Cardiovascular Pharmacology
Cardioprotection of salidroside from ischemia/reperfusion injury by increasing N-acetylglucosamine linkage to cellular proteins

https://doi.org/10.1016/j.ejphar.2009.04.012Get rights and content

Abstract

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. In the present study, we reported the cardioprotection of salidroside from ischemia and reperfusion. 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 uM) was added 24 h before ischemia/reperfusion was induced. Treatment with salidroside markedly improved cell viability from 64.7 ± 4.5% to 85.8 ± 3.1%, decreased lactate dehydrogenase (LDH) release from 38.5 ± 2.1% to 21.2 ± 1.7%, reduced cell apoptosis from 27.2 ± 3.2% to 12.2 ± 1.9%, significantly improved cardiomyocytes glucose uptake by 1.7-fold and increased O-GlcNAc levels by 1.6-fold, as well as reducing cytosolic Ca2+ concentration compared to untreated cells following ischemia/reperfusion. Furthermore, the improved cell survival and the increase in O-GlcNAc with salidroside were attenuated by alloxan, an inhibitor of O-GlcNAc transferase. These results suggested that salidroside significantly enhances glucose uptake and increases protein O-GlcNAc levels and this is associated with decreased cardiomyocytes injury following ischemia/reperfusion.

Introduction

There is growing recognition that the O-linked N-acetylglucosamine (O-GlcNAc) on serine and threonine residues of nuclear and cytoplasmic proteins is a highly dynamic posttranslational modification that plays a key role in signal transduction pathways. Numerous proteins have been identified as targets of O-GlcNAc modification including kinases, phosphatases, transcription factors and metabolic enzymes. Studies have shown that cells exposed to various stress stimuli exhibit elevated O-GlcNAc levels and this promotes cell survival (Yang et al., 2006, Zachara et al., 2004). It has recently been reported that preischemic treatment with glucosamine protects against ischemia and reperfusion injury in isolated rat hearts (Norbert et al., 2007) and this protection is associated with elevated levels of O-GlcNAc on nuclear and cytoplasmic proteins. The mechanisms underlying the cardioprotective effect associated with increased protein O-GlcNAc levels have yet to be determined. However, previous studies have shown that it may be mediated partly by changes in Ca2+ homeostasis (Liu et al., 2006, Nagy et al., 2006).

Rhodiola rosea is a popular medicinal plant found in mountains at high altitudes and has long been used in traditional Tibetan medicine system as an adaptogen to enhance the body's resistance to fatigue and to extend human life. Salidroside (p-hydroxyphenethyl-β-d-glucoside, chemical structure shown in Fig. 1), a major active ingredient isolated from the plant R. rosea, has been used in the treatment of diabetes, hypertension, fatigue and hypoxia (Kucinskaite et al., 2004, Wang et al., 2003). Recently, salidroside was reported to be capable of protecting SH-SY5Y cells against hydrogen peroxide-induced cell apoptosis in a dose-dependent manner (Zhang et al., 2007) and protecting the PC12 cells against hypoglycemia/serum limitation-induced cytotoxicity (Yu et al., 2008). Studies demonstrated that salidroside has protective effects on myocardial ischemia/reperfusion injury in isolated rat heart (Yan et al., 2008). However, the mechanisms have yet to be elucidated.

This study worked on protective effects of salidroside on cardiomyocyte ischemia/reperfusion injury. It was found that salidroside treatment attenuated ischemia/reperfusion injury, stimulated glucose uptake and elevated protein O-GlcNAc levels. Furthermore, alloxan, an inhibitor of O-GlcNAc transferase (OGT) (Konrad et al., 2002), markedly decreased the levels of O-GlcNAc and blocked the protection of salidroside.

Section snippets

Materials

Salidroside (purity 99%) was purchased from National Institute for the control of pharmaceutical and biological products (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) was purchased from GIBCO (Auckland, NZ). Alloxan, Hoechst 33342 and [3H]-2-deoxyglucose were purchased from Sigma (St. Louis, MO, USA). Anti-O-GlcNAc antibody CTD110.6 was purchased from Covance (Princeton, NJ, USA). Fluo-3/acetoxymethyl ester (Fluo-3/AM) was purchased from CalbioChem (San Diego, CA, USA). 1- to

Salidroside prevented against ischemia/reperfusion-induced decrease in cell viability

Cardiomyocyte survival following ischemia/reperfusion was assessed by MTT assay (Fig. 2A). It was found that ischemia/reperfusion induced significant decrease in cell viability to 64.7 ± 4.5% as compared to control cells, while salidroside treatment resulted in a significant increase in cell survival, restoring cell survival to 85.8 ± 3.1% at the end of reperfusion. However, treatment with salidroside plus alloxan markedly decreased cell viability to 66.5 ± 3.8%. Our preliminary experiments showed

Discussion

Salidroside has been reported to have a wide range of pharmacological properties, including anti-inflammation, anti-hypoxia, anti-oxidative and hepatoprotective effects (Kanupriya et al., 2005, Kucinskaite et al., 2004). This study aimed to explore the protective effects of salidroside against ischemia/reperfusion injury in cardiomyocytes. In the present study, treatment of cardiomyocytes with salidroside significantly improved cell viability, decreased LDH release, attenuated cells apoptosis,

Acknowledgement

The authors are grateful to Dr. Xin L. Ma (Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, USA) for language editing.

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