| Parkinson's disease(PD),which manifests as extrapyramidal dysfunction,is a common degenerative disease that affects the central nervous system.Its main feature is the loss or denaturation of dopamine(DA)neurons in the substantia nigra(SN).Existing therapeutic drugs do not prevent remaining DA neurons from continuing to degenerate or die.Therefore,it is very necessary and meaningful to find a neuroprotective agent that can effectively block the progression of PD.Cordycepin,an active component,isolated from Cordyceps militaris(Northern Cordyceps sinensis).In our previous study,we had found that cordycepin could effectively protect DA neurons in rotenone-induced PD rats and PC 12 cells,which might be associated with the improvement of mitochondrial dysfunction.The neuroprotective effects of cordycepin have to be further confirmed in other models.And it is necessary to conduct in-depth research on the associated mechanisms.Objective:To investigate the protective effects of cordycepin on DA neurons in MPTP-induced mice and to explore whether it plays a neuroprotective role by inhibiting endoplasmic reticulum stress(ERS)and/or neuroinflammation.Methods:The mice were randomly divided into the control group,the model group with MPTP treatment,and the cordycepin treatment groups(5.0,10.0,20.0 mg/kg).PD model was established by intraperitoneal injection(i.p.)of MPTP(30 mg/kg),once a day for 5 days.The open-field test,pole test and rotarod performance test were used to assess the motor function.The numbers of TH-,Iba-1-,GFAP-positive neurons and the level of DAT were investigated by immunohistochemistry.DA,5-HT and their metabolites were measured by HPLC-ECD.TUNEL staining was used to observe the morphology and quantity of apoptosis.SOD,GSH-Px activities and MDA levels were determined by spectrophotometry.The protein expression of TH,Caspase 3,Cleaved-caspase 3,GRP78,CHOP,Caspase12,iNOS and NF-?B p65 were detected by Western blot,and the mRNA expression levels GRP78,CHOP,TNF-a and IL-1? were measured by RT-qPCR.The contents of TNF-? and IL-1? in the blood were determined by ELISA.Results:1.The protective effects of cordycepin on DA neurons in MPTP-induced miceCompared with the control group,the distance and time of mouse movement and the latency time on the rod were decreased in the model group,whereas the pole-climbing time was increased(all P<0.01).The TH-positive cells were decreased in the model group(P<0.01).A significant decrease in the TH protein expression was observed in the model group(P<0.01).The level of DAT was significantly decreased(P<0.01).The levels of DA,5-HT and their metabolites were significantly reduced(DA,DOPAC and HVA,P<0.01;5-HT,P<0.05)in the striatum of model mice.Compared with the model group,cordycepin increased significantly the distance of mouse movement at the dosage of 10.0 mg/kg(P<0.01).And it also shortened the pole-climbing time(all P<0.01)and prolonged the latency time on the rod(5.0 mg/kg,,P<0.05;10.0 and 20.0 mg/kg,P<0.01)significantly at three dosages.The results indicated that cordycepin could improve the dyskinesia occurred in MPTP-induced mice,and the effects of 10,0 mg/kg cordycepin were the most significant among three dosages.When mice were treated with cordycepin(5.0 and 10.0 mg/kg),the number of TH-positive cells(P<0,01)and TH protein expression(5.0 mg/kg,P<0.05;10.0 mg/kg,P<0.01)were significantly increased.Cordycepin increased the level of DAT in the striatum at the dosage of 5.0 and 10.0 mg/kg,respectively,compared with those in the model group(both P<0.01).Cordycepin treatment resulted in a significant increase of the levels of DA(5.0 and 10.0 mg/kg,P<0.01;20.0 mg/kg,P<0.05),5.0 and 10.0 mg/kg cordycepin increased the levels of HVA(P<0.05),and 10.0 mg/kg cordycepin also increased the levels of 5-HT(P<0.01).As was mentioned above,the effects of 10.0 mg/kg cordycepin were the best,and its effects at the dosage of 20.0 mg/kg-were the weakest among three dosages.The results suggested that cordycepin could reduce the MPTP-induced loss of DA neurons within a certain dose