Melatonin attenuates the focal cerebral ischemic injury by inhibiting the dissociation of pBad from 14-3-3.

It has recently been reported that melatonin protects neuronal cells from damage by enhancing Akt activation, thus mediating antiapoptosis signals. However, there is little information regarding the effects of melatonin on the activation of genes further downstream in the Akt signaling pathway in ischemic brain injury. This study investigated whether melatonin modulates the antiapoptotic signal through Akt and its downstream targets, Bad and 14-3-3. Adult male rats were treated with melatonin (5 mg/kg) prior to middle cerebral artery occlusion (MCAO). Brains were collected at 24 hr after MCAO and infarct volumes were analyzed. Our results confirm that melatonin significantly reduces infarct volume and decreases the positive reaction of TUNEL staining in the cerebral cortex. Signal pathway activation was measured by phosphorylation of Akt at Ser(473) and Bad at Ser(136) using Western blot analysis. Melatonin prevented the injury-induced decrease of pAkt and pBad levels. However, melatonin did not affect the expression of 14-3-3, which acts as an antiapoptotic factor through interaction with Bad. Immunoprecipitation analysis showed that the interaction between pBad and 14-3-3 increased in the presence of melatonin, compared to that of control animals. Our findings suggest that melatonin prevents cell death because of brain injury and that these protective effects are mediated through maintaining the interaction between pBad and 14-3-3, thus blocking activation of the apoptotic pathway.