| Background:Peritoneal dialysis(PD)is one of the alternative treatments for patients with end-stage renal disease(ESRD).About 11% of ESRD patients receive peritoneal dialysis in worldwide.With the improvement of PD technology and the enhancement of peritoneal infection control,peritoneal dialysis-related peritoneal fibrosis has became the main reason for PD patients to withdraw from peritoneal dialysis,which greatly limits the development and promotion of PD.The epithelial-mesenchymal transition(EMT)of peritoneal mesothelial cells is the key factor of peritoneal fibrosis,and it is reversibility.Therefore,it is of great significance to reverse EMT on the protection of peritoneal mesothelial cells and reduction peritoneal fibrosis.Zinc is an indispensable trace element in the body,which is one of the most abundant mineral elements participating in enzyme activities,and is also the basis of nucleic acid metabolism,protein synthesis and transcription factor structure.The balance of zinc is essential to maintain the normal physiological function of the body.If the balance was broken,it would lead to the occurrence of related diseases.Animal models and some clinical studies suggest that zinc deficiency may be associated with fibrosis of chronic inflammatory diseases,including liver,myocardial,cystic fibrosis and so on.While Zn supplementation can exert multiple functions,including inhibition of fibrosis,anti-oxidation,anti-inflammatory and anti-apoptosis.In some fibrotic animal models and patients with chronic inflammatory disease,zinc treatment can improve the condition caused by zinc deficiency.Zinc ions can not be free to pass through the cell membrane and need to be completed with the help of zinc transporters.As one of the zinc transporters,SLC30A/ZnTs,mobilize zinc from the cytosol into the extracellular space and the lumens of intracellular compartmentsand in reduction the Zn in the cytoplasm.ZnTs not only directly participate in the metabolism of zinc in cells,but also affect the occurrence and development of diseases through complex mechanisms,such as tumors and fibrosis.In the previous study,we found that high glucose(HG)peritoneal fluid induced EMT in rat peritoneal mesothelial cells by promoting the production of reactive oxygen species(ROS),and zinc supplementation could inhibit ROS and reverse the high glucose-induced EMT of rat peritoneal mesothelial cells.And the ZnTs are abundant in rat peritoneal mesothelial cells and play an important role in the regulation of the metabolism and function of peritoneal mesothelial cells.However,the influence and mechanisms of Zn on peritoneal dialysis-related peritoneal fibrosis models in vivo and in human peritoneal mesothelial cells(HPMCs)are still unknown.The effects and mechanisms of ZnTs on EMT of high glucose-induced HPMCs still need to be further explored.Objetiv: To investigate the effects of Zn and ZnTs on EMT of high glucose-induced HPMCs,and further explore influencing mechanism,in order to provide a new scientific basis for effective inhibition of EMT and peritoneal fibrosis.Methods: On the basis of previous work,this study observed the role of Zn on EMT and peritoneal fibrosis in vivo and in vitro.In vivo,the model of peritoneal fibrosis-related peritoneal fibrosis was established to detect the peritoneal ultrafiltration function and glucose transfer capacicty.Hematoxylin-eosin(HE)staining and modified Masson trichrome staining were used to observe the degree of peritoneal fibrosis.Immunohistochemistry and Western Blot were used to detect the expression of E-cadherin,Vimentin and Collagen in rat peritoneal tissues.In vitro,the model of HPMCs EMT was used to observe the migration function by transwell co-culture system.By using Western Blot and immunofluorescence to detect the expression of EMT markers(E-cadherin,Vimentin),and using Flow cytometry to detect the expression of reactive oxygen species(ROS).The qRT-PCR was used to detect the expression of ZnTs mRNA,and the TSQ probe was used to detect the change of Zn in HPMCs.The Golgi apparatus was located by the red fluorescent probe of Golgi apparatus.The liposome transfection method was used to transfect the HPMCs.Immunofluorescence and Western Blot were used to detect the effects and related mechanisms of Zn and ZnT5 on EMT of high glucose-induced HPMCs.Results: The results were included in vivo and in vitro.The vivo experimental results1.The effects of Zn on the peritoneal histomorphology of rats: HE and MASSONstaining showed that the peritoneal thickness increased in the high glucose group,the subepithelial matrix was loose,a large number of collagen fibers deposited,epidermis cells were exposed,and inflammatory cells infiltrated.The Zn chelating group further aggravated the above the changes of peritoneal morphology in rats.However,the peritoneal morphology