美托洛尔抑制缺氧／复氧诱导的心肌细胞凋亡

Protective effects of Metoprolol on myocardial cell apoptosis of neonatal mouse after hypoxia/reoxygenation

目的探讨美托洛尔对缺氧／复氧（hypoxia reoxygenation，H／R）引起小鼠乳鼠心肌细胞凋亡的影响及其可能机制。方法原代培养小鼠（C57BL／6）乳鼠心肌细胞，并随机分为：①对照组（Control，Con），在正常培养液（含10％胎牛血清、1％青一链霉素的DMEM）中正常环境（37℃，95％空气-5％CO2）培养12h；②美托洛尔组（Metoprolol，Meto），5ixmol／L美托洛尔预处理心肌细胞24h后正常培养12h；H③／R组，在无糖无血清一无氧培养液中，95％N2-5％CO2的无氧环境培养4h，然后在正常培养液中正常环境培养8h；④Meto＋H／R组，5μmol／L美托洛尔预处理心肌细胞24h后再行缺氧复氧处理。各组细胞接受不同处理后，台盼蓝检测心肌细胞存活率，TUNEL染色检测心肌细胞凋亡率，免疫细胞化学染色检测细胞色素C（cytochromec，cytc）浓度，caspase-3活性检测试剂盒检测caspase-3活性，钙蛋白酶（calpain）活性检测试剂盒检测calpain活性。结果与对照组比较，H／R组心肌细胞存活率显著降低（91．67±4．38）％vs．（60．09±5．68）％，P〈0．05；细胞凋亡率显著升高（6．60±0．53）％vs．（15．95±2．01）％，P〈0．05；cytc释放显著增加（2．55±0．28）ng／μg prot vs．（5．60±0．56）ng／μgprot，P〈0．05；caspase-3活性升高（0．26±0．04）pmol/μgprot vs．（0．83±0．08）pmol／μgprot，P〈0．05；calpain活性（荧光强度）显著增高（113．23±4．29）vs．（222．04±16．86），P〈0．05。与H／R组比较，Meto＋H／R组心肌细胞存活率显著升高（60．09±5．68）％vs．（71．82±6．25）％，P〈0．05；凋亡率显著降低（15．95±2．01）％郴．（10．72±1．93）％，P〈0．05；cytc释放减少（5．60±0．56）ng／μgprot vs．（3．59±0．46）ng／μgprot，P〈0．05；caspase-3活性降低（0．83±0．08）pmol／lxgprot vs．（0．61±0．07）pmol／μgprot，P〈0．05；calpain活性（荧光强度）显著降低（222．04±16．86）vs．（170．62±13．26），P〈0．05。结论美托洛尔能抑制H／R诱导的心肌细胞凋亡，其机制可能与抑制cytC释放，降低caspase-3和calpain活性有关。

Objective To investigate the effect of metoprolol on hypoxi：~/reoxygenation （H/R） - induced cell apoptosis in primary neonatal mouse cardiomyocytes and to clarify the underlying mechanism. Methods Primary neonatal mouse cardiomyocytes from C57BL/6 are randomly （random number） divided into four groups： ①Control group （Control, Con）, in which cardiomyocytes were incubated with routine medium [ a DMEM medium containing 10% fetal bovine serum （FBS） and 1% streptomycin/penicillin] for 12 hours in a specific environment （a 37 ℃ and 5% CO2 humidified atmosphere）;②Metoprolot group （Meto）, in which cardiomyocytes were pretreated with 5 μmol/L metoprolol for 24 hours, and then continuously cultured for another 12 hours in the routine medium and in a specific environment; ③H/R group, in which cardiomyoeytes were incubated with glucose-free and serum-free DMEM medium in hypoxia environment （95% N2 and 5% CO2 ） for 4 hours, and then were returned to the specific environment with the routine medium for 8 hours; ④Meto ＋ H/R group, in which cardiomyocytes were pretreated with 5 μmol/L metoprolol for 24 hours, and then exposed to H/R treatment. The cell viability and apoptosis were separately detected by trypan blue and TUNEL staining. The concentration of cytochrome e （ cyt c） was assayed by using cyt c immunocytochemistry. The caspase-3 and calpain activity were separately determined using easpase-3 and calpain activity detection kit. Results Compared to the control group, there was significant decrease in cell viability in the H/R group [ （91.67 ±4.38）% vs. （60.09 ±5.68）%, P〈 0. 05 ] and there was remarkable increase in numbers of apoptotic cardiomyocytes [ （6. 60 ± 0. 53 ） % vs. （ 15.95 ± 2. 01 ） %, P 〈 0.05 ] in the H/R group. Meanwhile, after cardiomyocytes exposed to H/R, there were significant increases in eyt c release [ （ 2. 55 ±0. 28 ） ng/μg protein vs. （ 5.60 ± 0. 56 ） ng/μg protein, P 〈0. 05], in caspase-3 activity [ （0. 26 ±0. 04） pmol/μg protein vs. （0. 83 ±0. 08） pmol/μg protein, P 〈 0. 05 ] and in calpain activity （intensity） [ （ 113.23 ± 4. 29） vs. （222. 04 ±16. 86）, P 〈 0.05]. However, pretreatment with metoprolol significantly reduces H/R-induced loss of cell viability （60.09±5.68）% vs. （71.82±6.25）%, P〈0.05] andapoptosis [ （15.95±2.01）% vs. （10.72± 1.93 ） %, P 〈 0.05 ]. In addition, metoprolol pretreatment significantly suppresses cyt c release [ （5.60 ± 0. 56） ng/μg protein vs. （3.59 ±μ 0. 46） ng/μg protein, P 〈 0. 05 ] and markedly inhibits the caspase-3 [ （0. 83 ± 0.08） pmol/μg protein vs. （ 0. 61 ±0. 07 ） pmol/μg protein, P 〈 0. 05 ] and calpain activity （intensity） [ （222. 04 ±16. 86） vs. （ 170. 62 ±13.26）, P 〈 0. 05 ] compared with H/R-treated cells. Conclusions Metoprolol can protect neonatal mouse cardiomyocytes against H/R-induced apoptosis, which might be associated with inhibition of cyt c release and suppression of caspase-3 activity as well as calpain activity.