Abstract
Catalpol, an iridoid glucoside, has been reported to inhibit apoptosis of neuron and endothelial cells. In the present study, we investigated the mechanism of catalpol-mediated cardioprotection. The rat embryonic ventricular myocardial cell line (H9c2) cells were first incubated with catalpol, and then exposed to hydrogen peroxide (H2O2). The concentration of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were all determined by using commercially available kits. Apoptotic cells were assessed by Hoechst 33258 and Annexin V-fluorescein isothiocyanate binding assay. Synthesis of Bcl-2, Bax, cytochrome c and caspase-3 were analysed by real-time semiquantitative reverse transcription-PCR and Western blotting. We observed that apoptosis in H9c2 was associated with increased Bax, cytochrome c, caspase-3, decreased Bcl-2 activity after 24h of H2O2 exposure. Catalpol pretreatment afforded a marked protection against the above H2O2-mediated cytotoxicity and apoptosis in H9c2 cells. Moreover, the catalpol pretreatment led to a great reduction in H2O2-induced MDA release and increased SOD. These findings indicated for the first time that pretreatment of H9c2 cells with catalpol can be against H2O2-induced apoptosis, and the protective effect of catalpol involves the mitochondrial-dependent caspase pathway and is associated with increased Bcl-2 and decreased Bax expression.
Highlights
Apoptosis or programmed cell death plays a key role in the maintenance of homoeostasis of both normal development and many pathologic conditions
Effect of catalpol on the viability of H9c2 exposed to H2O2 The results in Figures 1(A) and 1(B) showed that incubation of H9c2 cells with 100 μM H2O2 resulted in a dramatic decline of cell viability and Lactate dehydrogenase (LDH) levels, which decreased to 64.90 +− 7.01 % and 665.69 +− 217.07 unit/l respectively
Effect of catalpol on cellular superoxide dismutase (SOD) content, MDA level in H9c2 exposed to H2O2 As shown in Figures 2(A) and 2(B), compared with the control (17.34 +− 3.58 unit/mg · protein), treatment of H9c2 cells with 100 μM of H2O2 for 24 h caused significantly less activities of Effect of catalpol on apoptosis in H9c2 exposed to H2O2 Round-shaped nuclei with homogeneous fluorescence intensity is shown in the control group (Figure 3A)
Summary
Apoptosis or programmed cell death plays a key role in the maintenance of homoeostasis of both normal development and many pathologic conditions. Oxidative stress is a well-known factor that promoted apoptosis [1], and implicated in the pathophysiology of a number of cardiovascular disorders such as ischaemia-reperfusion injury [2], atherosclerosis [3], chronic heart failure [4], hypertrophy [5] and hypertension [6]. Modification of apoptotic pathways to attenuate cardiomyocyte damage induced by myocardial oxidative stress is a major area of clinical interest. The first iridoid glycoside isolated and identified from the radix of Rehmannia glutinosa Libosch, has been shown to have many important and extensive pharmacological action, including hypoglycaemic [7], diuretic [8], anti-cancer [9], antispasmodic, anti-inflammatory [10] and anti-apoptotic [11], based on in vitro and in vivo pharmacodynamic experiments. The effects of catalpol on the oxidative stress have not been elucidated in cardiac muscle cells
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