of the Zn group was significantly improved.2.The effects of Zn on the peritoneal function of rats: The results showed that the peritoneal ultrafiltration of the high glucose group decreased and glucose transfer capacicty increased.Zn chelating group intensified the above changes.The peritoneal ultrafiltration function of Zn group increased and glucose transfer capacicty decreased.3.The effects of of Zn on the peritoneal tissue EMT of rats: Immunohistochemical analysis and Western Blot showed that the expression of Vimentin and Collagen increased,and the expression of E-cadherin decreased in high glucose group.Zinc chelating group further intensified the above changes.The expression of Vimentin and Collagen decreased,and the expression of E-cadherin increased in Zn group.The vitro experimental results1.The effects of of Zn on morphological changes of high glucose-induced HPMCs:Epithelial-like cells and spindle cells were found in the high glucose group.A large number of spindle-forming fibroblasts were seen in the Zn chelating group.The morphological changes of HPMCs in high glucose group and Zn chelating group suggested EMT occurred in HPMCs,which could be partially reversed after Zn supplementation.2.The effects of Zn on the migration of high glucose-induced HPMCs: The number of HPMCs transferred to submembrane increased in high glucose group.The chelating group increased further,while the number of HPMCs cells decreased in Zn group.The activity of HPMCs migration in high glucose and Zn chelator group enhanced,indicating that HPMCs had the characteristics of mesenchymal cells.That meaned that EMT occurred in HPMCs.3.The effects of Zn on EMT of high glucose-induced HPMCs: Immunofluorescence analysis and Western Blot showed that the expression of E-cadherin decreased and the expression of Vimentin increased in HG group.The Zn chelating group further aggravated the above changes.However,the expression of E-cadherin increased and theexpression of Vimentin decreased in Zn group.4.The effects of Zn on ROS of high glucose-induced HPMCs: High glucose could increase ROS of HPMCs and promote oxidative stress,and the Zn chelating group further increased ROS.While the Zn group significantly decreased ROS.5.The effects of Zn on Nrf2 signal pathway of high glucose-induced HPMCs: High glucose promoted the expression of nuclei Nrf2,NQO1 and HO-1 in HPMCs.Compareing with the HG group,the expression of nuclei Nrf2,NQO1 and HO-1decreased in Zn chelating group,Zn group further increased the expression of these proteins,suggesting that Zn may exert anti-oxidative stress through activation of Nrf2 signaling pathway in HPMCs.6.The effects of high glucose on ZnTs of HPMCs: The qRT-PCR showed that the ZnTs existed in normal HPMCs,and ZnT5 increased significantly after high glucose stimulation,suggesting that ZnT5 might be involved in the EMT of high glucose-induced HPMCs.7.The effects of ZnT5-siRNA and Zn transport of high glucose-induced HPMCs: High glucose increased the expression of ZnT5 mRNA,which mediated Golgi zinc influx and decreased intracellular zinc ion concentration,and promoted EMT of HPMCs.8.The effects of ZnT5-siRNA on EMT of high glucose-induced HPMCs: Western Blot results showed that ZnT5-siRNA group could significantly increase E-cadherin expression and decrease Vimentin expression of HPMCs,suggesting that ZnT5-siRNA could reverse EMT of HPMCs.9.The effects of ZnT5-siRNA on TGF-β/Smad pathway: Western Blot showed that the expressions of TGF-β1 and p-Smad2/3 in ZnT5-siRNA group were significantly lower than those in HG group,and p-Smad7 expression was significantly higher than that in HG group.Conclusion:1.High glucose peritoneal fluid could cause changes of peritoneal morphology and function in rats.The peritoneal mesothelial cells of rats turned EMT.2.Zinc deficiency further aggravated the changes of peritoneal morphology and function induced by high glucose peritoneal dialysis in rats and promoted peritoneal fibrosis in rats.Zinc supplementation could partly reverse the changes of peritoneal morphology andfunction and inhibit peritoneal fibrosis induced by hyperglycemic peritoneal dialysis in rats.3.High glucose could induce EMT of HPMCs.Zinc deficiency could promote EMT of HG-induced HPMCs,while zinc supplementation could partially reverse the EMT of HPMCs4.The mechanisms of zinc inhibiting the EMT of HG-induced HPMCs is:(1)Zinc could inhibit the EMT of HG-induced HPMCs by inhibiting oxidative stress.(2)Zinc could inhibit the EMT of HG-induced HPMCs by activating the Nrf2 pathway.5.ZnTs existed in HPMCs.The expression of ZnT5 was related to the EMT of HG-induced HPMCs.6.ZnT5 gene silencing could attenuate the the EMT of HG-induced HPMCs.7.ZnT5 gene silencing could reverse the EMT of HG-induced HPMCs by regulating TGF-β/Smad signaling pathway. |
PDF Full Text Request