Cardioprotection Of Salidroside From Ischemia/reperfusion Injury By Increasing N-acetylglucosamine Linkage To Proteins In Vitro

Objective: The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is increasingly recognized as an important posttranslational modification that modulates cellular function. Recent studies suggested that augmentation of O-GlcNAc levels increase cell survival following stress. Salidroside, one of the active components of Rhodiola rosea, shows potent anti-hypoxia property and protective effects on myocardial ischemia/reperfusion injury in isolated rat heart. However, the mechanisms have yet to be elucidated. In the present study, we reported the cardioprotection of salidroside from ischemia and reperfusion and discussed the relationship between protective effect of salidroside and O-GlcNAc levels.PartⅠ:Protective effects of salidrosides via increased protein O-GlcNAc on ischemia and reperfusion injury in cardiomyocytesMethods: Cardiomyocytes were isolated from 1-to 2-day-old Sprague- Dawley rats. Within 2 days of isolation, cardiomyocytes were exposed to 4 h of ischemia and 16 h of reperfusion, and then cell viability, apoptosis, glucose uptake, ATP levels and cytosolic Ca2+ concentration were determined, and O-GlcNAc levels were assessed by Western blotting. Salidroside (80 M), salidroside (80 M) plus alloxan (2.5 mM) were added 24 h before ischemia/reperfusion were induced.Results: Compared to untreated cells following ischemia/reperfusion, treatment with salidroside markedly improved cell viability from 64.7±4.5% to 85.8±3.1%, decreased LDH release from 38.5±2.1% to 21.2±1.7%, reduced cell apoptosis from 27.2±3.2% to 12.4±1.9%, reduced cytosolic Ca2+ concentration from 147.2±10.5% to 121.9±9.9%, increased ATP content from 17.9±2.8 mol/g protein to21.3±3.5 mol/g protein, as well as significantly improving cardiomyocytes glucose uptake by 1.7-fold and increasing O-GlcNAc levels by 1.6-fold. However, alloxan, an inhibitor of O-GlcNAc transferase that should block the formation of O-GlcNAc, markedly decreased cell viability to 66.5±2.9%, increased LDH release to 39.3±1.6%, increased cell apoptosis to 26.2±2.4%, elevated cytosolic Ca2+ concentration to 138.5±9.2%, and reversed the increase in O-GlcNAc levels seen with salidroside to 98.8±9.8%. Conclusions: Salidroside significantly improved cell viability, decreased LDH release, attenuated cells apoptosis, as well as attenuating cytosolic Ca2+ elevation, stimulating glucose uptake and increasing O-GlcNAc levels compared to untreated cells following ischemia/reperfusion. Alloxan prevented salidroside-induced increase in O-GlcNAc and also blocked the protective effects of salidroside. These findings suggested that cardioprotection of salidroside was associated with enhanced glucose uptake and increased protein O-GlcNAc levels. PartⅡ:Cardioprotective effects of salidroside with increased O-GlcNAc on ischemia and reperfusion injury rat hearts in vitroMethods: Rat hearts were excised and perfused at a constant pressure of 75 mm Hg with KHB equilibrated with 95% O2 and 5% CO2, and then were subjected to 30 min of global, no-flow ischemia followed by 30 min of reperfusion. There were three experimental groups (n=6):①I/R groups: ischemia and reperfusion;②Salidroside groups: salidroside (80 M) added 20 min before the start of ischemia;③Alloxan groups: salidroside (80 M) plus alloxan (2.5mM), an inhibitor of OGT, added 20 min before the start of ischemia. Cardiac function, ATP content, LDH release, cardiomyocyte apoptosis and ultrastructural alterations were examined.Results: There was no significant difference in contractile function among three groups before ischemia. After 30min of ischemia and 30min of reperfusion, salidroside significantly increased cardioal functional recovery compared with the I/R group. The recovery rates of LVDP in I/R groups, salidroside groups and alloxan groups were 76.4±5.2%, 73.8±5.2% and 85.9±4.7%, respectively; The recovery rates of +dP/dt were 75.2±6.3%, 72.9±5.8% and 88.2±6.1%, respectively; The recovery rates of -dP/dt were 79.1±4.4%, 74.4±7.2% and 87.4±6.8%, respectively. Treatment with salidroside markedly reduced cell apoptosis from 21.3±3.5% to 9.5±2.6%, significantly increased O-GlcNAc levels by 1.9-fold compared to untreated hearts following ischemia/reperfusion. However, alloxan, an inhibitor of O-GlcNAc transferase, blocked the increase of O-GlcNAc induced by salidroside, markedly increased cell apoptosis to 19.8±3.1%. Electron microscopy studies demonstrated better cardiomyocyte structure and mitochondrial integrity in salidroside group than those in I/R group and alloxan group.Conclusions: Salidroside showed protective effects from ischemia and reperfusion injury in perfused rat isolated hearts. It increased hemodynamic recovery rate, attenuated cardiomyocytes apoptosis, and increased O-GlcNAc levels. Alloxan prevented the salidroside-induced increase in O-GlcNAc and also blocked the protective effects of salidroside. Salidroside cardioprotection from ischemia and reperfusion injury in perfused rat hearts was associated with increased O-GlcNAc.