内源性及外源性一氧化氮诱导心肌细胞预适应的延迟保护作用

Exogenous or endogenous nitric oxide pretreatment induced delayed protection of cardiomyocytes

目的 研究内源性及外源性一氧化氮(NO)是否诱导心肌细胞预适应延迟保护作用及其可能的机制。方法取体外培养的新生大鼠心肌细胞,分为如下各组:(1)阴性对照组(C组);(2)NO供体S-亚硝基-N-乙酰青霉胺(SNAP)500μmol·L-1组;(3)L-精氨酸(L-Arg)1 mmol·L-1组;(4)NOS抑制剂(L-NAME)1 mmol·L-1与L-Arg 1 mmol·L-1共同作用于心肌细胞组(L-NAME+L-Arg组);(5)缺氧预适应组(HP组);(6)L-NAME 1 mmol·L-1作用细胞后再行缺氧预适应组(L-NAME+HP组);(7)cGMP阻断剂亚甲基蓝(M8)50μmol·L-1加SNAP 500μmol·L-1组;(8)MB 50μmol·L-1加L-Arg 1 mmol·L-1组;(9)MB 50μmol·L-1加HP组;(10)缺氧复氧损伤组(H/R组)。(2)-(9)组细胞在给予干预因素24 h后使细胞经历6 h缺氧及3 h复氧,H/R组不予任何处理直接使其缺氧61h复氧3h。分别检测心肌细胞存活率及乳酸脱氢酶(LDH)活性,以判定心肌细胞损伤程度。结果SNAP处理心肌细胞24 h后使其缺氧复氧损伤减轻,表现为与单纯H/R组细胞比较其LDH活性显著降低,细胞存活率显著提高(P<0.01);缺氧预适应和L-Arg处理细胞24 h后均可显著减轻心肌细胞缺氧复氧损伤(P<0.01),但此作用可被NOS抑制剂L-NAME所拮抗;SNAP、L-Arg和HP减轻缺氧复氧心肌细胞损伤的作用均可被cGMP阻断剂所取消。结论

Objective Ischemia preconditioning provides early and delayed protection against ischetnic injury. It has been shown that nitric oxide(NO) exerts protective effects on cardiovascular system. The purpose of this study was to determine if exogenous or endogenous NO pretreatment could provide delayed protection of neonatal rat cardiomyocytes. Methods Neonatal SD rats (1-3 days) were used in this study. Cardiomyocytes obtained from beating heart were cultured and incubated for 72 h. The study was composed of ten groups: (1) no medicine was added to the cell medium and the cardiomyocytes were subjected to no hypoxia/reoxygenation (control group);(2) NO donor, S-nitroso-N-acetylpenicillamine (SNAP) 500 μmol.L-1 was added to cell medium 24 h before H/R( hypoxia 6 h followed by 3 h reoxygenatiori) (SNAP group); (3 )MO precusor, L-arginine 1 mmoL L-1 was added to cell medium 24 h before H/R (L-Arg group); (4) nitric oxide synthase(INOS) antagonist, L-NAME 1 mmol.L-1 and L-arginine 1 mmol.L-1 were added to cell medium 24 h before H/R (L-NAME + L-Arg group); (5) hypoxia preconditioning HP group) in which cultured cells were subjected to 60 min hypoxia followed by 30 min reoxygenation 24 h before H/R; (6) L-NAME 1 mmol.L was added to cell medium during hypoxia preconditioning 24 h before H/R ( L-NAME + HP group) ; (7) cGMP antagonist methylerie blue(MB) 50 μmol · L-1 was added to cell medium and one hour later SNAP 500 μmol·L-1 was added , after being incubated for 24 h the cells were subjected to 6 h hypoxia and 3 h reoxygenation ( MB + SNAP group) ; (8) MB 50 μmol · L-1 and L-arginine 1 mmol.L-1 were added to cell medium at 1 h interval 24 h before H/R (MB + L-Arg group); (9) MB 50 μmol · L -1 was added to cell medium during hypoxia preconditioning 24 h before H/R (MB + HP group) ; (10) cardiorayocytes underwent 6 h hypoxia and 3 h reoxygenation without any pretreatment (H/R group) . After H/R cardiomyocytes was examined for lactate dehydrogenase ( LDH) activity and cell viability.Results SNAP pretreatment protected cardiomyocytes from H/R injury as shown by reduced LDH activity and improvement in cell viability ( P < 0.01). Both HP and L-Arg pretreatment attenuated H/R injury to cardiomyocytes( P < 0.01), but this protective effect was inhibited by L-NAME. The protection provided by SNAP, L-Arg and HP was aboished by cGMP inhibitor MB (P < 0.01) .Conclusion Both endogenous and exogenous NO pretreatment can induce delayed protection of cardiomyocytes via cGMP pathway.