Uric Acid In Human Umbilical Vein Vascular Endothelial Cell Function In Vitro

Background.Uric acid is the product of metabolism of purine;however,a relatively higher level of serum uric acid in human is observed than that in any other mammals since a genetic mutation is detected in human genomes.The level of serum uric acid is reported to be associated with many systemic diseases such as vascular and metabolic diseases in recent years.The incidences of cardiovascular disease and metabolic diseases are related to the elevation of serum uric acid in general population,reported by clinical and epidemiological researches.However,different and diverse opinions rise from laboratory researches on the effects of serum uric acid on the endothelial function.Some reported that the endothelial dysfunction could be induced by inhibiting the NO production by hyperuricemia and then initiate the process of arthrosclerosis. Another group of research indicated that it was ROS,which was generated as a by-product from xanthine by xanthine oxidase during the production of uric acid, caused the endothelial dysfunction.A third opinion thought the serum acid can erase the ROS and thus regarded as a protective factor in maintaining the endothelial function.Finally,another group of study indicated that a lower level of serum uric acid could provide protective effect on vascular endothelium while a higher level of serum uric acid could accelerated the destruction of vascular endothelial,especially when it had already been injured.Objective.To determine the production of ROS and the expression of eNOS mRNA in Human Umbilical Vein Endothelial Cells(HUVEC) stimulated by uric acid solution with stratified concentrations for a certain duration in vitro,in order to study the effects on endothelial dysfunction by uric acid with different concentrations.Methods.MTT test for cell viability on HUVEC stimulated by uric acid of stratified concentrations in different time point.Confocul Fluorescent Microscopy scanning for ROS production in HUVEC after a 48h co-culture in uric acid at stratified concentrations.Flow Cytometry test for ROS production in HUVEC after a 48h co-culture in uric acid at stratified concentrations.RT-PCR amplification for the test and comparison of eNOS mRNA expression in HUVEC after a 48h co-culture in uric acid at stratified concentrations.Real-Time PCR amplification and detection for the comparison of eNOS mRNA expression in HUVEC stimulated by uric acid of stratified concentrations.Results.HUVEC viability had no significant decrease with the increase of uric acid(0,5,10,20,30mg/dL) in 8h and 12h;however,significant differences among groups of different uric acid concentrations were discovered after 24h and 48h culture of HUVEC.The mean intensity of ROS expression in HUVEC in vitro after 48h co-culture with uric acid was significantly positively related to the concentration of uric acid applied.The increase of eNOS mRNA production in HUVEC in vitro after 48h co-culture with uric acid was significantly positively related to the increase of uric acid concentration.Conclusion.In this study,viability of HUVEC in vitro had significant difference between groups after 24h and 48h co-culture with uric acid of different concentrations.ROS expression increased with the elevation of exogeneous uric acid concentration in HUVEC in vitro,indicating that endothelial function may be impaired by uric acid in a higher concentration.eNOS mRNA production incrased with the increase of exogenous uric acid concentration.A response as an antioxidant reaction against the activation of ROS should be taken into concern.