Effects of high glucose and insulin on the mRNA expressions of TGF-β1 , and TIMP-1 of hepatic stellate cells in rat / 中国医师杂志

Objective To explore the mechanism of diabetic hepatic fibrosis by observing effects of high glucose and insulin on expressions of transforming growth factor beta 1 (TGF-β1) and tissue inhibitor of metalloproteinase (TIMP-1) mRNAs of hepatic stellate cell (HSC) in rat.Methods Hepatic stellate cell lines in vitro were administrated by different dose of glucose without insulin and glucose with insulin for 72 h,and mannitol was chosen as hyperosmosis control group.Real time fluorescent quantitation polymerase chain reaction (RT-FQ-PCR) was used to determine expressions of TGF-β1 and TIMP-1 mRNAs of HSC in each group.Results Expressions of TGF-β1 and TIMP-1 mRNAs of HSC in each group could be detected and showed no significant difference among groups (P ＞ 0.05).However,in general,expression of TGF-β1 mRNA in glucose with insulin group decreased and TIMP-1 mRNA increased.Conclusions The mRNA expressions of TGF-β1 and TIMP-1 in HSC could not be induced by high glucose alone,high dose of insulin may increase expressions of HSC TIMP-1 and reduce TGF-β1 expression.The main mechanisms of diabetic hepatic fibrosis may not be TGF-β1 pathway but be closely related to TIMP-1 pathway.

Effects of glucose on apoptosis of endothelial progenitor cells from human peripheral blood / 中国医师杂志

Objectives To investigate the effects of glucose on apoptosis rate of cultured endothelial progenitor cells (cEPCs). Methods The peripheral blood of healthy adults was isolated by density gradient centrifugation, and mononuclear cells (MNCs) were inducted to differentiate at cultured conditions.EPCs were identified by Dil-acLDL and FITC-UEA-1 as double fluorescent-positive cells. The effectsof glucose at different concentrations on apoptosis rate of the harvested EPCs were measured by fluorescent microscope and fluorescence-activated cell sorting (FACS) after staining with Annexin V-FITC and PI. Results No significant differences were observed in apoptosis rate between samples treatedwith 5.6mmol/l glucose and 11. 1 mmol/l. P ＞0. 05). 25.5 mmol/L glucose enhanced the EPCsapoptosis rate in a time-dependent manner( P ＜0. 05). Conclusion High concentration glucose can accelerate apoptosis rate of EPCs in a time-dependent manner.

Effects of high levels of glucose on the expression of adiponectin receptors in human kidney proximal tubular cells / 中国医师杂志

Objective To investigate the effect of in vitro high glucose stimulation on the expression of adiponectin receptor (adipoR) in human kidney proximal tubular cells.Methods The HK-2 cells were cultured in the low glucose DMEM culture medium containing 10% fetal bovine serum until the cells were adherent and 80% confluence. After cultured in the serum-free DMEM for 24 hours, these cells were stimulated with glucose-containing 1mg/ml, 2mg/ml, 4mg / ml, 6mg/ml, 8mg/ml serum-free DMEM for 48 hours. Then RT-PCR and western blot were used to analyze adipoR ( R1, R2) expression levels. The HK-2 cells were cultured respectively in high glucose (4mg/ml) , low glucose (1mg/ml) DMEM culture medium containing 10% fetal bovine serum to cultivate 0h, 12h, 24h, 48h, 72h, 96h, then RT - PCR was applied to analyze adipoR (R1, R2) mRNA expression levels semi-quantitatively. Results Two kinds of adiponectin receptor gene were both expressed in HK-2 cells, and the quantity of gene expression of adipoR1 (0. 63 ±0. 12) was 3. 9 times to adipoR2 (0. 16 ±0.03) , the difference was statistically significant ( P<0. 01). The different concentrations of glucose and different time of high glucose on HK-2 cells had no significant effect ( P>0. 05 ) on adipoR gene expression. Expression of adipoR 1 protein in HK-2 cells was detected by western blot, and it was not affected by glucose concentration ( P>0. 05).Conclusion adi-poR1 and adipoR2 gene were both expressed in HK-2 cells, and the adipoR1 was the major one, which suggested that adipoR1 played a more significant role in kidney disease. The expression of adipoRl/R2 of HK-2 cells was not affected by high glucose concentration.

The effect of hyperglycemia on the expression of Toll-like receptor 4 in renal tubular epithelial cells / 中国医师杂志

Objective This study was designed to explore the effect of hyperglycemia on the expression of Toll-like receptor 4 (TLR4 ) in renal tubular epithelial cells and its significance in diabetic nephropathy. Methods In vitro cultured renal tubular epithelial cells ( NRK-52E) were divided into LG group (cultured in 5mmol / L glucose DMEM) and HC group (cultured in 25mmol / L glucose DMEM). Cells were harvested at different time points. Immunohistochemistry, Rt-PCR, Western Blot were used to detect TLR4 protein and mRNA expression, and the levels of IL-6 and TNF-α from the cell culture supernatant were determined by EL1SA assay. Results After 6 hours, there was increased expression TLR4 mRNA in HC group, which appeared to be maintained for 24 hours and began to decrease after 48 hours ( P < 0.05). TLR4 protein expression increased in HC group after 24 hours, and increased even further after 48 hours. Compared with LG groups, the difference had statistical significance ( P <0.05). In HG group, IL-6 and TNF-α expression in the supernatant from the NRK-52E culture were significantly increased ( P < 0.05) , and the expression of IL-6 and TNF-α was positive correlated with the expression of TLR4 protein ( r =0.799,0.820). Conclusion High glucose triggers an increase in expression of TLR4 in NRK-52E cells, itself leading to an increase in expression of inflammatory factors such as TNF-α and IL-6. In this way, TLR4 participates in the progress of diabetic nephropathy.

Effect of high glucose on the role of fibronectin, collagen Ⅳ and platelet activating factor of mesangial cells / 中国医师杂志

promoted by high glucose, which can enlarge the biological effect of PAF.

High glucose stimulate collagen Ⅰ synthesis in renal tubular epithelial cells via activated TGF-β/Smad signaling pathway / 中国医师杂志

Objective To investigate the molecular mechanisms that high glucose stimulate collagen Ⅰ synthesis in renal tubular epi-thelial cells. Methods Normal rat pwximal tubular epithelial (NRK52E) cells were cultured grown in RPMI-1640 medium and were divid-ed four groups: mannitol group, high glucose group, high glucose + neutralizing TGF-β1 antibody group, high glucose + IgG1 group. TGF-β1 in the supematant of cultured cells were measured by enzyme-linked immunosorbent assay (ELISA). Nuclear expression of p-Smad2/3 were examined by immunocytochemistry. Expression of collagen Ⅰ mRNA was detected by RT-PCR. Expression of collagen Ⅰ pro-tein was detected by Western blot. Results High glucose up-regulated the expression of collagen Ⅰ mRNA by time-dependent manor. Com-pared with mannitol group, high glucose markedly increased the level of TGF-β1 in supernatant of cultured cell after 24h and 48h and upreg-ulated p-Smad2/3 nuclear expression(t =4. 2, t = 3.25, P <0.01). Neutralizing TGF-β1 antibody inhibited high glucose- induced p-Smad2/3 nuclear expression, downregulated high glucose-induced collagen Ⅰ mRNA and protein expression(t = 3.12, t =3.02, P < 0.01). Conclusion High glucose stimulated collagen Ⅰ synthesis in renal tubular epithelial cells via activated TGF-β/Smad signaling path-way.