Cardiomyocyte Protection Of Salidroside Against Hypoxiainduced Apoptosis By Up-regulation Of HIF-1α Through PI3K/Akt Signaling Pathway

Cardiomyocyte apoptosis contributes to a variety of heart diseases such as myocardial infarction, cardiomyopathy, progressive pump-failure, arrhythmias as well as cardiac remodeling[1, 2]. Recent reports indicate that a variety of stimuli which are known to participate in the pathogenesis of heart failure have been shown to induce cardiomyocyte apoptosis, including hypoxia[3], ischemia and reperfusion[4], and oxidative stress[5]. Therefore, therapeutic strategies aimed at preventing or delaying cardiomyocytes apoptosis induced by these stimuli might be a reasonable choice for the treatment of related heart diseases. Recently, attention has been focused on searching for substances of cardioprotective potential which can protect the cardiomyocytes from the deleterious stimuli.Salidroside (p-hydroxyphenethyl-β-D-glucoside,,C14H20O7: 300.30), which is extracted from Rhodiola rosea L and has long been used as an adaptogen in traditional Tibetan medicine, has been reported to possess various pharmacological properties including resisting anoxia, anti-aging, anti-cancer, anti-inflammation, antioxidative, hepatoprotective and cardioprotective effects[6-11]. As reported by Maslova et al, salidroside can protect cardiovascular tissue from stress-induced catecholamine release and mitigate against adrenaline-induced arrhythmias in rats. The antiarrhythmic effect of salidroside is supposed to be secondary to the ability to induce opioid peptide biosynthesis, and to be related to the stimulation of petipheal kappa-opioid receptors[12-15]. Nevertheless, the mechanism underlying its cardioprotective effects remains unclear, especially at the cellular level.Therefore, this study was to investigate the protective effect of salidroside against hypoxia-induced cardiomyocytes apoptosis, and furthermore to explore the possible mechanisms involved by detecting the protein levels of HIF-1, VEGF, PI3K and Akt.Aims1. To investigate whether salidroside protects cardiomyocyte against hypoxia-induced apoptosis by preparing primary cultures of neonatal rat cardiomyocyte and mimicing the hypoxia injury in vitro.2. To explore whether salidroside increases the levels of HIF-1 and VEGF, a downstream protein of HIF-1, determine the possible mechanisms involved in the protective effect of salidroside against hypoxia-induced cardiomyocyte apoptosis.3. To determine whether salidroside increases the expression of HIF-1 via PI3K/Akt signaling pathway.Methods1. Primary cultured cardiomyocytes of neonatal rats were prepared and incubated in a hypoxia solution to mimic the hypoxia injury in vitro, and were divided into six groups: control group, hypoxia group, hypoxia+ different concentrations of salidroside pretreatment group (10,50,100μg/mL), and hypoxia+10^-7mol/L insulin group. 2. The cell viability and the activity of LDH,CK were measured by colorimetric method. The morphologic changes in cardiomyocytes were observed under an inverted phase contrast microscope and Hoechst 33258 staining. The apoptosis of cardiomyocytes was detected by flow cytometry and DNA fragmentation.3. The expressions of HIF-1αand VEGF, a downstream protein of HIF-1, were determined by immunofluorescence and Western blot.4. The hypoxia model was the same as that of the first part. 20μM LY294002, a specific inhibitor of PI3K/Akt, was added. Cell viability was assessed by MTT. The apoptosis of cardiomyocytes was detected by Hoechst 33258 staining and DNA fragmentation. The expressions of Akt, phosphorylation of Akt and HIF-1αwere determined by immunofluorescence and Western blotting.Results1. Hypoxia for 1h to 8h induced significant decreases in cell survival in a time-dependent manner. Exposed to hypoxia for 6h, there were only 47.7±10.1% viable cells as compared to the control cells. Therefore, hypoxia for 6h was used as a standard apoptosis induction in the subsequent experiments.2. Compared with Hypoxia group, pretreatment with different concentrations of Sal significantly decreased the morphologic changes in cardiomyocytes, attenuated hypoxia induced cell viability loss and apoptotic cell death (P <0.05) and dramatically decreased the activity of the LDH,CK (P <0.01). The Hoechst staining positive cells were significantly decreased with 100μg/mL salidroside pretreatment (P <0.01). Using flow cytometry, we observed that salidroside decreased cell apoptosis induced by hypoxia in a dose-dependent manner. Pretreatment with 100μg/mL salidroside also attenuated the typical DNA ladder formation induced by hypoxia. In addition, there were no statistically significant differences between 100μg/mL salidroside pretreatment group and 10-7mol/L insulin group whose protective effect of cardiomyocytes against apoptosis has been confirmed (P >0.05).3. Hypoxia activated the expressions of the HIF-1αand VEGF, and induced the translocation of HIF-1α. Pretreatment with 100μg/mL salidroside significantly up-regulated the protein levels of HIF-1αand VEGF.4. Pretreatment with LY294002, a specific inhibitor of PI3K/Akt, markedly abolished the protective effect of salidroside against hypoxia-induced cardiomyocytes apoptosis. Cell viability was significantly attenuated (P <0.01). The Hoechst staining positive cells and the typical DNA ladder formation were significantly increased (P <0.01). Pretreatment with Salidroside significantly increased the expression ofρ-Akt and HIF-1α, which were markedly abolished by pre-treating cells with LY294002.Conclusion1. Pretreatment with different concentrations (10, 50, 100μg/mL) of salidroside greatly protect cardiomyocytes against hypoxia-induced apoptosis in a dose-dependent way.2. The protection is possibly mediated through activating HIF-1αand VEGF expression and inducing the translation of HIF-1α.3. The protection is possibly associated with up-regulation of HIF-1αthrough PI3K/Akt signaling pathway.