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Objective:To investigate the regulation effect of endogenous nicotinamide phosphoribosyl transferase (Nampt) on the Vimentin expression of glomerular cells in high concentration glucose,and to clarify the mechanism of formation of diabetic kidney inflammation fibrosis.Methods:The C57/BL6 diabetic mice were selected and the kidney tissues were collected,and the wild C57/L86 mice were used as control group;the pathological section and tissue fluorescence staining were performed.The expression and location of endogenous Nampt and Vimentin in the glomerular cells were detected by immuno-focused technology.The HBZY-1 cells were randomly divided into 4 groups:low concentration of glucose (LG,0.56 mmol · L-1) control group,high concentration glucose (HG,200 mmol · L-1) group,HG +-FK866 group and HG+nicotinamide mononucleotide (NMN) group.In HG group,the cells were treated with FK866 (10 μmol · L-1) and NMN (1 mmol · L-1) for 24 h after cultured with HG for 5 d.The expression levels of Nampt,Vimentin,nuclear factor-kappa B p65 (NF-κBp65) and sirtuin type 1 (Sirt1) were detected by immunofluorescence and Western blotting methods.The expression levels of Nampt and Vimentin were detected by RT-PCR and Western blotting methods.Results:The shape and size of glomerulus had obvious atrophy of the mice in severe diabetic group compared with normal C57/BL6 mice group.The expression level of Vimentin in glomerular cells was increased with the increasing of endogenous Nampt (P＜0.01).When the HBZY-1 cells were cultured in HG condition,the exprssion levels of Nampt,Vimentin and NF-kB p65 were obviously increased while the Sirt1 expression levels was significantly decreased compared with control group (P＜ 0.01).The expression levels of Nampt,Vimentin and NF-κB p65 in glomerular cells in HG+FK866 HG+ NMN groups were singnificartyly decreased compared with control group (P＜0.01).Conclusion:The endogenous Nampt over-expression in glomerular cells can enhance the expression of Vimentin under high concentration of glucose stress through NF-κBp65 and Sirt1 signal pathway.
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Objective:To investigate the regulation effect of endogenous nicotinamide phosphoribosyl transferase (Nampt) on the Vimentin expression of glomerular cells in high concentration glucose,and to clarify the mechanism of formation of diabetic kidney inflammation fibrosis.Methods:The C57/BL6 diabetic mice were selected and the kidney tissues were collected,and the wild C57/L86 mice were used as control group;the pathological section and tissue fluorescence staining were performed.The expression and location of endogenous Nampt and Vimentin in the glomerular cells were detected by immuno-focused technology.The HBZY-1 cells were randomly divided into 4 groups:low concentration of glucose (LG,0.56 mmol · L-1) control group,high concentration glucose (HG,200 mmol · L-1) group,HG +-FK866 group and HG+nicotinamide mononucleotide (NMN) group.In HG group,the cells were treated with FK866 (10 μmol · L-1) and NMN (1 mmol · L-1) for 24 h after cultured with HG for 5 d.The expression levels of Nampt,Vimentin,nuclear factor-kappa B p65 (NF-κBp65) and sirtuin type 1 (Sirt1) were detected by immunofluorescence and Western blotting methods.The expression levels of Nampt and Vimentin were detected by RT-PCR and Western blotting methods.Results:The shape and size of glomerulus had obvious atrophy of the mice in severe diabetic group compared with normal C57/BL6 mice group.The expression level of Vimentin in glomerular cells was increased with the increasing of endogenous Nampt (P＜0.01).When the HBZY-1 cells were cultured in HG condition,the exprssion levels of Nampt,Vimentin and NF-kB p65 were obviously increased while the Sirt1 expression levels was significantly decreased compared with control group (P＜ 0.01).The expression levels of Nampt,Vimentin and NF-κB p65 in glomerular cells in HG+FK866 HG+ NMN groups were singnificartyly decreased compared with control group (P＜0.01).Conclusion:The endogenous Nampt over-expression in glomerular cells can enhance the expression of Vimentin under high concentration of glucose stress through NF-κBp65 and Sirt1 signal pathway.
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AIM:To study the expression of glycine receptorα1 subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptorα1 subunit in the neonatal rat myocardial cells.METHODS:Neonatal rat myocardial cells were cultured in vitro.The expression of glycine receptorα1 subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100 μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h.Subsequently, the cell viabil-ity was measured by CCK-8 assay, and the expression of glycine receptorα1 subunit was determined by Western blotting. RESULTS:The expression of glycine receptor α1 subunit in the neonatal rat myocardial cells was positively detectable by Western blotting.Compared with control group, no significant difference of the cell viability ( P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed.The expression of glycine receptor α1 subunit was in-creased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION:Glycine receptorα1 subunit exists in the neonatal rat myocardial cells.A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptorα1 subunit, but HG down-regulates the expression of glycine receptor α1 subunit in cultured neonatal rat myocardial cells.
