The Study On High Uric Acid Induces Endothelial Dysfunction Through HMGB1/RAGE Signaling Pathway

Background and objective:With the improvement of people’s living standard and the changes in people ’s diets,the morbility of hyperuricemia has showed an obvious rising trend in the world.Numerous epidemiological studies suggested that the levels of serum uric acid has a close connection with cardio-cerebral vascular diseases.Vascular endothelial dysfunction is the initiating link of cardio-cerebral vascular diseases.However,the effect of uric acid(UA)on vascular endothelial function is in dispute.High mobility group box 1(HMGB1)is an important inflammatory factor which is lately found,studies have showed that UA can activate endothelial cells and lead to the translocation and release of HMGB1,extrace llular HMGB1 can bind to its receptor and activate NF-κB signalling,which resulted in the release of proinflammatory cytokines and sustained the inflammtory response.Receptor for advanced glycation end products(RAGE)is a high affinity receptor of HMGB1,which expressed in a variety of cells,and imp lacated in many kinds of chronic diseases.So we deduce that HMGB1/RAGE signaling pathway may be involed in the process of UA-induced vasscular endothelial dysfunction.In this study,UA was used to interfere with human umbilical vein endothelial cells(HUVECs),and then observe the activation of HMGB1/RAGE/NF-κB signaling pathway.Then pretreat HUVECs with anti-RAGE antibody before UA stimulate,which can partly block the interaction between HMGB1 and RAGE,and observe the changes of UA-induced activation of HMGB1/RAGE system,the activation of NF-κB signaling and the release of proinflammatory mediates in endothelial cells.To analyze the mechanism of HMGB1/RAGE signaling pathway in UA-induced endothelial dysfunction,and to provide experimental data for the treatment of UA-induced endothelial dysfunction.Methods:Divided cells into different groups after cultivated with normal-glucose medium.The concentration gradient group was divided into five groups: 1、control group,2、5mg/dl UA group,3、10mg/dl UA group,4、20mg/dl UA group,5、30mg/dl UA group,intervention for 24 hours;the time gradient group was divided into four groups : stimulate HUVECs with 20mg/dl UA for 0,12,24,48 hours.Then extract cells RNA and protein,extract the cytoplasm and nuclear protein with nuclear-plasma protein extraction reagent,extract extracellular HMGB1 protein with the Millipore’s ultrafiltration centrifuge tube,real time PCR was used to analyze RAGE、HMGB1 and ICAM-1、VCAM-1 m RN A expression,western blot was used to observe the expression of cells RAGE、HMGB1、e NOS、ICAM-1、VCAM-1 and extracellular HMGB1、cytoplasm and nuclear NF-k B protein;Collect the cell culture supernatant,detect the content of NO with nitrate reduction method and analyze the expression of inflammation factors of IL-6 and TNF-α with ELISA.Adopt CCK-8 test to detect the effect of UA on cell viability and select the suitable intervention concentration and time.Further backward verificate the role of HMGB1/RAGE system play in UA induced endothalial dysfunction.Divided cells into four groups:1、control group,2、control Ig G group,3、20mg/dl UA+ control Ig G group,4、20mg/dl UA+anti-RAGE antibody group.Real time PCR was used to analyze RAGE、HMGB1and IC AM-1、VCAM-1 mRNA expression,western blot was used to detect the expression of cells RAGE、HMGB1、e NOS、ICAM-1、VCAM-1 and extracellular HMGB1、cytoplasm and nuclear NF-k B protein;N itrate reduction method was used to detect the content of NO;ELISA was used to analyze the expression of IL-6 and TNF-α.Result:1.e NOS protein expression and NO level in high UA group significantly lower than control group(P<0.01)in HUVECs,which showed that high concentration of UA can cause endothelial dysfunction.2.RAGE、HMGB1 mRNA expression significantly higher than control group(P<0.05),RAGE protein expression significantly higher than control group(P<0.05),but with the time increased,until 48 hour,HMGB1 protein expression decreased(P<0.01),instead extracellular HMGB1 level increased(P<0.01),which showed that high concentration of UA induced overexpression of RAGE and HMGB1,at the same time promote the release of HMGB1 protein.3.Nuclear mobility of NF-κB in high UA group significantly higher than control group(P < 0.01),and adhesion molecule ICAM-1 and VC AM-1 expression significantly higher than control group(P<0.01)and cytokine IL-6 and TNF-α level in high UA group significantly higher than control group(P<0.01).4.Anti-RAGE antibody can partly block the interaction between HMGB1 and RAGE.The activation of HMGB1/RAGE system partly supressed in anti-RAGE antibody pretreated group compared with high UA group(P<0.05),eNOS protein expression and NO level in anti-RAGE antibody pretreated group significantly higher than high UA group(P<0.01),nuclear mobility of NF-κB in anti-RAGE antibody pretreated group significantly lower than high UA group(P<0.01),as well as the adhesion molecule ICAM-1、VC AM-1 and cytokine IL-6、TNF-α(P<0.01).Conclusions:The results of this study suggest that,high concentration of UA can induce vascular endothelial dysfunction,which maybe through stimulating endothelial cells overexpression of RAGE,while promoting the release of HMGB1 protein,which bind to RAGE receptor and then activate HMGB1/RAGE signaling pathway,and further induced the activation of NF-κB,then induce the overexpression of proinflammatory mediates and lead to the development of inflammation reaction,utimately resulted in the endothelial function.This provide some experimental datas for the mechanism about UA-induced endothelial dysfunction.