Mechanism Study Of The Protective Effect Of Baohuoside I Against MPTP/MPP~+-induced Dopaminergic Neurons Injury

Parkinson’s disease（PD）is the second most common neurodegenerative disease,which is characterized by progressive degeneration and loss of dopaminergic neurons and the accumulation of Lewy bodies in the substantia nigra（SN）.The pathogenesis of PD is extremely complex including mitochondrial dysfunction,oxidative stress et al.Epidemiological investigation shows that PD has gender differences,and the prevalence rate of males is significantly higher than that of females,indicating the neuroprotective effect of estrogen.Current studies suggest that estrogen can exert neuroprotective effects through genomic and non-genomic pathways.The genomic pathway is related to the activation of the classical intracellular estrogen receptor（ER）,while the non-genomic pathway is mediated by G protein-coupled estrogen receptor（GPER）on the plasma membrane.However,the long-term use of estrogen may cause serious side effects on the human body.Therefore,we’ll screen the phytoestrogen with estrogenic activity from traditional Chinese medicine and systematically study its neuroprotective mechanism.It will be of great significance for the popularization and application of traditional Chinese medicine.Baohuoside I is one of the main pharmacological active components of Herba Epimedii,which has estrogen-like activity.Many studies have confirmed that Baohuoside I has many pharmacological effects,such as anti-tumor and anti-osteoporosis and so on.In the nervous system,Baohuoside I can antagonize the cognitive impairment caused by oxidative stress.However,the neuroprotective effect of Baohuoside I on dopaminergic neurons and the possible mechanism have not been reported.Objective:1.To investigate whether Baohuoside I can antagonize the injury of dopaminergic neurons induced by the neurotoxin.2.To investigate whether the neuroprotective mechanism of Baohuoside I is related to estrogen receptor signal pathway.Methods:1.The neuroprotective effect of Baohuoside I was studied by using 1-methyl-4-phenylpyridinium（MPP⁺）-induced cell injury in SH-SY5Y cells.2.MTT assay was used to detect the effect of different concentrations of Baohuoside I（0.01,0.10,1,10,20μmol/L）on cell survival and the optimal concentration of Baohuoside I against MPP⁺-induced cell injury.3.Western blot was used to detect the protein expressions of Bcl-2,Bax,caspase-3 andα-synuclein as well as the blocking effect of GPER specific blocker G15 and ER specific blocker ICI182,780.4.GPER^+/+（wild-type）mice and GPER^-/-（gene knockout）mice were injected intraperitoneally with neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）to establish PD mice model.Baohuoside I（10 mg/kg）was administered intragastrically to observe its protective effect on dopaminergic neurons.5.The rotarod test and pole test were used to detect the behavioral function of mice.6.The protein expressions of Bcl-2,Bax,TH andα-synuclein in SN of mice were detected by western blot.Results:1.Mechanism study of Baohuoside I against MPP⁺-induced SH-SY5Y cell injury（1）MTT results showed that low concentrations of Baohuoside I（0.01,0.10,1μmol/L）had no significant effect on cell viability,while high concentrations of Baohuoside I（10,20μmol/L）significantly damaged the cell（P<0.001）.MPP⁺treatment caused obvious cell injury（P<0.001）,while pretreatment with 0.01,0.1,1μmol/L Baohuoside I significantly inhibited MPP⁺-induced cell injury（P<0.01,P<0.001）.（2）Western blot results showed that compared with the control group,the protein expression of Bcl-2 was decreased significantly（P<0.05）and the protein expression of Bax was increased significantly（P<0.01）.0.1μmol/L Baohuoside I pretreatment significantly inhibited the decrease of Bcl-2 protein expression induced by MPP⁺（P<0.05）.0.01μmol/L and 0.1μmol/L Baohuoside I pretreatment significantly antagonized the increase of Bax protein expression induced by MPP⁺（P<0.05,P<0.001）.In the follow-up experiment,0.1μmol/L Baohuoside I was selected.（3）To investigate whether the neuroprotective effect of Baohuoside I is related to signaling pathway mediated GPER or ER,the cells were pretreated with G15 or ICI182,780.The results showed that the protective effect of Baohuoside I on the protein expressions of Bcl-2,Bax and caspase-3 induced by MPP⁺was significantly decreased in the presence of G15（P<0.05）,but it was not affected by ICI182,780.Baohuoside I could significantly inhibit the MPP⁺-induced increase ofα-synuclein protein expression,which could be blocked by both G15（P<0.05）and ICI182,780（P<0.001）.The results suggest that GPER plays a major role in the anti-MPP⁺neurotoxicity of Baohuoside I on the cellular level.2.Mechanism study of Baohuoside I against MPTP-induced dopaminergic neurons injury in PD miceTo further clarify the role of GPER in the neuroprotective effect of Baohuoside I against PD,Baohuoside I was administered orally and the protective effects on dopaminergic neurons in wild-type and GPER gene knockout PD model mice were observed.（1）The results of the rotarod test showed that there was no significant difference between GPER^+/+and GPER^-/-mice in the control group.Compared with the control group,MPTP could significantly cause dyskinesia in GPER^+/+and GPER^-/-mice（P<0.001）,and GPER gene knockout did not affect the sensitivity of mice to the neurotoxin MPTP.Compared with the MPTP group,the dyskinesia of GPER^+/+mice was significantly improved after administration of Baohuoside I（P<0.01）,but there was no significant improvement in GPER^-/-mice.（2）The results of the pole test showed that the T-turn time of GPER^+/+and GPER^-/-mice in the MPTP group were significantly extended compared with the control group（P<0.05,P<0.001）,and the T-total time was also extended（P<0.01,P<0.05）.Compared with the MPTP group,the T-turn time of GPER^+/+mice was significantly shortened after administration of Baohuoside I（P<0.05）,but there was no significant improvement in GPER^-/-mice.（3）The results of western blot showed that the protein expression of Bax andα-synuclein were significantly increased（P<0.001）,while the protein expression of TH and Bcl-2 was significantly decreased（P<0.001）in SN of GPER^+/+and GPER^-/-mice in MPTP model group.Baohuoside I could antagonize the changes of the target proteins in the SN of GPER^+/+mice induced by MPTP,but the neuroprotective effect of Baohuoside I was significantly attenuated in GPER^-/-mice.Conclusions:Baohuoside I can inhibit MPTP/MPP⁺-induced neuronal apoptosis and protect dopaminergic neurons by activating the estrogen signaling pathway.Estrogen membrane receptor GPER is involved in the major neuroprotective effect of Baohuoside I.This study provides new experimental data for the neuroprotective target and the possible mechanism of Baohuoside I.