一氧化氮供体预处理对乳鼠心肌细胞的延迟保护作用及其机制

目的　探讨一氧化氮模拟预处理延迟保护作用的机制。方法　体外培养新生大鼠心肌细胞 ,实验分为 :①阴性对照组 (Normal组 ) ;②SNAP组 :一氧化氮 (nitricoxide ,NO)供体S 亚硝基 N 乙酰青霉胺 (S nitroso N acetyl 1 ,1 penicil lamine ,SNAP ,5 0 0 μmol·L-1 )与心肌细胞共育 2 4h ;③Che +SNAP组 :蛋白激酶C拮抗剂白屈菜季铵碱 (chelerythrinechlo ride ,Che ,1 0 μmol·L-1 )处理心肌细胞 30min后 ,加入SNAP与心肌细胞共育 2 4h ;④PDTC +SNAP组 :核因子κB(NF κB)特异性阻断剂PDTC(1 0 0 μmol·L-1 )处理心肌细胞 30min后 ,加入SNAP与心肌细胞共育 2 4h ;⑤缺氧复氧损伤组 (H/R组 ) :心肌细胞缺氧 6h ,复氧 3h。②～④组部分取细胞爬片以免疫组织化学法观察SNAP对热休克蛋白 70 (HSP70 )表达的影响 ;其余细胞经历H/R损伤后 ,检测心肌细胞存活率及乳酸脱氢酶 (LDH)活性。结果　在正常心肌细胞免疫组织化学法未检测到HSP70的表达 ,心肌细胞经过H/R损伤后 ,可检测到少量HSP70阳性细胞 ,其阳性染色A值为 94 6± 9 1 ,心肌细胞培养上清LDH活性为 (2 1 90 5± 1 5 1 7)U·L-1 ,细胞存活率为 5 1 7%± 4 6 % ,细胞损伤较正常组加重 (P <0 0 1 ) ;SNAP处理细胞后 2 4h ,HSP70阳性细胞数增多 ,

Myocardial protection and mechanism of nitric oxide donor preconditioning on neonatal rat cardiomyocytes

AIM To investigate late cardioprotective effects and mechanism of nitric oxide(NO) donor preconditioning on neonatal rat cardiomyocytes. METHODS Cultured neonatal rat cardiomyocytes were divided into five groups: ①Negative control;②SNAP group: Cardiomyocytes were treated with NO donor SNAP(500 μmol·L -1 )for 24 h;③Che+SNAP group: After pretreated with protein kinase C antagonist chelerythrine chloride(Che, 10 μmol·L -1 ) for 30 min,cardiomyocytes were treated with SNAP(500 μmol·L -1 )for 24 h;④PDTC+SNAP group: After pretreated with nuclear factor κB(NF κB)specific antagonist PDTC(100 μmol·L -1 ) for 30 min,cardiomyocytes were treated with SNAP(500 μmol·L -1 )for 24 h;⑤H/R group. Cells underwent H/R injury (6 h hypoxia followed by 3h reoxygenation). Cells in ②～④ group were divided into two parts: cells growing on slides were used to detect HSP70 protein expression; the rest underwent H/R injury before LDH and cell viability detection. Expression of heat shock protein 70(HSP70) was detected by immunohistochemistry combined with computer image analysis. LDH activity and cell viability were detected to evaluate injury of cardiomyocytes. RESULTS HSP70 positive cells were not detected by immunohistochemistry in normal cardiomyocytes. After H/R injury, HSP70 positive cells were detected with a A value of 94 6±9 1. Meanwhile cell injury was aggravated with a higher LDH activity of ( 2 190 5 ±151 7) U·L -1 and a lower cell viabity of 51 7%±4 6% compared with normal cells( P <0 01). Pretreatment with SNAP for 24 h significantly increased the expression of HSP70 protein with a A value of 165 9±9 3, higher than that of H/R group( P <0 01). Cell injury was reduced with LDH activity of (690 8±53 9) U·L -1 and cell viability of 88 9%±6 5% compared with H/R group( P <0 01). Pretreatment with Che or PDTC antagonized SNAP in duced expression of HSP70 protein and its protective effects on H/R induced injury of cardiomyocytes compared with SNAP group( P <0 01). CONCLUSION NO could induce late myocardial protection by induction of HSP70 protein expression through PKC NF κB signaling pathway.