三磷酸腺苷敏感性钾通道介导一氧化氮对缺氧/复氧鼠心肌细胞损伤的保护作用

Protective effect of nitric oxide on myocardial cells from anoxia/reoxygenation injury in rat via activation of KATP channels

目的 研究三磷酸腺苷敏感性钾通道 (KATP)在一氧化氮 (NO)对缺氧 /复氧心肌细胞损害中的保护作用。方法 采用细胞缺氧 /复氧损伤模型 ,培养细胞随机分为 5组 :A组 ,正常对照组(培养 3h) ;B组 ,格列本脲 (glybenclamide ,Gly ,商品名 :优降糖 )预处理组 ,加入终浓度为 10 μmol/LGly后培养 3h ;C组 ,单纯缺氧 /复氧组 (A/R缺氧 2h ,复氧 1h) ;D组 ,一氧化氮预处理组 ,加入S 亚硝基 已酰青酶胺 (S nitroso n acetyl penicillamine ,SNAP)使其终浓度为 1mmol/L ,预处理 4 0min后缺氧复氧 ;E组 ,Gly +SNAP预处理组 ,加入终浓度为 10 μmol/LGly ,培育 2 0min后再加入SNAP使其终浓度为 1mmol/L ,预处理 4 0min后缺氧复氧。与复氧后测定细胞存活率 ,培养液中乳酸脱氢酶、肌酸激酶含量 ,细胞内丙二醛及游离Ca2 + 的变化。结果 与正常组相比 ,Gly处理组和正常组各指标无明显差别 ;与正常组相比 ,单纯缺氧 /复氧组乳酸脱氢酶、肌酸激酶、细胞内丙二醛水平显著升高 (P<0 0 1) ,与正常组相比 ,细胞存活率显著降低 (P <0 0 1)及发生明显钙超载 (P <0 0 1) ;1mmol/LSNAP预处理组明显减轻上述变化 (P <0 0 1) ;而与NO预处理组相比 ,Gly +SNAP预处理组取消了NO组上述保护作用 (P <0 0 1)。

Objective To evaluate the roles of KATP channels in nitric oxide (NO)-induced protection in anoxia/reoxygenation injury of neonatal rat cardiomyocyte. Methods An anoxia/reoxygenation model of myocardial cells of neonatal SD rats wa s established. The cells were randomly divided into 5 groups: group A (control), without any treatment; group B, preconditioned with Gly by adding Gly into cult ure medium with a final concentration of 10 μmol/L; group C (anoxia/reoxygenat ion), 2 h anoxia followed by 1 h reoxygenation; group D, preconditioned with NO by adding SNAP into culture medium with a final concentration of 1 mmol/L (for 40 min); group E, preconditioned with gly+SNAP by adding glybenclamide into cul ture medium with a final concentration of 10 μmol/L (for 20 min), and then addi ng SNAP into culture medium with a final concentration of 1mmol/L (for 40 min). The rate of cell viability , the activity of creatine kinase (CK) , Lactic dehy drogenase (LDH), the contents of cellular malondiadehyde (MDA), and the free Cal cium([Ca_2+]i) were measured in each group after reoxygenation. Results (1) No difference of the measurement was found between group B and group A_(P>0.05); (2) There was a significant increase of LDH, CK, MDA, intracellular free Ca_2+(P<0.01)and a significant decrease of cell viability(P<0.01)in group B compared with group A; (3) There was a significant decrease of LDH, CK, MDA, intracellular free Ca_2+(P<0 .01)and a significant increase of cell viability(P<0.01)in group D comp ared with group B; (4) No protective effects of NO were found in group E compar ed with group D(P<0.01). Conclusions Activation of KAT P channels is involved in mediating protective effect of NO in anoxia/reoxygenat ion injury of neonatal rat cardiomyocyte,mainly via reducing intracellular Ca _2+ overload and lipoid hyperoxidation.