Melatonin, a natural product of the pineal gland, has been shown to protect against ischemic stroke, but the molecular mechanisms underlying its protective function are not fully understood. In the present study, we tested whether melatonin could protect against ischemia–reperfusion (I/R) injury to rat brain by targeting the autophagy pathway. The I/R brain injury was induced by the established rat transient middle cerebral artery occlusion model. We found intraperitoneal injection of melatonin can ameliorate rat brain injury as evidenced by multiple morphological and behavioral criteria, such as infarct size, neurological score, serum creatine kinase, and lactate dehydrogenase content, as well as pyknotic-positive cells. Further studies revealed that the beneficial effects of melatonin is through targeting the autophagy pathway by inhibiting expression of beclin-1 and conversion of LC3, as well as activating the PI3K/Akt pro-survival pathway. To further confirm this finding, the autophagy pathway was activated by lentiviral mediated beclin-1 delivery and the PI3K/Akt pathway was inhibited by a pharmacological inhibitor, LY294002. In both manipulations, the beneficial effects of melatonin were greatly abolished. Taken together, our study suggested melatonin plays a protective role against I/R brain injury by inhibiting autophagy and activating the PI3K/Akt pro-survival pathway.

Background/Aims: Ischemic stroke is a leading cause of long-term disability. To date, there is no effective treatment for stroke. Previous studies have shown that Ginkgo biloba extract has protective effects against neurodegenerative disorders. In this present study, we sought to test the potential protective role of an active component of Ginkgo biloba extract, bilobalide, in a rat model of middle cerebral artery occlusion (MCAO). Methods: A rat model of MCAO was used to test the potential protective effects of Bilobalide B on stroke protection. TTC staining was performed to evaluate infarct size of the brains. Neurological deficit score was measured to reveal the effects of the treatments on animal behavior and cognition. Immunohistochemical staining and transmission electronic microscope analysis were performed to measure the cellular responses to drug treatment. Western blotting and ELISA were performed. The expression of Cleaved- Casepase 3, Beclin-1, p62 and LC3I/II were quantified, and the Phosphorylation of eNOS and Akt were evaluated. The ratio of Bcl-2/ Bax was determined to reveal the molecular pathways that are involved in the drug treatment. Results: We found that intraperitoneal delivery of various Bilobalide doses during ischemia can protect against brain injury, as evidenced by reduced infarct size and improved neurological scores after surgery. Histochemical analysis revealed that treatment with bilobalide can significantly reduce apoptosis, autophagy, and promote angiogeneis following ischemia/reperfusion injury to the brain. The performence of increased phosphorylation of eNOS and Akt suggested that bilobalide can activate Akt prosurvival and eNOS pathways to promote cell survival and angiogenesis, respectively. Conclusions: Our results suggested that bilobalide benefits stroke symptoms by reducing cell death pathways and promoting angiogenesis. As such, bilobalide may be a potential agent for improving self-repair after ischemic stroke.