Effects Of Sodium Hydrosulfied On Primary Human Umbilical Vein Endothelial Cells Cultured With High Glucose

[Objective] Hydrogen sulfide (H2S) is the third endogenous gaseous signal molecule besides nitric oxide and carbon monoxide, H2S has protective effect on endothelial cells and can reduce endothelial injury. Endothelial injury is the basis of diabetic vascular disease. Among many impairment endothelial factors, over high level of glucose is one of the main factors. High glucose toxicity can cause endothelial injury, which is the important reason of diabetic vascular diseases. Up to date, there is little research on whether exogenous H2S can ameliorate the damage by high glucose on endothelial cells. In this study, primary human umbilical vein endothelial cells (HUVECs) were cultured and identified by the presence of von Willebrand factor, and then we aimed to observe the effect of sodium hydrosulfide (exogenous doner of H2S) on HUVECs viability and the expression of cystathionine-γ-Iyase (CSE) when the cells cultured with high glucose.【Methods】0.125% trypsin and 0.01% EDTA perfusion method was used to isolate HUVEC, and then the gained cells were cultured in M199 medium containing 10% fetal bovine serum (FBS) in the 37℃,5% carbon dioxide incubator. All experiments were performed using cells bwteen passages 2 to 3.(1)When HUVECs reached 70% fusion, morphological observation were obtained by phase contrast microscope, and HUVECs were identified by detection ofⅧfactor.(2) When the cells reached 70%～80% fusion, medium containing 2% FBS was used instead of 10% FBS. The cells were treated for 72 h with 5.5 mmol/L glucose,25 mmol/L glucose,25 mmol/L glucose with 50μmol/L NaHS and 50μmol/L NaHS. MTT method was used for cells viability. Protein level of CSE was measured by western blot.[Results] (1)HUVECs could adhere to the plate and were monolayer, and they were small group distributed on the frist day, after 3 to 5 days, HUVECs were shaped like paving stone. And the cytoplasm of HUVECs were yellowish-brown identified by theⅧfactor, while the negative group were not.(2) MTT showed that compared to 5.5 mmol/L glucose group, cells viability of 25 mmol/L glcouse decreased 28.37%(P<0.05), while 50μmol/L NaHS with 25 mmol/L glucose group rose 30.3%(P<0.05) compared to 25 mmol/L glucose group, and there was no significant difference between 50μmol/L NaHS group with 5.5 mmol/L glucose group.(3) Western blot showed that 25 mmol/L glcouse could reduce the protein expression of CSE compared to 5.5 mmol/L glucose and 50μmol/L NaHS with 25 mmol/L glucose group could ameliorate this change that the expression of CSE protein was enhanced. Relative quantitative analysis of CSE showed that the expression of CSE protein in 25 mmol/L glucose group decreased 53.7%(P<0.05) compared to 5.5 mmol/L glucose group, while 50μmol/L NaHS with 25 mmol/L glucose group rose 19%(P<0.05) compared to 25 mmol/L glucose group.[Conclusion] (1) 0.125% trypsin and 0.01% EDTA perfusion method was feasible to obtain HUVECs and provided a reliable cell model.(2) High glucose could lower cells viability and decrease the expression of CSE in HUVECs, and NaHS could ameliorate the effect caused by high glucose.