Neuroprotective Effect And Mechanism Investigation Of Cordycepin Against Cerebral Ischemia/Reperfusion Injury In Mice

PartⅠ:Neuroprotective effect and mechanism investigation of cordycepin against cerebral ischemia/reperfusion injury in miceAim:To observe neuroprotective effect of cordycepin against cerebral ischemia/reperfusion ingury in mice and investigate its relating mechnisms.Method:Kunming male mice(postnatal 6-8weeks) were randomly divided into four groups:sham-operated group,model group, cordycepin low dose(10mg/kg),high dose(20mg/kg),. The bilateral common carotid artery occlusion (2VO) for 15 min was conducted in mice, and then subjected to a subsequent 4h period of ischemia reperfusion. The mice in cordycepin-treated groups were administered with cordycepin (10mg/kg 20mg/kg, respectively) orally once a day for a week before operation, whereas the sham and ischemia/reperfusion group were given the same amount of double distilled water(0.1ml/10g body weight). After transient cerebral global ischemia for 15min and 4h reperfusion period, the mice were deeply anesthetized and decapitated. Brain was quickly removed to collect the cerebrum for all biochemical assays. The fresh brain tissue was collected and homogenized with ACSF and centrifuged at 9680(xg) for 10 min at 4℃. The supernatant was collected and stored at-70℃until further processing.Results:1. Effect of cordycepin on morphologic changes in ischemic regions of hippocampus and cortex of mice suffering from bilateral carotid artery ligation for 15min and subsequent reperfusion for 4hours. In the cortex and hippocampus especially in hippocampal CA1 region.marked morphological changes were visualized in ischemia/reperfusion group:such as neuronal loss, nuclei shrinkage, and dark staining of neurons. The brain of ischemia/reperfusion group showed a significant cell loss of the cortex and the hippocampal CA1 area in comparison with the sham-operated group(P<0.01). Cordycepin (10mg/kg) treatment markedly reduced these pathological changes and attenuated ischemia/reperfusion-induced cell loss in the cortex and the hippocampal CA1 area.2. Effect of cordycepin on SOD activities and MDA levels in cerebrum of mice suffering from bilateral carotid artery ligation for 15min and subsequent reperfusion for 4hours. In sham-operated group, the level of MDA was 1.83±0.44 (nM/mg. protein), the activity of SOD was 93.54±12.94 (U/mg. protein) respectively. Ischemia/reperfusion group resulted in a significant increase in MDA level (up to 65.9%) and marked decrease in SOD activity(up to 48.5%) compared with those of sham-operated group (P< 0.05). Cordycepin (10 mg/kg) treatment attenuated these changes markedly in ischemia/reperfusion mice.3. Effect of cordycepin on the extracellular level of five kinds of amino acids such as glutamate and aspartate etc. in cerebrum of mice suffering from bilateral carotid artery ligation for 15min and subsequent reperfusion for 4hours. The content of 5 types of amino acid in the model group were increased markedly contrast with sham-operatcd group group.The increased percentage were as follows:aspartate(144%),glutamate(117%),glycine(328%), taurine(121%),γ-GABA (86%). (P< 0.05 vs sham-operated group)Whereas, in the cordycepin(10mg/kg)-treated group, the content of amino acids were decreased significantly contrast with model group.The decreased percentage in cordycepin(10mg/kg)group as follows:aspartate(39%), glutamate(28%), glycine(60%), taurine(23%),γ-GABA (36%). (P< 0.05 vs model group)4.Western blot analysis demonstrates the effect of cordycepin on the expression of MMP-3 in cerebrum of mice suffering from bilateral carotid artery ligation for 15min and subsequent reperfusion for 4hours. There was a marked increase in MMP-3 protein expression in the ischemia/reperfusion group.(p<0.05 compared with sham-operated group) MMP-3 was downregulated significantly by cordycepin treatment 10mg/kg).(p<0.05 compared with model group).5. Immunofluorescence analysis demonstrates the effect of cordycepin on the expression of IKKa and P65 in cerebrum of mice suffering from bilateral carotid artery ligation for 15min and subsequent reperfusion for 4hours. The expression of IKKa and P65 were increased significantly in the ischemia/reperfusion group.(p<0.01 compared with sham-operated group). They were downregulated markedly by cordycepin treatment(10mg/kg).