ABSTRACT
Objective In order to establish a basis for exploring diagnostic biomarkers of type 2 diabetes mellitus (T2DM), our research screened differentially expressed circRNAs in high glucose treated human umbilical vein endothelial cells ( HUVECs) and validated these circRNAs in both cell models and peripheral blood samples of T 2DM patients.Methods The research used HUVECs as experimental model . The control group (n=3) and high glucose group (n=3) were set up and treated under normal condition (5.5 mmol/L) and high glucose condition (30.0 mmol/L), respectively.The research utilized high-throughput sequencing technology to preliminarily screen differentially expressed circRNAs .Differentially expressed circular RNAs were validated in the endothelial cell model by PCR and real -time quantitative PCR ( q-PCR) techniques.Subsequently , in the peripheral blood samples of T 2DM patients ( n=32) and control individuals ( n=28 ), the differentially expressed circRNAs were further validated . Finally, t-Test, correlation analysis and ROC curve were used to analyze the experimental results .Results Total 1087 differentially expressed circular RNAs were screened by high-throughput sequencing;and of these , 554 were up-regulated, 533 were down-regulated.Six increased circRNAs were selected and validated in HUVEC model;and their PCR and q-PCR results matched with sequencing results .Further validation was conducted in peripheral blood samples for three most up-regulated circRNAs.This study found that the differential expression of hsa_circ_0031739 in peripheral blood samples was statistically significant ( 0.015 ±0.0025 vs.0.006 ±0.0013,P=0.0059 <0.05).The area under the ROC curve (AUC) was 0.730 and the expression level of hsa_circ_0031739 was positively correlated with blood glucose ( GLU ) and glycated hemoglobin ( GHb ) ( r=0.317, P=0.0137 <0.05;r=0.348, P=0.0064 <0.05 ) .Conclusion HUVECs were commonly utilized as cell models in the study of T 2DM, and differentially expressed circular RNA profiles existed afterhigh glucose treatment .The differential expression of hsa_circ_0031739 was significant in both high glucose treated HUVECs and peripheral blood samples of T 2DM patients, and the expression level of hsa_circ_0031739 was correlated with GLU and GHb , which has certain diagnostic significance.Therefore, hsa_circ_0031739 may become a new diagnostic biomarker for T2DM and be helpful for the comprehensive diagnosis and mechanism research of T 2DM.
ABSTRACT
Objective To establish cell models with different insulin sensitivity status and examine the expression of protein kinase B (PKB/Akt) in the same conditions. Methods 3T3-L1cells were cultured with different glucose concentrations (3.0, 5.5 and 50.0 mmol/L respectively).Insulin sensitivity, as presented by insulin-induced glucose transport rate in the cells, was tested by 3H-2-DG incorporation. PKB expression in cells was determined by RT-PCR. Results The insulin-induced glucose transport rate in 3T3-L1 cells was affected by different concentrations of glucose. The transport rate of 3H -2-DG in cells cultured in low glucose concentration was increased by 152% and 50% compared with high glucose concentration and control group, respectively. There was no difference in PKB mRNA expression among the three groups under the same conditions,whereas there was an obvious reduction of PKB expression in the three groups after the cells were treated with glucose and insulin. Conclusions 3T3-L1 cells cultured with different concentrations of glucose present the different insulin sensitivity status, but the difference of PKB mRNA expression has not been observed in the same condition.
ABSTRACT
AIM To investigate the inhibitory effect of pycnogenol on generation of advanced glycation end products (AGEs) in vitro. METHODS Advanced glycation end products were determined by fluorospectrophotometer in the medium of 1 mol?L -1 glucose and 5% bovine albumin incubated at 37℃, 50℃, and 70℃ for different times. The inhibitory effect of pycnogenol was confirmed by the same system incubated with or without pycnogenol at different concentrations. RESULTS The rate of generation of AGEs in vitro was related with incubation time and incubation temperature. The generation of AGEs was inhibited by pycnogenol in vitro. The inhibitory rate was 10%～80% dependent on concentration and incubation time of pycnogenol. The inhibitory effect of pycnogenol on generation of AGEs was almost the same as that of Aminoguanidine at the same concentration. CONCLUSION pycnogenol could significantly inhibit generation of AGEs in vitro.