ABSTRACT
Objective Saxagliptin regulates the level of blood glucose by selectively inhibiting high-performance dipeptidyl peptidase 4, but its action mechanism is not yet clear .This study was to investigate the effect of the novel hypoglycemic agent Saxaglip -tin on the expression of long non-coding RNA (LncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and its target gene products transforming growth factor-β1 ( TGF-β1 ) in human umbilical vein endothelial cells ( HUVECs) stimulated by high glucose. Methods HUVECs were cultured in with D-glucose (D-GS) at the concentrations of 5.5, 10, 20, and 30 mmol/L and Saxagliptin at 0, 0.01, 0.1, 1, and 10μmol/L.The best concentrations of D-GS and Saxagliptin were determined as 30 mmol/L and 1 μmol/L, respectively.The HUVECs were divided into four groups:control (5.5 mmol/L D-GS), Saxagliptin (5.5 mmol/L D-GS+1 μmol/L Saxagliptin ) , high glucose ( 30 mmol/L D-GS ) , and high glucose +Saxagliptin (30 mmol/L D-GS +1μmol/L Saxaglip-tin), all cultured for 24 hours.Then the expressions of MALAT1 and TGF-β1 mRNA in the cells were detected by qRT-PCR, that of the TGF-β1 protein determined by Western blot , and the level of TGF-β1 in the supernatant measured by ELISA . Results The expressions of LncRNA-MALAT1 and TGF-β1 were significantly increased in the high glucose group as compared with the control ( 8.65 ±0.70 vs1.00 ±0.00 and 1.36 ±0.07 vs 1.00 ±0.00, P<0.01) but markedly inhibited in the high glucose +Saxagliptin group in compari-son with the high glucose group (2.17 ±0.24 vs 8.65 ±0.70 and 1.15 ±0.02 vs 1.36 ±0.07, P<0.05). Conclusion High glu-cose can induce the overexpression of LncRNA-MALAT1 and its target gene products TGF-β1 in HUVECs and cause damage to the cells, while Saxagliptin can significantly suppress this effect .
ABSTRACT
Objective To investigate the in vitro effects of Treponema pallidum membrane protein Tpp17 on the permeability of endothelial barrier for further investigation on the immunopathogenesis of syphi-lis.Methods A cellular model of in vitro monolayer was established by using human umbilical vein endo-thelial cells ( HUVECs) .Cell-ELISA and a TMB kit were respectively used to measure the expression of VE-cadherin and the flux of horseradish peroxidase ( HRP) by monolayer HUVECs after stimulation with the re-combinant Tpp17 (rTpp17) protein.THP-1 cells stained with Calcein AM were added to the top of HUVEC monolayer in Transwell culture.Then, the numbers of THP-1 cells in the upper wells and beneath the HUVEC monolayer were counted by using a fluorescence microscope.The rTpp17 protein-treated HUVECs were fixed in 4%buffered paraformaldehyde and stained with rhodamine-phalloidin for observing the distri-bution of F-actin under a confocal laser scanning microscope.Results Compared with the control group, the expression of VE-cadherin in HUVECs was decreased, while the permeability of HUVEC monolayer was increased upon the stimulation with rTpp17 protein (P<0.05).Moreover, rTpp17 protein-induced F-actin redistribution and increased transendothelial migration of THP-1 cells were observed in rTpp17 protein-trea-ted HUVECs as compared with those of the control group (P<0.05).Conclusion Treponema pallidum membrane protein Tpp17 could suppress the expression of VE-cadherin and enhance the redistribution of F-actin, resulting in an enhanced transendothelial migration of THP-1 cells and an increased permeability of HUVEC monolayer.The Tpp17 protein might play an important role in the immunopathogenesis of syphilis.
ABSTRACT
Objective To determine the effect of Vibrio vulnificus cytolysin (VVC) inducing ap-optosis in human umbilical vein endothelial cell(HUVEC) and its possible mechanism. Methods The en-tire vvhA gene that encoding VVC from V. vulnificus strain GTC333 was amplified by PCR and sequenced af-ter T-A cloning. E. coli BL21DE3pET-42a-vvhA, a prokaryotic expression system of the vvhA gene, was then con-structed. Ni-NTA affinity chromatography was applied to purify the target recombinant protein rVVC, and SDS-PAGE plus Bio-Rad Agarose Image Analyzor were used to measure the output of rVVC and to determine the purity of rVVC extract. The activity of rVVC dissolving rabbit erythrocytes was detected by hemolysis test. DPNH chromotometry and TphBNa chromotometry were performed to examine the contents of LDH and K+ in the supernatants of rVVC-treated HUVEC cultures, respectively. The effect of rVVC inducing apepto-sis of HUVEC was detected by flow cytometry, rVVC was labeled with FITC and the location of FITC-labe-ling rVVC in HUVEC was observed by laser canfocal microscopy. Results The cloned whA gene had 96.09% and 98.26% similarities of nucleotide and amino acid sequences compared to the corresponding se-quences in GenBank. rVVC, with a dosage of 1 μg/ml, could dissolve rabbit erythrocytes (P<0.01). 10 μg/ml rVVC was able to promote the increases of K+ content (P<0.01) but no change of LDH content could be found in the cell supernatants. HUVEC was apoptotic after the cell was treated with 1～100 μg/ml of rVVC for 2 h. In the 5～240 min duration of co-incubation of FITC-labeling rVVC and HUVEC, the rV-VC gradually moved from surface to inner side of the membrane and then entered the cytoplasms. When FITC-labeling rVVC treated HUVEC for 30 min, most of the rVVC was found to be intracellular location. Conclusion rVVC has cytolytic activity. VVC has an ability to enter HUVEC and causes injury of HUVEC via inducing apoptosis, which may be the major pathogenic mechanism of VVC.