Effect of morphine preconditioning on expression of miR-133b-5p and Fas in rat cardiomyocytes subjected to hypoxia/reoxygenation

Objective To evaluate the effect of morphine preconditioning on the expression of miR-133b-5p and Fas in rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R). Methods Cardiomyocytes were isolated from healthy adult male Sprague-Dawley rats by using Langendorff perfusion.The cells were seeded into 24-well plates or 60 mm diameter dishes and randomly divided into 3 groups (n=24 each) using a random number table: control group (group C), group H/R, and morphine preconditioning group (group MPC). The cells in group C were cultured in normal culture atmosphere.In H/R and MPC groups, the cells were exposed to 95% N2-5% CO2 for 90 min followed by 120 min reoxygenation.In group MPC, the cells were cultured for 10 min in serum-free DMEM liquid culture medium containing morphine 1 μmol/L, and then were cultured for 30 min in morphine-free DMEM liquid culture medium before hypoxia.At 120 min of reoxygenation, the cells in 24-well plates were selected to detect the cell viability (by MTT), lactate dehydrogenase (LDH) activity in the culture medium, and cell apoptosis (by Hoechst 33234 staining). Apoptosis rate was calculated.Total RNA and protein were extracted from the cells in 60 mm dishes to detect the expression of miR-133b-5p and Fas mRNA (by quantitative real-time PCR) and Fas protein (by Western blot). Results Compared with C group, the cell viability was significantly decreased, LDH activity and apoptosis rate were increased, the expression of miR-133b-5p was down-regulated, and the expression of Fas mRNA and protein was up-regulated in H/R group.Compared with H/R group, the cell viability was significantly increased, LDH activity and apoptosis rate were decreased, the expression of miR-133b-5p was up-regulated, and the expression of Fas mRNA and protein was down-regulated in MPC group. Conclusion The mechanism by which morphine preconditioning reduces H/R injury to rat cardiomyocytes is related to up-regulation of the expression of miR-133b-5p and down-regulation of the expression of Fas. Key words: Morphine; Ischemic preconditioning; Myocytes, cardiac; Cell hypoxia; Oxygen; MicroRNAs; Antigens, CD95