The Neuroprotective Effects And Anti-apoptosis Mechanisms Of Cordycepin On Dopaminergic Neurons In PD Models Induced By Rotenone

Objective:To investigate the neuroprotective effects and anti-apoptosis mechanisms of cordycepin(Cor)on dopaminergic neurons in PD models induced by rotenone(Rot)..Methods:1.In vivoThe rats model of PD was established by Rot.Rot was injected subcutaneously(s.c.)at a dose of 1.0 mg/kg/d,two times at 12-h interval,for 30 days.The rats were divided into 5 groups:(a)control group(i.g.saline);(b)model group;(c)the low dose of drugs group(2.5 mg/kg Cor);(d)the middle dose of drugs group(5.0 mg/kg Cor);(e)the high dose of drugs group(10.0 mg/kg Cor);Cor was orally administered for 3 days before Rot injection.The rats were continuously treated with Cor for 33 days.The behavior changes were detected by behavior score and Grid experiment.The DA neuros morphology,the numbers of TH-positive neurons in SNpc,and DA,5-HT and their metabolites levels in the Str of PD rats were measured by HE staining,TH-immunohistochemistry,HPLC-ECD respectively.2.In vitroPD model was prepared by Rot(1.0 μmol/L)in PC12 cells.All experiments were divided into 5 groups:(a)control group(0.01%DMSO);(b)model group;(c)Three Cor-treated groups(0.4×10-6,2.0×10-6,8.0×10-6 μmol/L Cor).Cells were pretreated with Cor at different times,followed by incubating with Rot(1.0 μmol/L)for an additional 48 h.Cell viability was estimated by MTT assay.The rate of apoptosis,MMP and intracellular ROS level were analyzed by flow cytometry with Annexin V/PI,DCFH-DA and Rhodamine 123 respectively.The expressions of Bax,Bcl-2,Cytochrome-c in endochylema,Caspase-3,Cleaved caspase-3,Caspase-8,Caspase-9,Cleaved caspase-9 and Fas were determined by RT-qPCR and Western blotting.Results:1.Cor has neuroprotection against DA neurons injury in PD ratsIn model group,most of the rats exhibited tremor,unilateral rotation and even some rats showed unilateral hemiplegia after 30 days.Compared with the control group,the behavior scores were significantly increased and the grid descent latency were significantly prolonged(P<0.01).A few neurons with ambiguous cellular outline,smaller volume,bigger nucleus were observed and some eosinophilic cells were appeared by HE.Meanwhile,a considerable loss of TH-positive cells in the SNpc and the lower contents of DA,5-HT,DOPAC,HVA,5-HIAA in the Str were also observed when compared with control group(P<0.01).However,the behavioral impairment was improved after the administration of Cor.The behavior scores and the grid descent latency were significantly decreased in Cor groups(P<0.01).The changes of neurons morphology were partly improved and no eosinophilic cells appeared in Cor groups.Moreover,Cor markedly attenuated the loss of TH-positive neurons in the SNpc and significantly increased the contents of DA,5-HT,DOPAC,HVA,5-HIAA in Str compared with model group(P<0.05 or P<0.01).These suggested that Cor could have neuroprotection against DA neurons injury in PD rats induced by Rot.2.The anti-apoptosis mechanisms of Cor on Rot-induced PC12 cells(1)The effect of Cor on PD cells injury and apoptosis:Compared with control group,the viability of cells exposed to Rot with 1.0 μmol/L was significantly descended(P<0.01).The early and late apoptosis and total apoptosis rate in model group were apparently ascended(P<0.01)as well as the expression of Caspase-3 in mRNA and protein levels(P<0.01).When cells were pretreated with Cor(0.4×10-6,2.0×10-6 and 8.0×10-6 μmol/L)for 12,24 and 36 h respectively,the cell viability of each dose group(except for pretreatment with 0.4×10-6μmol/L Cor for 12 h)were increased obviously(P<0.05,P<0.01)and the groups pretreated with Cor for 24 h had the higher cell viability.After pretreated with Cor for 24 h,the early and late apoptotic cells and total apoptosis rate in the Cor groups were decreased significantly(P<0.01).There was no significant change in expression of Caspase-3 in mRNA and protein levels of cells pretreated with Cor when compared with model group.However,the Cleaved caspase-3 levels could be decreased significantly in Cor group(P<0.01).Cor at the dose of 8.0×10-6 μmol/L had the most obvious effect.Therefore,Cor could inhibit the cell apoptosis induced by Rot.(2)The effect of Cor on intracellular ROS levels:Compared with the control group,intracellular ROS levels in the Rot-treated cells were increased significantly(P<0.01).However,ROS levels were markedly decreased in cells pretreated with Cor(P<0.01).These results suggested that Cor could scaveng intracellular ROS.(3)The mitochondrial pathway related to the anti-apoptosis effects of Cor on Rot-induced cells:Compared to the control group,the expression of Bax and Caspase-9 in mRNA and protein levels were significantly increased(P<0.01,P<0.01).However,the ratio of Bax/Bcl-2 and the expression of Bcl-2 were significantly decreased(P<0.01)when cells exposed to Rot.The MMP in model group were significantly reduced(P<0.01)and the protein levels of Cyt-c in endochylem were significantly increased in PC12 cells exposed to Rot(,P<0.01).However,the expression of Bax,Caspase-9 in mRNA and protein levels were down-regulated and the expression of Bcl-2 were up-regulated in cells pretreated with Cor for 24 h(P<0.01)as well as the ratio of Bax/Bcl-2.Meanwhile the MMP in PC12 cells were markedly increased and the the protein levels of Cyt-c in endochylema were decreased significantly(P<0.05,P<0.01).These results indicated that the anti-apoptosis effects of Cor on Rot-induced cells could be related with the mitochondrial pathway.(4)The death receptor pathway related to the anti-apoptosis effects of Cor on Rot-induced cells:Compared to the control group,the expression of Caspase-8 and Fas in mRNA and protein levels were significantly increased in cells exposed to Rot(P<0.01).However,the expression of Caspase-8 and Fas in mRNA and protein levels were significantly down-regulated in cells pretreated with Cor(P<0.05,P<0.01),Cor at the dose of 8.0×10-6μmol/L had the most obvious effect.These results pointed that the anti-apoptosis effects of Cor on Rot-induced cells could be related with the death receptor pathway.Conclusion:Cor could inhibit DA neurons injury in PD models induced by Rot and the neuroprotective effect of Cor could be related with anti-apoptosis by suppressing oxidative stress,improving mitochondrial dysfunction via the mitochondrial pathway as well as the death receptor pathway.