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.