| Objective:To investigate the role and signal pathway of translationally controlled tumor protein in podocyte epithelial-mesenchymal transition in high glucose environment.Methods:Cell experiments:divided into four groups:normal control group(LG,5.6 mmol/1 glucose),LG+TCTP(-/-),high glucose group(HG,40 mmol/1 glucose),HG+TCTP(-/-),Then the expression of α-SMA,ZO-1,PI3K,p-AKT,p-mTOR,TSC2 proteins in each group was measured by Western blot.The expression of TCTP mRNA was detected by PCR,and ZO-1 was detected by immunofluorescence.a-SMAprotein expression.Animal experiments:Male DBA/2J mice were injected intraperitoneally with streptozotocin(STZ)40 mg/kg to establish a diabetic model.The mice were divided into four groups:normal control group,normal control group + TCTP(-/-)transfection group,diabetes group,and diabetes +TCTP(-/-)transfection group.Urine samples and kidney tissue collected after 6 weeks,Immunohistochemical staining was used to observe the expression of TCTP protein in each group.Immunofluorescence staining was used to observe the expression of Synaptopodin and TCTP protein.Morphological changes of podocyte were observed by electron microscopy.Results:1.High glucose-stimulated TCTP mRNA and protein expression in podocytes:Compared with LG,the expression of TCTP mRNA in podocytes of HG group was significantly higher(P<0.05),and the expression of TCTP mRNA peaked at 6 hours.Compared with LG,the expression of TCTP protein in podocyte of HG group was significantly increased(P<0.05),and the TCTP protein expression peaked at 24 hours.2.Relationship between the expression of TCTP protein and podocyte slit diaphragm protein:Compared with LG group,the expression of SD protein such as nephrin and ZO-1 was significantly decreased in HG group(P<0.05).Compared with HG group,the expression of SD protein such as nephrin and ZO-1 in HG+TCTP(-/-)group increased(P<0.05).3.The effect of TCTP protein on podocyte EMT:Compared with LG group,the expression of ZO-1 protein in podocyte was significantly decreased and the expression of a-SMA protein was significantly increased in the HG group(P<0.05).Compared with HG group,the expression of ZO-1 protein increased and the expression of a-SMA protein decreased in podocyte in HG+TCTP(-/-)group(P<0.05).4.The effect of TCTP on the expression of EMT related proteins:Compared with LG group,the expression ofp-mTOR,PI3K and p-Akt protein in HG group was significantly increased,and the expression of TSC2 protein was significantly decreased(P<0.05).Compared with HG group,the expression of p-mTOR,PI3K and p-Akt decreased and the expression of TSC2 protein increased in HG+TCTP(-/-)group(P<0.05).5.Mouse glomerular TCTP protein expression:Compared with the normal control group,the expression of TCTP protein in DM group increased in podocytes in diabetic nephropathy.6.Morphological changes of glomerular podocytes:In the normal control group,the podocyte foot processes are intact,whereas in the DM group,the foot processes are extensively fused,part of the podocytes fall off,and the basement membrane is exposed.In the DM+TCTP(-/-)transfection group,there was only partial foot process fusion,and the morphological changes were milder than those in the diabetic group.Conclusion:1.The expression of TCTP in podocyte was increased under high glucose condition;2.Inhibiting the expression of TCTP protein can improve the high glucose-mediated SD cell protein damage;3.Inhibition the expression of TCTP protein can improve the high glucose-mediated podocyte EMT process;4.TCTP mediates podocyte EMT through the PI3K-AKT-mToR signaling pathway. |
