Study On The Mechanism Of TGEA Infection Affecting The Activity Of Sodium Hydrogen Exchanger Subtype 3(NHE3)

Transmissible gastroenteritis(TGE)caused by transmissible gastroenteritis virus(TGEV)is a kind of highly contact intestinal infectious disease.The main clinical symptoms are fever,vomiting,severe diarrhea and dehydration.It belongs to class B infectious disease regulated by the world organization for animal health(OIE).TGEV mainly reaches the small intestine through the mouth and nose,and replicates in the intestinal epithelial cells,which disturbs the absorption and transport of nutrients and electrolytes(especially sodium ions)in the intestinal cavity,and increases the intestinal osmotic pressure,leading to diarrhea.Under physiological conditions,a large amount of Na~+ in the body is mainly absorbed by the membrane transport protein NHE3(Na~+ / H~+ exchanger 3)through the Na~+ / H~+ exchanger mediated neutral pathway,thus forming a permeability gradient and increasing the passive absorption of water by the intestine.Under basic conditions,like other membrane transporters,NHE3 circulates between plasma membrane and circulatory corpuscles,depending on early endosomes(EE)specific to Rab5 a and recycling endosomes(RE)specific to Rab11 a After reprocessing of endoplasmic reticulum(ER)and Golgi apparatus.When stimulated by the activities of key proteins such as EGFR / RSK2 or ezrin,NHE3 retranslocates from these recycled endosomes to plasma membrane;The components that can't be reused enter rab7 a specific late endosomes(LE)and lysosomes for protein degradation and clearance.Under pathological condition,the activity of NHE3 is closely related to the development of diarrhea.It has been proved that the normal differentiation and tissue results of intestinal tissues of mice treated with NHE3 gene knockout,NHE3 gene deletion or NHE3 inhibitor have been affected,and the absorption of Na~+,water has been significantly reduced and severe diarrhea has occurred.Therefore,studying the regulation mechanism of intestinal NHE3 activity has important practical significance for the future treatment of diarrhea or intestinal infection and inflammation.Under the infection of enterodiarrhea virus TGEV,does it affect the activity of NHE3 in intestinal epithelial cells,which leads to the physiological activity of Na~+ / H~+ exchange in intestinal epithelial cells,and finally leads to the disorder of intestinal electrolyte? In view of this,the small intestinal epithelial cell IPEC-J2 was used in this study as an infection model,the changes of sodium hydrogen exchange activity of NHE3,the expression of total NHE3 protein and plasma membrane surface protein,the expression and phosphorylation of Ezrin and RSK2 which stimulate the translocation of NHE3,the expression of NHE3,the autophagy of IPEC-J2 and the ubiquitination of NHE3 were studied under TGEV infection The mechanism of affecting the activity of NHE3 provides a theoretical basis,and also provides a new theoretical basis for revealing the Pathogenic mechanism of TGEV.Research contents are as follows:(1)Changes of Na~+ / H~+ exchange activity and expression of total protein in NHE3 after TGEV infection with IPEC-J2: after TGEV infection with IPEC-J2,H~+ exchange activity of NHE3 was detected by p H fluorescence probe bcecf-am at different time of TGEV infection.The results showed that,compared with the control group,the H~+ concentration in IPEC-J2 cells increased with the infection time,indicating that the H~+ exchange activity of NHE3 decreased gradually.The concentration of Na~+ in intracellular and extracellular fluid at different time points after TGEV infection was detected by flame atomic absorption spectrometry.The results showed that the concentration of Na~+ in intracellular and extracellular fluid decreased gradually with time,and that of Na~+ in extracellular fluid increased gradually with time.(2)The effect of TGEV infection on the translocation of NHE3 to plasma membrane: RTq PCR and Western blot were used to detect the m RNA level,protein and phosphorylation level of EGFR/RSK2 and Ezrin,the key proteins that stimulate the translocation of NHE3,respectively.The results showed that RSK2 and Ezrin protein and their phosphate levels decreased in the late stage of TGEV infection(48hpi and 72hpi),which indicated that TGEV could inhibit the key proteins RSK2 and ezrin of NHE3 translocation,so as to inhibit the translocation of NHE3 from cytoplasm to plasma membrane.The expression of NHE3 on the membrane of TGEV infected cells at different time points was detected by biotinylated membrane protein,immunofluorescence(IFA)and laser confocal technique.The results showed that compared with the control group,the amount of NHE3 on the cytoplasmic membrane of TGEV infection group decreased significantly,which indicated that TGEV infection could reduce the expression of NHE3 on the cytoplasmic membrane.(3)The change of NHE3 expression in vivo after TGEV infection: after TGEV infection with IPEC-J2,the different time points of infection were extracted by the in vivo extraction kit,and the protein expression of NHE3 was detected by Western blot.The results showed that the amount of NHE3 protein increased with the time of TGEV infection,which indicated that TGEV infection could stimulate the activity of the inner body and enhance the intracellular transport of NHE3.Immunofluorescence(IFA)and confocal laser were used to detect the protein expression of Rab11 a and NHE3 in the cytoplasm of TGEV infected cells at different time points.The results showed that Rab11 a and NHE3 increased significantly in the early stage of TGEV infection(0 hpi and 24 hpi),and decreased in the late stage of TGEV infection(48 hpi and 72 hpi),which further confirmed that the early stage of TGEV infection can stimulate the intracellular transport of NHE3.(4)Autophagy of IPEC-J2 cells and ubiquitination degradation of NHE3 in TGEV infection: after TGEV infection with IPEC-J2 at different time points,the autophagy test kit was used to evaluate the effect of TGEV infection on autophagy of IPEC-J2 cells.The results showed that the autophagy of IPEC-J2 cells increased significantly with the increase of TGEV infection time.Secondly,immunoprecipitation(IP)was used to detect the ubiquitination and degradation of NHE3 at different time points after TGEV infection.The results showed that with the increase of TGEV infection time,the ubiquitination of NHE3 in IPEC-J2 cells increased gradually,especially in the late stage of TGEV infection(48 hpi and 72 hpi).The results showed that the autophagy of IPEC-J2 increased in the late stage of TGEV infection,and it could stimulate the ubiquitination and degradation of NHE3.