吗啡预处理对大鼠心肌细胞缺氧复氧时miR-133b-5p与Fas表达的影响

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

目的 评价吗啡预处理对大鼠心肌细胞缺氧复氧时微小RNA-133b-5p（miR-133b-5p）与Fas表达的影响.方法 健康成年雄性SD大鼠心室肌细胞,接种于24孔板或60 mm培养皿培养,采用随机数字表法分为3组（n=24）：对照组（C组）、缺氧复氧组（H/R组）和吗啡预处理组（MPC组）.C组细胞正常培养,H/R和MPC组细胞缺氧90 min后复氧,MPC组于缺氧前采用含lμmol/L吗啡的无血清DMEM培养10 min,再换为无吗啡培养液培养30 min.于复氧120 min时采用MTT法测定心肌细胞活力,检测培养液乳酸脱氢酶（LDH）活性,采用Hoechst 33234染色法检测心肌细胞凋亡情况,计算细胞凋亡率;实时定量PCR法检测miR-133b-5p与Fas mRNA的表达,Western blot法检测Fas蛋白的表达.结果 与C组比较,H/R组心肌细胞活力降低,培养液LDH活性和细胞凋亡率升高,miR-133b-5p表达下调,Fas mRNA及蛋白表达上调（P＜0.05）;与H/R组比较,MPC组心肌细胞活力升高,培养液LDH活性和细胞凋亡率降低,miR-133b-5p表达上调,Fas mRNA及蛋白表达下调（P＜0.05）.结论 吗啡预处理减轻大鼠心肌细胞缺氧复氧损伤的机制与上调miR-133b-5p表达,下调Fas表达有关.

Objective To evaluate the effect of morphine preconditioning on the expression of miR-133b-Sp 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-Sp 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-regulatcd,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 cardiomyocytesis related to up-regulation of the expression of miR-133b-Sp and down-regulation of the expression of Fas.