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Objective To observe insulin synthesis and secretion in INS-1 under high glucose, and to clarify the effect of PTH1R. Methods After successful construction of recombinant PTH1R-siRNA vectors in INS-1 cell, insulin secretion and intracellular insulin content of control group, siPTH1R-Negative control group, PTHrP group, and siPTH1R group under 25 mmol/L glucose were measured by radioimmunoassay in INS-1 cell. Intracellular calcium were detected by Fluo-3/AM and the capability of glucose transport was calculated by assaying the uptake of [3H]-2-deoxy-D-glucose in cells.Results Compared with control group, and siPTH1R-NC group, PTHrP group showed increased capability of insulin secretion; PTHrP group had higher intracellular insulin levels than others; PTHrP group showed increased intracellular calcium; the uptake of [3H]-2-deoxy-D-glucose under high glucose after 48h of PTHrP group was increased(all P＜0.01). Conclusion There is a close relationship between PTH1R activation and insulin secretion and synthesis, PTH1R activation may be one of the protective mechanisms in maintaining function of β-cell under high glucose.
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AIM: To explore the effect of Snail1 siRNA on high-glucose induced tubular epithelial-to-mesenchymal transition (TEMT). METHODS: Subconfluent renal tubular epithelial cells were incubated in serum-free DMEM for 24 h to arrest and synchronize the cell growth. Then cells were treated with normal glucose (5.5 mmol/L D-glucose) or high glucose (25 mmol/L D-glucose) for 72 h. Meanwhile 19.5 mmol/L D-manntiol was used as high osmotic control. Snail1 siRNA was transfected into tubular epithelial cells. In parallel, cells were transfected with non-specific siRNA which served as the control data sets. Cells were then treated with 25 mmol/L D-glucose for 72 h. RNA and cell lysates were collected to determine the protein and mRNA levels of Snail1, TGF-β_1, α-SMA, vimentin and E-cadherin. RESULTS: Transfection caused the decreases in Snail1 at mRNA and protein levels by 62% and 68% respectively as compared to those in untransfected cells cultured in high glucose medium. Western blotting exhibited that Snail1 siRNA transfection restored E-cadherin protein expression by 61% compared to that in high-glucose-treatment cells, whereas it inhibited high-glucose-induced induction of α-SMA protein by 58%. Similarly, RT-PCR revealed that Snail1 siRNA transfection dramatically suppressed the high-glucose-induced mRNA expressions of α-SMA and vimentin by 72% and 61%, respectively, while E-cadherin mRNA increased by 53%. CONCLUSION: Our study provides direct evidence that Snail1 is able to control TEMT.
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AIM:To investigate the effect of high glucose on the expression of an endoplasmic reticulum stress marker glucose-regulated protein 78(GRP78),and explore its underlying mechanism.METHODS:(1) Human umbilical vein endothelial cells(HUVECs) were exposed to normal glucose(5.5 mmol/L) and high glucose(30 mmol/L) for 24 h,36 h or 48 h.Cell viability was determined by MTT method.Cell apoptosis was detected by flow cytometry analysis.The expression of proteins was evaluated by Western blotting analysis.RESULTS:After treated with high glucose for 24-48 h,the expression of GRP78 increased early but decreased at 48 h of incubation,while cyclooxygenase-2(COX-2) expression increased in a time-dependent manner.COX-2 selective inhibitor nimesulide inhibited high glucose induced changes of GRP78 expression and also inhibited high glucose induced cell apoptosis.CONCLUSION:Prolonged high glucose exposure changes the expression of GRP78 in a COX-2 dependent manner in HUVECs.
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0.05).The expression of glycine receptor ?1 subunit mRNA was increased(P
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AIM:To observe the expression of Smad7 and Smad ubiquition regulatory factor-Smurf2 in rat glomerular mesangial cells (GMC) stimulated by the high concentration of glucose,and to investigate the effect of the ubiquition on Smad signaling by adding MG132 as a proteasome differential inhibitor. METHODS:Cultured rat GMC were divided into normal group (the concentration of glucose:5.6 mmol/L),high glucose group (20 mmol/L,30 mmol/L,respectively),therapy group (30 mmol/L glucose with MG132). The expressions of Smurf2 and Smad7 in each group were measured by indirect immunofluorescence and laser scanning confocal microscope. RESULTS:(1) The expression of Smurf2 in GMC in normal group was weak (25.93?3.35) whereas the expression of Smad7 was strong (64.09?7.43). (2) The expression of Smurf2 in high glucose group was stronger than that in normal group (P
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Objective: To study how to activate transforming growth factor ? 1 (TGF ? 1) by coculturing bovine cerebral microvascular endothelial cells (BCMEC) and bovine cerebral microvascular smooth muscle cells (BCMSMC) in vitro , and to observe the effect of high concentration of glucose on active TGF ? 1 (aTGF ? 1) production by the cocultured cells. Methods: BCMEC and BCMSMC were cocultured, and compared with BCMEC or BCMSMC homotypically cultured. 3H TdR assays were used to measure the bioactivity of aTGF ? 1 in the conditioned media. Dot blots assays were used to evaluate the TGF ? 1 mRNA levels of the cultured cells. Then, the cocultured cells were cultured with 5.5 (normal level), 15 and 25(high level) mmol/L glucose for 24 h. The activity of aTGF ? 1 in the media was measured by the same way. Results: aTGF ? 1 was produced in the media of cocultured cells, but not in the media of homotypically cultured cells. Activity of aTGF ? 1 in the media with high concentration of glucose was significantly higher than that in the media with normal concentration of glucose. The dot blots assays implicated that both BCMEC and BCMSMC had TGF ? 1 mRNA expression. But there was no significant change after coculturing. Conclusion: TGF ? 1 can be activated by contacted coculture of BCMEC and BCMSMC in vitro , but the coculturing does not increase the TGF ? 1 mRNA expression. Cultured with high concentration of glucose can increase the aTGF ? 1 content in the media of the cocultured cells. [