Analysis Of Neurotransmitters And Metabolites In Brain Tissue Of PD Mouse Model Intervened By Salidroside

Parkinson's disease(PD)is a neurodegenerative disorder of central nervous system,which mainly occurs in the middle aged and elderly people.Its characteristic clinical manifestations are tremor,muscular rigidity,unsteady walking,etc.,and seriously affect patients' normal life.A loss of dopaminergic neurons lead to decrease of dopamine is the direct cause of PD,which generally considered associate with oxidative stress,aging,environmental impact,familial factors and so on.Current PD medication is levodopa replacement therapy,but long-term use alternative drugs also initiate resistance and side effects.Surgery,like deep brain stimulation,also has difficulties and risks.So Chinese medicine treatment of PD caused researchers' interests.Studies have shown that Salidroside(Sal),the active ingredient in traditional Chinese medicine Rhodiola,can effectively against oxidative stress and protect dopaminergic neurons.Therefore,the establishment of a rapid,controllable,efficient and stable method for determining the content of DA and related neurotransmitters(5-HTP?DOPAC?5-HIAA?HVA)before and after the intervention of Sal,is of great significance in studying neuroprotective effect of Sal on PD.This paper includes the following four sections:1.Literature reviewThe clinical symptoms,etiology,pathogenesis and treatment of PD were described in this chapter,especially the protective and therapeutic effects of Sal on nervous system.Commonly used content determination methods of neurotransmitters in recent years and their characteristics were also reviewed.2.Establishment and evaluation of MPTP-induced PD mouse modelMPTP-induced PD mouse model was established on the basis of that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)can lead to apoptosis of dopaminergic neurons.The behavioral and neuronal morphologic evaluation of this model was also carried out.Hanging and climbing pole test results showed that,there was obvious movement coordination disorder on mouse after the MPTP were injected.Intervened by Sal of different concentrations,the symptoms of PD were improved.The immunofluorescence histochemistry results showed that number of TH-positive cells and DAT-positive neurons in the substantia nigra of MPTP group was significantly reduced comparing with the control group.But after Sal of different concentration were injected,decrease of TH-positive cells and DAT-positive neurons slowed down,positively correlated with the concentration of Sal.This illustrated that Sal can significantly alleviate PD symptoms,inhibit the reduction of TH-positive cells and DAT-positive neurons and protect nervous system from PD.3.Establishment of analysis method of five neurotransmitters and metabolites in mouse brain tissueA qualitative and quantitative analysis method of five neurotransmitters and metabolites in mouse brain tissue by RP-HPLC-UV was established.A VP-ODS C18 column was employed and mobile phase A was water +0.1%TFA,mobile phase B was methanol +0.1%TFA,flow rate was 1.0 mL/min,detection wavelength was 280 nn.Five neurotransmitters and metabolites(DA,5-HTP,DOPAC,5-HIAA,HVA)were completely separated under the above conditions.Precision testing,stability testing and recovery testing results showed that the peak area and the concentration of reference substances had a good linear relationship in their measurement range,the recoveries were between 91.5%to 96.0%and the relative standard deviation was less than 5.0%.It indicated that this method was simple,reliable,reproducible and appropriate for the neural biochemical evaluation of MPTP-induced PD mouse model.4.Content determination and identification of neurotransmitters and metabolites in mouse brain tissueThe content of five neurotransmitters and metabolites in brain tissue of PD mouse model before and after the intervention of Sal were measured,which based on the established qualitative and quantitative analysis method of neurotransmitters and metabolites by RP-HPLC-UV.Compared with reference substances,the components(DA,5-HTP,5-HIAA and HVA)in samples was confirmed by LC-MS and ESI-MS.Results showed that the content of DA,5-HTP,5-HIAA and HVA in striatum of MPTP group was significantly lower than that of control group.After different concentration of Sal was injected,the content of each neurotransmitter increased and was proportional to the concentration of Sal.It indicated that Sal can significantly inhibit the content decrease of neurotransmitters in MPTP-induced mouse brain and have a certain neuroprotective effect on PD.