Effects of chiral 3-n-butylphthalide on apoptosis induced by transient focal cerebral ischemia in rats

Abstract

Aim: To investigate the effects of 3-n-butylphthalide (NBP) on apoptosis induced by transient focal cerebral ischemia in rats, compare the action potency of s-(-)-, r-(+)- and (+/-)-NBP, and clarify the enantiomer that played a main role.

Methods: DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay and gel electrophoresis. The expression of cytochrome c and caspase-3 protein was observed by Western blot analysis and immunohistochemistry. Middle cerebral artery was occluded for 2 h.

Results: Significant DNA fragmentation was detected at 24 h after reperfusion. This response was inhibited by s-(-)-NBP (5, 10 mg/kg i.p.). s-(-)-NBP 10 mg/kg almost completely inhibited DNA fragmentation, whereas r-(+)- NBP 10 mg/kg showed less effect. (+/-)-NBP (20 mg/kg) showed an inhibitory effect between that of s-(-)-NBP (10 mg/kg) and r-(+)-NBP (10 mg/kg). During the apoptotic process, cytochrome c was released into the cytosol and caspase-3 was activated. This effect was markedly inhibited by s-(-)-NBP, and the action potency of r-(+)- and (+/-)-NBP on the changes of cytochrome c and caspase-3 protein was similar to that on DNA fragmentation.

Conclusion: NBP, especially its s-(-)-enantiomer, could potently reduce the release of cytochrome c, decrease the activation of caspase-3, and inhibit DNA fragmentation after transient focal cerebral ischemia. Our findings on the beneficial effects of NBP on cerebral ischemia-induced apoptosis might have important implications for the study and treatment of ischemic cerebrovascular diseases.