吗啡预处理对H9 c2心肌细胞缺氧/复氧时microRNA表达的影响

摘要

目的：探讨吗啡预处理( morphine preconditioning, MPC)对 H9c2心肌细胞缺氧/复氧( hypoxia reoxygenation, H/R)时microRNA( miRNA)表达的影响,为明确其机制提供参考。方法培养H9 c2心肌细胞,随机分为3组( n=4)：①正常对照组(CON)：H9c2心肌细胞置于DMEM/F12细胞培养液常规培养；②缺氧/复氧组( H/R)：对心肌细胞行缺氧5 h/复氧1 h处理；③吗啡预处理组( MPC+H/R)：在H/R前,应用1μmol·L-1吗啡预处理10 min。处理结束后,采用CCK-8法检测细胞增殖、化学比色法检测细胞培养液中乳酸脱氢酶( LDH)活性、Annexin-V-FITC/PI双染法检测细胞凋亡、Western blot法检测细胞内Fas蛋白表达、荧光定量RT-PCR法检测细胞 miRNA 表达水平。结果与 CON 组比,H/R组细胞活力降低,LDH活性升高,细胞凋亡率增加, Fas蛋白水平升高(P<0．01)；MPC可明显提高细胞活力,降低LDH活性,抑制细胞凋亡,降低Fas蛋白水平( P<0．01)。qRT-PCR结果显示,与 CON 组相比,H/R 组 miR-133a-5p、miR-133b-5p、miR-664-1-5p、miR-6216和 let-7e-5p表达明显下调,而MPC能明显地抑制H/R对这些miRNA表达的下调作用(P<0．01)。结论吗啡预处理减轻H9c2心肌细胞缺氧/复氧损伤可能与调控 miR-133a-5p、miR-133b-5p、miR-664-1-5p、miR-6216和let7e-5p等miRNA表达有关。%Aim To evaluate the effects of morphine preconditioning ( MPC ) on the expression of microR-NAs ( miRNAs ) induced by hypoxia-reoxygenation (H/R) in H9c2 myocardial cells. Methods H9c2 cells were randomly divided into 3 groups ( n=4 each) as follows:control group ( CON) , hypoxia/ reoxygen-ation group ( H/R ) and morphine preconditioning group ( MPC+H/R) . The cells were cultured in nor-mal condition in CON group. The cells were subjected to 5 h hypoxia followed by 1 h reoxygenation in H/R group and MPC+H/R group. Specifically, the cells in MPC+H/R group were preconditioned with morphine with the final concentration of 1 μmol·L-1 for 10 min before H/R. After the treatment, CCK-8 was used to detect cell viability and chemical colorimetry was used to detect lactate dehydrogenase ( LDH ) activity in the culture medium. Cell apoptosis was assessed by An-nexin-V-FITC/PI flow cytometry. Relative expression of Fas protein was detected by Western blot. The ex-pression of miRNA in myocardial cells was analyzed by quantitative reverse transcription polymerase chain re-action ( qRT-PCR ) . Results Compared with CON group, the cell viability was significantly decreased, while the LDH activity, apoptotic rate and Fas protein expression were dramatically increased in group H/R (P<0. 01). However, MPC significantly increased the cell viability, whereas it decreased the LDH activity, apoptotic rate and Fas protein expression induced by H/R injury ( P < 0. 01 ) . The expressions of miR-133a-5p, miR-133b-5p, miR-664-1-5p, miR-6216 and let-7 e-5 p were markedly down-regulated by H/R as compared to CON group ( P <0. 05 ) , while MPC inhibited these miRNAs which were significantly down-regulated by H/R group ( P <0. 01 ) . Conclusion Morphine preconditioning might protect H9 c2 myocar-dial cells against H/R injury by regulating the expres-sion of miRNAs such as miR-133a-5p, miR-133b-5p, miR-664-1-5p, miR-6216 and let-7e-5p.