Study On The Effect Of P38MAPK Signal Pathway On High Glucose Induced Epithelial-Mesenchymal-Transition Of Human Renal Tubular Epithelial Cell

PartⅠConstruction of pshRNA Expression Vectors targeting p38MAPKObjective: To construct vectors for small interfering RNA (siRNA) targeting P38MAPKMethods: According to the designing principles of shRNA, a reverse repeated sequence of p38MAPK and a srambled siRNA with 9 bp hairpin spacer were designed and synthesized .The complement strain was obtained by annealing and inserting the vector containing green fluorescent protein sequence and U6 promotor to generate the shRNA expression vector targeting p38MAPK. Finally, the recombinant plasmids were identified by enzyme digestion and DNA sequencing analysis.Results: The recombinant plasmids were verified by sequencing analysis. The recombinant plasmids were denominated as pshRNA-p38MAPK1, pshRNA-p38MAPK2 and pshRNA-hk. Conclusions: Construction of the recombinante plasmid targeting the mRNA of p38MAPK gene was successful. The constuction of recombinant plasmid pshRNAp38MAPK will help further study on the role of P38 mitogen-activated protein kinases in high glucose–induced epithelial-mesenchymal transition. PartⅡThe effect of p38 MAPK on high glucose induced epithelial- mesenchymal - transition of human renal tubular epithelial cellObjective: To investigate the effect of p38 MAPK on high glucose induced epithelial- mesenchymal-transition in cultured human renal tubular epithelial cell (HK2).Methods: To investigated the inhibition of p38MAPK expression in human renal epithelial cells by siRNA,the recombinante plasmid were were stably transfected into the HK2 cells using Lipofectamine 2000 reagent.Human renal tubular epithelial cells of the strain HK2 were cultured and divided into 4 groups : (1) normal control group; (2) pshRNA-p38MAPK1 group; (3) pshRNA-p38MAPK2 group; (4) pshRNA-hk group. To further study the effect of p38 MAPK on high glucose induced EMT in cultured HK2 cell.Another HK2 cells were divided into 5 groups as follows: normal glucose group(NG), high glucose group(HG), Random pshRNA-hk transfected group(RG), pretransfected with pshRNA-p38MAPK for 24h group(24hG), pretransfected with pshRNA-p38MAPK for 48h group(48hG).The cell morphological changes were observed by inverted microscope and Electronic microscopy. The phosphorylation and total p38MAPK was assessed by western blotting. Electrophoretic mobility shift assays (EMSA) were performed to determine the activity of AP-1. Fibronectin synthesis were detected by enzyme-linked immunosorbent assay(ELISA).The proteins of E-cadherin, cytokeratin(CK),α-smooth muscle actin(α-SMA) and vimentin were determined by immunocytochemistry. The expressions of CK, vimentin and Snail mRNA were detected by RT-PCR. At the same time, expressions of E-cadherin andα-SMA were measured by Western blotting.Results: The results showed that constructing vector can be transfected effectively in HK2 cells and expressed successfully. SiRNA for p38MAPK inhibited the activation of p38MAPK induced by high glucose. The HK2 cells stimulated with high glucose became elongated and the number of microvilli decreased, while the number of rough endoplasmic reticulum increased. The levels of AP-1 binding activity were increased with the treatment of high glucose, but decreased by transfected with pshRNA-p38MAPK. Secreted fibronectin was significantly up-regulated by the stimulation of high glucose (54.89±2.30ng/ml vs. 11.25±1.31ng/ ml, P<0.05), while the level was markedly decreased after pretreated with pshRNA-p38MAPK. High glucose induced Snail mRNA expression,while the level significantly decreased by transfected with pshRNA-p38MAPK(P<0.05).Upon the stimulation of high glucose,the expression ofα-SMA protein significantly increased (P<0.05),while the the level markedly decreased by transfection of pshRNA-p38MAPK (P<0.05). When cells exposed to high glucose, a markly decrease in E-cadherin and cytokeratin expression were detected after 48 h, while it were significantly increased by transfected with pshRNA-p38MAPK (P<0.05). Cultured with high glucose, the levels of vimentin significantly increased , but reduced after pretransfected with pshRNA-p38MAPK(P<0.05).Conclusions: p38MAPK may play an important role in high glucose-induced EMT characterized by gain ofα-SMA and vimentin expression, loss of E-cadherin and CK expression in tubular epithelial cells. High glucose induces the expression of Snail by the activation of p38MAPK. The expression of Snail can downregulate E-cadherin expression and induce EMT. The siRNA-mediated down-regulation of p38 expression inhibited EMT and fibronectin synthesis induced by high glucose treatment. At the same time, this study suggests high glucose could induce the activation of AP-1 mediated by p38MAPK in tubular epithelial cells. However, It remains unclear that whether the activation of AP-1 take part in EMT induced by high glucose. PartⅢThe effect of AP-1 on high glucose induced epithelial- mesenchymal - transition of human renal tubular epithelial cellObjective: To explore the effect of AP-1 on high glucose induced epithelial- mesenchymal- transition in cultured human renal tubular epithelial cell.Methods: The cultured HK2 cells were divided into three groups as follows:normal glucose group (NG), high glucose group (HG), activator protein-1(AP-1) inhibited group (AG). The cell morphological changes were observed by inverted microscope and Electronic microscopy. The activity of activator protein-1(AP-1) was assessed with electrophoretic mobility shift assay (EMSA). Fibronectin synthesis was detected by enzyme-linked immunosorbent assay (ELISA). The proteins of E-cadherin, cytokeratin (CK),α-smooth muscle actin (α-SMA) and vimentin were determined by immunocytochemistry. The expressions of CK,vimentin and Snail mRNA were detected by RT-PCR. At the same time, expressions of E-cadherin andα-SMA were measured by Western blotting.Results: The HK2 cells stimulated with high glucose became elongated and the number of microvilli decreased, but the number of rough endoplasmic reticulum increased.The levels of AP-1 binding activity were increased by 79.22% with the treatment of high glucose, but decreased by 51.19% with the treatment of the inhibitor of AP-1. Secreted fibronectin was significantly up-regulated by the stimulation of high glucose (55.22±2.06ng/ml vs. 9.99±1.68ng/ml, P<0.05), while the level was markedly decreased after treated with AP-1 inhibitor (30.25±0.59 ng/ml vs. 55.22±2.06ng/ml, P<0.05). Upon the stimulation of high glucose,the levels ofα-SMA and vimentin protein significantly increased (P<0.05),while the the level markedly decreased by the treatment of the inhibitor of AP-1(P<0.05). When cells exposed to high glucose, a markedly decrease in E-cadherin and cytokeratin expression were detected after 48 h, while it was significantly prevented by the treatment of the inhibitor of AP-1 (P<0.05).Conclusions: The study shows that activation of AP-1 play a crucial role in high-glucose -induced EMT in tubular epithelial cells.High glucose induces the expression of Snail by the activation of AP-1. The expression of Snail can downregulate E-cadherin expression and induce EMT.