Time-effect Of Damage On Human Umbilical Vein Endothelical Cell Lines Induced By Salt Load And Its Mechanism

Objective:This study was conducted to investigate the time-effect of damage and mechanism on human umbilical vein endothelial cells (HUVECs) induced by high salt, in order to elucidate the role of salt in the development of cardiovascular disease which its endothelium function was obstructed such as hypertension.Methods:Vitro cultured human umbilical vein endothelial cell line, choose3-9generation used in the experiment. HUVECs were cultured in RPMI1640containing salt (NaCl final concentration increased by30mmol/L) at different times(6h,12h,24h,48h)as the test group; D-Hank’S fluid, with the same volume of test group, instead of NaCl, were added in the cell culture medium and cultured for48h as the blank control group; Cultured cells with equal milliosmol of mannitol instead of NaCl for48h, and detect the various indicators to exclude experimental confounding factors. Morphological change of cells was observed by H-E staining; The activity of cell growth was detected by colorimetry reaction with cell counting kit-8; The transudation content of LDH was observed by colorimetry reaction; The nitrite content of the culture medium was measured by nitrate reductase method; Biochemical method to detect the activity of NOS; The protein expression of eNOS and iNOS of HUVECs was detected by immunocytochemistry approach respectively; The mRNA expression of eNOS and iNOS of HUVECs was detected by RT-PCR approach respectively. Experimental data are expressed as mean±standard deviation, use the SPSS19.0statistical software for statistical analysis.Results:Salt load induced cell morphological change, inhibited the activity of cells, increased the transudation content of LDH on HUVECs in a time-dependent manner; Salt load increased the level of nitric oxide(NO) in the culture medium in a time-dependent manner, reaching the top at24hours after stimulation; Salt load inhibited the activity, protein and mRNA expression of eNOS, and induced the activity, protein and mRNA expression of iNOS both in a time-dependent manner, and the highest inducing effects appeared at24hours after stimulation. The detections of NO, LDH, eNOS and iNOS were unaffected in the presence of equal milliosmol of mannitol, which excluded osmotic effect.Conclusions:1、Salt load can directly induce injury on human umbilical vein endothelial cells.2、The mechanism of damage on human umbilical vein endothelial cell induced by salt load may be that:inhibiting the activity and expression of eNOS to decrease physiologic activity of NO, resulting in endothelial cell damage; Meanwhile, inducing the activity and expression of iNOS to increase the content of pathological NO, causing the emergence of metabolic disorder of NO and increasing the content of peroxynitrite, which aggravating the injury on endothelial cells.3、Salt load induce damage on endothelial cells through the approach mentioned above, which may be one of the mechanisms of salt-induced hypertension.