(p<0.05 compared with model group).Conclusion:1. Cordycepin is able to protect against cerebral ischemia/reperfusion injury. The protective effects are possibly involved in decreasing excitotoxicity of excitatory amino acids such as glutamate and aspartate. Meanwhile,cordycepin has been shown to interfere with the production of the MMP-3 as well as block free radicals in cerebral ischemia/reperfusion injury.2..Cordycepin has not neuroprotective effect at high dose (20mg/kg).The detailed mechanisms is not vet elucidated. PartⅡ:Neuroprotective effect and mechanism investigation of cordycepin against oxygen and glucose deprivation/reoxygenation injuryAim:To observe neuroprotective effect of cordycepin against oxygen and glucose deprivation/reoxygenation injury in mice and investigate its relating mechnisms.Methods:postnatal(six to eight weeks) health adult male mouse were randomly divided into five groups:control group,OGD group, cordycepin-treated groups (20μM,40μM,80μM).Whole brain slices were made by vibration slicer soon after the mouse were anesthetized by 10% Chloral Hydrate (350mg/kg,i.p.) and decapitated. The thickness of slice was 400μm.The brain slices were put in ACSF which keep inpouring 95%O2 and 5%CO2 and recovered there liveness for 30min in room temperature.Then the six-hole-board containing brain slices was put in 37℃thermostat-controlled water-bath for 30min.Then culture condition was substituted with free glucose ACSF inpouring 95%N2 and 5%CO2 and the brain slices were suffering from oxygen-glucose deprivation(OGD) for 15min. Brain slices were subjected to a subsequent 4h period of reoxygenation with 95%O2 and 5%CO2 in ACSF. TTC staining and PI staining were performed to evaluate the injury extent of brain slices. The incubation liquid in whole culture period was collected and utilized to detect LDH by kit,to detect five kinds of amino acid such as aspartate, glutamate, glycine, taurine, gamma-aminobutyric acid by high performance liquid chromatography(HPLC), to detect the content of IL-1βand IL-10 by ELISA kit. The tissue protein was extracted soon after brain slices tissue were collected. Western blot was utilized to detect the expression of IKKa,P65,P-ERK(1/2). Results:1.Effect of cordycepin on viability of brain slices suffering from OGD forl5min and reoxygenation for 4h. TTC staining and PI staining and LDH detection show that the injury extent of brain slices in OGD group was more severe compared with control group. Cordycepin at 20 and 40μM was able to lessen the injury extent of brain slices compared with OGD group, whereas 80μM had little effect.2.Effect of cordycepin on the release of cytokines in the incubation ACSF.ELISA detection show that the content of cytokines such as IL-1βand IL-10 in OGD group were increased significantly compared with control group. Cordycepin at 20 and 40μM and 80μM was able to downregulate the level of IL-1βmarkedly and at 40μM was able to upregulate the level of IL-10 markedly.3.Effect of cordycepin on the release of amino acids neurotransmitter in the incubation ACSF.HPLC detection show that the content of five kinds of amino acids neurotransmitter such as glutamate, aspartate, glycine, taurine, GABA in OGD group were increased significantly compared with control group. Cordycepin at 20 and 40μM was able to downregulate the level of glutamate and aspartate markedly and at 20μM was able to upregulate the level of taurine markedly.Cordycepin-treated groups were able to downregulate the levels of glycine and GABA at different extent.4.Effect of cordycepin on the expression of IKKa,P65,P-ERK (1/2) in brain slice tissues suffering from OGD forl5min and reoxygenation for 4h. Western-blot show that the expression of IKKa,P65 and P-ERK (1/2) in brain slice tissues of OGD group were increased significantly compared with control group. Cordycepin at 20,40μM and 80μM was able to downregulate the expression of IKKaand P65 markedly and at 20μM and 40μM was able to downregulate the the expression of P-ERK(1/2) markedly. Conclusions:1. Brain slice tissues were suffering from evident injury when subjected to OGD for 15min and reoxygenation for 4h. The content of IL-1β,IL-10 and excitatory amino acids and inhibitory amino acids in incubation ACSF were increased in OGD group. The expression of IKKa,P65 and P-ERK(1/2) in brain slice tissues of OGD group were increased significantly.1. Cordycepin was able to exert neuroprotective effect against OGD/reoxygenation injury. The mechanism maybe was related to downregulate the content of excitatory amino acids such as aspartate and glutamate and regulate the balance between excitatory amino acids and inhibitory amino acids in extracellular fluid. Downregulating the expression of IKKa,P65 and P-ERK (1/2) indicated cordycepin was able to block the inflammation pathway, decreasing the production of mediators of inflammation such as IL-1β.2. Cordycepin at 20 and 40μM was able to exert neuroprotective effect against OGD/reoxygenation injury, whereas 80μM had little effect.