Neuroprotective activity of some selected phytochemicals in ischemic stroke

Abstract

Ischemic stroke is a challenging disease with high mortality and disability rates, causing great economic and social burden worldwide. Neuroprotection that reduces the damaging events in neurons is an important therapeutic strategy for ischemic stroke. Phytochemicals show great potential in neuroprotection, with hundreds being reported to exhibit neuroprotection in preclinical research. This study aimed to screen several leading compounds from phytochemicals to provide the basis for the development of new neuroprotective agents. We evaluated the neuroprotective effects of 12 phytochemicals with oxygen and glucose deprivation (OGD/R) model and found that two stilbenoids: pinosylvin and cajaninstilbene acid (CSA) showed stronger activities. Hence, middle cerebral artery occlusion (MCAO/R) model, and molecular biological and biochemical experiments were applied to further examine their neuroprotective activities and functional mechanisms. Pinosylvin significantly decreased cell death in OGD/R-damaged cells and improved brain damages in MCAO/R rats, indicating its neuroprotective activity against cerebral ischemia and reperfusion (I/R) injuries. Mechanism studies showed that pinosylvin improved the mitochondrial function. Notably, mitochondrial protection of pinosylvin was achieved at least partially by enhancing PINK1/Parkin mediated mitophagy, as suggested by the elevated protein levels of LC3 II, Beclin1, PINK1 and Parkin after pinosylvin treatment. Besides, pinosylvin activated the Nrf2 pathway and upregulated the expressions of antioxidant enzymes to reduce the oxidative stress induced mitochondrial dysfunction. Hereby, pinosylvin exhibited neuroprotection by enhancing PINK1/Parkin mediated mitophagy and activating Nrf2 pathway to improve I/R-induced mitochondrial dysfunction. CSA possessed significant neuroprotective activity, as evidenced by the reduced cell death in OGD/R or t-BHP injured cells, and decreased brain damages in MCAO/R rats. Further studies indicated that the protective effect was achieved via the antioxidant activity of CSA, which decreased the oxidative stress and its related mitochondrial dysfunction in cells. Notably, Nrf2 was activated in cells and MCAO/R rats by CSA, and the inhibition of Nrf2 by brusatol weakened CSA-mediated neuroprotection. Furthermore, after applying a series of kinase inhibitors, CSA-induced Nrf2 activation was markedly inhibited by BML-275 (an AMPK inhibitor), implying that AMPK was the dominant kinase to regulate the Nrf2 pathway for CSA’s neuroprotective effects with enhanced AMPK phosphorylation observed both in vivo and in vitro. Accordingly, CSA exerted neuroprotection via activating the AMPK/Nrf2 pathway to reduce I/R-induced oxidative stress and mitochondrial disfunction. Based on the structures of pinosylvin and CSA, we then synthesized several novel compounds (stilbenoid-fused flavonoids) that combined a styryl group with naringenin. Results showed that these naringenin derivatives reduced t-BHP induced cell death, with compound 6 (possessing a fluorine in the styryl group) showing strong protective effect and low cytotoxicity. Further studies showed that compound 6 exerted higher neuroprotective efficacy than that of pinosylvin and CSA in MCAO/R rats, and AMPK/Nrf2 mediated antioxidation might be one of its neuroprotective strategies. In summary, this research found that pinosylvin and CSA possessed significant neuroprotective activities against cerebral I/R injuries mainly via mitophagy regulation and antioxidation, respectively, and the synthesized stilbenoid-fused flavonoid, compound 6 showed high neuroprotective efficacy. The findings provided new insight into the development of new drugs for ischemic stroke treatment.