Objectives To study the effects of Wedelolactone (Wed) on the protein expression of apoptosis-associated Bcl-2, Bax and PARP (89KD) in the primary cultured rat cardiomyocytes subjected to anoxia/reoxygenation injury.
Methods The primary cultured neonatal rat cardiomyocytes were pretreated with Wed (0.2, 2, and 20 µmol/l) or Wed (2 µmol/l) for 1 h, respectively, and subjected to anoxia for 3 h and subsequently reoxygenation for 2 h. Cell viability, Creatine kinase (CK) and lactate dehydrogenase(LDH) activity in medium were measured. Terminal deoxynucleotidyl transferase d-UTP nick end labelling (TUNEL) staining was performed using an In Situ Cell Death Detection kit on rat cardiomyocytes. The expression of Bcl-2 and the apoptotic protein Bax and PARP (89KD) were detected by Western blotting.
Results Compared with that of the control group, the numbers of TUNEL-positive nuclei were significantly increased in cardiomyocytes after 3 h of anoxia and 2 h of reoxygenation. Bcl-2 protein in cardiomyocytes decreased significantly (p<0.01) and the expression of Bax protein and PARP (89KD) in cardiomyocytes increased significantly after reoxygenation. (p<0.01), Cell viability decreased obviously after anoxia/reoxygenation (p<0.05). Compared with that of the anoxia /reoxygenation group, Pretreatment with different concentration Wed decreased LDH activity and increased the survival of the cells significantly (p<0.05). The expression of Bcl-2 protein in the Wed (2 µmol/l) groups increased significantly (p<0.05) and the expression of Bax protein decreased significantly (p<0.05).
Conclusions The Wed pre-treatment before ischaemia has antiapopotic effects on neonatal rats myocardial cells undergoing anoxia/reoxygenation, the underlay mechanism might be attributed to the up-regulated the expression of Bcl-2 gene and the inhibited the expression of Bax and PARP(89KD) gene expression.
*These authors contributed equally to this work.
This study was supported by the Natural Scientific Foundation of China (30972917), the Natural Scientific Foundation of Gongdong Province (8451008901000788) and the Medical Scientific Research Foundation of Guangdong Province (A2009163)