| Parkinson's disease(PD)is the second most common neurodegenerative disorder after Alzheimer's disease.The major clinical motor symptoms of PD include tremor on a rest,lowness of movement,rigidity and postural instability.Epidemiologic study has reported that PD affects more than 1% of the population over the age of 60,and the incidence of PD also increases with age.It is estimated that the number of PD patients in China will increase to 5 million in 2030,which is more than 50% of the total number of PD patients in the world.Since the etiology and pathological mechanism of PD have not yet been fully elucidated,effective treatments for PD is still limited.The pathological characteristics of PD include the selective loss of dopaminergic neurons in the substantia nigra,and the formation of Lewy bodies,whose major component is aggregated ?-synuclein in the dopaminergic neurons.Autophagy is the most important routes for cells to degrade the damaged organelles and long-lived proteins(such as ?-synuclein).Aging,genetic and environmental factors could cause autophagy dysfunction,which will not effectively remove damaged organelles and toxic protein aggregation;damaged organelles and toxic protein aggregation will further damage autophagy function,which thus forms a vicious circle and results in degeneration of dopaminergic neurons.Dysfunction of autophagy has been found in the brains of PD patients and animal models of PD.Also,PD-related cell and animal experiments have revealed that the overexpression of autophagy-related genes or small molecule autophagy inducers could effectively promote the clearance of ?-synuclein and protect dopaminergic neurons.Therefore,screening for new small molecule autophagy inducers is suggested to be an effective strategy for preventing or treating PD.In the previous studies,we have firstly identified an oxindole alkaloid inhibitor of the mammalian target of rapamycin(mTOR)--Corynoxine,from Chinese herbal medicine Uncaria rhynchophylla,which could promote the clearance of ?-synuclein via inducing autophagy in the PD cell models.However,the neuroprotection of corynoxine have not been evaluated in a PD animal model.In this study,we constructed a PD animal model in the 10-week-old male C57BL/6n mice which were orally administrated with rotenone.The classic mTOR inhibitor--rapamycin acted as a positive control of autophagy inducer,and the madopar acted as a positive control of PD treatment.All of the drug or compounds including corynoxine were orally administrated to the C57BL/6n mice.Firstly,all of the mice were randomly divided into 6 groups: the black control group(control group),rotenone-induced PD mice model group(rotenone group,30mg/kg),madopar group(rotenone+madopar group,1.95mg/piece),rapamycin group(rotenone+Rapmycin group,10mg/kg),low-dose corynoxine group(rotenone+Cory-L,5mg/kg),and high-dose corynoxine group(rotenone+Cory-H group,10mg/kg).And then the neuroprotection of corynoxine will be evaluated by the followed experiments.The pole test and the rotarod test are emoloyed to detect the motor function of mice at the end of the fifth and ninth weeks.High-performance liquid chromatography(HPLC)is employed to detect the concentration of dopamine in the striatum.Immunohistochemistry is employed to detect the tyrosine hydroxylase(TH)positive cells in the substantia nigra.And immunofluorescence is employed to detect the activation of microglia.The detailed results are as following:1.The pole test:At the end of the fifth weeks,the pole test was performed.There was no significant difference in the turning time between the control group and the rotenone group(F=3.082,P>0.05,n=13-18),however,the climbing time was significantly increased in the rotenone group compared with control group(F=9.746,P<0.001,n=13-18).There was no significant difference in the turning time between the madopar group and rotenone group(P>0.05,n=13-16),however,the climbing time of the madapor group was significantly shorter than that of the rotenone group(P<0.05,n=13-16).There is no significant difference in both of the turning time and climbing time between the other three groups and rotenone group(P>0.05,n=8-13).Also,there is no significant difference in both of the turning time and climbing time between madopar group and corynoxine groups(P>0.05,n=11-16),as well as between rapamycin group and corynoxine groups(P>0.05,n=8-11).At the end of the ninth weeks,the pole test was performed again.Both of the turning time(F=18.60,P<0.001,n=13-18)and climbing time(F=5.056,P<0.01,n=13-18)were