The Effect And Mechanism Of Protein Binding Uremic Toxins On Vascular Endothelial Cells

Objective:Chronic kidney disease（CKD）has become one of the most important public health problems endangering human health.More and more evidence indicated that cardiovascular events are the main cause of death for CKD population.Although multiple mechanisms are involved,more and more evidence shows that vascular endothelial cell damage is an important pathological characteristics for atherosclerosis and cardiovascular disease in patients with end stage renal disease.Recent studies demonstrated that the accumulation of uremic toxins in uremic patients is an important cause of cardiovascular events,among which protein-binding uremic toxins belongs to binding serum albumin.To date,more than 20 kinds of toxins have been found,most of which have a small molecular weight,but their molecular weight increases when they are in the binding state,which leads to difficulties in dialysis clearance.Accordingly,when uremic toxin is combined with serum protein,the protein structure changes,and it is easy to compete with the drug for binding sites,and at the same time,it will form a pair of droplets,which will cause severe damage to the body.So,in the uremic microenvironment,the effect of protein-binding uremic toxins on vascular endothelial cells remains poorly understood.Therefore,this study is intended to explore the effect of protein-bound uremic toxin on vascular endothelial cells in uremic patients,and analyze the exact mechanism of action affecting vascular endothelial cells in details.This study has very important clinical significance for finding new strategies for the treatment of cardiovascular diseases and improving the prognosis of uremic patients.Methods:Forty-two uremic patients with long-term maintenance hemodialysis and 31 healthy subjects from our hospital were selected.Ten ml of venous blood was extracted from uremic patients and healthy subjects,respectively,and the whole blood was centrifuged at low temperature to prepare serum protein powder.Human umbilical vein endothelial cells（HUVECs）were randomly divided into five groups:blank control group,normal group 1,normal group 2,uremia group 1 and uremia group 2.Normal saline,1%normal serum protein,2%normal serum protein,1%uremia serum protein and 2%uremia serum protein were respectively cultured.The cells of the five groups were incubated for 24,48and 72 hours,respectively.In this study,the cell proliferation ability（CCK8）,cell apoptosis rate（flow cytometry）,inflammatory factor（ELISA）in cell supernatant,oxidative stress（ELISA）in cell supernatant,endothelial cell contraction factor（ELISA）levels and PI3K/AKT/NF-κB signal protein expression（Western blot）were examined.Results:1.With the extension of time,the OD490 value（cell proliferation）of vascular endothelial cells in blank control group,normal group 1,normal group 2 and uremia group 1 showed an increasing trend,with p_time<0.001,indicating that there was statistical significance in the data difference at each time point.At 24 h,48 h and 72 h,OD490（cell proliferation）of vascular endothelial cells in uremic 1 group and uremic 2 group was lower than that in blank control group,normal 1 group and normal 2 group,OD490 of uremic 2 group was lower than that in uremic 1 group,p_group<0.001,indicating that there was statistical difference between groups at each time point.p_time*group>0.05,indicating that the time factor does not vary with different groups.2.With the extension of time,the apoptosis rate of blank control group,normal group 1,normal group 2,uremia group 1 and uremia group 2 showed a gradual increase trend,p_time<0.001,indicating that there was statistical significance in the data difference at each time point.At 24 h,48 h and 72 h,the apoptosis rate of uremia group 1 and uremia group 2 was higher than that of blank control group,normal group 1 and normal group 2,and apoptosis rate of uremia group 2 was higher than that of uremia group 1 and p_group<0.001,indicating that there was statistical difference between the groups at each time point.p_time*group<0.05,indicating that time factors vary with groups.3.With the prolongation of time,there was no significant difference in the levels of inflammatory factors（TNF-αand IL-1β）in the supernatant of blank control group,normal group 1 and normal group 2.Inflammatory factors（TNF-αand IL-1β）increased gradually in uremia group 1 and uremia group 2,with p_time<0.001,indicating that there was statistical significance in the data differences of each time point between uremia group 1 and uremia group 2.The levels of inflammatory factors（TNF-αand IL-1β）in the supernatant of uremia group 1 and uremia group 2 were higher than those of the blank control group,the normal group 1 and the normal group 2,and the levels of inflammatory factors（TNF-αand IL-1β）in uremia group 2 were higher than those of uremia group 1,and p_group<0.001,indicating that there were statistical differences between the groups at each time point.p_time*group<0.05,indicating that time factors vary with groups.4.With the prolongation of time,there was no significant difference in anti-oxidative stress factor SOD and oxidative stress MDA levels of cell supernatant among the blank control group,the normal group 1 and the normal group 2,and oxidative stress MDA in the uremia group 1 and the uremia group 2 increased gradually,anti-oxidative stress factor SOD decreased gradually,p_time<0.001,which indicated that there was statistical significance in the data difference of each time point between the uremia group 1 and the uremia group 2.Oxidative stress MDA level in the supernatant of uremia group 1 and uremia group 2 was higher than that of blank control group,normal group 1 and normal group 2,anti-oxidative stress factor SOD level was lower than that of blank control group,normal group 1 and normal group 2,and oxidative stress MDA level of uremia group 2 was higher than that of uremia group 1,anti-oxidative stress factor SOD level was lower than that of uremia group 1,p_group<0.001,which indicated that there was statistical difference between the groups at each time point.p_time*group<0.05,indicating that time factors vary with groups.5.With the prolongation of time,there was no significant difference in the level of ET-1 and no in the supernatant of the blank control group,the normal group 1 and the normal group 2.The level of ET-1 and no in the supernatant of the uremia group 1 and the uremia group 2 increased gradually,and the no decreased gradually.p_time<0.001indicated that there was statistical significance in the data difference of each time point between the uremia group 1 and the uremia group 2.The level of ET-1 in the supernatant of uremia group 1 and uremia group 2 was higher than that of blank control group,normal group 1 and normal group 2,the level of no was lower than that of blank control group,normal group 1 and normal group 2,the level of ET-1 in uremia group 2 was higher than that of uremia group 1,the level of no was lower than that of uremia group 1,p_group<0.001,which indicated that there was statistical difference between the groups at each time point.p_time*group<0.05,indicating that time factors vary with groups.6.With the prolongation of time,there was no significant difference in the levels of PI3K,p-Akt/Akt and NF-κB p-p65 between the blank control group,the normal group 1and the normal group 2,and the number of PI3K,p-AKT/Akt and NF-κB p-p65 vesicles in the uremia group 1 and the uremia group 2 increased gradually,with p_time<0.001,indicating that there was statistical significance in the data differences of each time point between the uremia group 1 and the uremia group 2.The levels of PI3K,p-AKT/Akt and NF-κB p-p65 in uremia group 1 and uremia group 2 were higher than those in blank control group,normal group 1 and normal group 2,normal group 1 and normal group 2,an the levels of PI3K,p-AKT/Akt and NF-κB p-p65 in uremia group 2 were higher than those in uremia group 1,with p_group<0.001,indicating that there were statistical differences between the groups at each time point.p_time*group<0.05,indicating that time factors vary with groups.Conclusion:Protein binding uremic toxins play an important role in vascular endothelial cells,which can inhibit endothelial cell proliferation,promote apoptosis,promote the release of inflammatory factors,enhance oxidative stress,and lead to vasomotor dysfunction.More interestingly,we demonstrated that protein binding uremic toxins may promote vascular endothelial cells injury through PI3K/AKT/NF-κB signal pathway,which provides theoretical evidence for finding new strategies for the treatment of cardiovascular diseases and improving the prognosis of uremic patients.