorbed in the intestinal lumen by osmosis,and when laxative substances act on the intestine,the function and/or expression of various functional entities present on the cavity membrane such as ion transporters,channels,and multiprotein junction complexes that make up the epithelial barrier are lost.This cascade of reactions leads to a disturbance in the balance of electrolyte absorption and/or secretion processes in the intestine disrupting the osmotic pressure gradient and leading to water retention and diarrhoea.The main physiological function of NHE3 is to transport extracellular Na⁺into the cell and extracellular H⁺out of the cells.Pre-laboratory studies have shown that TGEV infection of the pig small intestine replicates in a large number of IPEC-J2 cells,disrupting the Na⁺transport function of the epithelial cells,leading to an increase in intestinal osmotic pressure and promoting diarrhoea in piglets,but its exact mechanism of action is not yet clear.The main physiological role of CD44 is to mediate cell-cell and cell-extracellular matrix interactions and to regulate many biological processes,including cell adhesion,migration and proliferation,and it has been shown that CD44 can be involved in viral infection and that it can inhibit or promote viral replication during the pathogenesis of coronaviral disease.Based on the above principles,this project examined the changes in the quantity of CD44 and NHE3 under TGEV infection by RT-q PCR,flame atomic absorption and immunofluorescence at the cellular level;and then explored whether the quantity and activity of NHE3 were dose-dependent on TGEV;finally,by constructing CD44 overexpression vectors and interference vectors,the effect of CD44 changes on viral Finally,the effects of CD44changes on viral replication and NHE3 activity/quantity were investigated by constructing CD44 overexpression vectors and interference vectors.To elucidate the role of CD44 in Na⁺imbalance diarrhea caused by TGEV infection.Finally,Conivaptan was selected by computer-assisted screening to target the CD44 protein activator and was shown to be effective in preventing viral replication in vivo.The main studies are as follows:1.Transcriptomic analysis and validation of the small intestine of TGEV-infected pigsIn this chapter,through transcriptomic analysis of the intestinal tissues of TGEV-infected piglets in the pre-laboratory period,CD44 in the ECM pathway was screened out.By predicting the protein interactions between CD44 and NHE3,it was found that CD44 and NHE3 may be potentially linked,and the experiments also demonstrated that TGEV infection could inhibit NHE3 m RNA expression and promote CD44 m RNA expression.The experimental results in this chapter suggest that there may be a regulatory relationship between NHE3 and CD44.2.Effect of TGEV infection of IPEC-J2 cells on NHE3 and CD44In this chapter,the expression of NHE3 and CD44 was detected by RT-q PCR,Western blot with immunofluorescence,and the exchange of Na⁺concentration in the extracellular and extracellular fluids was detected by flame atomic absorption.The results showed that TGEV could down-regulate the expression of NHE3 and affect the uptake of Na⁺by IPEC-J2 cells.The above results indicated that TGEV could affect the concentration of Na⁺inside and outside the cells;and the total and plasma membrane protein levels of NHE3 were detected by infecting IPEC-J2 cells with TGEV of different infection complexes（MOI=0.5,MOI=1,MOI=2）,respectively,using Western blot and RT-q PCR;and by flame atomic The expression of NHE3 was found to be dose-dependent on TGEV,thus demonstrating that NHE3 plays an important role in the Na⁺imbalance diarrhea caused by TGEV.3.Effect of CD44 on NHE3 and TGEV replicationTo investigate the relationship between CD44 and NHE3,this chapter examined the effect of CD44 protein on NHE3 expression by constructing recombinant overexpression p EFGP-CD44 and interfering p LVX-CD44plasmids,and also detected the effect of CD44 protein on NHE3 expression by Western blot and immunofluorescence,and found that there was no direct regulatory relationship between CD44 and NHE3;under the condition of TGEV infection,the effect of CD44 protein on NHE3 expression was detected by regulating under TGEV infection,the expression of NHE3 protein was detected by modulating CD44,and the concentration of Na⁺inside and outside the cell was detected by flame atomic absorption,and it was found that CD44protein could cause significant downregulation of NHE3 plasma membrane expression and affect the exchange of Na⁺inside and outside the cell.Overexpression of CD44 protein inhibited TGEV replication,while interference with CD44 promoted TGEV replication;the above experiments demonstrated that CD44 protein could affect the amount and activity of NHE3by regulating TGEV replication.4.Screening and validation of CD44 small molecule activators.The sequence of CD44 protein was downloaded from NCBI and a protein model of CD44 was constructed using Trrosetta based on the protein sequence;the potential activator Conivaptan was screened for molecular docking of CD44 with candidate ligands using Smina et al.Cellular assays revealed that 4×10^-4mol/L of Conivaptan significantly In animal studies,piglets were fed Conivaptan followed by TGEV infection and no significant pathological changes were observed after autopsy.Using Western blot with absolute fluorescence quantitative PCR,it was found that Conivaptan reduced the expression of TGEV N gene on intestinal epithelial cells and also reduced the reduction of NHE3 after TGEV infection and effectively prevented the replication of the virus.Overall,this study demonstrated that CD44 can mediate the regulation of NHE3 by TGEV,thereby affecting Na⁺exchange,and screened for activators to attenuate TGEV-induced diarrhoea in piglets.In the present study,we aim to elucidate the regulatory relationship between CD44 and NHE3 during TGEV infection and to confirm the role of CD44 in TGEV-induced diarrhoea.This study will open up new ideas for the design of new anti-TGEV drugs targeting CD44 and the development of other drugs for coronavirus-induced diarrhoea.