Uric Acid Aggravates Myocardial Ischemia/reperfusion Injury By Activating ROS/NLRP3 Pyroptosis Signaling Pathway

Background Acute myocardial infarction(MI)has been reported as the deadliest complication of coronary atherosclerotic heart disease.Its essence is the imbalance between the oxygen supply and demand of the myocardium,and the ischemic myocardium can present a series of damage in function,form,and metabolism.At present,the most effective treatment method to reduce ischemic injury caused by MI is to restore blood perfusion.After reperfusion,a series of damages in various aspects such as function,structure,metabolism,and electrophysiological characteristics of myocardial cells have been further deteriorated,defined as myocardial ischemia/reperfusion(MI/R)injury.Therefore,exploration of the pathophysiological mechanism of MI/R injury has become an urgent problem that needs to be solved.This is of vital significance for reducing the risk factors of MI/R injury and promoting the recovery of myocardial injury to prolong the prognosis of patients with MI/R.Studies have found that MI/R injury is closely related to pyroptosis.More and more studies have shown that MI/R can activate the oxidative stress in myocardial cells and promote the activation of NOD-like receptor pyrin domain-containing protein 3(NLRP3)and promote the assembly of apoptosis-associated speck-like protein containing a CARD(ASC)and cysteine-containing aspartate specific proteinase-1(pro-cysteinyl aspartate specific proteinase-1,pro-caspase-1)and activated NLRP3 into mature NLRP3 inflammasomes.NLRP3 inflammasome has the ability to cleave Pro-caspase-1 into mature caspase-1,which promotes the activation and maturity of pro-gasdermin D protein(GSDMD)and pro-inflammatory cytokines interleukin 1β and 18(pro-IL-1βand pro-IL-18).GSDMD has the properties of pore-forming cells,and inflammatory factors produce a non-biological inflammatory response,thereby aggravating MI/R injury.Uric acid(UA),a metabolite of purine,has been found by a large number of studies to promote the oxidative stress response of cells and the production of reactive oxygen species(ROS).Related clinical studies have found that patients with high UA have a higher risk of cardiovascular disease and the prognosis after MI is even worse than ordinary people.In recent years,However,the mechanism of UA aggravates MI/R injury has not yet been clearly studied,and the role of the pyroptosis pathway mediated by NLRP3 inflammasomes needs to be further explored to provide valuable ideas for reducing myocardial injury in patients with reperfusion.Objective A mouse hyperuricemia model and a mouse model of myocardial ischemia-reperfusion were performed to observe the effect of high-level serum uric acid on myocardial ischemia-reperfusion injury.Hypoxia/reoxygenation(H/R)model of mice primary cardiomyocytes was established.The most suitable concentration of soluble uric acid was selected to pretreat cardiomyocytes before establish hypoxia/reoxygenation model to explore the effects of uric acid on hypoxia/reoxygenation injury and cell death at cellular and molecular levels.NLRP3 inflammatory body inhibitor and ROS scavenger were used to explore the specific mechanism and relationship between uric acid aggravating cardiomyocytes hypoxia/reoxygenation injury and NLRP3 mediated pyroptosis.Methods Kunming mice,6 weeks,were divided into these groups:(1)sham;(2)MI/R;(3)hyperuricemia + MI/R.Potassium oxonate(PO)suspension was intraperitoneally injected in 2 weeks to establish the hyperuricemia mice model,the solvent was intraperitoneally injected to the other two groups.Two weeks later,the mice were anesthetized and mechanically ventilated and the left coronary artery of the mice was ligated by thoracotomy for 30 minutes,and then untie the line.2 hours later,MI/R model was eatablished.All operations in the sham operation group were the same as those in the myocardial ischemia/reperfusion group except that the left coronary artery was not ligated.After reperfusion,both infarct size and cell death was detected.The intracardiac blood of mice was collected to detect the concentrations of lactate dehydrogenase(LDH),creatine kinase isoenzyme(CK-MB)and UA.The expression of pyroptotic protein were detected.The primary cardiomyocytes of C57BL/6 newborn mice were cultured with multi-concentration gradient of soluble uric acid concentration(0,100,200,300,400 mg / L).The cardiomyocytes viability was detected.The most suitable concentration of soluble uric acid was selected as the concentration of subsequent uric acid stimulation.Five groups:(1)control;(2)H/R;(3)UA + H/R;(4)UA + BAY + H/R;(5)UA + N–acetylcysteine(NAC)+ H/R.The cells were treated with corresponding treatment for 24 hours,then exposed to H/R condition(1% O2 / 94% N2/ 5% CO2)for 4 hours,and then reoxygenated for 2 hours to establish a stable hypoxia/reoxygenation model.After H/R,not only cell viability,AI,LDH,proptotic protein were detected as similar as in vivo,but the ROS level was analyzed by ROS kit.Results Compared to sham group,the infarct size of the mice underwent myocardial ischemia/reperfusion was significantly larger with the high level of LDH and CK-MB.The level of UA in the hyperuricemia mice was statistically different from other two groups which indicating that the establishment of hyperuricemia mouse model was successful.Compared with MI/R group,myocardial infarct size,LDH and CK-MB in hyperuricemia mice were significantly increased after myocardial ischemia/reperfusion.TUNEL fluorescence showed that myocardial cell mortality and apoptosis index(AI)increased.Hyperuricemia significantly aggravated MI/R injury and promoted the expression of pyroptotic signaling pathway proteins.100mg/L was used as the concentration of follow-up experiment.After the cardiomyocytes underwent H/R,cell viability decreased,AI increased,LDH level and ROS level increased,and pyroptotic signaling protein expression level upregulated.UA treatment aggravates the changes of injury indexes after H/R.After treatment with BAY,those changes aggravated by uric were reversed.After apply of ROS scavenger,the level of ROS was significantly decreased with the H/R injury deteriorated by uric were obviously alleviated.Conclusions Hyperuricemia not only promotes the injury and death of myocardial cells in mice after ischemia/reperfusion but also further promotes the expression of pyroptotic protein.Uric acid pretreatment further aggravated the injury of cardiomyocytes underwent hypoxia/reoxygenation,and also promoted the aggravation of pyroptosis,and the mechanism was proved by promoting ROS production,which activates NLRP3 inflammatory bodies and promoting the activation of downstream pyroptosis pathway.