Crocetin attenuates norepinephrine-induced cytotoxicity in primary cultured rat cardiac myocytes by antioxidant in vitro

Abstract

This study aims to investigate the protective role of crocetin, a natural antioxidant, against cytotoxicity produced by exposure to norepinephrine (NE) in primary cultured rat cardiac myocytes. Reactive oxygen species (ROS) and Ca²⁺ in cells were evaluated by fluorescence microplate reader using 6-carboxy-2′,7′-dichlorofluorescein and fluoro-3-acetoxymethyl ester, respectively. Lipid peroxidation was quantified using thiobarbituric acid-reactive substances. The activities of superoxide dismutase (SOD) and contents of glutathione (GSH) were detected by xanthine/xanthime oxidase-mediated ferricytochrome c reduction assay, and recycling effection of glutathione disulfide with GSH reductase and NADPH, respectively. The apoptotic cells were assayed by fluorescein diacetate (FDA)–ethidium bromide (EB) two-staining method. Intracellular accumulation of ROS, Ca²⁺, and products of lipid peroxidation resulting from NE were significantly reduced by crocetin. Preincubation of primary cultured rat cardiac myocytes with crocetin remarkably prevented the decrease in SOD activity and quantities of GSH induced by NE. The percentage of NE-induced apoptosis in the cells was decreased by FDA–EB two-staining assay after pretreated with crocetin. The results showed that crocetin may ameliorate NE-induced injury in cardiac myocytes by enhanced SOD activity and increased quantities of GSH, decreased lipid peroxidation and Ca²⁺ in cells, and apoptosis death ratio that may represent the cellular mechanisms for its cardioprotective role.

Keywords:

• crocetin
• norepinephrine
• cardiac myocytes
• intracellular Ca²⁺
• apoptosis
• oxidative stress

Acknowledgements

This research was supported by a grant from National Natural Science Foundation of China (No. 30701024), High-ranking Intellectual Special Assistant Foundation of Guizhou Province (No. TZJF-2006-13), Science and Technology Foundation of Guizhou Province (No. 2007-2133), Provincial Key Technologies R&D Program of Guizhou (No. 2007-1035), and Jiangsu Postdoctoral Science Foundation (No. 0801036B). The authors are grateful to Prof. Wen-Yong Huang for valuable comments on the manuscript.