The Effection And Mechanism Of Hydrogen Protecting Advanced Glycation End Products-induced Endothelial Cell Apoptosis

Background and ObjectiveIt is known that, type1and type2diabetes alike, with the gradual progression of thelong-term high blood glucose levels, can cause different degrees of complications invarious target organs, among which the cardiovascular disease is the most common andthreatening[1]. At present, the number of patients with type2diabetes has amounted to morethan150million worldwide, and is expected to grow exponentially in the next20years[2].Patients with diabetes-induced cardiovascular disease will significantly increase in number,leading to a heavier global medical burden. How to control cardiovascular complications isthe focus of current research.In fact，cardiovascular complications in diabetes mellitus are one of the leading causesof mortality. Vascular endothelial injury and dysfunction are the important earlypathological manifestations of these complications.[31]Increasing apoptosis is an earlymanifestation of endothelial injury, which will lead to endothelial dysfunction[32].Advancedglycation end products (AGEs) are the products of non-enzymatic glycosylation of theamino groups of macromolecules, such as proteins and nucleic acids[33]. In diabetic patients,sustained high blood glucose level significantly increases the production of AGEs[33].Epidemiological studies show that the presence of AGEs is highly correlated with diabeticcardiovascular complications. Previous studies have found that AGEs increase endothelialcell apoptosis and dysfunction[34]. In addition, according to our previous study, AGEsincrease apoptosis and dysfunction of endothelial precursor cells[35].The generation ofintracellular reactive oxygen species (ROS) increases in AGEs-induced cells, and oxidativestress and ROS production, in turn, contribute to AGEs-induced apoptosis of endothelialcells and endothelial precursor cells[36].As we know，removal of AGEs from the body is difficult. One strategy to reduce AGEs-induced endothelial injury is to antagonize AGEs-activated oxidative stress[32].Hydrogen (H2) is the smallest naturally occurring gas molecule. Studies have shown thatH2has antioxidant activities in living organisms. It can specifically neutralize the mostpotent oxidative free radicals (OH and ONOO-) and attenuate the superoxidant anion levelunder certain pathophysiological conditions[37]. Moreover, it is easy for H2to pass throughmembrane structures such as cell membranes and the mitochondrial membranes, where itcan neutralize intracellular ROS, thereby maintaining normal mitochondrial function andpreventing apoptosis[38]. Many studies have shown that H2or hydrogen-containing solutioncan alleviate ischemia-reperfusion injury[34.36.39]of the heart[40],brain[41.42], kidney, smallintestine, and liver[41]; and it can antagonize irradiation-induced cell injury[43]. Inhalation ofH2also slows down the growth of atherosclerotic plaque in apoE-/-mice[44]. Our previousstudies also found that hydrogen-rich saline prevented neointima formation after carotidballoon injury[45]. Oxidative stress plays an important role in ischemia-reperfusion injury,irradiation-induced injury, atherosclerosis and neointima formation[46]. The ability of H2toantagonize these pathological reactions is closely related to its anti-ROS effects. However,it has yet to be elucidated whether H2can ameliorate AGEs-induced ROS generation andapoptosis of endothelial cells.The objective of the present study was to determine whether the use of hydrogen-richmedium (HRM) can protect the endothelial cells from AGEs-induced apoptosis by detectingROS production and antioxidant-related gene expression.Materials and MethodsTwo to three year old Sprague-Dawley rats purchased from the Experimental AnimalCenter of the Third Military Medical University were used in this study. The thoracic aortawas removed from these rats, and endothelial cells (ECs) were isolated and cultured. Afterculturing ECs in the presence of AGEs and/or with HRM for24h, Annexin V/7-AAD andTUNEL staining were carried out to detect apoptosis. Intracellular ROS was detected withfluorescent probes and quantified with flow cytometry. Anti-oxidative enzyme (SOD andGSH-PH) expression was determined by real-time PCR analysis and enzymatic assay. Therelative expression levels of Bcl-2and Bax were analyzed by western blotting. Results and Discussion1. AGEs induced ECs apoptosis in a concentration-dependent manner. After24h,evaluation of the4EC groups, including AGEs (400μg/ml) group, HRM group, HRM+AGEs group, and normal control group, revealed that the cell apoptosis rates were notsignificantly different between the HRM group and the control group when AGEs wereabsent. However, the presence of AGEs increased the rate of ECs apoptosis, and theaddition of HRM reduced the apoptosis rate of AGEs-treated ECs.2. Treatment of ECs with AGEs significantly increased the generation of intracellularROS in a dose-dependent manner. However, HRM decreased significantly.3. AGEs significantly reduced the level of SOD mRNA. However, after HRMintervention, the SOD expression reduction caused by AGEs was significantly attenuated.AGEs treatment significantly increased the optical density of anti-oxidative enzymes. HRMtreatment was also able to partly reduce this effect.4. ECs co-cultured with AGEs showed a decrease in the Bcl-2/Bax ratio. ECsco-cultured with AGEs+HRM were shown to attenuate the AGEs-induced decrease in theBcl-2/Bax ratio.This indicate that endothelial cell apoptosis induced by AGEs occurs partlythrough the mitochondrial apoptotic pathway.ConclusionAGEs induce ECs apoptosis and oxidative stress in a concentration-dependent manner.HRM can antagonize the apoptosis and oxidative stress induced by AGEs. It plays arole in anti-oxidative stress and apoptosis by increasing SOD, GSH-PH and Bcl-2(inhibitorof apoptosis protein)/Bax (apoptosis-promoting protein) ratio and reducing ROS levels.