Study Of The Effect And Mechanism Of High Uric Acid On Endothelial Cell Adhesion By Microfluidic Chip Technique

Background: Uric acid(UA)is the final product of human purine metabolism,which mainly related to gout.Many epidemiological studies have shown that high UA is important risk factors for Atherosclerosis(AS).And UA is considered as risk factors of cardiovascular disease,such as hypertension and coronary heart disease.However,the multifactor analysis of some cohort studies disargee this conclusion.Therefore,the correlation between high UA and cardiovascular disease is unclear.These arguments urge us to explore the relationship and mechanism between high UA and cardiovascular diseases.In basic research,the pathophysiological mechanism of vascular injury caused by hyperuricemia is not clear.It is suggested that hyperuricemia can lead to vascular injury and endothelial dysfunction by over-activating renin-angiotensin system(RAS).(Pro)renin receptor [(P)RR] is a new and upstream member of RAS system,whether(P)RR can be activated by UA to induce endothelial dysfunction is not clear.At the beginning of AS,the key is the change of hemodynamics and the adhesion of leukocytes to vascular endothelial cells.Monocytes exhibit stronger deformability than other leukocytes in adhesion and migration.Therefore,to study the effect of endothelial cells on monocyte adhesion and migration is helpful to understand the mechanism of atherosclerosis and to provide more accurate and effective sites for clinical targeted therapy.Whether(P)RR can be activated by UA to induce endothelial dysfunction,increase adhesion,and promote the development of AS is not clear.Therefore,study of the effect and mechanism of high UA on endothelial cell adhesion,it is helpful to understand the mechanism of AS formation.Microfluidic chip technology is used to simulate blood flow state,control fluid station and observate real-time,save cell resources,which has great significance for studying endothelial cell adhesion function.In order to observe the dynamic process of mononuclear cells and endothelial cells adhesion,microfluidic system is used for simulating the the blood flow state.Aims: In this study,the effect and mechanism of high UA on endothelial cell adhesion function were investigated.And in the hemodynamic state,the effects and mechanism of low shear stress(LSS)and high UA on vascular endothelial cells were studied by microfluidic chip techniqueMethods: 1.The primary HUVECs was extracted and intervened by UA at different time points(0h,12 h,24h,36 h,48h)and different concentrations(0 mg/dl,6 mg/dl,9 mg/dl,12mg/dl).2.The most suitable time point and concentration was selected as the optimal condition.The expression of(P)RR and ICAM-1,P-P38 MAPK was detected by western blotting.And the number of adhesion between THP-1 and HUVECs was gauge by Transwell.3.The microfluidic channel was designed to study on the expression of(P)RR and ICAM-1 at LSS on HUVECs.4.In the microfluidic system,the expression of(P)RR and ICAM-1 was observed in LSS and high UA,and the adhesion function and mechanism of monocytes and endothelial cells was studied.Result: 1.The expression of(P)RR was significantly increased with 9mg/dl UA stimulation,compared with other groups.2.The expression of(P)RR increased at 12 th hour,reached the peak at 24 th hour after 9mg/dl UA stimulation,and decreased gradually after 36 hours which was still significantly higher than that of control group.3.Western blot analysis revealed the higher expression of(P)RR and P-P38 MAPK,ICAM-1 in UA(9mg/dl)group,compared with control.After the UA absorption was inhibited by probenecid,the expression of(P)RR and P-p38 MAPK,ICAM-1 decreased.4.Western blot analysis revealed the lower expression of(P)RR and P-p38 MAPK,ICAM-1,when(P)RR si RNA was used,compared with UA groups.5.In the microfluidic chip channel,the expression of(P)RR and ICAM-1 increased more significantly in the low shear stress than that in the static group.6.In the microfluidic chip channel,fluorescence analysis revealed the higher expression of(P)RR and ICAM-1 in UA(9mg/dl)and LSS group.7.In the microfluidic chip channel,LSS and higher UA(9mg/dl)increased the adhesion of THP-1 cells to endothelial cells.Conclusion: 1.(P)RR was expressed abundantly in HUVECs.2.UA increased the expression of(P)RR in a dose and time.When UA concentration was 9 mg/d L and stimulated time was 24 h,the expression of(P)RR was the highest.3.High UA probably activated p38-MAPK signaling pathway by up regulating(P)RR expression and resulting in increased expression of ICAM-1.4.The expression of(P)RR and ICAM-1 in HUVECs were increased in LSS,compared with the static state.5.In the low shear stress,(P)RR and ICAM-1 can be induced by high UA,resulting in the higher number of adhesions between HUVECs and THP-1,which may induces vascular endothelial dysfunction and the development of atherosclerosis.