Pharmacological Effects Of Ciclopirox Olamine And Compound A11 On Cerebral Ischemic Injury

Ischemic stroke is a common cerebral vascular disease with the characteristics of high morbidity,high mortality,high disability and high recurrence.It seriously threatens human health and brings a great burden to family and society.However,the current available drugs for clinical use are very limited.It is urgent to develop effective therapies for ischemic stroke.Cell-based phenotypic screening was applied to search for anti-ischemic stroke agents from the FDA-approved drugs,chemical synthesis small molecules and natural products.In the first part of this thesis,we evaluated the protective effects of anti-fungal drug ciclopirox olamine(CPX)against ischemic injury and explored its mechanisms.Firstly,we established the oxygen glucose deprivation(OGD)model on SH-SY5Y cell line to mimic the clinical ischemia/reperfusion injury and tested the pharmacological activity of 778 FDA-approved drugs.We found that the antifungal drug CPX conferred significant neuroprotection against oxygen glucose deprivation injury.CPX is an antifungal agent,which is widely used to treat superficial fungal infection,and its neuroprotective effects against cerebral ischemia/reperfusion injury have not been reported before.CPX effectively protected neuronal cells from oxygen glucose deprivation injury and oxidative stress in a dose dependent manner.Meanwhile,in rat MCAO model,CPX greatly reduced infarct volume,alleviated brain edema and improved neurological deficits in rats suffering from ischemia/reperfusion injury.In the long-term efficacy investigation,comparing with the single dose of CPX administered after reperfusion,administration of CPX once a day for 7 successive days significantly improved the motor function.Above all,CPX has advantages on efficacy and dosage comparing to the active control edaravone.We further evaluated the protective effects of CPX against the multiple pathological processes induced by ischemic injury including apoptosis,inflammation and blood brain barrier(BBB)disruption.The number of apoptotic cells in injured brain was also decreased by treatment with CPX,which were detected through Tunel staining and western-blot.CPX inhibited the activation of glial cells and down-regulated the expression of inflammatory cytokines in the brain tissue.CPX treatment effectively decreased the permeability of BBB induced by cerebral ischemia/reperfusion and up-regulated the expression of vascular endothelial tight junction proteins.To further explore the underlying protective mechanisms of CPX,we studied the relationship between the reported targets of CPX and its neuroprotective effects,and then employed the means of antibody array,phosphoproteomics and photoaffinity labeling combined with click chemistry in our research.The results have demonstrated that CPX increased the expression of HIF-1?in ischemic neuronal cells,but the protective mechanism is independent of up-regulation of HIF-1?.The results of antibody array indicated that CPX regulated AKT/GSK-3?signaling pathway,which plays a key role in cell survival and apoptosis.The results of phosphoproteomics further suggested that CPX treatment does not only regulated serine/threonine protein kinase but also the phosphorylation of cell cycle related proteins.We further provided evidence that CPX treatment prevented the cell cycle progression of microglia,which is related with inhibition of microglia activation.Moreover,we cooperated with medicinal chemistry group,photoreactive group was linked to CPX.After validation of the similar protective effects of the probe,we combined photoaffinity labeling in live cells with click chemistry to search for the targets of CPX under physiological conditions.In the second part of the thesis,we tested the neuroprotetive effects of 414chemical synthesis small molecules and 232 natural products,and found that diaryl acylhydrazone derivatives showed obvious protective effects against ischemic injury,especially the compound A11.We found that A11 confered neuroprotection against ischemic injury in a dose-dependent manner in in vitro ischemic stroke models:OGD,hydrogen peroxide(H₂O₂)and glutamate-induced neuronal cell injury models.More importantly,A11 treatment reduced infarct volume and ameliorated neurological deficits in rats suffered from middle cerebral artery occlusion(MCAO)in vivo.Moreover,A11 potently alleviated three critical pathological changes—apoptosis,oxidative stress,and mitochondrial dysfunction by inhibiting the cleavage of Caspase-3,down-regulation of p53 in nuclear,protection against H₂O₂ injury,decreased the level of intracellular ROS and increased the ATP level.Further mechanistic study suggested that A11 up-regulated the phosphorylation levels of AKT and extracellular signal-regulated kinases(ERK)in the same ischemic cellular model,indicating the involvement of joint activation of phosphoinositide3-kinase(PI3K)/AKT and mitogen-activated protein kinase(MEK)/ERK pathways.Specific inhibitors of MEK/ERK(U0126)and PI3K/AKT(LY294002)were used.The up-regulated phosphorylation levels of ERK and AKT in ischemic cells were remarkably suppressed by treatment with U0126 and LY294002 accompanied with the blockade of A11 induced neuroprotection.Our study for the first time reported the anti-ischemic effect of CPX and diaryl acylhydrazone derivative A11.The anti-fungal drug CPX robustly combatted the multiple pathological processes induced by ischemic injury.The neuroprotective mechanisms were related with regulating the apoptosis related signaling pathways and the cell cycle progression.The diaryl acylhydrazone derivative A11 revealed multifunctional capacities on ischemia-associated pathological processes.The mechanisms of its neuroprotection was through triggering both ERK and AKT signaling in ischemic neuronal cells.Our findings may provide important clues for the development of novel therapeutic strategy for ischemic stroke.