range.2.The protective mechanisms of cordycepin on DA neurons in MPTP-induced mice(1)Effects on cell apoptosis:TUNEL experiment results showed that the majority of the apoptotic cells in the SN were karyopyknosis and the bodies of cells were round or oval in the model group.The numbers of apoptotic dopaminergic neurons in the model group were found to be significantly greater than those in the control group(P<0.01).Western blot analysis revealed that the expression of caspase 3 and cleaved caspase 3 were significantly up-regulated,respectively,in the model group(both P<0.01).Compared to the model group,cordycepin induced significant decreases of apoptotic cells(5.0 and 10.0 mg/kg,P<0.01;20.0mg/kg,P<0.05).However,cordycepin(5.0,10.0,20.0 mg/kg)significantly down-regulated the level of cleaved-caspase 3 expression(all P<0.01),whereas the expression of caspase 3 protein showed no significant changes(P>0.05).The results suggested that cordycepin might reduce the apoptotic cells in the SN via inhibiting the activity of caspase 3.(2)Effects on antioxidase activity:Compared with the control group,the activities of SOD(P<0.01)and GSH-Px(P<0.05)were also reduced,and the level of MDA in the model group was significantly increased(P<0.01).Cordycepin markedly increased SOD and GSH-Px activities at the dosage of 5.0 and 10.0 mg/kg(all P<0.01),and also decreased MDA levels at the dosage of 5.0 and 10.0 mg/kg(both P<0.05).It was worth mentioning that cordycepin treatment with 20.0 mg/kg just displayed the similar improvement trend without reaching significance(P>0.05).(3)Effects on ERS:The levels of GRP78,CHOP and Caspase12 protein expression in model group were increased compared to the control group(all P<0.01).The levels of GRP78 mRNA and CHOP mRNA in the model group were markedly increased,compared to the control group(both P<0.01).Cordycepin treatment significantly down-regulated the levels of GRP78 protein(10.0 mg/kg,P<0.01;5.0 and 20.0 mg/kg,P<0.05),CHOP(5.0 and 10.0 mg/kg,P<0.01;20.0 mg/kg,P<0.05),and Caspase12(10.0 mg/kg,P<0.01;5.0 mg/kg,P<0.05).Cordycepin significantly decreased the expression levels of GRP78 mRNA at the dosage of 5.0(P<0.05)and 10.0 mg/kg(P<0.01),respectively,compared to the model group.Cordycepin significantly decreased the expression levels of CHOP mRNA at three dosages(5.0 and 10.0 mg/kg,P<0.01;20.0 mg/kg,P<0.05).Cordycepin treatment with 20.0 mg/kg also decreased the levels of GRP78 mRNA whereas there was no statistical significance(P>0.05).The results suggested that cordycepin could inhibit ERS.(4)Effects on inflammation:Compared with the control group,the microglial cells had more branches and shortened processes,and the astrocytes became more coarse and radial in the SN of the model group.The number of Iba-1 and GFAP immunopositive cells were increased significantly(both P<0.01).Likewise,the levels of iNOS and NF-?B p65 protein expression were increased(both P<0.01),and the levels of TNF-? mRNA and IL-1? mRNA were markedly increased(both P<0.01)compared to the control group.The levels of IL-1?(P<0.05)and TNF-a(P<0.01)also increased significantly in the blood.Compared to the model group,cordycepin induced significant decreases of Iba-1 and GFAP immunopositive cells(all P<0.01).Cordycepin treatment significantly down-regulated the level of iNOS and NF-?B p65 protein(5.0 and 10.0 mg/kg,P<0.01;20.0 mg/kg,P<0.05).Cordycepin significantly decreased the expression levels of TNF-? mRNA and IL-1? mRNA(all P<0.01).Cordycepin treatment resulted in a significant decrease of the levels of TNF-a(P<0.01)and IL-1?((5.0 mg/kg,P<0.05;10.0 mg/kg,P<0.01)in the blood.Among the above effects,10.0 mg/kg cordycepin had the strongest effects.The results suggested that cordycepin could reduce MPTP-induced neuroinflammation and peripheral inflammation.Conclusions:Cordycepin has protective effects on DA neurons in MPTP-induced PD mice within a certain dose range,which may be related to inhibiting cell apoptosis by the increase of antioxidase activity and the inhibition of ERS and inflammation... |
PDF Full Text Request