significantly increased in the rotenone group compared with control group.Compared with the rotenone group,both of the turning time and climbing time in madopar group were significantly shortened(P<0.001,n=13-16),the turning time of the other three groups(rapamycin group,low and high-dose corynoxine group)were also significantly shortened(P<0.001,n=8-13),however,there was no significant difference in climbing time between the other three group and rotenone group(P>0.05,n=8-13).There is no significant difference in both of the turning time and climbing time between madopar group and corynoxine groups(P>0.05,n=11-16),as well as between rapamycin group and corynoxine groups(P>0.05,n=8-11).2.The rotarod test:At the end of the fifth weeks,the rotarod test was performed.Compared with the control group,the movement time on the rotating rod was significantly shortened in the rotenone group(F=2.726,P<0.05,n=13-18);compared with the rotenone group,there was no significant difference in the movement time maintained by the other four groups(madopar group,rapamycin group,low and high-dose corynoxine group)of mice on therotating rod(P>0.05,n=8-16).There was no significant difference between madopar group and corynoxine groups(P>0.05,n=11-16),as well as between rapamycin group and corynoxine groups(P>0.05,n=8-11).At the end of the ninth weeks,the rotarod test was performed again.Compared with the control group,the movement time on the rotating rod was significantly shortened in the rotenone group(F=3.514,P<0.01,n=13-18).Compared with the rotenone group,the movement time maintained by the other four groups of mice on the rotarod was significantly increased(P<0.05,n=8-16).There was no significant difference between madopar group and corynoxine groups(P>0.05,n=11-16),as well as between rapamycin group and corynoxine groups(P>0.05,n=8-11).3.Detection of the concentration of dopamine and its metabolite in the striatumHPLC was employed to detect the concentration of dopamine and its metabolite content in the striatum.The results showed that the concentration of dopamine(F=4.551,P<0.05,n=8-11),as well as homovanillic acid(HVA)(F=6.264,P<0.01,n=8-11)and2-Hydroxyphenylacetic acid(DOPAC)(F=4.476,P<0.05,n=8-11)were significantly decreased in the rotenone group compared with control group.Compared with the rotenone group,the concentration of dopamine(P<0.05?P<0.01,n=5-10),as well as HVA(P<0.01?P<0.05,n=5-10)and DOPAC(P<0.001?P<0.05,n=5-10)were all significantly increased in the other four groups.There was no significant difference between madopar group and corynoxine groups(P> 0.05,n=6-10),as well as between rapamycin group and corynoxine groups(P>0.05,n=5-6).4.Detection the number of dopaminergic neurons in the substantia nigraImmunohistochemical staining was performed to detect the number of TH-positive cells in the substantia nigra of mice.The results showed that: the number of TH-positive cells in the rotenone group was significantly reduced compared with the control group(F=8.692,P<0.05,n=5-7).The numbers of TH positive cells in the other four groups were significantly increased compared with rotenone group(P<0.05,P<0.001,rapamycin group: n=3,other groups: n=5-6).There was no significant difference between madopar group and corynoxine groups(P>0.05,n=5-6),as well as between rapamycin group and corynoxine groups(P>0.05,rapamycin group: n=3,other groups: n=5).5.Detection of the number of active microglial in the substantia nigraImmunofluorescence staining was performed to detect the activation of microglia in the substantia nigra.The results showed that the number of activated microglia in the rotenone group was significantly increased compared with the control group(F=13.48,P<0.01,n=5-7).Compared with the rotenone group,the numbers of activated microglia in the other four groups were significantly reduced(P<0.01?P<0.001,rapamycin group:n=3,other groups: n=5-6).Compared with the rapamycin group,the number of activated microglia in the high-dose of the corynoxine group was significantly reduced(P<0.05,rapamycin group: n=3,other groups: n=5),and there was no significant difference between rapamycin group and low-dose of corynoxine group(P>0.05,rapamycin group:n=3,other groups: n=5).In summary,corynoxine play a neuroprotective effect on rotenone-induce PD mice model by improving the movement function,increasing the concentration of dopamine in the striatum,decreasing the loss of dopaminergic neurons and inhibiting the activation of microglia. |
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