Effects And Mechanism Of Uric Acid On Vascular Endothelial Cells

Uric acid(UA),as an end product of purine metabolism in humans,is one of the major antioxidants in plasma and is effective against oxidative stress caused by reactive oxygen species.However,in human evolution,due to the loss of the uricase gene,UA in human blood is about 10 times higher than in other mammals.It is defined as hyperuricemia when the human serum uric acid concentration is higher than 6.8 mg/dl.Hyperuricemia is associated with pathological processes of various cardiovascular diseases,such as hypertension,coronary heart disease,and atherosclerosis.Among them,hyperuricemia is closely related to hypertension.Some experts tend to regard hyperuricemia,or UA higher than physiological concentration,as an independent risk factor of hypertension.Hypertension,as a systemic disease,is closely related to the inflammatory state of blood vessels.Mechanisms of hyperuricemia involved in hypertension may be associated with the pro-inflammatory role of hyperuricemia,and the related mechanisms of which are including endothelial cell activation and dysfunction,oxidative stress and smooth muscle cell proliferation induced by uric acid.However,due to insufficient study on related mechanisms,there is still no conclusive evidence to support the use of uric acid-lowering drugs for the treatment of hypertension.High mobility group box-1(HMGB1),as an alarmin,is released during both infectious and sterile inflammation and activate the immune system to respond to cell damage.The NLRP3 inflammasome is a tripartite cytosolic complex formed of three proteins NLRP3,ASC and pro-CASP1,whose main role is to recognize non-microbial risk signals and induce a aseptic inflammation.Activation of the NLRP3 inflammasome requires two distinct signals.These two signals consist of a priming signal to induce transcription of both NLRP3 and pro-IL-1β,and a second signal that induces oligomerisation of the inflammasome,which cleaves up-regulated pro-IL-1β into matured IL-1β and releases it extracellularly to induce an inflammatory response.Is UA able to induce the release of HMGB1 in the blood vessels,and activate the NLRP3 inflammasome to induce vascular inflammation,thereby participating in the formation of hypertension?From this point of view,we propose research approach:A model of hyperuricemia-induced hypertension was established in rats to investigate whether hyperuricemia induces the release of HMGB1 and activates NLRP3 inflammasome in vascular.At the same time,in vitro studies were carried out to explore whether UA induces vascular inflammation by activating the HMGB1 and NLRP3 inflammasome of ECs and VSMCs,thereby participating in the formation of hypertension.Through the research,we are trying to provide a theoretical basis and new targets for new antihypertensive drugs.1.Mild hyperuricemia was developed by feeding oxonic acid(OA)to rats,and blood pressure was measured in rats.The results showed that hyperuricemia significantly upregulated systolic blood pressure in rats;The expression of HMGB1 in blood vessels and renal tissues were analyzed by immunohistochemistry.The results showed that hyperuricemia induced up-regulation of HMGB1 expression and release of HMGB1 into the cytoplasm in endothelial cells and renal tubular epithelial cells;The expression of NLRP3 in blood vessels and renal tissues was analyzed by immunohistochemistry.The results showed that hyperuricemia induced up-regulation of NLRP3 expression in blood vessels and kidneys;2.In vitro,real-time quantitative PCR was used to analyze the effects of UA on mRNA levels of inflammatory cytokine in human umbilical vein endothelial cells(HUVECs).The results showed that soluble UA induced HUVECs to express a variety of cell adhesion molecules and chemokines;3.Real-time quantitative PCR was used to analyze the effects of UA on the level of nitric oxide synthase(eNOS)mRNA in HUVECs.The results showed that soluble UA significantly down-regulated the expression of eNOS in HUVECs and possibly induced EC dysfunction;4.The effects of UA on cytoplasmic ROS levels in HUVECs and human aortic smooth muscle cells(HASMCs)was examined by a reactive oxygen species(ROS)assay kit.The results showed that soluble UA induced up-regulation of ROS levels in HUVECs and HASMCs;5.Immunofluorescence staining was used to analyze the effects of different concentrations and stimulation time of UA on the release of HMGB1 in HUVECs and HASMCs.The results showed that UA could not induce the release of HMGB1 from the nucleus at different concentrations and stimulation time;The effects of UA on the mRNA level of RAGE,an HMGB1 receptor,in HUVECs was analyzed by real-time quantitative PCR.The results showed that there was no significant change in RAGE mRNA levels;Real-time quantitative PCR and Western blot were used to analyze the effects of different concentrations and stimulation time of UA on HMGB1 mRNA level and protein expression in HUVECs and HASMCs.The results showed that UA stimulation from 6h to 48h and from 50μg/ml to 300μg/ml did not significantly affect the mRNA and protein levels of HMGB1;6.Immunofluorescence staining was used to analyze the effects of soluble UA on NF-κB,a crucial protein in the pathways of HMGB1 receptors.The results showed that the expression of NF-κB was not upregulated and there was no obvious nuclear import;7.Real-time quantitative PCR and Western blot were used to analyze the effects of different time and different concentrations of UA on the NLRP3 inflammasome priming in HUVECs and HASMCs.The results showed that UA stimulation from 6h to 48h and from 50μg/ml to 300μg/ml had no significant effect on NLRP3 inflammasome priming;The effects of soluble UA on the NLRP3 inflammasome activation in HUVECs was examined by immunofluorescence staining.The results showed that UA induced the formation of NLRP3-ASC complex in HUVECs.Our study successfully established a hyperuricemia-induced hypertensive animal model in vivo,and our findings support that hyperuricemia induced up-regulation of HMGB1 expression and release of HMGB1 into the cytoplasm in blood vessels and kidneys and induced up-regulation of NLRP3 expression,suggesting that UA may activate HMGB1 and NLRP3 inflammasome in vivo,thereby participating in the formation of hypertension.In vitro,soluble UA induces endothelial cell activation and production of ROS.However,UA had no significant effect on HMGB1 and did not induce NLRP3 inflammasome priming.Although UA induced the formation of NLRP3-ASC complex in HUVECs,it had no significant effect on IL-1β production.Therefore,in vitro,UA is unable to induce the activation of NLRP3 inflammasome.However,it cannot be ruled out that UA induces activation of NLRP3 inflammasome under the synergistic effect of other factors.In addition,in this study,the results of in vivo experiments are quite different from human cell experiments,which may be due to the differences in species and microenvironments in vivo and in vitro.