CAJ | 학술논문

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(C57 BL/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-链霉素的DMEM)中正常环境(37℃,95％空气-5％ CO2)培养12 h;②美托洛尔组(Metoprolol,Meto),5μmol/L美托洛尔预处理心肌细胞24h后正常培养12 h;③H/R组,在无糖无血清-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(cytochrome c,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/μg prot,P＜0.05;caspase-3活性升高(0.26 ±0.04) pmol/μg prot vs(0.83 ±0.08) pmol/μg prot,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)％vs(10.72±1.93)％,P＜0.05;cytc释放减少(5.60±0.56) ng/μgprot vs.(3.59±0.46) ng/μg prot,P＜0.05;caspase-3活性降低(0.83±0.08) pmol/μg prot vs.(0.61±0.07) pmol/μg prot,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cyt c释放,降低caspase-3和calpain活性有关.
목적 탐토미탁락이대결양/복양(hypoxia reoxygenation,H/R)인기소서유서심기세포조망적영향급기가능궤제.방법 원대배양소서(C57 BL/6)유서심기세포,병수궤분위:①대조조(Control,Con),재정상배양액(함10％태우혈청、1％청-련매소적DMEM)중정상배경(37℃,95％공기-5％ CO2)배양12 h;②미탁락이조(Metoprolol,Meto),5μmol/L미탁락이예처리심기세포24h후정상배양12 h;③H/R조,재무당무혈청-무양배양액중,95％ N2-5％ CO2적무양배경배양4h,연후재정상배양액중정상배경배양8h;④Meto+ H/R조,5μmol/L미탁락이예처리심기세포24h후재행결양복양처리.각조세포접수불동처리후,태반람검측심기세포존활솔,TUNEL염색검측심기세포조망솔,면역세포화학염색검측세포색소c(cytochrome c,cytc)농도,caspase-3활성검측시제합검측caspase-3활성,개단백매(calpain)활성검측시제합검측calpain활성.결과 여대조조비교,H/R조심기세포존활솔현저강저(91.67 ±4.38) ％vs.(60.09±5.68)％,P＜0.05;세포조망솔현저승고(6.60±0.53)％vs(15.95±2.01)％,P＜0.05;cytc석방현저증가(2.55±0.28) ng/ μg prot vs(5.60±0.56) ng/μg prot,P＜0.05;caspase-3활성승고(0.26 ±0.04) pmol/μg prot vs(0.83 ±0.08) pmol/μg prot,P＜0.05;calpain활성(형광강도)현저증고(113.23±4.29)vs.(222.04±16.86),P＜0.05.여H/R조비교,Meto+ H/R조심기세포존활솔현저승고(60.09±5.68)％vs.(71.82±6.25)％,P＜0.05;조망솔현저강저(15.95±2.01)％vs(10.72±1.93)％,P＜0.05;cytc석방감소(5.60±0.56) ng/μgprot vs.(3.59±0.46) ng/μg prot,P＜0.05;caspase-3활성강저(0.83±0.08) pmol/μg prot vs.(0.61±0.07) pmol/μg prot,P＜0.05;calpain활성(형광강도)현저강저(222.04±16.86)vs.(170.62±13.26),P＜0.05.결론 미탁락이능억제H/R유도적심기세포조망,기궤제가능여억제cyt c석방,강저caspase-3화calpain활성유관.
Objective To investigate the effect of metoprolol on hypoxin/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);② Metoprolol group (Meto),in which cardiomyocytes were pretreated with 5 μ mol/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 cardiomyocytes 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 c (cyt c) was assayed by using cyt c immunocytochemistry.The caspase-3 and calpain activity were separately determined using caspase-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 cyt 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 ± 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.