The Molecular Mechanisms Of NHE3 Dysregulation In Gallstone Pathogenesis

Objective: Gallstone disease seriously affects people’s health and quality of life.The incidence of gallstones is significantly elevated due to changes in dietary structure.Although the research on the pathogenesis of gallstone has made great progress,the underlying mechanism is still not very clear.It has been found that enhanced gallbladder absorptive function and the subsequent increased concentration of cholesterol,calcium ion and mucin in the bile is an important cause of gallstone formation.However,the gallbladder itself,an organ closely related to the occurrence and development of gallstone disease,has not received enough attention.The gallbladder is the organ that stores and concentrates bile,and its mucosa has strong absorptive function.The absorptive process of gallbladder bile is mainly achieved by the absorption of biliary ions and water.In the process,Na+/H+exchanger isoform 3(NHE3) plays an important role.NHE is an ion exchanger expressed in epithelial cells.Its function is to transport extracellular Na+ into the cell while excreting H+,which plays an important role in regulating electrolyte and acid-base balance.In the gallbladder epithelial cells,NHE3 transports Na+ into the cells to form an osmotic pressure gradient,and the water enter the cells along the gradient,to concentrate the bile in the gallbladder.An important study from Narins showed that the absorption of gallbladder mucosa was enhanced in the early stage of gallstone formation,and the activity of NHE3 was increased.The usage of NHE3 inhibitor amiloride significantly inhibited mucosal absorption and reduced gallstone formation rate.This study suggests that increased NHE3 activity and subsequent enhanced gallbladder absorptive function is an important cause of gallstone formation.Unfortunately,the role of gallbladder NHE3 regulation and dysfunction in the formation of stones is unknown.Based on previous studies,we propose that NHE3 dysfunction caused by lithogenic diet is an important cause of enhanced gallbladder absorptive function.Therefore,the main purpose of the study is to explore the molecular mechanism ofNHE3 dysfunction during cholesterol gallstone formation.Methods: We fed C57BL/6 mice with high cholesterol diet and study the expression of NHE3 in the gallbladder mucosa.RT-PCR was used to detect the transcription of NHE3 in the control group and lithogenic group.The protein expression of NHE3 in the gallbladder mucosa was detected by western blot.Immunofluorescence was performed to study the location of NHE3 in the gallbladder tissue.The protein expression of PKCα,NHERF1 and ezrin in the gallbladder mucosa was detected by western blot.The interaction of NHE3 with ezrin and NHERF1 was detected by immunoprecipitation.To explore the molecular mechanism of enhanced NHE3 activity in the early stage of gallstone formation,we stimulated cholangiocarcinoma cells with chenodeoxycholic acid(CDCA) and/or forskolin.The effects of CDCA on the expression of NHE3,PKCα,NHERF1 and ezrin were detected by western blot.The effect of CDCA on the interaction between NHE3 and ezrin was detected by immunoprecipitation.Results: In the control group,C57BL/6 mice had clear gallbladder bile,with a pale yellow color.The stone formation rate was zero.All C57BL/6 mice in the lithogenic group had thick gallbladder bile,and there were granular stones.The stone formation rate was 100%.RT-PCR showed that the transcription level of NHE3 in the gallbladder tissue of the mice in the lithogenic group was significantly higher than that of the control group(P < 0.05).There was no significant difference in the expression level of total NHE3 protein between the control and lithogenic group.The difference in protein and mRNA expression levels of NHE3 may be a result of decreased protein stability or reduced translation of NHE3 mRNA.Since phosphorylation can affect the exchange activity of NHE3,we further examined the level of PS552-NHE3.We found that the phosphorylation of NHE3 in the gallbladder tissue from the lithogenic group was significantly higher than that in the control group(P < 0.05),which was consistent with the abnormal gallbladder absorptive function.The single-layer columnar epithelial cells of the gallbladder mucosa express NHE3 and PS552-NHE3.NHE3 protein is expressed in both the apical membrane and the cytoplasm,which is consistent with the report that NHE3 can be transported between the apical plasma membrane and the cytoplasm.In addition,there was no significantdifference in total NHE3 protein expression between the lithogenic group and the control group.The expression level of PS552-NHE3 protein in lithogenic group was significantly increased.These results are consistent with our western blot data.The western blot results showed that there was no significant difference in the expression level of total ezrin in the lithogenic group,but the phosphorylation level of the ezrin protein was increased(P < 0.05).Moreover,PKCα and NHERF1 are increased in protein levels(P < 0.05).Immunoprecipitation was performed using NHE3 antibody and total protein of gallbladder mucosal epithelial cells.After transfection,immunoblotting analysis was performed by ezrin antibody.The results showed that the NHE3 immunoprecipitation complex contained ezrin protein.Immunoprecipitation was performed using ezrin antibody and total protein of gallbladder mucosal epithelial cells,and then subjected to immunoblotting analysis by NHE3 antibody after transfection.The results showed that the ezrin immunoprecipitation complex contained NHE3 protein.Immunoprecipitation was performed using NHE3 antibody and total protein of gallbladder mucosal epithelial cells,and subjected to immunoblotting analysis by NHERF1 antibody after transfection.The results showed that the NHE3 immunoprecipitated complex contained the NHERF1 protein.Immunoprecipitation was performed using NHERF1 antibody and total protein of gallbladder mucosal epithelial cells,and subjected to immunoblotting analysis by NHE3 antibody after transfection.The results showed that the NHERF1 immunoprecipitated complex contained NHE3 protein.The above co-immunoprecipitation experiments showed that NHE3 can bind to NHERF1 and ezrin and form a protein complex.It was found that under the synergistic effect of PKA agonist forskolin,CDCA can significantly enhance the expression of PKCα and NHERF1(P < 0.05),and activate ezrin in cholangiocarcinoma cells(P < 0.05).CDCA can also promote the formation of protein complexes between ezrin and NHE3.Conclusion: The high-fat diet can induce cholesterol gallstone formation in C57BL/6mouse.The stone formation rate is high and this method is both economical and reliable.It can be used in the experimental research on the cause of gallstone formation.The phosphorylation level of NHE3 is increased after the formation ofgallstones,which is consistent with the phenomenon of decreased gallbladder absorptive function.It also suggests that NHE3 dysfunction is associated with abnormal PKA signaling pathway activity.In gallbladder mucosal epithelial cells,NHE3 is expressed in both cell membrane and cytoplasm,suggesting that NHE3 transportation is an important mechanism in regulating its activity.The decreased mucosal absorption after the formation of gallstones may be related to the dysfunction of NHE3 caused by abnormal expression of ezrin,PKCα and NHERF1.The formation of NHE3 protein complex in gallbladder tissue is an important mechanism in regulating its ion transport activity.Abnormal absorptive function of the gallbladder mucosa is likely to be the result of its exposure to the lithogenic bile.CDCA is likely to inhibit the activity of NHE3 in gallbladder epithelial cells under physiological conditions;Decreased CDCA concentration in lithogenic bile may be an important cause of enhanced gallbladder absorptive function.Through systematic research of this project,we can deepen the understanding of the pathogenesis of gallstones,and provide theoretical and experimental basis for the prevention and treatment of cholesterol gallstone diseases.