Studies On Neuroprotective Effect And Its Mechanisms Of Osthole Against Acute Ischemic Stroke On Middle Cerebral Ischemia Occlusion

Ischemic cerebrovascular disease of the central nervous system is common and frequent diseases,is one of the three killer diseases,is the first disability factors, and its incidence, mortality and disability are increasing year by year. Therefore, the molecular mechanisms and treatment of cerebral ischemia-reperfusion are research focus for domestic and foreign scholars. Osthole (OST) is the main component of coumarin compounds,derived from the herb Cnidium,and is rich in natural resources. In recent years,a large number of experiments show that OST has anti-inflammatory,anti-clotting,dilates blood vessels,inhibits thrombosis,improves learning and memory,and has potential neuroprotective effect. In this study,we will discusse the neuroprotective effect of OST and its possible molecular mechanisms,as well as the relationship of the effect and endothelial nitric oxide synthase (eNOS) relevant pathway in rats by cerebral ischemia-reperfusion model. The aim of this study is to clarify the neuroprotective effect of OST and its possible mechanism on ischemic brain injury, which will provide a theoretical basis for OST to be used in the clinical treatment of ischemic brain injury. Part I Neuroprotective effects of OST in ischemic brain injuryObjective: To identify neuroprotective effects of OST in ischemic brain injury. Methods:Step One:Create a rat middle cerebral artery occlusion (MCAO) model of cerebral ischemia. Step Two: Male Sprague–Dawley rats were randomly divided into five groups: Sham group, MCAO group, pretreatment groups which were treated with osthole 10, 20 or 40 mg/kg (IP) 30 min before MCAO respectively. Twenty four hours after reperfusion, rats in all groups were evaluated neurological deficit score (NDS) by taking the form of double-blind. Then, three rats in each group were randomly selected for hematoxylin-eosin(HE) staining to observe morphological changes in ischemic brain tissue, three rats were randomly selected for 2% of the 2,3,5-triphenyltetrazolium chloride (TTC) staining to evaluate ischemic area of brain tissue, three rats were selected for the dry and wet weight to observe edema. Result:Pretreatment with OST significantly decreased the volume of infarction, NDS and edema compared with rats in the MCAO group at 24 h after MCAO. The histopathological changes of OST groups are also better than that of MCAO group (P < 0.01). Furthermore, the dose of 40 mg/kg of OST was more protective than the dose of 20 mg/kg and 10 mg/kg on the cerebral injury after MCAO. Conclusion:Pretreatment with OST can prevent neurons from the ischemia injury in rats with MCAO. The dose of 40 mg/kg of OST was more protective than the other dose on the cerebral injury after MCAO. Part II The potential mechanisms of nuroprotective effects of OST in ischemic brain injuryObjective:To observe some indices of anti-inflammatory and antioxidant when pretreatment with OST prevents neurons from the ischemia injury in rats with MCAO. Methods:Male Sprague–Dawley rats were randomly divided into three groups: Sham group, MCAO group and pretreatment with OST groups. Twenty four hours after reperfusion, the rats in each of the groups were subdivided into four subgroups consisting of 3 animals. The first subgroup was used for detecting MDA; the second subgroup was used for detecting GSH; the third subgroup was used for detecting MPO; and the fourth subgroup was used for detecting IL-1βand IL-8 by ELISA. Results:Pretreatment with OST significantly increased in GSH, and decreased MDA, MPO, IL-1βand IL-8 compared with rats in the MCAO group at 24 h after MCAO. Conclusion:The antioxidative action and anti-inflammatory property of OST may contribute to a beneficial effect against stroke.PartⅢThe molecule mechanisms of nuroprotective effects of OST in ischemic brain injuryObjective:To investigate the neuroprotective effect of OST on acute ischemic stroke induced by MCAO in rats and the underlying mechanism.Methods: Adult male Sprague-Dawley rats weighing 250 to 280g were subjected to 2h MCAO and pretreated with vehicle (20% Tween-80), osthole alone, or osthole plus NG-nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor), LY-294002 (a specific PI3K inhibitor) or GW9662 (a selective PPARγantagonist) at 30min before the transient occlusion. Neurological deficit scores (NDS) and infarct volumes of brain were assessed at 24h and 72h after MCAO. Neuronal apoptosis, NO concentration and activated caspase-3, iNOS, nNOS, eNOS, phosphorylated eNOS (p-eNOS), Akt, phosphorylated Akt (p-Akt), Tumor necrosis factor-α(TNF-α) and arginase I proteins expression were also determined at 24h after MCAO. Results:Pre-administration of osthole on rats with MCAO-induced acute ischemic stroke resulted in a significant decrease both in NDS and infarction volume and in the levels of neuronal apoptosis, activated caspase-3 and iNOS proteins expression at 24h after MCAO. In contrary, NO concentration and eNOS protein expression were increased. The neuroprotective effect of osthole was completely blocked by NOS inhibitor L-NAME. Furthermore, osthole-induced eNOS protein expression and phosphorylation was blocked by GW9662 and LY-294002 respectively. And the phosphorylation of eNOS had a greater effect on osthole neuroprotection than the expression of eNOS. Conclusion:Our results provided direct evidence that osthole attenuates cerebral ischemic neuroinjury via eNOS-dependent and NO-mediated anti-apoptotic action in which both eNOS protein up-regulation and phosphorylation are involved, with the latter being the primary pathway.